Progress in Medicinal Chemistry 12
Progress in Medicinal Chemistry 12
This Page Intentionally Left Blank
Progress ...
17 downloads
930 Views
21MB Size
Report
This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form
Progress in Medicinal Chemistry 12
Progress in Medicinal Chemistry 12
This Page Intentionally Left Blank
Progress in Medicinal Chemistry 12 Edited by G . P. ELLIS,
D.SC., PH.D., F.R.I.C.
Department of Chemistry, University of Wales Institute of Science and Technology, King Edward VII Avenue, Cardig, CF1 3NU
and G . B. WEST, B.PHARM., D.SC., PH.D., F.I.BIOL. Department of Applied Biology, North East London Polytechnic, Romford Road, London E l 5 4LZ
1975
NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM * OXFORD AMERICAN ELSEVIER PUBLISHING COMPANY, INC. - NEW YORK
0North-Holland Publishing Company - 1975, Ali rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or b y any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner.
LCCN: 73-86078 North-Holland ISBN for the series: 0 7204 7400 0 North-Holland ISBN for this volume: 0 7204 7412 4 American Elsevier ISBN: 0 444 10880 7
PUBLISHERS:
NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM NORTH-HOLLAND PUBLISHING COMPANY, LTD.-OXFORD
SOLE DISTRIBUTORS FOR THE U S A . AND CANADA:
AMERICAN ELSEVIER PUBLISHING COMPANY, INC. 52 VANDERBILT AVENUE, NEW YORK, N.Y. 10017
Printed in the Republic of Ireland
Preface We have pleasure in presenting the eight reviews which make up this volume. Six of these cover the application of physical, chemical and enzymological techniques to biological problems. Biochemists, pharmacologists and toxicologists are finding increasing use for methods which have hitherto been used mainly by chemists, and the aim of the first six reviews is to allow biologists to assess the potential value of the techniques in their own work. The reviews also serve to summarize recent progress and therefore should be of value both to those who are already active in the fields covered and those who are about to enter them. One of the most successful recent applications to biology has been the combined use of gas-liquid chromatography and mass spectrometry (Chapter 1 by A. M. Lawson and G. H. Draffan). Recent advances in the biological applications of column chromatography are reviewed in Chapter 2 by K. W. Williams and R. C. Smith. Nuclear and electron magnetic resonance spectroscopic techniques, which are so valuable in organic chemistry, are being applied to biological problems and are discussed in Chapters 3 and 4 (P. J. Sadler, and D. L. Williams-Smith and S. J. Wyard, respectively). Polarography also is being applied to the analysis of certain types of biological material (Chapter 5 by M. Biezina and J. Volke). Methods of determining nucleotides, in particular cyclic AMP and the enzymes concerned with its formation and breakdown, are surveyed in Chapter 6 by B. G. Benfey. The remaining two chapters are concerned with the fight against pathogenic bacteria. One of the most troublesome of these is Pseudomonas aeruginosa and the means by which its depredations may be minimised are reviewed in Chapter 7 by R. B. Sykes and A. Morris. Finally, J. Cs. JBszber6nyi and T. E. Gunda (in the first of two reviews) survey the multitude of penicillin and cephalosporin antibiotics which have recently been synthesized in this topical and important field. We wish to thank our authors for their work, the owners of copyright of diagrams for permission to reproduce and the publishers for their co-operation. G. P. Ellis G. B. West
April 1975
V
This Page Intentionally Left Blank
Contents 1. Gas-Liquid Chromatography-Mass Spectrometry in Biochemistry, Pharmacology and Toxicology A. M. Lawson, Ph.D. Division of Clinical Chemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, England G. H. Draffan, Ph.D. Department of Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Road, London, W12 OH5, England
1
2. Recent Advances in Column Chromatography K. W. Williams, B.Sc. Miles Laboratories Ltd., Slough, England R. C . Smith, Ph.D. Miles Laboratories Inc., Elkhart, U.S.A.
105
3. NMR Spectroscopy in Biological Sciences P. J. Sadler, M. A., D.Phil. (Oxon). Department of Chemistry, Birkbeck College, University of London, London, W C l E 7HX, England
159
191 4. Electron Spin Resonance in Medicinal Chemistry D. L. Williams-Smith, B.Sc., Ph.D., and S. J. Wyard, B.A., Ph.D., D.Sc. Physics Department, Division of Biological Sciences, Guy’s Hospital Medical School, London Bridge, London, SE1 9RT, England
5. Polarography in Biochemistry, Pharmacology and Toxicology M. Biezina and J. Volke J. Heyrovskj Institute of Physical Chemistry and Electrochemistry, Czechoslovak Academy of Sciences, Opletatova 25, 11000 Prague 1, Czechoslovakia
247
6 . Methods Related to Cyclic AMP and Adenylate Cyclase
293
B. G. Benfey, M.D., DipLChem. Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada vii
7. Resistance of Pseudomonas neruginosa to Antimicrobial Drugs R. B. Sykes, B.Sc., Ph.D. and A. Morris, B.Pharm., Ph.D. Glaxo Research, Greenford, Middlesex, England
333
8. Functional Modifications and Nuclear Analogues of p-Lactam Antibiotics-Part I J. Cs. Jhzberknyi, Ph.D. Institute of Organic Chemistry, L. Kossuth University, H -401 0 Debrecen, Hungary T. E. Gunda, Ph.D. Antibiotics Research Group of the Hungarian Academy of Sciences, Institute of Organic Chemistry, L. Kossuth University, H-4010 Debrecen, Hungary
395
Index
479
viii
Contents of earlier volumes VOLUME 5 POLYPEPTIDE ANTIBIOTICS OF MEDICINAL INTEREST-R. 0. Studer NON-STEROIDAL ANTI-INFLAMMATORY DRUGS-S. S. Adams and R. Cobb THE PHARMACOLOGY OF HEPARIN AND HEPARINOIDS-L. B. Jaques THE HISTIDINE DECARBOXYLASES-D. M. Shephard and D. Mackay PSYCHOTROPIC DRUGS AND NEUROHUMORAL SUBSTANCES IN THE CENTRAL NERVOUS SYSTEM-J. Crossland 6 THE NITROFURANS-K. Miura and H. K. Reckendorf 1 2 3 4 5
VOLUME 6 1 THE BRITISH PHARMACOPOEIA COMMISSION-G. R. Kitteringham 2 PHARMACOLOGICAL ASPECTS O F THE CORONARY CIRCULATION-J. R. Parratt 3 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTERESTPart I-C. C. Cheng 4 THE MECHANISM OF ACTION OF SOME ANTIBACTERIAL AGENTS-A. D. Russell 5 THE BIOSYNTHESIS AND METABOLISM OF THE CATECHOLAMINESM. Sandler and C. R. J. Ruthven 6 THE LITERATURE OF MEDICINAL CHEMISTRY-G. P. Ellis VOLUME 7 1 SOME RECENTLY INTRODUCED DRUGS-A. P. Launchbury 2 THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES-John H. Montgomery 3 THE CHEMISTRY OF GUANIDINES AND THEIR ACTIONS AT ADRENERGIC NERVE ENDINGS-G. J. Durant, A. M. Roe and A. L. Green 4 MEDICINAL CHEMISTRY FOR THE NEXT DECADE-W. S. Peart 5 ANALGESICS AND THEIR ANTAGONISTS: RECENT DEVELOPMENTSA. F. Casy 6 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTERESTPart 11-C. C. Cheng and Barbara Roth VOLUME 8 1 ORGANOPHOSPHOROUS PESTICIDES: PHARMACOLOGY-Ian L. Natoff 2 THE MODE OF ACTION OF NOVOBIOCIN-A. Morris and A. D. Russell 3 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTERESTPart 111-C. C. Cheng and Barbara Roth 4 ANTIVIRAL AGENTS-D. L. Swallow 5 ANTIFERTILITY AGENTS-V. Petrow 6 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA-R. M. Pinder 7 THE PROSTAGLANDINS-M. P. L. Caton ix
VOLUME 9 1 NATURALLY-OCCURRING ANTITUMOUR AGENTS-K. Jewers, A. H. Machanda and Mrs. H. M. Rose 2 CHROMONE-2- AND -3-CARBOXYLIC ACIDS AND THEIR DERIVATIVESG . P. Ellis and G. Barker 3 4-OXOPYRANOAZOLES AND 4-OXOPYRANOAZINES-MisbahulAin Khan 4 ISOTOPE TECHNIQUES IN THE STUDY OF DRUG METABOLISMY. Kobayashi and D. V. Maudsley 5 THE PHARMACOTHERAPY OF PARKINSONISM-R. M. Pinder 6 ADRENOCHROME AND RELATED COMPOUNDS-R. A. Heacock and W. S. Powell VOLUME 10 1 MEDLARS COMPUTER INFORMATION RETRIEVAL-A. J. Hartley 2 THE USE OF ENZYMOLOGY IN PHARMACOLOGICAL AND TOXICOLOGICAL INVESTIGATIONS-W. G. Smith 3 THE METABOLISM AND BIOLOGICAL ACTIONS OF COUMARINSG. Feurer 4 CARCINOGENICITY AND STRUCTURE IN POLYCYCLIC HYDROCARBONS-D. W. Jones and R. S. Matthews 5 LINEAR FREE ENERGY RELATIONSHIPS AND BIOLOGICAL ACTIONK. C. James 6 RECENT ADVANCES IN THE SYNTHESIS OF NITRILES-G. P. Ellis VOLUME 11 1 STEREOCHEMICAL ASPECTS OF PARASYMPATHOMIMETICS AND THEIR ANTAGONISTS: RECENT DEVELOPMENTS-A. F. CASY 2 QUANTUM CHEMISTRY IN DRUG RESEARCH-W. G. Richards and M. E. Black 3 PSYCHOTOMIMETICS OF THE CONVOLVULACEAE-R. A. Heacock 4 ANTIHYPERLIPIDAEMIC AGENTS-E.-C. Witte 5 THE MEDICINAL CHEMISTRY OF LITHIUM-E. Bailey, P. A. Bond, B. A. Brooks, M. Dimitrakoudi, F. D. Jenner, A. Judd, C. R. Lee, E. A. Lenton, S. McNeil, R. J. Pollitt, G. A. Sampson and E. A. Thompson
X
Progress in Medicinal Chemistry-Vol. 12, edited by G. P. Ellis and G. B. West @ 1975-North-Holland Publishing Company
1 Gas-Liquid ChromatographyMass Spectrometry in Biochemistry, Pharmacology and Toxicology A. M. LAWSON, Ph.D. Division of Clinical Chemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ G . H. DRAFFAN", Ph.D. Department o f Clinical Pharmacology, Royal Postgraduate Medical School, Ducane Road, London W12 OHS 2
INTRODUCTION INSTRUMENTATION AND TECHNIQUES Basic principles of mass spectrometry Ionization Mass analysis Detection and recording Inlet systems Combined gas chromatography-mass spectrometry GC-MS interfacing Derivative formation Type of columns Gas chromatography-high resolution mass spectrometry Computer methods Basic systems Data processing Computer-aided identification Computer-aided interpretation Special techniques Selected ion monitoring Stable isotopes Alternative methods of ionization
*
2 2 3 4 6 7 7 7 11 15 16 18 19 21 23 24 25 25 30 33
Present address: Inveresk Research International, Inveresk Gate, Musselburg, EH21 7UB, Scotland.
1
2
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
APPLICATIONS Biochemistry Amino acids Peptides Steroids Lipids Carbohydrates Inborn errors of metabolism Pharmacology and toxicology Drug metabolism and disposition Prostaglandins Biogenic amines Clinical and forensic toxicology Environmental toxicology
37 37 37 40 46 53 57 61 68 69 75 79 84 86
REFERENCES
88
INTRODUCTION Gas chromatography-mass spectrometry (GC-MS) combines a versatile method for the separation of mixtures of organic compounds with one of the most sensitive and generally effective techniques for their detection and structural characterisation. These capabilities are potentially of value in several fields and particularly in the biological sciences where there has been a rapid growth of interest in the applications of the technique since its commercial introduction in the mid-1960’s. This article considers some of these applications as they relate to biochemistry, pharmacology and toxicology with the intention of demonstrating the present scope of the methods in these areas. Principles of mass spectrometry and GC-MS are briefly described and emphasis throughout placed on developments concerned with the role of computers in data analysis, selected ion monitoring methods, the use of stable isotopes, and alternative ionization processes. The literature coverage is not intended to be complete and the reader is referred to general reviews [ 1-41 and to specialist journals, e.g. [5-71for a broader appreciation of current work. The proceedings of the principal symposia on various aspects of this field [&lo] and several excellent books are available covering GC-MS [ l l ] and the biochemical applications of MS [12, 131. INSTRUMENTATION AND TECHNIQUES BASIC PRINCIPLES OF MASS SPECTROMETRY
A mass spectrum is produced by ionizing the molecules of a compound
A. M. LAWSON AND G. H. DRAFFAN
. 3
and separating and recording the positive ions which are formed. In general the relative abundance of the different ions are characteristic of a particular compound and are reproducible for a given set of instrumental conditions. In addition, the total number of ions formed from a sample is directly proportional to the sample concentration. Thus, both qualitative and quantitative measurements are possible with the technique. A further important aspect of the abundances of ions is their relationship to the initial structure of the sample molecule. It is often possible to deduce this structure, or elements of it, by comparison of the MS fragmentation reactions of related compounds. Examples of mass spectra are shown in Figures 1.6, 1.12 and 1.13. The reasoning used in the interpretation of a mass spectrum is based on the accumulated knowledge from the rationalisation of fragmentation mechanisms of known compounds and supported by labelling studies and the accurate mass measurement of ions. Detailed information of such mechanisms can be found in specific textbooks [ 141 and throughout the literature. There are several textbooks which discuss the physical principles involved in mass spectrometry and the fundamental aspects of mass spectrometer design and operation [15-17]. However, it is worthwhile considering briefly the way in which the mass spectra of organic compounds are produced and recorded, together with the instrumentation and techniques most commonly employed in GC-MS. Ioonization
The requirement that the compound to be analysed must be in the vapour phase in the ion source confers a considerable limitation on the range of compounds which can be studied. The ion source operates at a pressure in the region of lo4 to 10" mm Hg and at temperatures up to 350°C. The temperature is maintained at a level which prevents adsorption of the sample but which is not high enough to create thermal decomposition. The usual method of ionizing organic molecules, after suitable introduction and vaporisation, is electron impact ionization. (Further ionization methods will be discussed in Special techniques, p. 33). This is accomplished by an electron of sufficient energy colliding with a sample molecule (M) and forming a positive ion by removal of an electron i.e. M+e+M"+2c In the process the bombarding electron transfers excess energy to the ion which, if sufficient,results in the instability of atomic bonds leading to the formation of fragment ions. Although a variety of atomic and
4
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
molecular species can be formed in these processes, only the range-of positive ions produced is commonly considered. This is achieved by employing a positive source potential which neutralises negative ions and has no effect on neutral, radical or molecular entities. -Figure 1.1 shows the arrangement of an electron impact ionization assembly. Electrons are produced by the heated filament, collimated by a magnetic field from a small pair of magnets and passed through the impact region to be collected, if not in collision, by the anode. The ions formed by bombardment are repelled out of the source and accelerated by the positive ion block voltage normally of several thousand volts.
anode ion source block -Yi-:&?m
b fi
object slit I
+
++ ion beam
Figure 1.1. Schematic drawing of electron impact ion source
Mass analysis Magnetic deflection and quadrupole filtration are the two principal methods of mass analysing or separating the positive ions with respect to their mass-to-charge ratios. Time-of-flight instruments are also suitable for GC-MS use but they have several disadvantages which limit their application. In magnetic deflection (the most widely used method), the accelerated ions are focused by the magnetic field (Figure 1 . 2 ) but are deflected by different amounts dependent on their momenta and charge. The mass-to-charge ratio is related to the other parameters by the equation: m H2R2 e - 2V
where R = magnet radius V = accelerating voltage H = magnetic field
Changing either H or V alters the deflection path of the ions. In normal
A . M. LAWSON A N D G. H. DRAFFAN V
lighter ions
heavier ions magnet (H)
Figure 1.2. Ion flight path in 60" sector magnetic instrument
operation V is kept constant, while the magnetic field is varied over a sufficient range to allow all ions to be consecutively focused on a single collector. The mass resolution of magnetic instruments is expressed as M/AM where AM is the mass difference between mass M and the next higher mass from which it is being separated. An overlap of the two peaks leading to a 10% valley has been selected arbitrarily for a working definition of unit resolution. Several factors affect the practical resolution attainable by a single sector instrument such as the object and collector slit dimensions and the field radius. However the ultimate resolution is limited by the kinetic energy spread of monoisotopic ions and the angular dispersion of the beam. A radial electrostatic field can be used to counteract these factors to give higher resolutions. Figures 1.3a,b show the electrostatic analyser and magnetic sectors in the Nier-Johnson and Mattauch-Herzog geometries for high resolving power. The latter can focus ions in a plane and permit photographic detection. a
p-3
b
collector
analyser
--I fB
IS
-I-
collector y
Figure 1.3. Double sector geometries a) Nier-Johnson and b) Mattauch-Herzog
6
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
The quadrupole filter separates ions by virtue of their stability in the alternating electric fields created in a square array of four electrodes (Figure 1.4) by particular combinations of radio frequency ( U ) and d.c. ( VO)voltage applied to diagonal pairs of the electrodes. A small voltage is used to introduce the ions into the quadrupole filter in the overall direction of the Z-axis. The ratio U l V , is held constant but increased in amplitude to allow increasingly larger ions to survive passage through the filter (see [18] for review). An advantage of quadrupole analysis in GC-MS applications is the ease and speed with which U and Vo can be manipulated. Y Z
X
Figure 1.4. Quadrupole assembly showing hyperbolic fields
The resolution achievable by a quadrupole filter depends on the selection of the U / V , ratio and can thus be adjusted throughout the mass range. For this reason the resolution varies throughout a scan of the mass range, with AM in the resolution equation remaining constant.
Detection and recording The low ion currents of the ion beam and the requirement of fast scan rates for GC-MS necessitates the use of a high current amplification detection device. Secondary electron multipliers are employed for this and achieve gains in the region of lo6. The output from the multiplier is amplified and passed to a recorder with adequate frequency response to cope with the rapidly acquired mass spectrum. Oscillographic recorders are suitable with a series of galvanometers operating at attenuations which allow signals varying by three to four orders of magnitude to be recorded and measured. Many instruments have electronic devices to superimpose a mass scale on the record. In a later section the recording and manipulation of spectra by digital computer are discussed.
A. M. LAWSON A N D G . H. DRAFFAN
- 7
Inlet systems Although this article is concerned with gas chromatographic introduction, a variety of other inlet systems are available which are often necessary for handling biomolecules. Direct introduction probe. The solid sample to be analysed is placed in a gold or glass crucible at the tip of a probe which can be introduced through a vacuum lock into close proximity with the ionizing electron beam. The temperature of the probe tip is controlled to maintain a steady evaporation rate of the sample. Although the direct probe gives rise to mixed spectra when more than one component with similar vapour pressures is present, its utility in handling certain samples is indispensible. It is an efficient method of sample introduction with respect to sample utilization, and it can handle material both too labile or too nonvolatile for other methods. Several instances of its use are referenced in the applications section. Reference inlet. This is a small heated reservoir from which reference materials can be leaked into the ion source for use as mass calibration standards or to assist in resolution and focusing adjustments. Batch inlet. This classical method of sample introduction is still a very useful and necessary inlet for both liquids and gases giving a steady sample flow rate. However it requires fairly large samples of materials which are thermally stable and of adequate vapour pressure. Mixed spectra result from multicomponent samples. COMBINED GAS CHROMATOGRAPHY-MASS SPECTROMETRY
GC-MS interfacing The interpretation of a mass spectrum is more readily made when it can be assumed that all the major ions are derived from a single molecular species. Samples of biological origin are seldom obtained pure and frequently many components in the final extract are of interest. Gas chromatography is well established as a versatile means of resolving complex mixtures of structurally related compounds, and the major attraction of the mass spectrometer to the medicinal chemist is that it can operate on line to a gas chromatograph. While the commercial interfacing of GC and MS is now commonplace, the link between the two remains the most critical stage in the combined operation and a likely source of trouble. An in depth review by McFadden [ 113 covers most practical and
8
i
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
theoretical aspects of GC-MS interfacing. More general review articles of GC-MS technique are also available e.g. [4,19,20]. This section summarises the characteristics of the interfaces most commonly employed in biomedical GC-MS. Samples sufficiently volatile for gas chromatography are readily handled by mass spectrometry. The two instruments are chiefly incompatible in the vast difference in operating pressures: 760 mm Hg at the column exit and 10-5-10-6mm Hg in the analyser of the mass spectrometer. There are two solutions; the vacuum system can be designed to accommodate a substantial fraction of the column effluent or a ‘molecular separator’ can be employed to enrich sample relative to carrier gas effecting pressure reduction prior to transmission to the ion source. A flow split at the column exit and a direct link to the mass spectrometer provides the simplest means of sample introduction. However, it is only recently that trends in instrument design have included provision for high speed pumping of the ion source so that carrier gas flows of 5-8ml/min can be accepted without loss of spectrometer sensitivity and resolution. A high conductance differentially pumped system allows all of the effluent from most types of capillary columns to be utilised and the discard rate from the lower flow packed columns may be acceptable [21]. Where wide bore, high flow rate, packed columns are used, or where the pumping configuration is of conventional design, a molecular separator must be employed as a means of sample enrichment. Assuming that the resulting pressure is within the operable limits, the most important parameter in separator evaluation is the yield or transfer efficiency, defined as the fraction of sample leaving the column which reaches the ion source. Three types of unit are in common use and several variants of each are commercially available. Enrichment may be achieved by; 1) fractionation of the gas stream by diffusion from an expanding jet; 2) removal of either sample or carrier gas by diffusion through a semi-permeable membrane; or 3) differential effusion through a porous tube or narrow slit. The two stage jet separator designed by Ryhage [20,22,231 is represented in Figure 1 . 5 ~The . column effluent is forced through a constriction, d l , into a chamber evacuated to viscous flow conditions. Helium, as carrier gas, diffuses from the line of the expanding jet and the heavier organic molecules of the sample, enriched in the core of the jet, are collected by the orifice d2.A similar enrichment process occurs between d, and d4. Performance depends critically on dimensions, alignment, flow rate and pumping configuration. A transfer efficiency of 40% was deter-
A. M. LAWSON AND G . H. DRAFFAN
Enriched sample TO MS
From GC
h Y
Porous
restriction
To pump
Spiral cavity
@
Figure 1.5. Molecular separators a) Two stage jet separator due to Ryhage Typical dimensions are: d, = d3 = 1 l Y cm, d2 = 2.5 x 1F’cm, d, = 3 x 1F2 cm, I, = 1.5 X 1 P cm, I, = 5 x 10-’ cm. The first chamber is evacuated with a mechanical pump and the second by diffusion pump. 6 ) Silicone membrane separator[26]in which the GCeffuent is channelled in a spiral path in contact with the membrane surface. c) Watson-Biemann fritted glass separator [28]
mined between 27 and 50ml/min for an early version of the two stage separator [23]. This type remains restricted under patent to one instrument company, but single jet versions are now also commercially available, e.g. [24]. Sample transfer can be higher, however the single stage units are generally used at lower column flow rates unless the mass spectrometer has a high conductance pumping system. The utility of jet separators in a wide range of application has been proved. Semi-permeable barrier separators may be based either on preferential diffusion of carrier gas through a membrane as in the Teflon separator [25] or on preferential diffusion of sample in the silicone rubber separators. The former type has not found general application but there are several variations of the silicone membrane separator (for original design references see [ l l ] . The principle is illustrated in a version shown in Figure 1.5b [26].Organic materials are removed from the gas stream by passage through a thin (0.0025 cm) barrier of dimethyl silicone polymer in which
10
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
inorganic carrier gases have a low solubility. Sample transfer efficiency can be as high as 90% but it is impossible to obtain an optimum performance for a range of unknowns. Thus, for highest solubility and therefore efficiency,the temperature should be low, a recommended 50°C below the boiling point [26], but for high diffusion rates and therefore sharp chromatographic peaks the temperature should be as high as possible. These separators can be used with capillary effluents but some loss of chromatographic resolution should be anticipated [27]. In the Watson-Biemann effusion separator [28] (Figure 1.5c), the chromatographic effluent enters a porous glass tube through a restriction which reduces the pressure sufficiently to establish conditions of molecular flow through the fine (approx. cm) pores of the glass. The flow rate through the porous wall for each component in the effluent is then proportional to the partial pressure difference and inversely proportional to the square root of the molecular weight. Both factors favour removal of helium as carrier gas, and higher molecular weight organic sample is enriched in the gas passed to the mass spectrometer. There have been many design variations using the principle of effusive removal of carrier gas (for original references see [l I]) and efficiencies range between 10 and 50%. Fixed geometry results in a critical gas flow dependence, and an interesting (commercially available) modification is the variable conductance separator [29] in which effusion occurs through a small adjustable slit between two plates. The slit separator is reported to operate efficiently over a wide range of flow rates. GC-MS interfacing merits serious attention when efficient sample transfer is to be achieved consistently. The situation is less problematic now than it was four or five years ago when biological chemists, reluctant to speculate in interface development, frequently limited their attention to one company whose patented jet separator was the most reliable version available. Elsewhere, the gas chromatograph tended to be regarded as a troublesome alternative inlet added as an afterthought. All commercial systems now function. However, it should be noted that most units have a defined range of flow rates for optimum performance. Further, variation in the transmission of sensitive components may be encountered. Where it is intended to apply GC-MS to a broad spectrum of compound classes and to use both packed and capillary columns, one flexible solution is to consider an instrument with high speed pumping capacity suitable for direct coupling, and to retain one of the fixed geometry separators as an alternative inlet. The predicted ideal path for minimum sample loss, when handling labile
A. M. LAWSON AND G. H. DRAFFAN
11
compounds, is the shortest possible direct ‘line-of-sight’ link between column exit and ion chamber, unencumbered by separator, valves or flow restrictors. For some applications, by-pass valves are desirable; for example, detection of minor constituents can be impeded by the admission of solvent or major components to the ion source producing prolonged background effects. Attention should be paid to the type and siting of valves as potential sources of sample loss and, as a general rule, the interface should be kept as simple as possible. Individual systems are best checked by careful consideration of dilution curves for labile compounds. In such an evaluation of sample degradation. GC conditions must first be at their optimum as the interface is commonly blamed when the chromatography is at fault.
Derivative formation The preparation of chemical derivatives is standard practice in gas chromatography in order to increase the volatility of polar compounds, provide a guide to identification by incrementing retention time, and introduce responsive groups for use with selective detectors. In GC-MS, volatility and good chromatographic performance should ideally be combined with an informative mass spectrum. Thus derivatives may be employed ;i) to provide further structural information when the spectrum of an unknown cannot be fully interpreted or; ii) to increase sensitivity and specificity when the mass spectrometer is used as a selective gas chromatographic detecto; in an ion monitoring mode. Applications taken from the prostaglandin field serve to illustrate these two approaches while many further examples for specific compound classes are cited throughout this article. The identification of an unknown component in a biological extract may result from the recording of a single mass spectrum and the achievement of a good library match. Much more commonly a single spectrum serves only to indicate possible structural features and may not even establish a molecular weight. Based on whatever knowledge of the sample is available from the initial class separation, microchemical techniques and complementary derivatives are selected. A meticulous approach to identification based on the use of multiple derivatives is illustrated in the establishment of the dicarboxylic acid (1) as the major urinary metabolite of prostaglandin Fz, in man [301. Some sixteen derivatives were prepared for GC-MS in deduction and confirmation of the structure, and comparison of the spectra of derivatives ( 2 ) , (3) and (4)
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
12 0 R'
cx,",,, \.
(1) R ' = ~ 2 H =
\
(2) R1 = TMS, R2= Me (3) R1 = TMS-dg, R2= Me (4) R' = TMS, R2= Et
CO;!R2
CO, R2
6R'
j j o
I ' I
L!?
COzR'
(5)R' = R 2 = H, X = 0 (6)R ' = Me, R2= AcO, X = NOMe (7) R'= Me, R2= TBDMS, X = NO-TBDMS
I
6 R2
I
bR2
indicate the type of information obtainable from these low resolution spectra. Methylation and trimethylsilylation, (a standard opening manoeuvre in the investigation of a polar compound) gave a derivative with a probable moiecular ion of low abundance at m/e 502 (2). Trimethylsilyl (TMS) derivatives rarely give prominent molecular ions but commonly provide indirect evidence of molecular weight by the presence of fragment ions at M-15 (Me) or M-90 ( M e S O H ) . The presence of these ions together with M-(2 x 90) supported the assignment of the molecular ion and suggested a di-TMS derivative. A diol was confirmed by a shift of 18 a.m.u. in the molecular ion of the deuterated (ds) TMS derivative (3) and a dicarboxylic acid by a shift to mle 530 (28 a.m.u.) in the ethyl ester spectrum (derivative (4)).The presence of a keto function was established by the formation of a methoxime derivative. Location of the ketone at C-11 was suggested by the a-cleavage ion a (CO(CHz)4C02Me),incremented 14a.m.u. in the ethyl ester and unshifted in the d,-TMS spectrum. A side chain ketone and dihydroxycyclopentane was further
A. M. LAWSON AND G .
H. DRAFFAN
13
supported by the base peak in each of the spectra, M-(90+b +H), ascribed to p -cleavage with respect to C-1 1, with hydrogen transfer to the fragment containing the keto group. Of interest in this scheme is the use made of a deuterated TMS derivative. This is now a commonly applied means of establishing the total number of reactive groups in a molecule. Hydroxyl, amine, thiol, amide, phenol and acid functions all react with silylating agents. Stable isotope labelled derivatives in general should be considered as offering advantages over homologues (methyl/ethyl, acetyl/propionyl) where it is desirable that fragment ions are incremented in mass without the risk of altering pathways of fragmentation. The provision of structural information by recording a complete spectrum is the conventional role of organic mass spectrometry. The mass spectrometer may also be employed as a selective GC detector (a technique discussed in detail in the Section on Special techniques, p. 25) when one or more ions considered to be characteristic of the compound of interest may be monitored as the sample elutes. When maximum sensitivity is required, the practice is to search for a derivative which will either suppress or have a strong directing influence on fragmentation providing an abundant, preferably high mass ion as the signal for detection. The use of t -butyldimethylsilyl (TBDMS) derivatives for this purpose has recently been investigated [31] since spectra are frequently dominated by facile loss of the t-butyl group providing M-57 base peaks. A further example from the prostaglandin field illustrates the potential importance of these derivatives. A current assay for prostaglandin E2 (5) [32] involves methylation, 0-methyl-oxime formation and acetylation providing the derivative (6) in which the fragment ion M-60 (loss of MeCOOH) is monitored. Although this ion is the base peak, it accounts for only 4% of the total ion current (4% 2). An alternative derivative is obtained by oximatjon with hydroxylamine, followed by formation of the TBDMS ether yielding the ester (7) in which M-57 is 24% 2. Monitoring this ion, 10 pg of PGEz injected on to the gas chromatograph is detectable. A more striking example [31] of fragment ion stabilisation by loss of the t-butyl radical was observed with the methyl ester, oxime, TBDMS derivative of 1S-oxo-13,14-dihydroprostaglandinFZa (8), the major blood metabolite of PGF,,. There are two reaction products, the syn- and anti-oximes, and both show relatively simple spectra (Figure 1.6). In the spectrum of one of the isomers (lower panel of Figure 1.6), greater than 95% of the total ionization is accountable by t-butyl loss. A search for stabilising derivatives of biomolecules, often intrinsically labile on electron impact, provides a novel test of ingenuity for organic mass spectros-
'1
669
1
700
0
100
I
200
I
L
300
I
m/e
400
500
60 0
I
700
Figure 1.6. Electron impact spectra of isomeric (synlanti) oxime-t- butyldimethylsilyl derivatives of 13,14-dihydro-f5-ketoPGF,,. Both as the base peak (reproduced from reference 1311) spectra are dominated by loss of the t-butyl radical giving m / e 669 (M-57)
A. M. LAWSON AND G . H. DRAFFAN
15
copists. An alternative approach lies in the use of the ‘soft’ ionization methods, as discussed in the Section on Alternative methods of ionization (P. 33).
Types of columns In GC-MS when dealing with multicomponent samples the choice of the column type depends largely on the separation required. Ideally each homogeneous peak should have base line separation from all others to give mass spectra which present the best chance of either interpretation or comparative identification. Other important considerations are sample utilisation, loading factors and the concentration range of the components requiring analysis. As all column types can be interfaced with a mass spectrometer using the correct pumping and coupling arrangements, the GC column and conditions can be selected on their merit. Packed columns. Packed columns were introduced by Martin in 1952 [33]. Since then, they have been developed for application to compounds derived from biological sources [34,35] and are now the most widely used columns for gas phase chromatography in this field. Important factors contributing to this are the availability of a range of good thermostable liquid phases, methods of coating appropriate supports [35] and the simplicity of use. The analysis of high boiling and sensitive compounds is not satisfactory on packed metallic columns due to irreversible adsorption and decomposition in some cases. Properly treated glass columns are less prone to these effects and hence preferable for general biological samples. A variety of column dimensions has been used, most commonly 0.2-0.4 cm ID and from 1-5 m long. The flow rate of carrier gas (helium) in a packed column has an optimum setting for maximum separation efficiency. However, the proportion of a sample peak reaching the ion source across the GC-MS interface is also flow dependent. A compromise between column separation efficiency and sample transfer is not often difficult to reach. A 2 m column with a flow-rate of 25 ml/min operates at 2000-3000 theoretical plates. Although many hundreds of stationary phases are available the majority of separations are carried out on only a handful of different phases. The choice of phase is, of course, dependent on the components to be resolved but consideration has to be given to the column bleed rate. The common phases and details of bleed characteristics are described elsewhere [I 11.
16
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
Columns of between 0.1 cm and 0.06 cm ID, using carefully graded small particle size support material can be prepared up to 30 m in length [36]. Although these micropacked columns have theoretical plate values much higher than packed columns (15000 plates for 5 m length) their adequate preparation is difficult and as a result they are still not widely used. This problem may be overcome in the future and with high efficiency and high sample loads they may be preferable to their rival capillary columns in some applications. Capillary columns. Open tubular or capillary columns permit a relatively uniform distribution of liquid phase on the column surface and an unrestricted flow of carrier gas. These columns can thus be made of much longer lengths than packed columns and have more theoretical plates per unit length. The catalytic inertness of glass columns for high temperature work is an attractive advantage over metal capillaries. However, improved methods of coating the glass surface have been necessary to avoid degradation of the liquid film. Several types of surface modification have been employed [37,38] such as etching or deposition of very fine inert supports before application of the phase. The latter are referred to as support coated open tubular (SCOT) or by others as porous layer open tubular (PLOT) columns. They have a higher sample capacity than liquid coated columns due to the greater amount of phase per unit length. Several recent papers have described modified methods of column preparation [39-411 using Silanox 101 as the support. It is evident that many workers in GC-MS use columns of inadequate efficiency to handle complex mixtures and attempt to improve this situation by careful analysis of the MS data. This undoubtedly can lead to omissions and inaccurate identifications and it seems likely that in the future capillary columns, once their preparation is routine, will be very widely used. Figure 1.7 shows the chromatographic profiles of a derivatised urine steroid extract on packed and capillary columns. The vastly improved resolution in the latter instance is immediately obvious.
Gas chromatography -high resolution mass spectrometry (GC-HRMS) In high resolution mass spectrometry a mixed spectrum of an internal standard, normally perfluorkerosene, and of the sample is taken. Using the known masses of the internal standard, the accurate mass values of the sample ions are calculated. Their elemental compositions can then be determined by searching for the best fit of combinations of atomic mass
A. M. LAWSON AND G . H. DRAFFAN
17
Figure 1.7. Separation of trirnethylsilyl ethers o f infant urinary steroids on 1) packed (9f t x 4 m m , 2% SE-30, 190-27S"C programmed at t.S"C/min) and 2) open iubular (80f t x 0.3mm, OV-101, 160-270°C programmed at 2S0C/min f o r 30min and l°C/min for 2Smin) columns
values for each accurate mass. Such calculations are conveniently undertaken by computer, as they are too time consuming by hand. The final output from the HRMS scan is the list of the peaks present and their intensities, accurate mass values and elemental compositions. Although knowing ion compositions is no guarantee of identification of the compound in every case, when coupled with other chemical and physical information a structure can usually be assigned. The peak matching technique [42] is the other frequently used method for making accurate mass measurements. It utilises the relationship between the ratio of the accelerating voltages required to focus a reference mass and a sample ion at the same point on the detector, and the ratio of the masses, i.e.
18
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
When an unknown compound requires identification, if it is easily purified, it would be introduced on a direct insertion probe and accurate measurements made by peak matching or computer scan. However GC-HRMS is necessary when the unknown is a component of a complex mixture. In HRMS the resolution and scan speed limit the accuracy of mass measurement. Sufficient samples need to be taken by the computer recording system across each ion peak to establish good peak shapes and hence accurate peak centres. The duration of a GC peak in the source can be too short to allow a complete mass scan while satisfying the required statistical conditions. The usual solution is to limit the mass scan. Some of the problems of electrical detection are not present in photographic recording [43] which has considerable potential in GCHRMS particularly when using capillary columns. The photographic plate integrates all ions in the spectrum simultaneously and gives greater sensitivity than electrical detection for GC peaks of short duration. Photoplates have the attendant problems of handling and development and require further expensive equipment for evaluation. This has placed them outside the interest of many workers who argue that the long term solution for GC-HRMS lies in improved detection and computer interfacing systems. It is significant that the majority of biological applications of MS are successfully undertaken with low resolution instruments (approx. R P 1000). Among the reasons for this is that often only known compounds for which reference standards are available, require to be detected or identified. However, when a completely unknown compound is encountered, accurate mass measurement is often the most effective way of elucidating the structure. Such studies can be undertaken by single sector instruments operating at medium resohtion (approx. RP < 10000) which, although less sensitive and accurate than larger machines, give adequate results as long as no insufficiently resolved multiplets are encountered. The application of GC-HRMS to the SIM technique (see Selected ion monitoring, p. 25) has the advantage of increasing specificity when handling multicomponent mixtures, but suffers from decreased sensitivity and the requirement for more costly equipment. Several examples are cited in later sections. COMPUTER METHODS
Over the last few years, the coupling of a computer or computing facilities to GC-MS systems has become commonplace and indeed essential in
A. M. LAWSON AND G. H. DRAFFAN
. 19
laboratories handling large numbers of multicomponent samples. This has contributed to a wider acceptance and use of GC-MS, especially by those involved with biologically derived material. The reasons for using a computer for acquiring and processing mass spectra are self-evident and include a saving in time and effort coupled with improved accuracy and efficiency. It can be argued that, for a small dedicated computer, the overall costs are reduced when the capital outlay is set against the salaries of personnel to carry out the task manually. The fallacy in this argument is that a laboratory in a research environment with a GC-MS computer system is inclined to tackle a greater number of problems with samples of higher complexity. The real cost saving is in increased efficiency and one has to decide if this capacity is necessary in relation to the objectives of the laboratory. The primary role of the computer system monitoring the spectra from a GC-MS run is to assign mass values to each ion peak, subtract background peaks where necessary, correct for intensity bias and then print out or graphically present normalised spectra. These form the basis of an MS analysis. Other important uses of the computer in the manipulation of this data are discussed later.
Basic systems Commercial data systems are now available for all the principal GC-MS instruments although a number of independent centres have developed their own systems to suit individual requirements [44]. These range from low cost off-line systems to dedicated mini computers linked to timeshared central computers. Many of these systems are in advance of commercial packages but the different capabilities of their configurations are outside the scope of this article. (For a review of computerised data acquisition and handling see [45]).However it is useful to consider some of the hardware components and requirements of a small on-line computer system (Figure 1.8) for low resolution GC-MS work. Analogue signals from the electron multiplier (SEM) generated by a magnetic scan are converted to digital form before passing to the central processing unit (CPU). The digitisation rate of the analogue to digital (A/D) converter in the interface is selected in conjunction with both the scan rate and the resolution of the MS. In high resolution MS this is particularly important in obtaining good digital peak shapes for accurate peak centre determinations. Other electronic features such as the band pass and impedance of the intermediate amplifier have to match the
20
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
Mass Spectrometer
Voltage
,
Computer C ont r ol
Data Storage
Programme Storage Output Devices
Figure 1.8. Components of basic computer system for GC-MS
sample rate and input impedance respectively of the AID converter. In most systems the interface contains a switching device to multiplex other signals from the MS such as the total ionization and magnetic field sensing voltages used in certain aspects of data processing. Directed by an operator from an input terminal, the data are processed in the CPU under the control of programmes stored in a peripheral device. Most of the early systems are not capable of real time mass conversion and initially store raw data in the partially reduced form of intensities and peak centre times. After acquisition the latter are converted to mass values using a mass calibration table, generated from a scan of known standard masses. On a quadrupole instrument the standard masses are stored in conjunction with the rod voltages in the calibration table. This greatly facilitates the speed of mass conversion, making it achievable in real time and allows the display of a spectrum immediately after the scan. When multiple scans are made, the total processing time is considerably reduced, a fact which has been recognised in current computer system design for magnetic instruments and real time mass conversion is now widely available. Peripheral data storage devices have to satisfy the requirement of fast access time with the capacity to store a sufficient volume of data. Magnetic tape systems are normally used although considerable time saving can be achieved with disc units. Software programmes are initially loaded into their storage facility from prepared paper or magnetic tape.
A. M. LAWSON AND G. H. DRAFFAN
.2 1
The choice of output devices is often governed by their high cost. A flexible configuration in a laboratory, heavily involved in GC-MS, should probably include a teletype, a visual display with hard copier, a fast bar graph plotter and, if possible, a line printer.
Data processing Several standard programmes are essential in all computer systems for GC-MS application. These range from programmes for data acquisition, mass calibration, spectrum subtraction and presentation of the spectra in various forms on a specified output device. An increasing variety of others for more specialised uses are available. Among these are programmes for the processing of data for multiple spectra, obtained by repetitively scanning the MS during the GC run [46,47]. The several hundred spectra which can result from repetitive scans are best stored on fast access discs for rapid processing. Individual magnetic tape decks, although leading to an increase in processing time, have greater capacity and permit permanent storage of data when necessary. The first step in analysing these data is to identify the position of each scan in the chromatographic trace. This is achieved by plotting the scan number against the summation of the ion intensities for each scan. The resulting reconstituted total ionization chromatogram can then be presented on a video screen (e.g. Figure 1.9, H) and from this, the scans of particular interest can be selected and printed out. The repetitive scan mode of operation when investigating complex chromatograms of largely unknown material has the advantage that the sample need only be run through the GC-MS once. A further important aspect is that the data can be examined in total and the changing intensities of individual ions or related groups of ions followed throughout the run. The resulting output is commonly termed a ‘mass chromatogram’ [48]. The general approach can be demonstrated with reference to a recent study of a complex mixture of methyl esters of fatty acids isolated from an acid catalysed methanolysis of cat brain galactocerebrosides [49]. The mixture was treated with a trimethylsilylation reagent to convert hydroxy functions to the corresponding TMS ethers, and then submitted to GC-MS using repetitive scanning. Different classes of fatty acids were detected by searching the accumulated data for the presence of ions characteristic of these classes. Figure 1.9 shows some of the resulting mass chromatograms. The location of three different 2-hydroxy acids
22
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
h A
I
G
383
I I
H
0
411
40
80
120
160
200
t. i.c.
240
280
Scan number
Figure 1.9. Mass chromatograms from a fatty acid extract of cat brain galactocerebrosides (TMS-methyl ester derivatives) run on a 2 m X 3 mm column of 3% EGSS-X on Supelcoport 100/120, programmed from 130-200°C. The chromatograms are of m/e 382 (A), m/e 354 (B), m/e 326 (C) and m/e 74 (0).The (M-59)' ions at m/e 411 (E) for methyl 2-trimethylsilyloxytetrucosanoate, at m/e 397 (F) for methyl 2-trimethylsilyloxytricosanoate and m/e 383 (G) for methyl 2-trimethylsilyloxydocosunoateas shown with the reconstructed TIC chromatogram (H)(adapted from reference 1491)
was found by plotting the ion intensities of the (M-59)' ions together with the trimethylsiloxy ion, rnle 73 (not shown), (E, F and G in Figure 1.9). Individual normal acids were identified by plotting the molecular ions (A, B and C in Figure 1.9) and ions rn/e 74 and 87 (not shown). The choice of ions which may be informative is not often an easy one and depends on a considerable pre-knowledge of the fragment ions from
A. M. LAWSON AND G . H. DRAFFAN
. 23
the compound class under study. A useful selection is easier to make in retrospect after detailed study of a sample, but once made can be applied to similar samples in the future. Nevertheless, when the sample is unknown or contains unsuspected components, objective principles are necessary for ion selection. One approach [48] uses the computer to calculate the highest normalised intensity and the sum of normalised intensities of each mass throughout the entire chromatogram. The former indicates, for a given mass, the spectra in which the mass is important and the summed intensities orders the importance of the mass relative to all scans. The masses for mass chromatograms can then be chosen from the important ions, which are normally related to homologous series or common structural features, and from the significant ions, which are present in only a few spectra and are generally characteristic. Although the use of the repetitive scanning technique t o generate profiles of compound classes in physiological tissue and fluids has value in the preliminary stages of an investigation and specialised instances, the ultimate level of sophistication is the automatic identification and quantitation of every component in the mixture. Quantitative aspects are discussed in relation to the SIM method, see Selected ion monitoring (p. 25). The present situation regarding the identification of peaks by comparison of the MS scan with a reference file is encouraging in cases where there is adequate GC resolution. However, the mixed spectra resulting from poor separation of peaks create problems especially when analysing the minor component of a mixed peak. The maximum use of the existing GC resolution can be made by monitoring a predetermined number of fragment ion currents [ e g . 501. When an increase in these intensities followed by a decrease is detected a peak maximum is found and as long as these maxima are separated by at least one scan the relative retention values of each can be assigned. Suitable subtraction routines can then give corrected spectra. The value of retention data calculated by the computer is increasingly recognised [50-531. A greater degree of specificity in the identifications is achievable and more efficient use of the library file can be made by searching only for specific compounds in small retention zones. Computer-aided identification A large computer is required for fast and efficient searching of extensive mass spectral data files. Using a small computer, the search time is normally much longer due to the limited blocks of data which the C P U
24
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
can handle at any one time. Nevertheless, such systems are infinitely preferable to manual searching. Several methods are available to reduce the total data from a mass spectrum to a form which can be quickly and usefully employed when dealing with many thousands of spectra. One method is the selection of a fixed number of the most intense peaks in the spectrum and another is the encoding of a fixed number of the most intense peaks in specified mass ranges over the spectrum (e.g. 3 mass values every 20 mass units). The latter has the potential advantage that peaks of low intensity at the higher mass values, which may have structural importance, are often retained in the encoded spectrum. Programme algorithms are considered in detail elsewhere e.g. [54]but in general a similarity index or correlation coefficient has to be calculated for each fit due to variations in spectral data. The latter arise from the differences in spectra on different instruments or under different conditions, from additional components in unresolved GC-peaks and from discrepancies due to concentration changes. A yeslno answer can be more closely approached when all spectra are unique and completely reproducible. The best fits within specified correlations are normally printed out and further criteria have to be applied to these to obtain the final answer. This would be straightforward assuming the correct spectrum was on file. In cases where this is not so, some indication of the class and type of compound may be suggested from the list of best fits. Although individual laboratories find it useful to compile their own reference library files, access to very large collections of mass spectra and to published data [55] is essential. A compilation of many thousands of spectra by the Aldermaston Mass Spectrometry Data Centre and the Division of Computer Research and Technology at the National Institutes of Health [56-58] has been made available commercially. The file can be searched in a number of ways using an interactive conversational mass spectral search system via a teletype and acoustic link over telephone lines.
Computer-aided interpretation The interpretation of an unknown mass spectrum, to a point where a structural assignment is possible, is in many cases difficult without a considerable background knowledge of the mass spectral fragmentation mechanisms and pathways of many classes of compounds. Broadly, two classes of systems have been developed for computer-
A. M. LAWSON A N D G. H. DRAFFAN
25
aided interpretation. Both require large computing facilities and programming effort. In the first e.g. [59],the computer correlates the spectra in the reference file with specific structural features to obtain ‘pattern classifiers’. These are applied to the data of the unknown compound to determine the probability of the related structural features being present in the molecule. Although this method is useful, a large data base is necessary to develop sufficiently reliable pattern classifiers. In the second system, the computer is programmed to follow very similar reasoning processes to the mass spectroscopist by making available to it the extensive body of knowledge currently known about mass spectral behaviour [60,61]. The reduction of the latter to a computer programme represents considerable effort and requires up-dating as further MS data becomes available. McLafferty, Venkataraghavan, Kwok and Pesyna 1621 have described a program they call STIRS (Self-Training Interpretative and Retrieval System) which combines aspects of these two approaches with normal library searching. A variety of spectral data classes which indicate certain structural features is derived from previous knowledge of mass spectral correlations. These data classes are then matched against the file of reference compounds. When a particular structural feature is found to be common to a number of the selected compounds this indicates this feature in the unknown. By combining the available information such as molecular weight and elemental composition with the structural features indicate by each of the data classes, it is hoped to identify the unknown. SPECIAL TECHNIQUES
Selected ion monitoring When low resolution spectra are recorded at the typical rapid scan rates necessary in GC operation, only of the order of 1-5msec is spent in registering each m/e value. By dedicating the mass spectrometer to the detection of either just one or a limited number of selected ion currents, integration is achieved and much less sample is required to produce a response. Detection limits are typically in the range 0.1-1 pmol, this high sensitivity being obtained at the expense of some of the detailed information contained in the complete mass spectrum. Rapid switching between mle values is possible, and, with the incorporation of simple channel separation and smoothing circuits, the output may be in the form shown in Figure 1.10 in which continuous profiles are obtained for each mass
26
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
tfrnin)
5
0
Figure 1.10. Chromatograms obtained by monitoring total ion current (tic) and m/e 617 (M), 603 and 602 (M-Me) in the detection of the pentafluoropropionyl derivative of the alkaloid salsolinol in an extract of urine from a Parkinsonian patient. Response ratios for 617/602 and 6031602 at the indicated point on the chromatograms correspond to these ratios in the authentic substance (adapted from reference [90])
monitored. Application depends on a prior knowledge, or at least a firm prediction of the fragmentation pattern of the samples chromatographed. The method may then be applied as originally described [63] to the resolution of unresolved GC peaks, or extended [64] to the qualitative identification of trace components in complex extracts. Identification involves measurement of peak height or peak area for each ion channel at the expected retention time on the chromatograms. By judicious choice of the masses monitored, significant fragments and their relative abundance
A. M. LAWSON A N D G . H. DRAFFAN
. 27
can be determined thus providing the main elements of the ‘mass spectrum. With the incorporation of appropriate standards, ion monitoring may also be used to obtain precise quantitative data. This important and flexible technique, referred to inter alia as accelerating voltage alternation (AVA) 1631, mass fragmentography [64], selected or multiple ion monitoring and multiple ion detection (MID) is now widely applied throughout biological research. There have been several recent reviews of application and methodology [65-681. T h e proliferation of nomenclature has been pointed out and a case presented for the use of the term ‘selected ion monitoring’ (SIM) as adequately describing the technique [68, 691 a suggestion adopted throughout the present article. Instrumentation. The early development of ion monitoring methods in association with gas chromatography [63,64,70] was done with magnetic sector instruments and much of the recent refinement in technique has been with this design [71-781. Referring to Figure 1.2 and the basic equation mle = H2R2/2V,switching the focus between two or more ions involves alteration of either the magnetic field ( H ) or the accelerating voltage (V). Although magnetic field switching is being developed by some companies, current methods focus the selected ions by maintaining H constant and adjusting V. Commercially available systems now allow for recording of at least four ion current profiles within a mass range of 10-30% and with a typical switching rate of four masseslsec. The mass range in ion monitoring with voltage alternation depends in part on system design, quoted limits being for example 20% [71] approximately 30% [73,75] and SO% [72]. The useful range in practice is determined by the extent of the sensitivity decrease encountered at the lowest voltage (highest mass) and of the change in ion focus which may result at the extreme of the range [68,721. In these respects, the quadrupole design (see earlier discussion of principles) has a decided advantage when used for ion monitoring [66,79]. Switching may be achieved throughout the entire mass range without loss of sensitivity and with rapid stabilisation of the DC and RF fields. An authoritative discussion of the finer points in instrumentation and of the relative merits of quadrupole and magnetic instruments in SIM may be found in the most recent review of the topic [68]. The current trend in SIM technique is toward computer control of the mass spectrometer with concurrent software development allowing data reduction as peak areas and channel ratios. The process of manually distinguishing several ion profiles and of making measurements from an
28
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
oscillographic recording or a multichannel pen recorder is not only tedious but open to subjective error. It is also inflexible in that the choice of channel amplification prior to sampling commits the operator to a single version of hard copy for his measurements. A variety of systems has now been described for initial selection of operating parameters (masses, cycle time, filtering) and subsequent display and manipulation of the data for use with both magnetic [73-781 and quadrupole instruments [SO]. Approaches to automatic focus correction in magnetic instruments include monitoring and compensation for field drift [78] and an ingenious programme involving generation of a sweep across the nominal voltage for each mass selected, sensing of the peak value and adjustment of the accelerating voltage before sampling the signal [74]. A different approach involves narrow mass range repetitive magnetic scanning which will accommodate any slight variation in focus [Sl j. Computer generated voltage variation (up to 2000 V) has also been described and advantages pointed out for this method as an alternative to computer control of the standard mass spectrometer power supplies and switching circuits [75]. It is important to place these recent developments in perspective since the vast majority of laboratories obtain satisfactory data ‘by hand’. However, where there is a heavy commitment to routine assay or a special need for high precision, some degree of automation with computer involvement should be considered. Quantitative measurement by SIM. The rapid growth of interest in SIM is in large part attributable to its potential as a precise measuring technique where more conventional methods lack either sensitivity or specificity. Quantitative assay from biological material is most reliably achieved by measurement of sample response relative to that of a reference compound (internal standard) added at the earliest stage in the separation sequence. Tn an extension of normal gas chromatographic practice, the reference may be a structural analogue of the sample. A common fragment ion may be monitored e.g. [82,83j or different ions used for sample and reference response e.g. [84,85]. In principle, any assay based on flame ionization detection may be extended to SIM with improvement in selectivity and detection limit. There is, however, a further, unique possibility when the mass spectrometer is the detector in that the internal standard can be a stable isotopically labelled form of the sample. The stable isotopes of C , H, N and 0 and their natural abundance are listed in Table 1.1. This method, stable isotope dilution, ensures essentially no distinction between sample and standard throughout the recovery sequence and usually only minimal differences in gas
A. M. LAWSON AND G. H. DRAFFAN
29
Table 1.1. STABLE ISOTOPES OF HYDROGEN, CARBON, NITROGEN AND OXYGEN 1861
Element
Atomic mass (amu1
Natural abundance (atom %)
‘H *H ‘*C !’C I4N ”N ‘“0
1.00783 2.01410 12.00000 13.00335 14.00307 15.0001 1 15.99491 16.99914 17.99916
99.985 0.015 98.89 1.11 99.63 0.37 99.759 0.037 0.204
370
I8O
chromatography. Final differentiation and ratio measurement is made by the mass spectrometer. Measurement by stable isotope dilution was introduced for quantitative SIM using a deuterated methoxime derivative of prostaglandin El [87]. At about the same time, a method was described for measurement of nortriptyline in plasma based on dilution with the deuterium-labelled drug [88]. Where possible, the compound itself should be synthesised with incorporation of label in preference to the use of labelled derivatives; losses in all stages of recovery may then be standardised. Thus, the use of tetradeutero analogues [89] has superceded the initial derivative procedure for assay of the parent prostaglandins. The practice in general has been to multiply-label the internal standard achieving as high an isotopic enrichment as possible, since the sensitivity is in part limited by the residual unlabelled content in setting a ‘blank’ value (see Stable isotopes, p. 30). A substantial excess (10-1000-fold) of the labelled compound can be employed when required as a protective carrier for labile material in extraction and can also provide some improvement in detection limit where adsorptive losses are encountered in carrier-free gas chromatography e.g. [87,90]. Sensitivity. When operated in an ion monitoring mode as a GC detector, a mass spectrometer with standard electron multiplier detection and signal amplification is theoretically capable of producing a response to samples of less than 10-”mol (1 femtomole) [68]. In practice such limits have not been reached due to the combined effects of variable statibnary phase and instrument background, sample degradation in gas chromatography or, in isotope dilution, residual blank contributions from
30
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
carrier. Nevertheless, exceptional sensitivity has been reported, for example, 1 pg (3 x mol) for chlorpromazine [64, 651 1 pg (8 x mol) for chromium [92]. mol) for chloroform [91] and 0.5 pg (1 x As noted in a previous section, for the highest sensitivity in SIM, it is desirable to select derivatives or a mode of ionization which results in a single ion in the spectrum taking the maximum possible fraction of the total ion current. Further, the highest sensitivity is obtained in single ion monitoring and signal-to-noise ratio deteriorates as the number of ions monitored increases. In contrast to the absolute detection limits attainable with pure samples, in quantitative determination from biological material lower sensitivity and precision are usually encountered due to manipulative loss and slight variations in the co-extracted background. Depending on the complexity of the assay, published data generally record precisions of 1-20% for sample quantities in the 5 to 0.1 ng range. When using the isotopically labelled analogue as the internal standard sample size, isotopic abundance and precision are interdependent. Further discussion of mass spectrometric methods of isotope ratio determination, including SIM, is provided in the Section on Stable isotopes. Stable isotopes This section provides a brief guide to biological applications of stable isotopes and to isotopic abundance measurement using mass spectrometry. Shortly after the development of fractionation techniques for the isotopes of carbon, hydrogen, oxygen and nitrogen in the 1930’s, applications of the heavier less-abundant isotopes (Table 1.1) as tracers in intermediary metabolism began to emerge. Stable isotopes have been used to follow incorporation into a metabolic pool and to monitor the fate of a particular atom or molecule in biosynthesis and catabolism, in studies of enzyme kinetics and isotope effects and in quantitative assay using isotope dilution. Further, since there are no long lived radioactive isotopes of oxygen and nitrogen, tracer studies with these elements must be undertaken with the stable isotopes. The use of stable isotopes in general is rapidly expanding. This is attributable to increased production, variety and lower cost of highly enriched substrates and to advances particularly in GC-MS methods of measurement. The following publications relating to biological research using stable isotopes may be consulted: early metabolic studies [931; general application [94-981; phar-
A. M. LAWSON A N D G. H. DRAFFAN
.
31
macology [99]; clinical pharmacology and drug metabolism [66, 100, 1011; biological isotope effects of deuterium [98, 99, 1021; deuterium toxicity [102]; preliminary evaluation of 13Ctoxicity [97]. The proceedings of two recent conferences [94,98] provide a useful guide to trends in the use of stable isotopes in biomedical research. The impetus here is provided by a growing concern over the use of radiotracers in clinical investigation and the continued requirement to study metabolic balance in patient categories, for example in children, where radiotracer use is restricted. In this context, deuterium-labelled bile acids have been employed in studies of the cause and treatment of gallstones [103], in metabolic studies following administration of intermediates [ 1041 and in a determination of bile salt kinetics in newborn infants [lOS]. Among several other preliminary reports is the use of deuterium-labelled f o l k acid as a means of determining pool size and turnover [ 1061 deuterium-labelled metabolites in studies of alanine and glucose turnover [lo71 and [ 1,2-”C] glycine in the investigation of children with several types of inborn errors of metabolism Cl081. Applications of stable isotope labelling in drug metabolism are also being explored as complements to the standard use of 3H and 14C(see later section). Isotope ratio measurement. Studies with stable isotopes depend on separation of a reaction product and determination of isotope ratio as a measure of dilution or incorporation of label. Mass spectrometry, while not the only means of determining isotope abundance can offer higher precision, accuracy and sensitivity than other methods and can also locate the site of the isotope in the molecule. It is, however, impossible to combine each of these functions optimally in a single instrument design. A fundamental problem is the natural background of the heavier isotopes which imposes a limitation on the range of dilution accessible. Thus, since the natural abundance of I3Cis about 1 . I 1%, to measure a 1 in lo3dilution of I3C in a one carbon molecule requires the ability to detect an alteration in abundance to 1.22% (10% change) which is beyond the capacity of mass spectrometers used in conventional scanning mode. The highest precision (but not necessarily accuracy) is obtained by the indirect method of sample isolation and total combustion to a gas, usually hydrogen, nitrogen or carbon dioxide, followed by determination of mass ratio in a dual inlet, double collector mass spectrometer [96]. Precision (*0.001%) is achieved by long sampling times (0.5’-2 min), comparison with a standard gas mixture and the use of milligram quantities of material. In contrast, by recording a complete mass spectrum, a wealth of structural information is available but at the expense of precision. This is
32
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
the standard approach used in organic mass spectrometry in the elucidation of fragmentation mechanisms with specifically labelled molecules, and in tracer studies where dilution is low [95,96]. One means of improving precision in direct isotope ratio analysis (without sample degradation) is to scan repetitively over a narrow mass range. This concept is taken to a logical conclusion in a highly sophisticated computerised scheme involving signal averaging at high resolution using a double focusing mass spectrometer [109]. In measuring the ''N2 content of creatinine in a turnover study involving administration of doubly 15 N-labelled creatinine, 64 electric sector scans (in 40 sec) are averaged through the M + 2 multipiet at a resolution of 15000. The 13C,'s0component of the multiplet provides the internal standard. High dilutions may be distinguished, the limit being 0.002 atom % excess I5N2 with sample consumption of about 25 pg. An alternative approach to direct analysis of isotope ratios is in selected ion monitoring (see also Selected ion monitoring, p. 25) maximising the dwell time on the masses of interest and applicable to much smaller samples. A number of systems have recently been described employing both magnetic [72-781 and quadrupole [79,80, 1101 instruments. A common aim has been improved performance in the ratio determination obtainable by ion monitoring GC-MS. An assessment of potential and limitations has been made using a quadrupole mass spectrometer with a data system enabling peak area integration and ratio measurement. The results of several series of experiments are summarised in Figure 1.11 which shows the dependence of percent accuracy (difference from the calculated value) on sample size and isotope abun-
0.01
0.1
1.0
10
1w
ISOTOPE ABUNDANCE
Figure 1.11. The eflect of sample size and isotope abundance on the accuracy of isotope ratio measurements in GC-MS using a quadrupole instrument and computer data system. The curves are based on nunierous separate studies (reproduced from reference [ I 101)
A . M. LAWSON A N D G. H. DRAFFAN
33
dance. Mixtures of benzene-d6 (mle 84) in benzene (mle 78) were employed in a dilution study in the range 0.03% to 77%. The precision was 0.2-0.5% for abundances greater than 1% with a marked deterioration (attributable to poor ion statistics), below 0.1% abundance to 5% precision at the highest dilution used. In studying the effect of sample size on precision, the methyl ester pentafluoropropionyl derivative of p bromophenylalanine (9) was used, monitoring the m/e 344 and 346 bromine isotope peaks of the M-59 fragment ion. At the 1 pg level, precision was 1.5% while in the nanogram range 0.15% precision was obtained. Equally impressive data on both accuracy and precision are reported for a variety of samples isolated from biological sources prior to GC introduction. p -BrC6H4CH2CH(NHCO-C2F5)C02Me
(9)
It is apparent that, for the majority of studies undertaken with stable isotopes, ion monitoring GC-MS affords the best compromise solution in the inter-relationship between accuracy, isotope dilution and sample size. A degree of structural information and specificity in terms of isotope location in the molecule is also retained as several fragment ions may be monitored in each analysis. Alternative methods of ionization Electron impact (EI) ionization involves the bombardment of vaporised sample with electrons usually in the energy range 12-70eV. Energy transfer is such that the initially formed molecular ions, fragment to give a spectrum reflecting bond cleavage and rearrangement, providing the detailed information on which structural elucidation is based. The molecular ion, the single most useful observation in compound identification, may be absent or of low abundance. Further, when mass spectrometry is used for high sensitivity in selective detection, the production of many fragment ions can be a disadvantage. The chemical ionization (CI) process, now the most widely used complement to EI, results from ion-molecule reaction and occurs with much less energy transfer. Fragmentation is thus both modified and reduced. Generally, an indication of molecular weight is provided and the spectrum may also reveal structural features unnoticed in the conventional E I spectrum. Field ionization (FI) is an alternative low energy ionization process which may produce an enhanced relative abundance of the molecular ion. Both CI and FI have the same requirement as in E I in that the sample must be in the vapour
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
34
phase. The recently introduced technique of field desorption (FD) therefore promises to be of immense value since non-volatile compounds may be ionized. General reviews of ionization processes are available [ l l 1-1 131 and developments in chemical ionization [114-1161, field ionization [ 1 17, 1181 and plasma chromatography [ 1191 are periodically surveyed. Chemical ionization. Chemical ionization spectra result from ionmolecule reaction between the ionic products of a high pressure reagent gas, commonly methane, with a low pressure sample gas. Because of the low abundance of the sample, almost all of the initial ionization by electron impact is of the reagent gas. When methane is ionized at a source pressure of 1 mm Hg, the normal EI products CHI' and CH: react with neutral CH, molecules producing a plasma in which CH: (48% Z) and CzH: (41% 2) are the principal species available for further ion-molecule reaction:
CH: CH:
+ CH, -+ CH: + CH, + CH, -+ CzH: + Hz
The ions CH: and CzH; are strongly acidic and react with the sample molecule chiefly by proton transfer (as Bronsted acids) or by hydride abstraction (as Lewis acids); stable addition products can also be formed:
CH: + M+MH'+ CH, C2H: + M + MH' + C2H4 CzHZ+ M + (M - H)' + CzHs CzH: + M + (M + CzH5)'
proton transfer proton transfer hydride abstraction addition reaction
While fragmentation, depending on the stability of the quasi-molecular ion (M H)' is encountered, an increased relative abundance of these species is to be anticipated in comparison with the molecular ion region in EI (e.g. Figure 1.12). Since total ionization of sample is of the same order of magnitude, a gain in both selectivity and sensitivity can result in CI. The reagent gas is not limited to methane, and among the alternatives are isobutane and ammonia, providing a choice of milder ionizing conditions. Thus in the methane CI spectrum of atropine (10) [120], protonation of the ester by CH: ions, a powerful proton donor, is followed by fragmentation to m/e 124 (81% Z) as the base peak. Using NH: as the reactant species,
*
o.co.cHm.cH20H (10)
A. M. LAWSON AND G. H. DRAFFAN (a) E l
(b) C I
,6
i5% P
3
z40 J
w D !
I
20
99
0 M
1W
180
m ie
200
2%
m ie
Figure 1.12. a ) Electron impact and b) part of the methane chemical ionisation spectra of the drug metoclopramide. In EI, the molecular ion is not observed while in CI the quasimolecular ion is the base peak [I311
protonation of only the most basic group in the molecule, the tertiary amine, results in an abundant quasi-molecular ion (72% 2). The value of ammonia as a reagent for trace detection of basic compounds, particularly many drugs and drug metabolites, has been pointed out [120]. In a chemical ionization mass spectrometer, the vacuum system must be optimised to pump large volumes of gas and the ion chamber designed to maintain a high pressure of the order of 1 mm Hg, of the reagent. The large fast pumping systems required in CI are those now being applied in GC-MS technology (see earlier section) and while the early GC-CT work was done with quadrupole instruments [l IS] which are more readily adaptable to high pressure operation, most of the manufacturers of sector instruments have now produced GC-CT systems. It is desirable that the two modes of operation, ET and CI, should be readily interchangeable in a single instrument. A broader appreciation of CI in organic mass spectrometry and of subtleties in the use of reagent gases may be obtained from the reviews cited. The literature in the biological sciences in considerable. Some recent examples serve to indicate scope in application: biogenic amines [121]; drugs and metabolites [116, 120, 1221 and [123] in which a listing of
36
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
the major peaks in over 300 methane CI spectra is provided; prostaglandins [ 1241; steroids [ 1251 and polychlorinated biphenyls [ 1261. Atmospheric pressure ionization. A mass spectrometer in which sample ionization is achieved at atmospheric pressure (API) in an external source has been described [ 127, 1281 and applications reported [ 1291. The source of electrons is a "Ni foil and samples, injected in organic solvent, are swept through the reaction chamber (ion source) in a stream of nitrogen carrier gas. Ions and neutral molecules then enter a quadrupole mass analyser via a 25 p m diameter aperture. Continuous analysis of the ions, either positive or negative, formed in the reaction chamber, may be achieved by repetitive scanning or by selected ion monitoring. The ion-molecule reactions in the production of sample ions involve the carrier gas and the solvent. Thus the quasi-molecular ion for a compound injected in benzene as solvent may result from the following sequence:
N; +2N2+N: + N , N: + CaH,+ C,H,' + 2Nz charge transfer C6H: + M + MH' + C6H, protonation In a study of MH' detection limits for 2,6-dimethyl-4-pyrone introduced in benzene, 0.15 pg produced a response in selected ion monitoring [ 1281 and 25 pg was detected in scanning using signal averaging [ 1271. In corporation of APT in both liquid and gas chromatographic systems is being investigated. The technique also allows the analysis of biological extracts without a separation stage prior to ionization. Field ionization (FI) and field desorption (FD). Field ionization occurs in the vapour phase in a strong electrical field (107-10sV/cm) usually produced by a blade or wire held at a high positive voltage (7-10 kV). The energy transfer to the molecule during field ionization is about 12-13 eV, close to the ionization potential of most organic molecules. A consequence in many cases is promotion of the relative abundance of the molecular ion. The method has been most valuable for the direct introduction of polar compounds [117] and while it can also be used in conjunction with gas chromatography [130], GC-FI has not found wide application. The absolute sensitivity in FI is 10-100 times less than in ET or CI and technical problems arise in field emitter preparation, stability and memory. For these reasons and because of the restricted number of commercial sources available, FI has not thus far proved as popular as CI as a complement to electron impact in biological research. In a recent review, technical developments are emphasised which may broaden the potential application of FI [ 1 181.
A . M. LAWSON AND G . H. DRAFFAN
-
37
There is currently much interest in field desorption (FD) which describes the process of field ionization from the adsorbed state with desorption as an ion. Field desorption takes place at lower temperatures than are normally required for evaporation of a molecule and since there is also only a low energy transfer in the ionization process itself, molecular ions of high intensity are formed. A number of underivatised polar compounds of low volatility have been investigated without thermal decomposition; these include nucleosides and nucleotides [132], pesticides [ 1331 and glycosides [ t 341. The method has also been extended by combining pyrolysis with FD to distinguish the five bases and some of the nucleotide fragments of deoxyribonucleic acid [ 1351.
APPLICATIONS BIOCHEMISTRY
The applications of mass spectrometry in the general field of biochemistry are numerous and only human biochemical studies and some medicinal and clinical biochemistry are discussed here. These have been further reduced by selecting a limited number of compound types and dealing with investigations within these classifications. Although some important compound classes have had to be omitted, those selected are areas where GC-MS has made, and continues to make, a meaningful contribution. GC-MS is increasingly employed in studies of inborn errors of metabolism. The potential importance of the developing GC-MS methods in this subject, not only for studying the disorders themselves but in applying them to the investigation of normal endogenous metabolism and finally to the diagnosis of diseased conditions, has warranted the inclusion of a separate section on this topic.
Amino acids Amino acids were first studied as their esters by mass spectrometry in the late 1950’s and early 1960’s [136-1381. Although many free amino acids can be directly sublimed and give useful spectra, some decompose on heating while others, most noteably arginine and cystine, pyrolyse. These undesirable features prompted the search for derivatives which would permit either direct or reservoir introduction into the mass spectrometer of as many of the biological amino acids as possible. This was prior to the
38
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
GC-MS combination instruments and the emphasis in these studies was principally in obtaining interpretable spectra. The low volatility of amino acids also prevents their analysis by gas chromatography and in this connection many methods for obtaining derivatives have been described, the most common being esterification of the acid and acylation of the amino group, e.g. [ 139-1411. Derivatives such as the N-trimethylsilyl trimethylsilyl esters [142] N,O-isopropyl esters [143,144], Ntrimethylsilyl butyl esters [145], phenylthiohydantoins [146], and N dimethylaminomethylene alkyl esters [ 1471 have also been investigated with varying degrees of success. Much of the effort in preparing gas chromatographic derivatives was specifically aimed at a GC method for analysing mixtures of amino acids obtained from peptide hydrolysates or other sources. However, no quantitative method, without some disadvantage, has yet been demonstrated for the complete separation of the twenty protein amino acids on a single column. Nevertheless, some attempts approach this ideal, for example, Gehrke and Takeda [ 1481 using N-trifluoracetyl-butyl esters and Zanetta and Vincendon [149] with isoamyl N(0)-heptafluorobutyl ester derivatives. The analysis of trimethylsilylated methylthiohydantoins [150], the silylated products of Edman degradation, by GC is less satisfactory as only 14 of the 20 amino acids have adequate stability. The coupling of mass spectrometry with G C methods adds specificity to the identification of the individual components of these mixtures, increases sensitivity, offers quantitation even in difficult cases, and has particular advantages over other techniques when a new or unusual amino acid is present. There are many examples of the identification of such amino acids in natural product chemistry [l-4,1511. The mass spectral fragmentation reactions of a number of derivatives used in GC-MS have been reported including a series of esters of the N-trifluoroacetates [ 1521 and the pertrimethylsilyl derivatives [ 153-1 551 and their carbon-13- [156] and deuterium-containing analogues [157]. General reviews of the mass spectra of the common amino acids and their derivatives are also available [151, 1581. An example of the sensitivity range which can be achieved by GC-MS is illustrated by the detection and semiquantitative determination of N 6-monomethyllysine and lysine in myosin hydrolysates isolated from heart cultures [159]. There are 620 residues of lysine to one of methyllysine in myosin. Operating in the selected ion mode these two compounds as their TFA-butyl derivatives can be estimated in a single run of injected sample from 4 pmol of myosin.
A. M. LAWSON A N D G. H. DRAFFAN
39.
The increasing availability of amino acids labelled with stable isotopes makes possible a variety of metabolism studies by MS and in addition provides ideal internal standards for quantitative analysis. This latter aspect is made use of in a computerised method for the quantitation of 12 amino acids in biological fluids [ 1601. The mass spectrometer monitored by the computer follows selected ions for each of the 12 amino acids and their deuterated internal standards introduced as their TFA-butyl derivatives. The computer programme analyses the data, subtracting background, detecting peaks, identifying these peaks and finally quantitating each natural amino acid against its internal standard. The method covers only 12 amino acids at present due to the restricted availability of internal standards but its extension to additional compounds in the series is possible. However, arginine, cysteine and histidine are unstable to the GC conditions used. The procedure has been applied to the analysis of control urine and that from a patient with maple-syrup-urine disease. The results from 5 separate analyses in each case gave standard deviations of less than 10% of the mean. This was routinely achieved where the level of an amino acid was about 1 ng. The advantages of using standards labelled with stable isotopes are discussed in the Sections on Selected ion monitoring and Stable isotopes (pp. 25 and 30). Complete separation of the components of the amino acid mixture on the column is not a necessary condition for the quantitation as long as the ions selected for monitoring have no contribution at these values from the non-resolved components. The mass spectrometer used in this work was a quadrupole instrument which permitted rapid switching between masses over the entire mass range. Although it is possible to switch magnetic machines over similar ranges, stepping through 24 masses every 2 s e c as in this case, is not yet possible. The precision and speed of analysis compares favourably with other methods currently in use and its sensitivity should make its further development worthwhile for situations where only small samples are possible or where amino acid concentrations are low. Most work to date on the mass spectrometry of amino acids has employed electron impact ionization. Although this process gives spectra with adequate structural information for most applications in many instances the molecular ions are of very low intensity. As intense M + 1 ions are given by all amino acids using chemical ionization, e.g. [161, 1621 there is little doubt that this process will find wide application in the future [163].
40
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
Peptides Mass spectrometry has been applied to the structure elucidation of peptides and proteins for some time. Depending on the problem, it is used as both a primary and a complementary technique and although many difficulties still remain it is now an established means of determining amino acid sequences. As instrumental methods and new derivatives have developed, the different approaches employed, with particular combinations of chemical pretreatment and sample introduction to the MS, have been dictated by the size and type of the peptide and the information required. Several factors affect the volatility and stability of a peptide derivative, not least of these being the number and nature of the constituent amino acids. Heterocyclic and aromatic amino acids reduce volatility while those containing sulphur tend to decrease the thermal stability. Small naturally occurring peptides which are not derived from proteins often contain only aliphatic amino acids which lack functional groups in the side chains. Peptides of this type of up to about ten amino acids, after conversion to suitable derivatives, are amenable to analysis by mass spectrometry, e.g. [164]. A variety of derivatives has been reported and include N-trifluoroacetyl peptide esters [ 136,1651, N-acetyl peptide esters 1166-1681, aromatic N-acyl peptide esters [169-1721, and permethylated N-acyl peptides [173]. The principal modes of the electron impact induced fragmentation of these peptide derivatives are well established and have been summarised in recent reviews 1174, 1751. Although the spectra of the permethylated derivatives [I761 are perhaps the simplest and easiest to interpret and are now frequently used, the N-acyl peptide esters have been widely and successfully employed. Most small peptides derived from protein sources contain a variety of polar and non-polar amino acids and as such are more difficult to handle. Nevertheless appreciation of the problems and considered chemical manipulation have allowed their MS elucidation [ 177-1 841 although a practical limit of about six amino acids in the peptide is reached before degradative procedures become advisable. An important step before sequencing a peptide by mass spectrometry is whenever possible to obtain the amino acid content by hydrolysis and conventional column chromatographic analysis. This assists in the selection of any chemical pretreatment and with the spectral interpretation. A variety of small peptides has been identified by a combination of methods and include 5 - 0x0 - L - prolyl- L - histadyl- L - prolinamide (2-pyrollidone-5-carboxylyl-
A. M. LAWSON A N D G. H. DRAFFAN
-
41
histidyl-proline amide) as a hypothalmic thyrotropin (TSH) releasing factor (TSF) of ovine origin [185], tentoxin [186] and others, in addition to many peptides isolated from enzymic and hydrolytic action from large oligopeptides. Figure 1.13 shows the spectrum of a hexapeptide [181] as its permethylated derivative with the ions indicating the amino acid order. The rapid decrease in the intensity of the important sequence-determining ions at higher mass is evident and a principal reason why MS often requires several times as much material as the common micro wet chemical methods for sequencing small peptides. The chemical ionization mass spectra of some N-acyl permethylated simple peptides show a much more even distribution of the sequencing peaks and hence require a lower sample level than for EI spectra [187]. This may well prove of value in the future. CT has also been applied directly to peptides [188] where up to six amino acids units have been introduced by the direct insertion probe. Another approach to the sequencing of small peptides or peptide
t
THR-L ,YSO . Mc-
H SI533
5/s Figure 1.13. Mass spectrum of acetylated/permethylated hexapeptide: Val-Leu-Ala-His-ThyLys (reproduced from [ISI])
42
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
terminal sequences has been the MS identification of the residues of the amino acids released by several cycles of Edman degradation, followed by preparation of derivatives and MS analysis of the remaining peptide. The N-methyl- and N-phenylthiourea derivatives of the N-terminal amino acid of a peptide thermally rearrange in the MS ion source to give the thiohydantoin derivative of the terminal amino acid and the shortened peptide [189]. This has been suggested as a sequencing method for the first four acids after which point interference from side products becomes excessive. The first 34 residues of human parathyroid hormone (PTH) [190] were identified from the CT mass spectra of their phenylthiohydantoin derivatives by repeated Edman degradations on a Beckmann Sequencer 11631. In a simiIar way as part of the characterisation of ovine hypothalmic luteinizing hormone-releasing factor (LRF), the trimethylsilyl derivatives of the phenylthiohydantoins from Edman degradation were confirmed by MS [191]. Gray (1921 proposed a method in which by acetylating the initial protein the terminal peptide liberated by specific enzymic degradation was isolated and its permethylated derivative sequenced by mass spectrometry. Although the work described to this point, with the exception of the PTH and LRF studies, has not directly involved gas chromatography, its importance to protein structure determination has warranted its inclusion. The emergence of combined GC-MS did not immediately make an impact in peptide analysis as most workers were intent on extracting sequence information from the mass spectrum of the intact peptide or as large part of it as possible. Such peptides in general were too involatile for gas chromatography. More recently the problem has been tackled in the reverse manner. By hydrolysing the peptide or protein to mixtures of very small peptides containing between two and four amino acid residues and by effecting their separation by GC, the initial sequence can be deduced by identifying the small peptides by MS. The overall amount of peptide required for this procedure is less than when treating the undegraded peptide. This approach has been elegantly demonstrated by Biemann and co-workers [174]. They have adapted one of their earlier methods of modifying peptides [ 1931 by esterification, N-terminal acetylation and then reduction with LiAIH, (or LiAID,) to give the polyamino-alcohol. These steps are preceded by chemical or enzymatic hydrolysis of the protein to di, tri- and tetrapeptides and concluded by 0-silylation of the alcohol and side chain acid or hydroxyl function (see Scheme 1.1).
-
A. M. LAWSON AND G. H. DRAFFAN
Protein
Peptides
e. g. for dipeptide
R'
I
43
(Conventionally separated and individually hydrolysed to a mixture of di-, tri- and tetra-peptides. )
R2
I
I
NH2 CH CO NH CH C02H
R'
I
1 1
Acetylation Esterification
RZ
I
CH3 CO NH CH CO N H CH C02CH3
R'
Li A1 H4 (Li A1 D4)
R2
I
I
CH3 CH2 NH CH CH2 NH CH CH20H
R'
I
(C2H5)2N "MS
R2
I
CH2 NH CH CH2 OTMS
Scheme 1.1.
The mass spectra of the resulting pol yamino alcohols are relatively straightforward with the sequencing information coming from the fragments X and Y. The presence of amino acids such as arginine, histidine, tryptophan and sulphur containing amino acids can be handled without modification. An alternative derivative, the perfluoroalkyl polyamino alcohol TMS, has recently been claimed 21941 to have higher volatility with abundant and intensity-balanced sequence determining ions. The reconstructed total ionization chromatogram of the trimethylsilylated polyamino alcohols, prepared by the outlined procedure, from the acid hydrolysis of the C-terminal cyanogen bromide fragment of actin is shown in Figure 1.14 [51]. The identification of the peaks was achieved by
Thr-Lys Ser - 1le Val
-
Pro-Ser /
Srr. Ile
\
Glu-Alo
i',,)I
I Aec-?he
Ale- Gly-Pro
Glu*-Glu ASp*-Gtu
Ile-TIy-Lys
0
20
a
50
Bo
100
1x1
143
160
180
SPECTRUM
200
Thr-Lys-Glu
Z3
ZU2
ES3
2M
ZOO
33
Iw
363
INDEX NUMBER
Figure I 14 Total ionizutiorr plot of 0-TM.7 cleri~cllioesof polyatnino alcohols ohfainrd b y trpatment of an arid hydrnlyAute of un Eicosnpeptide (C-terminal cyattogen hromide frugiiient of a r t i n ) (reproduced from /Sf])
A. M. LAWSON A N D G. H. DRAFFAN
45
computer analysis of both GC retention indices and the mass spectra. The reassembly of the identified small peptides into the original sequence of the C-terminal peptide is also made by computer using the identified peptides and the amino acid composition as the input data. In this case, no small peptide existed to indicate the histidine-arginine link but the N-terminal end of the molecule was identified as the TMS phenylthiohydantoin derivative and allowed the sequence to be obtained (see Figure 1.15).This structure agreed with an independent analysis by conventional techniques [195]. TrpIle TrpIle-Thr Ile-Thr-Lys Thr-Lys Thr-Lys-Glu Lys-Glu Glu-Glu Glu-Glu-Tyr Glu-Tyr Glu-Tyr-Asp Tyr-Asp TY- AspGlu Tyr-AspGlu-Ala AspGlu AspGlu-Ala Glu-Ala Ala-Gly-Pro Gly-Pro Ro-Ser Ser-Ile Ser-He-Val Ser-Ile-Val-His Ile-Val Arg-Lys Lys-AEtCys Lys-AEtCys-Phe AEtCys-Phe 5
10
15
20
A: TrpIle-Thr-Lys-Glx-Glx-Tyr-AspGlx-Ala-Gly-~~~r-lle-V~-His-Arg-Lys-AEtCys-Phe
Figure 1.15. Oligopeptides identified by GC-MS-computeras their 0 - T M S polyamino alcohol deriratives from acid hydrolysate of C-terminal cyanogen bromide fragment of actin. The reassembled eicosapeptide i s shown (A) (reproduced from 1511)
The utility of the GC-MS data in this application depends greatly on suitable chemical and enzymatic procedures being available. Careful selection of the hydrolysis agents is important. The dipeptidylaminopeptidase I (DAP I) is an enzyme which catalyses removal of the dipeptide from the unsubstituted NHz termini of a polypeptide [196]. This method of hydrolysing peptides for GC-MS has been studied by several investigators, e.g. [197, 1981. A study employing DAP I [197] has been described where the
46
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
dipeptides produced by hydrolysis are converted to N-trifluoroacetyl methyl esters and submitted to GC-MS. A second DAP I hydrolysis is carried out after removal of the N-terminal amino acid by an Edman degradation. The identity of this set of peptides is then matched with the first and the overlaps determined. This procedure was demonstrated with a tetradecapeptide isolated from tryptic hydrolysis of cytoplasmic aspartate aminotransferase. Caprioli, Seifert and Sutherland [I991 have developed this approach and resolved several of its attendant problems. One difficulty is the inability of DAP I to hydrolyse peptide bonds involving proline. A combination of DAP TV [200] and a sodium and liquid ammonia reaction allows this problem to be overcome [201]. Employing the method, these authors were able to sequence porcine insulin A chain (21 amino acids of known sequence) using the N-perfluoropropionyl dipeptide methyl ester derivatives. The considerable ambiguity of the primary sequence generated from the overlapping dipeptide data can frequently be completely eliminated by considering the order of appearance of a few of the dipeptides during hydrolysis. It is clear that the application of GC-MS in protein structural studies has advanced significantly in the last few years but it is still in a development stage, particularly in the sense that its routine use to sequence large completely unknown peptides has still to be achieved. The total system installation cost to engage in the work carried out by Nau, Kelley and Biemann [51] is high and prohibitively so for many laboratories. Nevertheless, the speed and sensitivity of GC-MS techniques are often superior to conventional methods and will undoubtedly contribute significantly to future progress in protein analysis.
Steroids The marked impact of GC-MS on the analysis of steroids is strongly reflected in the comprehensive volume of published literature on the subject. It has become an indispensible technique for their structural elucidation and identification in extracts of the complex biological mixtures in which they are found. Recent reviews [2,202-2041 provide a useful summary of the general progress in the field. Considerable effort has been given to an understanding of the basic electron impact fragmentation mechanisms of many classes of steroids [204,205] which have subsequently been applied to the structure determination of naturally occurring known and unknown steroids.
A. M. LAWSON AND G . H. DRAFFAN
47
Suitable derivatives to render most steroids more stable and volatile for gas chromatography and to improve their mass spectrometric properties have been developed. The trimethylsilyl ethers [206] or the methoxime-trimethylsilyl ethers of hydroxy keto steroids [207] have been widely used and their mass spectra extensively studied [204]. A comparison of the GC-MS behaviour of several derivatives of some of the adrenocortical hormones has been made [208], including methoximetrimethylsilyl ethers, dimethylsiliconides, methyl boronates, oxetanones and acetonides. The recently described improved preparation of enoltrimethylsilyl ethers of corticoids 12091 should also prove of value. The role of GC-MS in investigations of bile acids follows a closely similar path to other steroids. Their mass spectra have been discussed in detail e.g. [210,2111 in addition to their gas chromatographic behaviour. GC-MS in abnormal bile acid metabolism has been reviewed [212] and the investigation of their basic biosynthesis, metabolism [213] and presence in biological fluids in controls [214,2151 and diseased states [216] has been reported. To demonstrate the utility of GC-MS in the steroid field, several general areas, restricted mainly to examples in human biochemistry, have been selected for brief discussion. The presence of steroids in animals, plants and micro-organisms is widespread and of importance but cannot be considered here. Identification of individual steroids. The sensitivity and definitive nature of GC-MS application to this aspect has immeasurably widened the scope and level at which problems can be tackled. There is an ever increasing number of examples of the identification by GC-MS of previously unknown steroids in human urine, blood, faeces, amniotic fluid, foetal tissue and bile. Their eludication all contribute to a more complete understanding of the steroid biochemistry of the developing and adult human. This is particularly true with respect to steroid metabolism in the newborn where the maturing and developing enzyme systems involved give rise to differences from the adult in the types of steroids and their quantitative levels [217]. The changing pattern of urinary steroid excretion during infancy has been studied by several workers using GC-MS e.g. [218,219], with the decline of 3p-hydroxy-Sene steroids as 3p -hydroxysteroid dehydrogenase activity increases in the adrenals being most noticeable. Metabolites of cortisol increase during this period [219]. In addition to the quantitatively important urinary 16-hydroxylated-3p hydroxy-Sene steroids in the neonate [220-2221, GC-MS was used to identify a further group of steroids [223,224], four of which were
48
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
metabolites of placental progesterone received before birth together with several androstenetetrols and androstenetriolones. Subsequently the major androstenetriolones were more fully characterised as 16/3,18dihydroxy-DHA (i.e. 3/3,16/3,18-trihydroxyandrost-5-en17-one) and 15/3,16a-dihydroxy-DHA and the androstenetetrols, corresponding to reduction of the 17-carbonyl to a 17P-hydroxyl group [225]. The identification of androstenetriolones and androstenetetrols with 15 and 16 hydroxyl groups supports the hypothesis [226] that these compounds may be precursors of 15a -hydroxyoestriol and other oestetrols. Additionally, the probability that the 16,18-dihydroxy-dehydroandrosteroneis aromatized by the placenta to a 18-hydroxyoestriol was strengthened by demonstrating the presence of this compound in pregnancy urine [227]. GC-MS has been similarly used to give a fuller understanding of in vivo foetal steroid metabolism by studying directly the endogenous content of the adrenals [228,229], testes [230], liver [228,229] and lung [231] in addition to body fluids [232,233] in early and mid-term foetuses. A growing number of oestrogens and related metabolites in various body fluids, e.g. [234,235] and in placental tissue [236] have been identified. In the latter study, care was taken to remove as much foetal blood as possible to minimise contamination of the extracts. In addition to the three principal oestrogens, oestrone, oestradiol and oestriol, other unconjugated oestriols, 16-hydroxyoestrone, 16-oxo-oestradiol, 2methoxyoestrone, 16-epioestratriol, 17-epioestriol and 15a -hydroxyoestrone were identified and semiquantitated. These results coupled with the levels of foetal and maternal oestrogens help to establish the complex interrelationships in steroid metabolism obtained in the mother, foetus and placenta. The nature of exogenous steroids introduced as drugs have also been established. The major human urinary metabolites of two progestational drugs, dimethisterone and norethisterone were identified by GC and MS [2371 as have metabolites of a number of other commercial preparations (e.g. norgestrel [238], norethynodrel [239], nilevar [240] and dianabol [241I). Detection and quantitation of steroids. The confirmation of the steroid content of a biological sample is a routine procedure in many GC-MS laboratories. The development of the human urinary steroid ‘profiles’ [242] as a method for detecting disturbance of steroid metabolism by pharmacological agents and disease, relies on the MS identification of endogenous steroids. The greatly improved resolution available by the introduction of open tubular glass capillary columns for steroid separa-
A. M. LAWSON AND G. H . DRAFFAN
49
tion, e.g. [243] and their use in combination with the mass spectrometer is already proving a significant advance. When a computer data system is available the most convenient method of acquiring data is by the repetitive scan technique (see Data processing, p. 21). The mass spectra of any GC peak in the chromatogram can then be studied after the run and identified from computer spectral files, when previously encountered, or printed out for manual interpretation. The computer evaluation of the masslintensity data from a repetitive scan run can be illustrated by recent work on the disulphate fractions from a plasma sample from a patient with choriocarcinoma [50]. A scan number and retention value were assigned to the maxima of each GC peak and the potential molecular ion with its associated general structure suggested. A series of characteristic fragments was then searched for their changing intensities plotted out with respect to scan number (see Figure 1.16).This
Figure 1.16. Compuferprint of mas.s chromatograms of significant ions in the analysis of a steroid disulphate fraction o f plasma from a patient with choriocarcinoma (reproduced from [501)
permitted the identification of 5-androsten-3@,17a-diol (scan 27), 5androstene-3/3,17@-diol (scan 34), 5[-pregnane-3a,20a-diol (scan 56), 5pregnene-3@,20a-diol(scan 69) and 5a-pregnane-3/3,20a-diol (scan 70-71). The last two steroids are elevated in this fraction compared with controls. Although the repetitive scanning method is extremely useful it may lack adequate sensitivity to permit detection of the characteristic ions at low concentrations. In these cases the monitoring of selected ions is necessary to improve the detection limit. The principle of identifying
50
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
compounds from only a few of their fragment ions is now widely used in steroid analyses and, in addition to proving the presence of known endogenous steroids, can allow unknown drug metabolites to be detected [240,241]. In the past, quantitative measurements have been made from the gas chromatographic trace and the MS used only to confirm the identification. Although this is still a satisfactory method in many instances, possible interference and lack of sensitivity when dealing with extracts of biological material containing complex steroid mixtures can make a basic GC analysis inadequate. GC-MS using SIM has the advantage of giving a high degree of specificity to the estimation and often avoids the need for extensive isolation steps. A number of reports describing the measurement of steroids in this way using an isotopically labelled or other suitable internal standard have been published. 15a-Hydroxyoestriolwas determined in unpurified extracts of phenolic compounds from pregnancy urine by monitoring the intensity of the ion m/e 191 in the spectrum of the trimethylsilyl derivative [244]. Synthetic 4methyl-15a -hydroxyoestriol served as an internal standard as it is chemically similar, gives rise to the same large fragmentation at m/e 191 and is resolved from 15a -hydroxyoestriol on GC. Adlercreutz and Hunneman [245,246] have used single ion monitoring to quantitate twelve oestrogens in pregnancy urine with high sensitivity. As little as lOpg was sufficient to permit analysis. Oestrone and oestradiol have been measured using [6,7-2H2]-labelled standards [247] while by administration of P-[4-2H]-oestradiol into the maternal circulation and oestrogen production rates in normal pregnancy can be estimated [248]. In the latter example, the administration of a single isotope-labelled precursor gives only sufficient data for a partial understanding of oestrogen biosynthesis in pregnancy and considerably more information may be obtained by using precursors with alternative labelling. However, this approach does make possible highly sensitive studies in vivo in a situation where radioactive labels are ethically inadvisable. Testosterone [249,250], cholesterol [2511, aldosterone [252] and tetrahydroaldosterone [253] assay methods have all been presented. In essence, any steroid can be estimated by SIM methods as long as a suitable derivative and an adequate internal standard are available. However before embarking on such a procedure, the analyst must be convinced that a particular assay cannot be usefully made by a cheaper and faster method. Although GC-MS has a great range of specificity and adequate sensitivity, both of which make the development of an assay
A. M. LAWSON AND G . H. DRAFFAN
51
relatively easy, it has the disadvantage of a slow sample through-put and hence a high cost per sample. This may well be prohibitive for bulk routine samples with the current commercial instrumentation. Metabolism studies. GC-MS is a powerful technique for following and identifying the metabolic products from the in vitro incubation of tissue preparations with steroid substrates. Examples of such studies include the 16a-hydroxylation of 18-hydroxydeoxycorticosterone by human adrenal gland [254], the aromatization of 3p,15p, 16p-trihydroxyJandrosten-17-one by placental homogenates [255], and the demonstration of lp, 126, 6a and 6 p hydroxylase enzyme activities in microsomal preparations of human foetal hepatic tissue [256]. In the latter study, testosterone was used as substrate and in addition to the hydroxylated metabolites isolated, several other testosterone derivatives indicated the presence of 3a, 3p and 17p -hydroxysteroid oxidoreductase in the adrenal gland preparation. The application of the twin ion technique [257] is also of importance in metabolism studies. The doubly labelled steroids [4-I4C+ 7 + ‘HO441androstenedione and [4-I4C+ 7p-2H042]-testosterone,were incubated with human placental microsomes and the resulting metabolites quantitated by counting 14C and identified by GC-MS [258]. The identified metabolites 17/3,19-dihydroxyandrost-4-en-3-one,19-hydroxyandrost-4en-3,17-dione, 17p-hydroxy-3-oxo-androst-4-en-3-one, 3,17-dioxoandrost4-en-19-al, oestradiol-17P and oestrone were easily recognisable from the double sets of relevant ions in their spectra due to the mixture of hydrogen and deuterium substitution at C-7. Hence the presence of the aromatizing enzymes in the placental preparation and the intermediates in oestrogen biosynthesis were confirmed. Clinical applications of steroid identification. GC-MS has been used in a research role up to the present time in the study of the metabolism involved in the function of steroid endocrine systems. When these are abnormal or deranged by related biochemical systems, the resulting changes in both the qualitative nature and quantitative levels in the different body compartments are important. Although GC analysis in some situations may be adequate, GC-MS is considerably more powerful for assessing many of the disorders of adrenocortical steroid biogenesis and reaching a clinical diagnosis. As the routine application to patients of such methods is carried out in only a few specialised laboratories, their ultimate general utility remains to be decided. Steroid 21-hydroxylase deficiency is the most commonly encountered form of the adrenogenital syndrome and has been widely studied using a
52
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
variety of methods [259]. An evaluation of the plasma and urinary concentrations of the steroids present enables this defect to be confirmed and differentiated from 3p -hydroxysteroid dehydrogenase deficiency [260]. GC-MS is ideally suited for an investigation of this kind and has enabled the identification of 5-androstene-3p,l7p -diol, 5a -pregnane3P,20a-diol and 3p, 17a-dihydroxy-5p-pregnan-20-one, the latter of which had not previously been found in patients with a steroid 21-hydroxylase deficiency [260], in addition to the increased amounts of metabolites expected in the condition. Virilization normally results from this autosomal recessive disorder as a consequence of large amounts of androstenedione which are generated from excessive 17-hydroxy-progesterone, the substrate of the defective enzyme. The androstenedione is then principally converted to testosterone in peripheral tissue. Virilization also results in an 1l p -hydroxylase defect from excessive testosterone. 17Ketosteroids and 11-deoxy compounds are elevated, particularly 1 1 deoxycortisol (Reichstein’s compound-B). Recently 5p-pregnane-3a, 17a,20a,21-tetrol and its 20p isomer were identified by GC-MS in a patient with this defect [261]. There are three other recognised varieties of disorders leading to congenital adrenal hyperplasia, including 3p-hydroxysteroid dehydrogenase deficiency. Patients with this defect have grossly elevated levels of 3P-hydroxy-5-ene steroids in their plasma and urine. Complete lack of the enzyme is incompatible with life; however, an incomplete deficiency has been reported [262,263] where GC-MS identification of saturated CI9and Czl steroids revealed that some enzyme activity was present in the liver. An interesting example of applying GC-MS is the diagnosis of the condition of a patient presenting symptoms of hyperkalemia and hyponatreamia [264]. Although these indicated a possible lack of aldosterone, Figure 1.17 shows, in addition to a grossly abnormal urinary steroid excretion, that tetrahydroaldosterone, which as its glucuronide is the principal urinary metabolic product of aldosterone, was present at abnormal levels. This suggested that, rather than an adrenal hyperplasia, the patient was suffering from an end organ unresponsiveness to aldosterone. The reduced excretion of 3p-hydroxy-5-ene steroids may be due to the depletion of the suggested precursor pool of pregnenolone resulting from overproduction of aldosterone. Only by a technique such as GC-MS can the wide range of steroids present be assessed both quantitatively and qualitatively with positive identification of abnormal constituents. In clinical problems of the type described the extremely comprehensive information that is obtained by combined GC-MS is often instrumen-
A. M. LAWSON A N D G. H. DRAFFAN
53
3
IS
Figure 1.17. Profile analysis of urinary steroids in a normal infant and one with a form of renal-tubule unresponsiveness to aldosterone. The following compounds were identified as their methoxime-TMS ether derivatives on a 25m glass capillary OV 101 column programmed from 160°C at 2.5"C/min for 40 min, l"C/min for 15min and final temperature 260°C. (1) t6a-hydroxy D H A : (2) 16-ox0 -androstanediol; (3) 5-androstene-3P,16a,17P-triol; (4,5) androstenetriolones; (6) 16a-hydroxypregnenolone: (7) 5-pregnene-30,2Oa,21 -trio/; (8) tet(10) cortolone; (It) tetrahydro 'corn rahydrocortisone; (9) 5-pregnene-3P,16a,20a-tetrol; pound A ' ; (12) tetrahydrocorticosterone; (13) tetrahydroxyaldosterone (adjoining peak hexahydro 'compound A ' ) ; (14) ID-hydroxycortolone and IS internal standard cholesterol butyrate(reproducedfrom[264])
tal in clarifying the case and its wider application to more generalised conditions may be developed in the future.
Lipids The number of classes of compounds which can be grouped under the general heading of lipids is too large to allow their individual detailed consideration. Some have been more fully studied than others and are best treated separately e.g. prostaglandins, steroids. The MS fragmentation modes of fatty acids and esters have been discussed extensively in the past and are well summarised by Odham and Stenhagen [265].A variety
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
54
of other reviews has appeared dealing with the MS and GC-MS of lipids [104,266-2751, two of which are restricted to complex lipids [269,275] and the latter covering only GC-MS of glycerolipids and sphingolipids. As lipids occur in mammalian systems and throughout the plant and animal kingdom in materials containing multicomponent mixtures of compounds, prior separation is required for their investigation. Many chromatographic methods for isolation and class separation are used [276] and the individual molecular species are detected by various means including gas chromatography. The addition of MS, with its potential for positive compound characterisation, to the existing methods in lipid research has led to significant advances. Many striking examples demonstrating the utility of GC-MS in identifying lipids or the products from their chemical or hydrolytic treatment are to be found in the analysis of the lipid fractions from animal and plant origins (e.g. see references in reviews). GC-MS has been responsible for the identification of a variety of unusual lipids associated with diseased conditions. Hydroxyoctadecadienoic esters of cholesterol for example, have been isolated from aortal atheroma placques 12771 and branched chain and odd numbered fatty acids identified in the glycerolipids of brain, spinal cord and sciatic nerve [278] from a patient with methylmalonic aciduria. The latter compounds are thought to arise by the replacement of malonyl CoA with methyl malonyl CoA, and acetyl CoA with propionyl CoA at certain stages of fatty acid synthesis. In these and other examples, the lipids need to be hydrolysed to permit the identification of the constituent fatty acids. As the class of lipids is usually known from the separation procedure used, the nature of the fatty acids may allow the characterisation of the complete molecule. However, volatilisation of the intact lipid into the mass spectrometer when possible would be preferable, particularly when it is present in a mixture and separation of the components is first made by GC . Triglycerides have been investigated by MS with direct probe introduction [279,2801 and in the general case (1 l), series of diagnostic ions such as (M - RCOz)', (RCO + 74)', (RCO + 128)', and RCO' allow the individual substituents R', RZ and R3 to be identified although their location with respect to each other on the glycerol moiety cannot be concluded. CH,.O.COR'
I
CH.0.COR2
I CH,.0.COR3
(1 1)
A. M. LAWSON AND G . H. DRAFFAN
55
GC can be used to determine the carbon number of a triglyceride, e.g. [281] but with GC-MS it is possible to obtain, from a scan of the GC peak at a particular carbon number containing several triglycerides, the fatty acid content of the mixture at that carbon number and hence their possible combination in the triglycerides [282]. Glycerophospholipids are present in a variety of tissues in the body but more particularly in nerve and brain. They are involved in several fundamental biochemical processes and much more work remains to be done to clarify both their complete function and structural content. As a class, they are fatty acids esters of esterified glycerophosphoric acid (12) and eliminate the phosphate ester group under GC conditions [283,284]. R10CH2CHOR2CH20.P02.0R3
(1 2)
A similar result is obtained by heating the phospholipid in diphenyl ether with a trace of water E28.51 or by enzymic dephosphorylation [286]. The diglycerides produced can be silylated and both the location on the glycerol chain and the identity of the fatty acids determined by GC-MS. Glycerophospholipids which can be analysed in this way [285] include the cephalins (R3= ethanolamine), phosphatidylserine [287], phosphatidylinositol, and phosphatidylcholine. As part of a study into the accumulation of triglycerides in the liver resulting from ethanol metabolism, a procedure has been described to measure the deuterium incorporation from administered [I, 1-*H2]ethanol into individual fatty acid and glycerol moieties of phosphatidylcholines [288]. The latter were isolated from bile of bile fistula rats and after hydrolysis and separation of the 1 ,Zdiglycerides as the trimethylsilyl derivatives, the deuterium content of the glycerol and fatty acid parts of the molecule was determined by GC-MS. Further careful manipulation to the 1-trimethylsilyl-3-perdeuterotrimethylsilylethers of the 2-monoglycerides permitted GC-MS measurement of the deuterium excess at different carbon atoms of the glycerol moiety. In this way, both the fate of hydrogen atoms of ethanol and the extent to which they are transferred to different lipid fractions can be followed. The glycerophospholipids have also been studied by deacylation and the characterisation of the resulting phosphate containing compound [289,290]. Cicero and Sherman [290] applied this procedure to mono-, diand triphosphoionsitides from rat hrain and assayed these compounds as their trimethylsilyl derivatives after deacylation (13). Cardiolipin was similarly treated to give deacylcardiolipin TMS (14) [291].
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
56 TMSO
OTMS
OTMS
OTMS (1 3) a, R’= R2= TMS, deacylmonophosphoinositide b, R 1 = TMS, R2= PO(TMS)2, deacyldiphosphoinositide c, R 1 = R 2= PO(TMS),, deacyltriphosphoinositide
TMSOCHz~CH~CHz~O~POO.CH2~CHz~CH~CH~~O~PO~O~CH~~CH~CH~~OTM
I
OTMS
I
OTMS
I
OTMS
I
OTMS
deacylcardiolipin -TMS
I
OTMS
(14)
Sphingophospholipids, like glycerophospholipids, are normally dephosphorylated before conversion to derivatives and GC-MS analysis. Elimination of the phosphate ester from the parent molecule (e.g. (15) where R most often represents acid residues such as stearic, palmitic, eignoceric or nervonic) gives a ceramide (16) and some anhydroceramides [285]. As their TMS derivatives, ceramides can be gas chromatographed and their mass spectra used to confirm the sphingosine long chain base and the acyl group of the amide [292] from the ions M - A , M-B, M - ( C + l ) and M-15 (17). GC-MS has proved an efficient method for exploring the complex mixture of sphingomylins in human plasma [293]. They are first hydrolysed to ceramides, fractionated on TLC as their diacetates, converted to 1,3-di-O-trimethylsilyl ethers, and then identified. Ceramides from sphingomyelin fractions of beef brain and human plasma [283] have also
Me(CHz),zCH=CHCHOHCH(NH.COR).CH,.OH
OTMS
NHCOR
(16)
A. M. LAWSON AND G. H. DRAFFAN
57
been investigated. The methanolysis of sphingomyelin [294] from plasma allowed the long chain bases sphinga-4,14-dienine, hexadecasphing-4enine and heptadecasphing-4-enine, to be identified. Extensive GC-MS studies of synthetic ceramide TMS derivatives [292,295,296] and free ceramides in plasma [297] have been made. They have also been identified in human aorta [298] and identified and quantitated in human platlets [299]. Glycosphingolipids or cerebrosides, which are ceramides with the terminal hydroxyl linked by a glycosidic bond to a sugar moiety, can be handled by MS and GC-MS [285]. Among the sources of glycosphingolipids, characterised with the help of MS, are the membranes of human erythrocytes [300], CSF [301] and from the pancreas of a patient with Fabry’s disease [302]. The biosynthesis of cerebrosides via the ceramide pathway has been studied by the in vitro incubation of deuterium labelled N-(2’-Dhydroxyhexadecanoy1)-sphingosine with mouse brain microsomes and a UDP-galactose regenerating system [303]. Both galactosyl and glucosyl ceramides were formed. Their conclusive analysis by GC-MS as the trimethylsilyl derivatives of both the intact molecules, and after degradation to ceramides permitted the deuterium content to be measured and substantiated the proposed pathway. However, direct probe insertion into the MS is more often used for the characterisation of monoglycosylceramides, e.g. [304-3061 although there is a limit to the number of carbohydrate units which can be handled before pyrolysis becomes a major concern [307] and the maximum mass range of the MS is reached [308]. The pyrolysis limit can be extended to about six or seven sugar units by employing methyl polyether derivatives [309] which have lower molecular weights and are more volatile than acetates of trimethylsilyl derivatives.
Carbohydrates The general literature concerning fragmentation mechanisms, ionization methods, structure elucidation, identification, derivative behaviour and other aspects of MS investigations of carbohydrates has been well established over the last 10 to 15 years. Radford and DeJongh [310] have reviewed the recent applications of such studies, with emphasis on those of interest to the biochemist. In mass spectrometry similar spectra often result from configurational
58
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
isomers and this detracts from its utility in dealing with carbohydrate structures where such isomers abound. However, both careful comparative examination of the spectra and the application of GC-MS help considerably in overcoming this difficulty, e.g. [31 I]. Such problems are less common in structural isomers and other closely related compounds where MS has found wide utility in distinguishing many of the features of carbohydrates e.g. cyclic from alicyclic forms [312], furanose from pyranose [313,314], pentose from hexose [311,312], aldose from ketose [314,315], substituent position [312,316,317] etc. The three derivatives which have received most attention for GC and MS are the TMS ethers, the acetates and the methyl ethers. The spectra of the last named have been longest studied and perhaps best understood but the ethers are the most lengthy to prepare while the acetates, although easier to make, give more complex spectra. TMS ethers also give complicated spectra but are readily synthesised on a small scale and on balance appear to be the most generally useful. Several other derivatives have been investigated such as methoxime trimethylsilyl ethers [318], boronates [319,320], and trifluoroacetates [314,321]. As the advantages of the different derivatives vary with the compound type and the nature of the problem, their selection should be made in context. The reader can refer to the mass spectral features of the principal derivatives used for monosaccharides 13101 as a guide to deciding on the most suitable derivative for the application. The anomeric forms derived from equilibration of aldoses give rise to multiple peaks when trimethylsilylated and gas chromatographed [311]. A method of overcoming this problem, assuming that mutarotation itself is not under study, is to modify the aldose. It can be oxidised and lactonised to the aldonolactone, for example, and characterised as its TMS derivative [3221. Alternatively for the identification of aldoses and alditols, more use may be made in the future of the separations achievable on open tubular columns of the poly-0 -acetylaldonic nitriles (1 8) produced from aldoses and the poly-acetyl esters from alditols [323]. Figure 1.18 shows the separation of 32 assorted polyols and aldoses. A more common procedure for aldoses is their reduction with sodium borohydride to alditols and submission to GC-MS after conversion to TMS ethers [3241, permethyl ethers, acetates o r trifluoroacetates. This method was successfully employed in studies of the mechanism of conversion of deoxythymidine diphosphate D-glucose to deoxythymidine 4-0x0-6deoxy-D-glucose by an oxidoreductase from E. coli [325]. An in-
A. M. LAWSON AND G. H. DRAFFAN
59
CH = N O H
?20H+
Me OH
A c ~ o- - A
Ho$I
t
OH
'
C=N ~
o OAc $ ~
-fOAc
OH
CH2 OH
CH20Ac
p -D -glucose
ALDOSES 8 POLYOLS WOHL 8 ACETYL DERIVATIVES SE-30 TP IYMIN. (64m x 0.3rnrn)(I7O0C)
I
10
20
30
40
50
60
TIME-MIN
Figure 1.18. Separation of polyacetyl and Wohl derivatives of polyols and aldoses respectively on a 60m SE 30 capillary column (1"Clmin from 170°C). (1) glycerol, (2) D-erythrose, (3) L-threose, (4) 2-deoxy-D-ribose, (5)erythritol, 16) L-threitol, (7)D-ribose, (8) D-arabinose, (9) D-xylose, (10) 2-deoxy-D-ribitol, (1 1) 2-deoxy-D-glucose, (12) 2-deoxy-Dgalactose, (13) ribitol, (14) D-arabinitol, (15) xylitol, (16) 3-0-rnethyl-D-glucose, (17) D-mannose, (18) D-glucose, (19) D-galactose, (20) 2-deoxy-D-glucitol, (21) 2-deoxy-Dgalactitol, (22) 3-deoxy-D-glucitol, (23) myo-inositol, (24) D-mannitol, (25) D-glucitol, (26) (28) D-glycero-D-gluco-heptitol, (29) D-glycero-Dgalactitol, (27) D-glycero-D-gulo-heptose, mnnno-heptitol, (30) L-glycero-D-gulo-heptitol,(31) meso-glycero-gulo-hepfitol and (32) D-gfycero-D-galacto-heptitol (reproduced from [323])
tramolecular hydrogen transfer from C , to C6 during the formation of the hexose was demonstrated by incubation of dTDP-D-glucose4d as substrate and the determination by GC-MS of the location of the deuterium in the alditol acetate from NaBH4 reduction and acetylation of the resulting deoxyhexose (19). The intramolecular nature of the transfer
~
~
60
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
-
was apparent from analysis of the products of incubating a mixture of dTDP-D-glucosedd and dTDP-d-glucose-6dz with the enzyme. Evidence for the mechanism of conversion of dTDP-D-glucose to dTDP-Lrhamnose and incorporation of hydrogen from the medium was obtained from similarly devised experiments [325,326]. CH20H
DPN+
H
,
0
H
O
R
H,OH H,OH
H20
R H,OH H,OH
dTDP-D-glucose
The characterisation of disaccharides can be approached in a similar way. An elegant application of this was recently described [327] where two disaccharides of previously unknown sequences were isolated from urine and their identity determined with the help of GC-MS. One of these was 2-0-a -L-fucopyranosyl-D-glucose (20) which on acid hydrolysis yielded L-fucose and D-glucose. Treatment with sodium borodeuteride prior to hydrolysis gave L-fucose and D-glucitol-I-d (which indicated the sequence). Further confirmation of the structure was obtained from the mass spectrum of the permethylated disaccharide, alditol (21), produced by methylation of the borodeuteride reduced product. Hydrolysis of this yielded 2,3,4-tri-O-methyl-L-fucose and 1,3,4,5,6-penta-O-methyl-Dglucitol-1-d which were also identified by GC-MS. 2 - 0 - a-L-Fucopyranosyl-D-glucose and L-fucosyl-myo-inositol which were previously isolated and identified [328] are apparently characteristic disaccharides in normal human secretors of blood group ABH substances. 3-0-a-DXylopyranosyl-D-glucose was also shown to be present although its relationship to secretory status is not known. CHD OMe
OMe OH
OMe
OH I
CH20Me
A. M. LAWSON AND G. H. DRAFFAN
61
The GC-MS of higher saccharides is also possible but becomes increasingly difficult as the molecular weight increases and volatility decreases. Their derivatives (acetates, trimethylsilyl ethers [329] or methyl ethers), give spectra which indicate, in addition to the number of sugar units from the molecular weight, some information about the sequence and contents. Spectra with sequence ions for acetylated pentasaccharides, where the reducing end is coupled with a stabilising and sequence directing aglycone (phenylflavazole [330] and N-arylglycosylamine [331]), have been presented. Such compounds require direct probe introduction and probably represent the upper limit with the acetate derivatives. In the absence of suitable sequencing peaks, controlled hydrolysis of the polysaccharide to a mixture of smaller identifiable oligosaccharides is necessary for a complete structural analysis. The individual sugar moieties present can be determined by complete hydrolysis of the polysaccharide and identification of the monosaccharides obtained. One procedure [332] of this type is the exhaustive methylation of the polysaccharide followed by hydrolysis to a mixture of monomeric methylated sugars. The unsubstituted hydroxyl groups indicate the positions of glycosidic linkage and by reduction and acetylation to yield the corresponding partially methylated alditol acetates [333]they can readily be identified. This and similar methods have been used to identify a number of oligosaccharides from natural sources 12,3341. One example of some clinical interest is the structural analysis of (Y -D-mannopyranoside-(1 + 3)-p -D-mannopyranoside-(1 + 4)-a-acetamido-a -deoxy-D-glucose in the urine from patients with a lysosomal storage disease, mannosidosis [335].
Inborn errors of metabolism An ever increasing number of disorders of intermediary metabolism are known which result from an inherited single enzyme defect [336-3381. Most of these are of autosomal recessive character and although the individual incidence of the homozygous state is low, the overall occurrence of inherited metabolic disease is much higher. A variety of methods has been used in the study and screening of inherited enzyme defects. Partition chromatography on paper with ninhydrin staining for identifying amino acids [338], TLC and more recently gas chromatography, e.g. [339-3411 have all been employed extensively. However the positive characterisation of compounds by GC-MS has been a very powerful additional technique. It has been used in
62
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
examining disorders involving metabolism of lipids, amino acids [342], steroids, and carbohydrates but has perhaps found greatest utility in those in which organic acids accumulate in the blood and are excreted in the urine. The human disorders of this type fall roughly into two categories shown in Table 1.2 [343]. There are organic acidurias associated with an amino acidemia or amino aciduria and organic acidurias that manifest only as an organic acidemia or organic aciduria. Several of the latter are of recent discovery due to the application of GC-MS. It is likely that further disorders of this type and of other types will be found as the range of compounds that are studied is extended. GC-MS has been applied in many ways to investigations of inherited metabolic diseases but its use can be grouped into two broad, and to an extent overlapping, approaches. Table 1.2. CHARACTERISED DISORDERS OF INTERMEDIARY METABOLISM THAT PRESENT WITH AN ORGANIC ACIDURIA A. Associated with an aminoaciduria or aminoacidaemia
Disorder
Principal organic acids detected
Branched-chain ketoaciduria (Maple-syrup-urine disease)
2-0~0-4-methylpentanoic,2-0x0-3methylpentanoic, 2-0x0-3-methylbutanoic (2-oxoisovaleric) 2-Hydroxyphenylacetic, 3-phenyllactic, 2-phenylpyruvic, madelic, 4-hydroxyphenyllactic, 4-hydroxyphenylpyruvic Arginiosuccinic 4-Hydroxyphenyllactic, 4-hydroxyphenylpyruvic, 4-hydroxyphenylacetic Iminoglycine 2-0x0-4-methiolbutanoic Methylmalonic
Phenylketonuria
Argininosuccinic aciduria Tyrosinaemia, tyrosinosis
Iminoglycinuria Hypermethioninaemia Hornocystinuria with methylmalonic aciduria (Methylmalonic aciduria Type 11) Histidinaemia Imidazole-lactic, imidazole-pyruvic, imidazole-acetic B. Without an associated aminoaciduria or aminoacidaemia Alcaptonuria Congenital lactic acidosis Methylmalonic aciduria, Types I, I11 and IV* Oast House disease
Homogentisic Lactic, pyruvic, 2-oxoglutaric Methylmalonic, 2-methylcitric, propionic 2-Hydroxybutyric
A. M. LAWSON AND G. H. DRAFFAN
63
Table 1.2. (contd.) Disorder
Principal organic acids detected
Orotic aciduria Short chain fatty acidaemia Isovaleric acidaemia
Orotic Butanoic, hexanoic 3-Methylbutanoic (isovaleric), 3-methylbutanoylglycine, 3-hydroxy-3-methylbutanoic Glucaric, glucuronic P yroglutamic Propionic, 3-hydroxypropionic, 2-methylcitric, 3-hydroxy-3-methylglutaric 3-Methylcrotonic acid, 3-methylcrotonylglycine, 3-hydroxypropionic, 2-methylcitric, 3-hydroxy-3-methylbutanoic Glycollic, glyoxylic, oxalic
Glucaric aciduria Pyroglutamic aciduria Propionic acidaemia (ketonic hyperglycinaemia) p -Methylcrotonylglycinuria
Glycollic aciduria (Primary hyperoxaluria Type I) L-glyceric aciduria (Primary hyperoxaluria Type 11) Benzoic aciduria Refsum’s disease Fructose 1,6-diphosphatase deficiency Pyruvate decarboxylase deficiency Pyruvate carboxylase deficiency (Y -Methylacetoacetyl CoA thiolase deficiency
*
L-glyceric, oxalic Benzoic Phytanic Pyruvic, lactic, 2-oxoglutaric Pyruvic, lactic Pyruvic, lactic 2-Methylacetoacetic, 3-hydroxy-2-methylbutanoic, N-tiglylglycine
Methylmalonic aciduria, Type I: methylmalonyl CoA mutase deficiency Type 11: 5’-deoxyadenosylcabolamindeficiency Type IV: methylmalonyl CoA racemase deficiency
In the first instance, it is used to confirm a diagnosis when dealing with a patient with a metabolic problem. When the routine chemical screens indicate the presence of abnormal metabolites or raised levels of the usual metabolites, then MS can positively confirm their identity and thus a diagnosis, or, should it be a new inborn error, point to the metabolic pathways which are affected. In many of the organic acidurias described, the defect leads to gross excretion of one or more compounds and, as quantitative extraction is not necessary for their detection, simple solvent or other methods are normally used. In some instances it is vital that analysis be carried out quickly so as to indicate the most suitable clinical management of an acutely ill patient. In the study of p -methylcrotonylglycinuria [344], first reported in 1970
64
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
[345], the volatile fatty acids were isolated by steam distillation and
p -methylcrotonic acid recognised by gas chromatography. The presence of P-hydroxyvaleric acid, P-methylcrotonylglycine and tiglyl glycine were confirmed by GC-MS which, in addition to pointing to the primary defect, indicated that the isoleucine metabolic pathway was also affected. In these two cases and a more recently reported patient [346], the clinical presentation of the disorder was different. This makes diagnosis difficult particularly when variants of the same enzyme defect are possible and this suggests that more extensive screening procedures are warranted. GC-MS should play a strong complementary role in such investigations. In known metabolic states and disorders, the nature of metabolites excreted at abnormal levels has been identified by GC-MS. Examples of this are adipic and suberic acids found in urine from ketotic patients 13471, 2-hydroxybutyric acid from patients with lactic acidosis [348], and methylcitric acid (2-hydroxybutan- 1,2,3-tricarboxylic acid) [349] in a case of propionic acidemia [350,351]. In the latter instance, the methylcitric acid is thought to be due to the condensation of accumulated propionyl CoA with oxaloacetate [349]. Increased amounts of odd-numbered fatty acids present in the tissues of these patients due to the involvement of the propionyl CoA in fatty acid synthesis, have also been characterised [278]. A deficiency in a -methylacetoacetyl CoA thiolase enzyme in the isoleucine pathway prevents the conversion of a methylacetoacetyl CoA to propionyl CoA and acetyl CoA [352,353]. The resultant urinary excretion of large amounts of 2-hydroxy-3-methylbutanoicacid ( a methyl-p -hydroxybutyric acid) and an excess of a -methylacetoacetate and often tiglyl glycine are readily detected and identified by GC-MS. GC-MS has applications in establishing GC screening procedures by confirming derivative formation and location of metabolites, keeping a future check on the methods and when required, making positive identification in samples of detzcted disorders. An example of this was reported [3541 in the GC monitoring of urinary acids for the recognition of organic acidemias in connection with maple-syrup-urine disease. Three patients with an intermittent form of the disorder exhibited excretion of 2hydroxy-3-methylbutanoic and 3-hydroxybutyric (GC-MS), in addition to a -ox0 acids and other metabolites. The positive confirmation of metabolite structural assignments allows the more confident use of GC as the principal technique in a screening procedure. The second approach to studying metabolic disease is the establishment of normal base-line profiles of the volatilisable metabolites (either free or as derivatives) in the human physiological fluids. In this, the GC
A. M. LAWSON AND G . H. DRAFFAN
65
and the MS play complementary roles. The resolution of the former is necessary to separate the complex mixture of components of the different classes of compounds and the MS is indispensable in identifying these compounds. In addition, a computing facility is almost essential to cope with the large data flow. The principal aim in developing these profiles is to permit the recognition of abnormal patterns which can be directly related to profiles from known disorders. When an unusual profile cannot be related to one of the latter, it provides the initial data to indicate the biochemical pathways which are disturbed and the nature of the molecular defect. The concept of metabolic profiling for investigating possible pathological situations and the effect of drugs was introduced in 1971 by Horning and Horning [242]. A number of laboratories have embarked on programmes incorporating this basic approach to assess its usefulness in screening and studying inherited diseases. Many restrict themselves to a single class of compound or even to a group within a class. At this stage in the development of these procedures, most workers carry out preliminary GC evaluation to reduce the number of complete GC-MS runs necessary. Jellum, Stokke and Eldjarn have been very active in the metabolic profile approach over several years and routinely investigate perhaps the most comprehensive number of compound types [341]. They screen eight fractions from a urine extract (also plasma and CSF if necessary) by GC and submit samples which show irregularities to GC-MS for further analysis. Spectra are identified off line from a remote terminal of a large, fast central computer. They have had considerable success with their methods, independently reporting three new disorders [345,355,356] and are able to detect about 40 of the approximately 150 documented inborn errors of metabolism. Extremely complex profiles are obtained if no class separation is attempted [357]and even with the aid of high resolution, the individual identification of constituents is difficult. More importantly, however, fairly large changes in the levels of many components may go unnoticed because of the limited dynamic range achievable. More sensitive monitoring is undoubtedly possible by initial class separation. Due to the growing interest in organic acidurias, urinary acids have been studied at some length. Although many of the acids present have been identified, many have not, often because of inadequate GC resolution or poor extraction. A series of aldonic and deoxyaldonic acids were recently detected [358] using a DEAE extraction method [3591. The greater sensitivity which is possible by more extensive fractionation has
66
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
been demonstrated by the identification of several branched-chain dicarboxylic acids in urine by carrying out additional TLC separation steps [360]. Despite the need for quantitative data a very limited amount is available in the literature. There are a number of reasons why this is so [361]. One study [361] reports levels of a number of acids measured in 21 subjects or, a controlled diet. Although these data are useful, they have the limitation of being estimated from material isolated by solvent extraction, a method known to lack quantitation [343]. Figure 1.19 shows a typical normal acid profile [358] and for comparison one from a patient with propionic acidemia [362]. Greatly improved separations are possible by using open tubular capillary columns [40] and it is likely that such columns will be widely used for this purpose in future. Organic acids have also been studied in plasma, e.g. [341], amniotic fluid [3631 and CSF 13641. Several other classes of compounds have received attention in profile analysis, most notably steroids (see above under Steroids) and amino acids [340,341]. A number of reports suggesting the possibility of detecting metabolic disease from profiles of the volatile constituents of human urine and breath have been made 136.5-3671 but the value of these profiles has yet to be demonstrated convincingly, although the profile from a diabetic patient showed a marked variation from the normal [367].
9.11
56
R, 1
0
1
4
1
1
8
1
1
12
1
1
16
1
1
20
1
1
24
1
1
28
I
I
32
I
I
36
I
I
40
I
I
44
A. M. LAWSON AND G . H. DRAFFAN
67
3
I
0
4
I
I
8
I
I
12
I
I
16
1
1
20
1
1
24
I
I
28
I
I
32
I
I
36
I
I
40
=Ft
I
44
48
Figure 1.19. a) Profile of the acidic components, as ethoxime-TMS derivatives, extracted from urine from a normal subject, (10% OV 101, il0"-285"C). Peak identifications are ( I ) sulphate; (2) phosphate; (3) 3-deoxytetronic acid; (4) 2-deoxytetronic acid; (5)erythronic acid; (6) threonic acid; (7) a deoxypentonic acid; (8) a deoxypentonic acid; (9) hippuric acid; (10) a pentonic acid; ( I t ) arabinoic acid; (12) citric acid; (13) a deoxyhexonic acid; (14) glucono-1,5-lactone; (15) undecandioic acid (internal standard); (16) glucuronic acid; ( I 7) a hexonic acid; (18)gluconic acid; (19) saccharic acid; (20) uric acid; (21) tetracosane (internal standard); (22) hexacosane (internal standard). b) Profile o f acidic components, as ethoxime-TMS derivatives, extractedfrom the urine of apatient with propionic acidaemia (10% OV 101, 110-28.i"C). Peak identifications are (1) lactic acid; (2) hydroxybutyric acid isomer; (3) P-hydroxypropionic acid; (4)sulphate; (5) P-hydroxybutyric acid; (6)methylmalonic acid; (7) 0-hydroxyisovaleric acid; (8) hydroxyvaleric acid isomer; (9) phosphate; (10) succinic acid; (11) giyceric acid; (12) adipic f maiic acids; (13) tetronic acids; (14) 3-hydroxy-3methylglutaric acid; (15) Chydroxyphenyl acetic acid; (16) citric acid; (17) methylcitric acid; (18) 4-hydroxyphenyllactic acid; (19) undecandioic acid (acidic internal standard); (20) glucuronic acid; (21) uric acid; (22) tetracosane (internal standard); (23) hexacosane (internal standard)
An important aspect of metabolite profiles is the study of known inborn errors where the qualitative and quantitative interrelationship of the affected metabolites can be studied. A recent example of this is the comparative study of three diseases, p -methylcrotonylglycinuria, propionic acidemia and methylmalonic aciduria [362]. The advantage of considering a complete class of compounds in a single experiment is that biochemical markers for a disorder can be detected in the context of any variations in other components. This is particularly important in monitor-
68
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
ing the progress of treatment or the effects of metabolic loading tests. In the latter connection, dietary restriction of the branched-chain amino acids and a load of leucine in a patient with maple-syrup-urine disease leads to an excretion of 4-methyl-2-oxopentanoic ( a-ketoisocaproic), 3methyl-2-oxopentanoic (a-keto-p-methylvaleric) and 3-methyl-2oxobutanoic ( a -ketoisovaleric) acids [368]. These changes are readily followed from the profile of the silylated oximes. Similar types of loading studies in patients with a methylacetoacetate CoA thiolase deficiency [353] and PKU [369] were conveniently monitored by GC and GC-MS. Although considerable progress has been made in the metabolic profile approach, a number of problems remain to be overcome. Many of these centre around the fluctuations in component composition, not from metabolic disorders, but brought about by other influences. These are principally due to diet and the metabolic variations in individuals in relation to activity. Drugs can also affect the excretion levels of compounds, in addition to the production of their own metabolites. These factors all make quantitative data difficult to obtain and evaluate. Careful statistical analysis of the results are necessary and a population of 500 subjects, grouped in age and sex, has been studied with a view to obtaining a suitable data base for urinary organic acids [370]. The application of GC-MS to the study of inherited metabolic disorders and pathological conditions is undoubtedly of expanding interest but it remains to be seen how effective it might be as a routine measure in the clinical diagnostic situation. PHARMACOLOGY AND TOXICOLOGY
Applications of GC-MS under the general heading of pharmacology now extend from the qualitative characterisation of drug metabolites to the highly specific and sensitive assay of a wide range of substances which may mediate physiological response. GC-MS applied in both pharmacology and toxicology has recently been comprehensively surveyed [66]. In the following sections, studies involving prostaglandins and biogenic amines are treated in some detail. The potential of stable isotope tracers employed in the investigation of drug metabolism and disposition is also considered, and emphasis throughout is placed upon the use of selected ion monitoring, reflecting the widespread adoption of this technique. The identification of drugs and toxic substances in poisoning cases as well as aspects of GC-MS anplied in environmental toxicology are discussed under separate headings.
A. M. LAWSON AND G. H. DRAFFAN
69
Drug metabolism and disposition Mass spectrometry may have several roles in the study of drug metabolism. Its most widely employed function is in aiding the qualitative identification of a foreign compound often tentatively distinguished as a drug metabolite by some complementary means. Commonly, this may follow from the use of radiotracer techniques in the prior definition of a chromatographic pattern of metabolites. A survey of the metabolic fate in several animal species normally precedes investigation in man. Identification depends upon the interpretation of the mass spectrum of the parent drug and a close comparison with that of the metabolite. Low resolution mass spectrometry combined with microchemical methods in derivative preparation is often sufficient to define the structure. However, the additional information content of high resolution spectra can be of value, as is illustrated in the characterisation of the metabolites of diazepam [371] and chlordiazepoxide [372]. Where it is possible to predict likely metabolites and a full interpretation of the parent drug spectrum has been made, the mass spectrometer can be used as a specific detection system in selected ion monitoring (SIM) mode (see Selected ion monitoring, p. 25). The early impetus provided by the development of ‘mass fragmentography’ in the identification of chlorpromazine metabolites [64] has led to many further applications [65,66,68]. The extension of SIM to quantitative drug measurement with the incorporation of internal standards e.g. [88] has again opened up new possibilities in pharmacokinetic studies in clinical pharmacology, a topic discussed separately below. The considerable literature on the application of mass spectrometry in drug metabolite identification has been periodically reviewed [2,66, 101,373-3751. The following examples are cited as representative studies of the metabolism of drugs in common clinical use. A detailed investigation of the metabolic fate of propranolol(22) by GC-MS methods has led to the characterisation of 16 metabolites in man and dog e.g. [376,377]. The glycol (23) which is structurally related to the central muscle relaxant, mephenesin (24), may contribute to some of the CNS effects observed with propranolol [377]. Nortryptyline metabolism in man has been investigated by ion monitoring methods with the identification of three metabolites [378]. Dihydrodiols have been identified in man as metabolites of a number of drugs containing a phenyl ring, such as diphenylhydantoin [379] phenobarbitone [380] and methsuximide [381]. Since the dihydrodiols are probably formed via epoxide intermediates, their iden-
70
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
(22) R = NHPr' (23) R = OH
(24)
tification is of particular importance in view of the toxic effects associated with certain epoxides. The placental transfer and subsequent metabolism in newborn infants of a number of drugs including phenytoin, barbiturates, diazepam and caffeine have been studied by GC-MS-computer methods [382,383]. Other metabolic investigations in man include the identification of an epoxide formed from carbamazepine [384], a dihydro derivative of digoxin [385] and a pharmacologically active hydroxy metabolite of glutethimide found to accumulate during glutethimide intoxication [3861. The final stage in the detoxication of drugs in the body frequently involves conjugation with glucuronic acid. Glucuronides are generally characterised only by enzymic cleavage, a technique which does not define the site of conjugation and which may be inconclusive because of incomplete hydrolysis. They can be identified intact by GC-MS, following chemical modification to increase volatility. Permethylation [387], TMS ether-TMS esters [388], and TMS ether-methyl esters, e.g. [389,390] have been employed for this purpose. TMS formation from glucuronides, ester-linked through an aromatic carboxy function in the aglycone, may result in partial exchange to give the fully modified aglycone [388]. A similar exchange reaction with aryl sulphate conjugates has been observed [391]. The EI spectra of the glucuronide derivatives are often relatively uninformative, showing low abundance molecular ions and fragmentation dominated by the glucuronic acid moiety. The advantages of CI in the identification of oxazepam and lorazepam glucuronides as the TMS derivatives have recently been demonstrated [392] and spectra contained abundant quasi-molecular ions and diagnostic fragments due to the aglycones. Although the water-soluble conjugates are pharmacologically inactive, their direct identification may still be of importance in defining the total metabolic fate of a drug. A general attraction of GC-MS-computer methods is the possibility offered for the analysis of complex mixtures and the avoidance of extensive sample purification. Several hundred spectra can be acquired by repetitive scanning and in the absence of complementary evidence,
A. M. LAWSON AND G . H . DRAFFAN
71
distinguishing a minor component as a drug metabolite may be a nearly impossible task (see also discussion on mass chromatograms, in Section on Data processing, p. 21). Where the parent drug contains a chlorine or bromine atom, the presence of pairs of ions with the correct isotope ratio can provide a marker. A predicted fragment ion may also serve to focus attention on a+particular component. For example, the m/e 191 ion (MesSiO-CH=OSiMe,) characteristic of TMS derivatives of vicinal diols has been used in search procedures to detect dihydrodiols of aromatic drugs e.g. [381,393]. Such predictions are also a prerequisite in the use of SIM methods. An alternative is to create artificially a diagnostic isotope pattern in the spectra of metabolites by administering a stable isotopically-labelled form of the drug, one aspect of the use of stable isotopes as tracers discussed in the following section. Stable isotopes as tracers in drug metabolism. General reference has been made (Stable isotopes, p. 30) to the use of stable isotopically labelled tracers in biological research. In studies of drug metabolism, administration of a mixture of the drug and its appropriately labelled counterpart can lead to the rapid recognition of metabolites in complex mixtures admitted to the mass spectrometer from the presence of characteristic doublets in the spectra. Isotope labelling can contribute to the interpretation of spectra, provide a means of distinction between a possible drug metabolite and an endogenous compound, and can be used to obtain quantitative pharmacokinetic data. Aspects of these topics have been reviewed [loo, 1011. The value of heavy isotope labelling is illustrated in an investigation of the metabolite pattern of (+)- propoxyphene in man 13941. Three different deuterium labelled forms of propoxyphene were prepared, d, (25), d, (N-CD,), and d, (N-CHD,). In the initial screen, a 1: 1 mixture of undeuterated propoxyphene (do) and its d, analogue was administered, D
D
D
D (25)
and a series of extractions of urine at different pH values were designed to recover all likely classes of metabolites. Each fraction was then examined by GC-MS-computer methods and spectra recorded for every GC peak. The presence of doublets at mle 91/98 arising from the benzyl
12
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
fragment (C7H,, C,D7) indicated propoxyphene metabolites and provisional interpretation was then confirmed following administration of the d, and d, forms. Propoxyphene dz or d, can be dosed without admixture with do in a search specifically for N-demethylation, since random loss of a methyl group resulted in a 1 : 1 mixture of labelled and unlabelled molecules in these metabolites. Hydroxylation in the benzyl group rather than the 2-phenyl ring was established by the observation that one deuterium atom was lost when hydroxymetabolites were formed from propoxyphene-d7. Eight metabolites were identified by these means following single oral doses of the drug. The same principle was employed in an investigation of the metabolism of the thienopyrimidine (26) [395] labelled with 60% enriched "C (*) in
position 2 of the pyrimidine ring. In this case. fractions recovered from thin layer chromatograms were admitted via the direct insertion probe. The temperature was raised providing a step-wise distillation with the mass spectrometer scanning repetitively until the predicted doublets, in an approximate 6 : 4 ratio, emerged indicating a drug metabolite. For further examples of the tracer technique using stable isotopes, reference may be made to work on nortriptyline, e.g. [396,397], barbiturates [3981 and cambendazole [399]. The identification of isopropylamine as an active metabolite of propranolol (22) has been confirmed following administration of propranolol-d6 (-NHCH(CD,),) which thus allowed distinction between the drug metabolite and endogenous isopropylamine [400]. An attractive, but as yet unexploited, possibility is the use of heavy isotope tracers in quantitative studies of bioavailability. For example, in the assessment of different formulations, comparison can be made in the same individual by co-administration of unlabelled and labelled variants. The principle, as well as potentially wider application of simultaneous
A. M. LAWSON AND G. H. DRAFFAN
73
quantitative measurement of different isotopic forms, is illustrated in a determination of the turnover kinetics of acetylcholine [401]. The d4form was administered to rats and then recovered, together with do, which in this case is the endogenous substance. Both do and d4 were measured in SIM assay using acetylcholine-d, as the internal standard. Similarly, prostaglandin Fz,-d, has been used to standardise simultaneous measurement of PGF,,-do and d4 [81,402]. Where isotopically labelled compounds are employed as tracers, biological isotope effects may be encountered. There have been several reports of primary isotope effects in the metabolism of deuterated drugs (for review see [403] and in general, metabolic rate is slower when a bond to deuterium rather than to hydrogen is cleaved during metabolism. Such effects have been observed, for example in the hydroxylation of butobarbitone 14041 and in the N-demethylation of morphine [405].A marked deuterium isotope effect has been reported in the 3-hydroxylation of cotinine-d2 (27). In this case a k d k , ratio of about 6 was determined by
6
mass spectrometry of the hydroxy metabolites following administration of a mixture of cotinine-do and d, [406]. The observation of such effects can be of value in defining the rate limiting step in metabolic deactivation, e.g. [404]. However, where isotope effects are undesirable and deuterium must be used, incorporation should be in metabolically secure positions. Alternatively, effects are less likely to be significant with the carbon, nitrogen and oxygen isotopes. An increasing use of stable isotopically labelled drugs as tracers, particularly in clinical pharmacology, may be anticipated [94,98]. However, it should be noted that deuterium and I3C do not replace tritium and I4C in all aspects of their tracer function. The stable isotopes can provide enhanced specificity in studying the fate or origin of particular groups in the molecule and can open the possibility of investigation in patient and volunteer categories where radiotracer use is now restricted. The applications thus far reported must still be considered to represent exploratory work on potential and technique. In contrast, the opposite approach, that
74
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
of stable isotope dilution (see below and Section on Selected ion monitoring, p. 25) is firmly established as a uniquely precise and sensitive means of quantitative drug determination. Quantitative drug assay by GC-MS. The principle of SIM used as a quantitative measuring technique with the inclusion of internal standards has been discussed in detail (see Selected ion monitoring, p. 25). References to its use in drug assay are listed in Table 1.3. Where labelled reference compounds have not been available, homologues or substances analogous to the component of interest have been used as standards, e.g. [83,409,85,413,414,418]. In stable isotope dilution, the incorporation of several deuterium atoms has been the most widely employed method, reflecting in part the greater selection and lower cost of synthetic precursors. However, labelling with I3C may have some advantage in eliminating the possibility of either isotope exchange or differential adsorptive effects during analysis. Assays based on [2,4,5-'3C& Table 1.3. DRUGS DETERMINED BY SELECTED ION MONITORING GC-MS USING INTERNAL STANDARDS
Conipound
References
Amphetamine Barbiturates (Amylobarbitone [83] pentobarbitone, phenobarbitone and other barbiturates [4081 Carbamazepine Codeine Diazepam Diphenylhydantoin Ethanol Imipramine Indoramin Guanethidine Lidocaine Morphine Nortriptyline Phentermine Piribedil Salbutamol Terbutaline A'-Tetrahydrocannabinol Thiothixene
407* 83,408**
* **
Using deuterium labelled standards Using "C labelled standards
409 410* 408* 408**,411* 412* 85
90* 413 414 410*,415* 88*,416 417 418 419* 420* 421* 422*
A. M. LAWSON A N D G . H . DRAFFAN
75
diphenylhydantoin, phenobarbitone and pentobarbitone have now been reported 14081. GC-CI has been used in an assay for morphine [415] and API 1408,1281 (see Alternative methods of ionization. p. 33), holds particular promise as a means of drug measurement by direct (without GC) injection of biological extracts. Drug measurement by repetitive scan techniques under computer control e.g. [408,811 has been evaluated as an alternative to SIM. GC-MS methods provide greater specificity and in many cases sensitivity when compared with more conventional techniques. They offer increased scope for the study of pharmacokinetics and of plasma concentration in relation to biological effect. SIM assay has been applied to the investigation of placental transfer of lipid soluble drugs and their subsequent elimination in the newborn (barbiturates, diphenylhydantoin, caffeine, pethidine and diazepam [ 122,4081 diphenylhydantoin [4111; amylobarbitone and 3'-hydrox yamylobarbitone [83,4231). A comparison of the elimination kinetics of amylobarbitone in mothers and their newborn infants has been made following administration of the barbiturate to the mothers shortly before delivery r4231. While the half-life of the drug in the neonates was 2.5 times that in the mothers, the results implied an appreciable ability to metabolise the barbiturate in the first few hours of life. This was confirmed by the determination of the plasma concentration of the hydroxy-metabolite which increased after birth (Figure 1.20). Similarly, it has been concluded that diphenylhydantoin can be effectively metabolised immediately after birth [411].
Prostaglandins As a class of hormones, the prostaglandins are characterised by a widespread occurrence in animal tissues and a high biological potency. They are considered to have fundamental roles in cell processes in health and disease and their human pharmacology and potential as therapeutic agents continue to be intensively investigated [424]. Because of the low concentrations and rapid turnover of prostaglandins in most body fluids, sensitive techniques for their determination in vivo are required. Mass spectrometry played an important role in the work of structural elucidation of the six primary prostaglandins of the E and F series (for references to early work see [425] and GC-MS remains one of the definitive chemical methods for the study of prostaglandin biosynthesis and metabolism in man. While eight classes of prostaglandins are now recognised, namely. A. B ,C.D,E,F,GandH.theEandFseries(e.g.PGEz(5)andPGFr,,(28))arein
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
76
I
Newborn Sa. A
Amylobarbitone
A
Hydroxyamylobarbitone
1.0
ti=17.4h 2
0.1
k4.6
-
10
M
30 40 M 60
t(h)
Figure 1.20. The elimination of amylobarbitone and its metabolite determined by SIM in 50-100p1 plasma samples from a newborn infant following a single therapeutic dose of the drug to the mother 1.5 hr before delivery. Time zero is time of birth. The sharp rise in the plasma concentration of hydroxyamylobarbitone confirms the capacity of the infant to metabolise the placentally transferred barbiturate (reproduced from reference 14231) HO i
I
A0
I I
I
OH
general terms the most widely active and have been the most studied. M S and GC-MS. A variety of derivatives have been used to protect labile functional groups of the free prostaglandins prior to gas chromatography and direct fragmentation in GC-MS. The 9,l l-p-ketol grouping of the E series is particularly sensitive in vapour phase analysis and commonly 0 -methyloxime TMS ether derivatives or 0 -methyloxime acetates have been employed in GC-MS (e.g. [426]). An alternative approach is to dehydrate the ketol under controlled conditions to the more stable enone analogues of the B series [427,428]. The cyclic n-butylboronate of the 9,ll-diol in the F series when used with methyl ester TMS ethers confers good GC properties and some simplification in
A . M . LAWSON A N D G. H. DRAFFAN
,
77
mass spectra [429]. This combination of derivatives has been employed in an SIM assay of PGF2, [4301. Alternative hydroxy derivatives include trifluoroacetates 14311 and t -butyldimethylsilyl (TBDMS) ethers 1311. The TBDMS function, particularly when introduced following oxime formation, can result in extremely simple spectra due to loss of the t-butyl radical (see Derivative formation, p. 1 1 and Figure 1.6). A detailed study of the mass spectra of representative members of the A, B, E and F series of prostaglandins as TMS ester and TMS ether derivatives has been reported in a series of publications [432-4351. High resolution mass measurement contributed to the interpretation of fragmentation patterns. Chemical ionization (CI) spectra of prostaglandin derivatives using methane and isobutane as reagent gases have been discussed and the potential of CI in structural and quantitative studies pointed out [436]. However, almost without exception, original structural determinations and all routine quantitative assay methods for both parent compounds and metabolites have been based on low resolution electron impact mass spectrometry. The recent identification of two new 19-hydroxy PGEs in human semen [437] (see below) has similarly been based on low resolution GC-MS methods. Biosynthesis and metabolism. Prostaglandins of the E and F series are derived from unsaturated fatty acid precursors. Mechanistic studies (for review see [438]) provide an excellent example of the value of mass spectrometry and of stable isotopes in determining biosynthetic pathways. The C-9 and C-1 I oxyfunctions were shown to originate in the same molecule of oxygen by in vitro biosynthesis in a mixture of I6O2and ''0:. Either two atoms of "0 or two atoms of "0 were incorporated with no 16 0-'"0 contribution. Endoperoxide intermediates were therefore postulated as precursors in PGE and P G F synthesis. These theories have now been directly confirmed by the isolation of 15-hydroperoxy-9a,ll a peroxidoprosta-5,13-dienoic acid (PGG:) (29) and 15-hydroxy-9a,l l a peroxidoprosta-5,13-dienoic acid (PGH,) on very brief incubation of arachidonic acid with sheep vesicular gland microsomes [439,440] PGG, is envisaged as the first stable product of 'prostaglandin synthetase' [440].
I
OOH
78
GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
The structures were confirmed by an elegant series of chemical transformations to known prostaglandins for validation by GC-MS which was also employed in estimating the time course of product formation. The significance of the endoperoxides is emphasised by their much greater potency than PGE, in contraction of the rabbit aorta strip and by the finding that they both cause human platelet aggregation and are released during thrombin-induced aggregation. Biological inactivation mechanisms and metabolic end products of prostaglandins E and F in man have been investigated by GC-MS methods following tracer doses of 'H-labelled PGE, [441,442] and PGF2, [30,443-4451. Twelve metabolites of PGF2, have been identified so far. However, key intermediates in terms of the quantitative estimation of PGF,, turnover (see below) are the major blood metabolite, 15keto,l 3,14-dihydro-PGF2, (8) [444] and one of the principal urinary metabolites, the 16-carbon diacid (1) [30]. The identification of the urinary metabolite [30] illustrates an exhaustive approach to structural elucidation using multiple derivatives and as such has been discussed in the Section on Derivative formation (p. 11). Two new prostaglandins, 19-hydroxy PGE, and 19-hydroxy PGE2have been identified as the major prostaglandins of human semen at an average total concentration of 100 pglml [437]. This recent finding is particularly notable since semen, a rich source of prostaglandins, was one of the first body fluids to be investigated in the classical early work. A new approach to isolation of the PGEs was employed involving oximation of unextracted semen preventing the facile degradation of the 9,ll-ketol grouping to the A or B series. Identification was based both on extraction of the stabilised derivatives for study by GC-MS and on oxidative degradation to known cleavage products. Quantitative determination of prostaglandin synthesis in man. The development of methods for the accurate determination of prostaglandin turnover in vivo are of considerable importance if direct evidence is to be obtained for their involvement in human pathogenesis. Such techniques are also required for the evaluation in man of the many agents shown to inhibit synthesis in animal studies and in vitro [446]. Ideally the levels of the primary prostaglandins should be monitored in blood or plasma. However, it has been suggested [447] that recorded levels do not reflect prostaglandin synthesis in vivo but, at least in part, represent platelet biosynthesis during blood sampling, together with possible contributions from, for example, non-enzymic cyclization. Synthesis inhibitors may be added to freshly drawn blood, but even so the predicted basal level of
A. M. LAWSON AND G. H. DRAFFAN
79
2pg/ml for PGF,, in peripheral human plasma is well below existing detection limits [447]. Radioimmunoassay is potentially the most sensitive of the methods now in use, although GC-MS is inherently a more specific approach. GC-MS methods are based on stable isotope dilution techniques using PGE,-d4 and PGF,,-d, as internal standards [89]. While a variety of different derivatives and techniques in instrumentation have been reported [32,81,76,430] the present practical limits are 100-200pg/ml for PGE, and PGFz, in whole blood [32]. Because of the technical problems associated with measurement of the primary prostaglandins in blood, attention has turned to metabolite measurement as an alternative means of monitoring turnover in man. Quantitative GC-MS assays have been developed for the common metabolite of PGEl and PGE, in human urine [448] and for the corresponding PGF metabolite (1) [449] using SIM with deuterated derivatives as internal standards. They have been applied, for example, in determining inhibition of PGE synthesis following therapeutic doses of indomethacin and aspirin [448] and in monitoring increased P G F synthesis during pregnancy [450]. The major blood metabolite of PGF,, (8) is measurable by GC-MS using stable isotope dilution techniques [32] and by radioimmunoassay [451]. However, the concentrations (of the order of 50 pg/ml) are close to the detection limits. This metabolite which is not released during blood sampling has been proposed as the compound of choice for monitoring PGF2, synthesis in vivo by analysis in peripheral plasma [447].
Biogenic arnines GC-MS has found wide application in studies of monoamines in both animal models and in human neuropharmacology [452]. Interest has centred on the use of selected ion monitoring in the determination of trace amounts of the amines, their metabolites and related substances with a possible function as neurotransmitters. The SIM approach complements established assay methods such as gas chromatography with electron capture detection (ECD), fluorimetry or enzymic assay. A check on specificity is afforded and in many cases enhancement in sensitivity and precision of measurement can be obtained. Method development, principally relating to estimation of central amine turnover, is noted in this Section and an outline of work on human depression serves to illustrate the potential of GC-MS to the study of CNS dysfunction. Chemical modification is required prior to GC-MS analysis, and with
80
G A ~ ~ L I Q U ICHROMATOGRAPHY-MASS D SPECTROMETRY
ion monitoring methods in mind, derivatives providing relatively simple
EI spectra with abundant, preferably higher mass ions are favoured. Perfluoroacylation has been the most widely adopted method both for the primary amines (e.g. [84,453]) and for metabolites (e.g. [454-4581). Spectra of TMS derivatives have been reported and employed in ion monitoring assay [459]. Isothiocyanates [460] and, in direct insertion probe assay at higher resolving power, dansyl derivatives [461] have also been used. Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [S41. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyldopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported 14621. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. In studies in man, less direct approaches are necessary and the concentrations of metabolites in CSF have been used to estimate central catecholamine turnover. Several GC-MS assays have been developed for the determination of homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid), the major metabolite of dopamine [457,458,464,465]. Satisfactory precision is reported for measurement in human CSF with the heptafluorobutyryl methyl ester derivative and HVA-d5 as the internal standard [457]. In an alternative SIM approach, CSF and plasma HVA have been determined using iso-HVA (3-hydroxy-4-methoxyphenylacetic acid) as the reference with gas chromatographic separation as the ethyl ester derivatives [465]. Endogenous iso-HVA in CSF has been determined as 4 give rise to
-
MUSCLE
111
T 18 55
79 121
111 I N V
VI
, n
Figure 3.1. " P N M R spectra of intact muscle from the hind leg of the rat, recorded at 129MHz, 20°C. Peak assignments are I, sugar phosphate and phospholipid; 11, inorganic phosphate; I l l , creatine phosphate; IV, y A T P ; V, a-ATP; Vl, P-ATP. T is the time (min) after excision o f the muscle. The shifts show that ATP is almost entirely complexed to Mg" and ageing of the muscle is accompanied b y a fall in p H (monitored by changes in frequencies of phosphate and A T P resonances) and creatine phosphate concentration (from [71)
P. J. SADLER
161
broad signals; these include I4N ( I = l), 23Na,' T l , 39K ( I = 3/2) and "0 and ZsMg(Z = 5/2). The technique is an insensitive one but with modern pulse methods on Fourier transform (FT) spectrometers, a reasonable signal can be observed from 0.2 ml of a 1 mM solution of protons ('H) in about 15 minutes. When a sample is placed in a magnetic field of 23000 Gauss (2.3 T), proton resonances are detected by applying a radio-frequency of 100MHz. All protons (e.g. aromatic, alkyl, NH) resonate within about 0.001 MHz of this frequency. For "C nuclei in this field, the required radio-frequency is 25 MHz and a sweep width of about 0.01 MHz will detect all resonances. Each resonance can be characterised by; (i) a chemical shift-that is, the exact resonance frequency, a reference peak being conveniently set at zero and a scale of parts per million used (0.001 MHz at 100 MHz is 10 ppm); (ii) coupling constants-resonances are split through interactions with other nuclei (e.g., 'H with 'H, or 'H with "C) which are up to about five bonds away, and the coupling constants can usually be measured from the splittings; (iii) a spin-lattice relaxation time--,, describing the return of the nuclear magnetism to equilibrium after the radio-frequency is switched off, and; (iv) a spin-spin relaxation time-T,, related to the width of the resonance signal. In this article, examples are chosen from recent literature to highlight progress in the NMR study of biological materials. Many emphasise the need for a careful choice of experimental procedure andlor instrumental design. The field is now expanding and too large to cover comprehensively, but several review articles on previous work are available: e.g. enzymes [9-121, protein structure [ 13-16], interactions of biological molecules [ 17-19], and medicinal chemistry [20]. Applications to molecular structure determinations are particularly plentiful and are considered first. Particularly noteworthy here are those experiments which are providing internuclear distances at the Angstrom" level of resolution for molecules in solution, as there is certainly a need to complement solid-state structural data obtained by X-ray crystallography. Many biological processes are triggered by the interaction of ions and small molecules with macromolecules; these studies form the second section. I3C Spin-lattice relaxation times are providing information about molecular mobility and sequential motion, thus increasing our knowledge of the behaviour of macromolecules which do not have the rigid frameworks of small molecules. However, other types of NMR experi-
*
1A=0.1nm.
162
NMR SPECTROSCOPY IN B I O ~ O G I C A LSCIENCES
ments can also investigate mobility, as is illustrated in the third section. Finally, work on intact tissues is described. Here the conclusions are often speculative, but it is undoubtedly an achievement to obtain NMR data from this type of material, and this will encourage much future activity. MOLECULAR STRUCTURE DETERMINATION ISOTOPIC LABELS
Biosynthetic pathways Biosynthetic pathways can be conveniently elucidated by employing specifically enriched I3C precursors. The sites and extents of incorporation into metabolites are simply determined from their I3C NMR spectra without recourse to tedious degradations required with radioactive l4C tracers. I3C Sources are readily available [21]. The I3C spectrum of the purified metabolite is compared with the same product having I3C in natural abundance (1.1%). Broad-band proton noise decoupling is used and five times the longest Ti (or at least three times) should be allowed between pulses so that the FT spectra are unaffected by relaxation effects. Streptomyces showdoensis incorporates 95% 1-[I3C]or 2-['3C]acetate into showdomycin (1) in the maleimide ring only [22], and this confirms earlier 14C findings. The exclusive incorporation of 1-[l3C]acetateat C-1, and 2-[I3C]acetate at C-2, C-3 and C-4 can be explained with reference to the Krebs cycle and the malic enzyme. I3C Spectra on 0.1 M solutions of labelled showdomycin in 13 mm tubes required 6 hr accumulation time. Furthermore, "C-labelled alanine, proline, glycine and serine have been fed to the bacterium Serratia marcescens [23] to trace the origin of the carbon atoms in its tripyrrole metabolite, prodigiosin (2). The methoxy group is probably derived from methionine whereas the C-2 methyl is from alanine.
P. J. SADLER
163
When a doubly-labelled precursor such as 13CH3'3C02Nais incorporated, then 13C-13Ccouplings appear in the spectrum of t h e metabolite unless C-C bond fission has occurred. This is the case for dihydrolatumcidin [24] from Streptornyces reticuli, and the couplings were found to be + CH,"CO2Na) was weaker when a mixed labelled acetate (13CH3COzNa used. Acetate incorporation can also be studied by tritium ('H) NMR. It is more sensitive to detection than "C and its radioactivity can be used as a simultaneous monitor. A good signal-to-noise ratio has been obtained [25] from as little as 1 mCi of 3H in penicillic acid (3), biosynthesised by PeniciZZiurn cyclopiurn. ['HI + Acetate (460 mCi) has been incorporated with 7% efficiency. 'H Chemical shifts and coupling constants are predictable from 'H spectra and direct information about stereospecific hydrogen labelling can be obtained.
Assignments Biosynthetic deuteration of all but specific amino acid residues is a useful aid to assignment of peaks in the complex proton spectra of proteins [26]. An alternative approach to spectrum simplification is to introduce I9Fas a label for specific residues. When observing 19Fresonances, there are no problems from HO'H signals and there is a Iarger chemical shift range compared with 'H, and spectra can be run in H 2 0 instead of 'H20. For example, fluorotyrosine alkaline phosphatase (monomer mol. wt., 43 000) can be isolated from E. coli grown in a medium containing rn-fluoro-DLtyrosine (10% yield of control enzyme). At least 10 resolved I9F resonances are obtained [27] (Figure 3.2), there being 11 tyrosines per monomer. In 6 M guanidine hydrochloride, all have the same chemical shift as expected upon denaturation. The fluoroenzyme shows a slightly different renaturation behaviour (pK of fluorotyrosine is 2 units less than tyrosine) which must be taken into account when deductions about tyrosine environments are extrapolated to the control enzyme. Synthetic modification is also possible: trifluoroacetylation of haemoglobin at cysteine p93 gives a single I9Fresonance whose chemical shift depends on the ligand state and conformation of the p chains [28].
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
164
"F-ENZYME
48
50
52
7
54
56
58
60
62
64
j E X T(PPMI CF,COOH
Figure 3.2. I9FNMR spectrum of Puorotyrosine alkaline phosphatase 25 mg/ml in 0.3 M Tris-HCI buffer. Each of the I 1 fluorotyrosines per monomer has a different chemical environment, and gives rise to a separate peak in the spectrum (from [27])
The complete assignment of the I3C spectrum of the cyclic decapeptide antibiotic gramicidin (4) has been achieved via biosynthetic enrichment studies [29]. Bacillus brevis, for example, was grown on a minimal medium containing a 1 mM supplement of a 10% "C-enriched amino acid (Leu, Val, Phe or Pro), and I3C Satellites in the 'H spectrum and selective decoupling aided the assignment. Unequivocal assignments of the NH proton resonances from oxytocin ( 5 ) employed [30] the synthetic *H-and '5N-substituted derivatives: 2-["N]Tyr, 3-[I5N]Ile and 8-[a-'H]Leu oxytocin. The appropriate NH resonance is split into a doublet (-"NH-CH-, I = for I5N, JlS,_, = 93 Hz) or broadened (-NH-CZH-, I = 1 for 'H but J2,., very small). L,PRO-L-
D.P,H E
VAL, LYRN
L-LFU L- LEU D+HE LOR,N LVAL- L- PRO ( 4 f Grarnicidin S
(5)
Two of the four histidine C-2 protons of ribonuclease A (mol. wt. 13700) exchange completely with 'H when incubated in 'H20 at 37°C at pH 8.5 for 5 days and their resonances disappear from the spectrum. This leads to their unequivocal assignment [3 I]. Other groups such as OH and SH exchange their protons very rapidly
P. J. SADLER
1 65
for deuterons but NH exchanges are often slow enough to give observable resonances. These can be good probes for future structural information. NH PROTONS
N H resonances often appear in a spectrum when the substance is dissolved in H,O instead of 'H20. Proton exchange is then slow, especially when it is hydrogen-bonded or lies in a pocket inaccessible to bulk solvent. Hydrogen-bonded and tryptophan-NH protons can usually be located to low field, conveniently several ppm outside the main spectral envelope. Continuous wave (CW) methods are used to record proton spectra when H,O is used as solvent, because the large signal from (55 M) H 2 0 overloads the computer store of an FT spectrometer. With the modification developed by Dadok and Sprecher [32] (called correlation spectroscopy), the overall signal-to-noise ratio from the CW experiment is greatly improved. Five of the 6 tryptophan-NH resonances of lysozyme (mol. wt. 14400) in H 2 0 are found at 10-12ppm from DSS" [33]. These protons take several hours to exchange with 'H when dissolved in 'H,O. Their differential exchange rates and response to substrates are an aid to assignment. Eleven protons of bovine pancreatic trypsin inhibitor take more than 4 months to exchange with 'H in *H,O at p H 7 [34,35]. Evidently some domains of this small protein (58 amino acids) are extremely tightly folded. NH Resonances from tryptophan, arginine and histidine have been observed for myoglobin 1361 in the range of 10-15 ppm. Deoxyhaemoglobin, dissolved in H,O at pH7.4, has a resonance at 14ppm which disappears upon ligation of the protein 1371. It has been used [38] as an indicator of quaternary structure as it is unaffected by unpaired iron-spin or porphyrin ring-current. An exchangeable proton of ribonuclease A titrates with a pK, of 5.8 and has been assigned [39] to the NH of the active-site histidine-119. A low field resonance can be observed for chymotrypsin in H,O [40] and its pH dependence (15 to lSppm, pK, 7.2) and response to chemical modification suggests that this is the hydrogen-bonded proton between His-57 and Asp-102 at the active site.
*
DSS is Me,SiCH2CH2CH,SOjNa+,the methyl proton reference peak is at Opprn. Positive shifts are to low field.
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
166
Resonances at 11-15 ppm from exchangeable protons of ribonucleic acids can be used as structural probes. 18 1 of the 21 ring NH groups of tRNAEh,',,,involved in base-pairing (6) through H-bonding can be assigned
*
t$pswr (;o HNHOfi
y\sugar
N
I
Sugar
Sugar
G*C
A.U
(6)
[41] in the proton NMR spectrum at 300 MHz ( 1 4 mg in microcell, pH 7 (Figure 3.3). Guanine:cytosine, G.C, (11 to 13 ppm) and adenine:uracil, A.U, (13 to 15 pprn) NH resonances are shifted by the ring currents of nearby bases, and calculations [42] indicate that the solid-state clover leaf structure is appropriate in solution. NH Resonances disappear when the helices melt and Mg2+helix stabilisation is readily observed by NMR [43] but optical melting points are higher than the temperature range over which NH resonances broaden and disappear [441. T,,,for the double helix of d(A-AC-A-A) with d(T-T-G-T-T), for example, is 28°C but resonances have disappeared by 9°C; T is thymine. Besides these nitrogen bases, many other aromatic groups produce ring currents. The most important are the amino acid side chains of phenyl-
n
N l j REGION
GC
A-U P '
I
1
14
I
I
I
12
PPm
Figure 3.3. ' H 300 MHz N M R spectrum of intact yeast tRNAPhe.This region contains the resonances ascribed to ring N H protons, and they can be further assigned to individual base-pairs (from [41J)
P. J. SADLER
167
alanine, tyrosine and tryptophan, and the porphyrin moiety. Resonances from nuclei within a few Angstroms of these rings are shifted from their normal positions, and this chemical shift change can be related to the position of these nuclei with respect to the ring. A movement of 0.1 A gives a shift of about 0.1 ppm. As an approximation, the ring current shift is considered to originate at the centre of the aromatic ring, although it extends over the whole of the conjugated framework of the molecule. The origin of the perturbation of resonances produced by paramagnetic probes, on the other hand, is well-defined and the effects can again be quantified. PARAMAGNETIC PROBES
Some metal ions (e.g. Mn”, Fe3+)and free radicals (e.g. semi-quinones, nitroxides) shift and broaden resonances from nuclei which come within several Angstroms of them, as they are paramagnetic. Paramagnetism is due to unpaired electrons on the metal ion or free radical and can often be detected by electron spin resonance (ESR) experiments. Those which give detectable ESR signals at room temperature produce the largest broadening of nuclear resonances (e.g. MnZ+,Cu2+,Cr3+, Gd3+ and free radicals-broadening probes). Metal ions which show ESR signals only at low temperatures usually produce their largest effect on the NMR chemical shift of a nucleus (Cozi, Eu3+,P?). These are the shift probes. Broadening corresponds to a shortening of the nuclear spin-spin relaxation time, T 2 ;the spin-lattice relaxation time, TI, which unlike T2cannot be measured directly from the spectrum, is usually shortened as well. When the unpaired electrons are localised on the metal ion or free radical, it is relatively straight-forward to obtain geometrical information about the surrounding nuclei from the through-space (dipolar) interaction equations. When there is delocalisation of unpaired electron spin density onto these nuclei, there will also be through-bond contributions to the effect observed. In general, through-space and through-bond effects have to be separated. Through-space effects predominate in several cases and structural information about these molecules in solution is directly obtainable. Through-bond effects provide unpaired electron density maps (e.g. in haems). Metal ions Paramagnetic metal ions are often an intrinsic part of biological materials (e.g. Fe in globins, Mo in nitrogenase). Alternatively, diamagnetic metal
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
168
ions can be isomorphously replaced by paramagnetic ions (e.g. Ca” by Eu3+,Znz+by Co2+or Mn”) often with retention of biological activity. Thirdly, when a specific binding site exists, the ions may be added to the biological material as an extrinsic NMR probe of structure. The large decreases in spin-lattice relaxation times of nuclei surrounding Mn2+(3dS,five unpaired electrons) can be interpreted to give structural information. The ion is widely used in all three of the above categories. Table 3.1 illustrates some recent experiments. Many are concerned with relaxation-enhancement studies. The spin-lattice relaxation times of water (‘H or ”0)are usually enhanced when water is bound to a MnZt-macromo1ecule, compared with an identical solution without the macromolecule. It is often possible to calculate Mn2+hydration numbers, Mn2+-waterbond lengths, and solvent accessibilities [ 111. Table 3.1. Mn” AS A STRUCTURAL (RELAXATION) PROBE Material
Resonance
Intrinsic Concanavalin A Pyruvate carboxylase Isomorphous replacement Carboxypeptidase A Pyruvate kinase Phosphoglucomutase Carbonic anhydrase
Extrinsic E. coli ribosomal RNA EnoIa se
Adenosine triphosphatase Phosphorylase*
**
Comment on Mn2’
‘’C(sugars) 1~,i70(~20) ‘H,’’C(pyruvate)
*
f45,46,4%53]
*
[54,551
‘H(H20),”F(F-) ‘H(H,O),”C,’H,”P (pyruvate) ’H,”P(methyl phosphonate) ’H(H,O,N-acetylsulphanilamide, acetate
35%(Zn2+)** 100%(Mg2’)
[56,58] [54,56,59]
5%(Mg2+)
[601
4.5%(Zn2’)
[61,62,121]
‘H(H,O) ‘H(H,O),’H,”P (phosphoenolpyruvate analogues ‘H(H,O)
Binds to phosphate Binds at active site
[631 [641
Competes with Mg2’
[651
One site per protomer Enhances tetramer formation
1661
‘H(H,O) ‘H(H2O)
~~
* See text. ** Activity
Reference
of Mn” derivative compared with (native metal ion).
1671
P . J. SADLER
169
The work on Concanavalin A (Con A ) has elegantly illustrated that NMR methods on solutions can be used to obtain structural data complementary to that from X-ray methods on crystals. Con A monomers (mol. wt. 26000) contain one Caz+and one Mn2+ion and bind one sugar molecule ( a-D-gluco- or a -D-mannopyranosides). Brewer, Sternlicht, Marcus and Grollman [45,46] concluded from l3C NMR that the distance between the MnZ+ion and a bound glucopyranoside sugar (7) is about l o & which contrasts with the X-ray finding of 20A. This prompted noQoMe OH
( 7 )
further X-ray studies, and it now seems clear [47] that the iodophenyl sugar derivative used in the X-ray work is bound by the iodophenyl group and not the sugar ring. The carbon atoms of the sugar were enriched to 14% I3C for the NMR work (higher levels of enrichment give rise to 13C-”C spin-spin couplings in the spectra). The ‘H-noise decoupled 13C resonances (10 mM sugar) broadened but did not shift in the presence of Con A (0.9mM), and TI values were selectively shortened. Diamagnetic corrections (using Zn-Con A) were applied, and paramagnetic contributions to the relaxation rates (TT;) were obtained. The Mn” to sugar carbon distances ( r ) are given by equation (1)
T;; =
constant
r6
which assumes that the magnetic interaction is through-space (dipolar). Analogous measurements using natural abundance ‘3C have also been made 1481. There appears to be one water molecule bound to Mnz+in Con A and this can exchange with solvent water. This was the conclusion from ‘H spin-lattice relaxation rate studies with Con A dimers, 0.9 mM in 0.1 M phosphate buffer pH 5.6 [49]. A residence time of 2.5 p s e c and MnZ+-H distance of 2.7 A were calculated after the evaluation of five parameters. Addition of a sugar had no effect on the relaxation rates and the sugar, therefore, does not bind to the metal. However, removal of the Ca2+ion decreases the residence time of that H 2 0 molecule by a factor of ten [50]. In the crystal, the two ions are 5 A apart. A novel NMR experiment of Barber and Carver [Sl] has shown that
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
170
both the polypeptide chain and the Ca2+ion restrict access of solvent to the Mn2+ ion. When Mn2+ was added to de-metallised Con A, the 'H relaxation enhancement of H 2 0 gradually decreased for more than 2 hr. This can be accounted for by the slow wrapping of residues 12-22 around Mn", a process accelerated by Ca2+ which derives ligands from this loop." Further NMR work [53] confirms that Ca" is merely a catalyst for the formation of the Mn2+site. Mn" (or Co2+,Ni2+,Zn2+)creates the sugar binding site. The chicken liver enzyme pyruvate carboxylase also has a natural requirement for Mn". The distances between Mn" and the carboxyl and carbonyl carbon atoms of pyruvate (8) in the enzyme-pyruvate complex
(8)
have been determined [54]from TI measurements of 'H-decoupled I3C resonances of 35% enriched 1-[I3C]-and 2-['3C]pyruvate (60 mM, 20 p M Mn" bound to enzyme) (Figure 3.4). The paramagnetic contribution to the relaxation rate was 3.5-fold greater for the carboxyl than for the carboxyl carbon. The Mg2+-enzyme was used as diamagnetic control (from Mn2+deprived chickens, Mg2' and Mn2' are often good isomorphous replacements). The carbonyl carbon is 7.1 from Mn compared with 8.5 A for the carboxyl carbon. Pyruvate is therefore in the second co-ordination sphere of the metal ion and not bound to the metal by the carboxyl group as in the inorganic Mn2+-pyruvate complex. These conclusions do not agree with those from TIstudies of the methyl protons of pyruvate [ 5 5 ] . The discrepancy has been attributed to uncertainties in T ~ the , correlation time characterising the dipolar interaction. The conand a 10% error gives a 2% error in stant in equation (1) is a function of 7,-, calculated distances. Accurate geometrical information, therefore, from this type of experiment requires reliable estimates of 7,-(see [9] p. 241). The sharp ESR signals from free Mn2+provide a means of estimating the concentration of Mn2+bound to a macromolecule in a solution used for NMR work. Signals from bound Mn2+are usually very broad. Gd3+and Eu2+(4f') can also be used as broadening probes. Trivalent lanthanide (rare earth) ions, like Ca2+(which is also of a similar size) bind
*
They have now assigned 5 of the 6 His C-2 peaks in the 220 MHz 'H spectrum of Con A, using a third metal (lanthanide) binding site as additional probe [52].
P. J. SADLER
NFT
N FT
171
u&
&
42
Figure 3.4. ’H-Decoupled I3CNMR spectra of 35% enriched l-[”C/- and Z-[”C/pyruuate, in the presence of (A) Mn ’+-pyruvatecarboxylase, and (B) Mg”-pyruvate carboxylase. Each spectrum, except the normal Fourier transform (NFT) spectrum, is the result of applying two radiofrequency pulses to the sample; the delay between them is indicated in sec. From measurements of peak heights the TI values can be calculated, and d resonance has zero intensity at about 0.69 T,. Note that Mnz’ shortens TI,and has a diflerential eflect on the two carbon atoms (from 1541)
172
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
to negatively-charged oxygen atoms. Thus, Gd3' can substitute for Caz+in the formation of a ternary complex with staphylococcal nuclease (single polypeptide chain, mol. wt. 16900) and nucleotide inhibitor. From measurements of the spin-spin relaxation rates of protons and spin-spin and spin-lattice relaxation rates of "P nuclei of bound 3',5'-thymidine diphosphate, it was concluded [68] that the metal ion-nucleotide atom distances in solution correspond closely to those found by X-ray crystallography. Other lanthanide ions, notably Eu3+and Pr3+,can be used as shift probes. When there is a through-space interaction
Av
= constant x
(3 cos2 8 - l)/r3
(2)
between the metal ion and the nucleus, equation (2) describes the shift (Av)in terms of the distance between them ( r ) and the angle made by the internuclear vector and a symmetry axis passing through the metal. Proton resonances of the nuclease itself can also be studied, and metal-histidine distances have been obtained in this way [69]. Lanthanides have been extensively tested as structural probes with lysozyme. Water [70], inhibitor [71] and protein [6] resonances have all been observed. The native enzyme does not have a natural requirement for metal ions, but they bind at the active site between the carboxyl groups of Asp-52 and Glu-35. The most exciting aspect of this work is the correlation between the 3-dimensional structure of lysozyme in solution and the crystal, and this is beginning to emerge from observations of protein resonances (Figure 3.5).This is made possible by considerable improvements in the resolution of the complex 'H NMR spectrum (950 protons!) of lysozyme obtained by using a high frequency FT instrument (270 MHz) together with on-line computer manipulation of the spectrum to give sharper peaks (deconvolution, [72]). The computer can also readily calculate difference spectra; thus, Gd3' broadens those resonances from nuclei nearest to it, and difference spectra in the presence and absence of Gd3+show only resonances from those nuclei (effect decreases rapidly as r-6). Thus, as the concentration of Gd3+bound to lysozyme is gradually increased, the first peaks to appear in the difference spectrum are from Val-109 and Ala-110. The conformations of nucleotides have been probed by lanthanides [73]. Furthermore, adenosine triphosphate (ATP)-lanthanide complexes are strong enough to carry the metal on to enzymes. Thus, Gd3+ coinpetitively inhibits Mg2+-(yeast)-phosphoglycerate kinase (mol. wt. 47000) and selectively broadens histidine 'H resonances [74].
P. J. SADLER
3
2
1
173
0
-1
PPm
Figure 3.5. The methyl region of a 27OMHz ' H N M R spectrum of human leukaemia lysozyme ( 5 m M , 55°C). The upper trace is the normal spectrum, the lower trace (a) is a convolution difference spectrum. Note the additional fine structure present in (a) which aids peak assignment (from [72])
Iron is usually present in haem- and non-haem proteins in a paramagnetic ferrous or ferric state. The exception is low-spin Fez+,as in oxy-FeZ+myoglobin or haemoglobin, which is diamagnetic. Shifts and broadenings of the resonances of nearby nuclei are usually caused by a combination of through-space and through-bond effects, and a separation is therefore required before both geometrical and electron density information is obtained. Low-spin Fe3+globins have been extensively studied, especially the cyanide derivatives, on account of the large paramagnetic shifts and smaller broadening which they produce compared with high-spin Fe3+or Fez' derivatives. Much of the work is aimed at improving the understanding of the co-operativity of oxygen uptake by haemoglobin, and the
174
NMR SPECTROSCOPY I N BIOLOGICAL SCIENCES
mechanism of haem-haem interaction. Paramagnetically-shifted resonances are observed only from the ferric chains of the hybrids (a3+CNp2+02)2, ( L Y ~ + O ~ P ~and + Cthe N ) spectrum ~ of a2P2tetramer is not a simple combination of those from isolated (Y and P chains [75,76]. Definite small variations in spectra are found in a comparison of spectra of haemoglobins from humans, sheep, cows and rabbits [77]. The 'H NMR spectra of genetically determined haemoglobin variants assist the identification of those paramagnetically-shifted resonances which are indicators of quaternary structure: (see haemoglobin J. Capetown [78], M. Milwaukee [79], M. Iwate 1801, Kansas [Sl]). Separate I3C resonances are observed from I3CO bound to the a and /? chains of the tetramer [82]; haemoglobin M Iwate does not bind CO on the a-chain haems, so an assignment is possible. The study of cytochrome c (mol. wt. 12200) does not centre on haem ligation studies, since His-18 (N) and Met-80 (S) are firmly bound in the fifth and sixth positions, but upon the passage of electrons when the protein is reduced from the paramagnetic low-spin Fe3+to the diamagnetic low-spin Fez+ state. A wealth of information is available from high frequency 'H NMR spectra of the protein [83]. Elegant double resonance procedures have been used to correlate resonances in the spectra of reduced and oxidized forms [84], and paramagnetically-shifted resonances are affected by addition of cytochrome c peroxidase [85] and iron hexacyanides [86]. Fast electron transfer between Fez+ and Fe3+ cytochrome c produces chemical exchange effects in the I3C spectra of mixtures of the two species [87] (Figure 3.6). The close agreement between the positions of shifted lines in the spectra of Fe3'-cytochrome c -557 and the proteins from vertebrates indicates that the electronic structures of their haems are similar [88]. CoZt is available as an isomorphous replacement for Fez' in haems (cobaltmyoglobin, for example, still binds oxygen) and structural information has been obtained about cobalt porphyrin molecular complexes with steroids [891 and caffeine [90] in solution. The PCH2 groups of cysteines attached to the FelS clusters of ferredoxins have been identified [91] by comparison of their 'H spectra with model compounds [92]. When 50% reduced, the spectra are suppositions of peaks from oxidised and reduced species; hence electron exchange between the two forms must occur very slowly. In general, those resonances which are shifted through interaction with a paramagnetic ion are readily identified in an NMR spectrum: their shifts are inversely temperature-dependent.
P. J. SADLER
CYT c
175
i 14
ppm from CS2
Figure 3.6. The aromatic region of the 15 MHz 13C N M R spectrum of cytochrorne c; about 10mM, in phosphate buferpH6.7, 41°C. (A) Fe3'-cytochrorne c ( B ) 9: J mixture of Fez'-: Fe"-cytochrome c (C) Fez+-cytochromec. Resonances from carbon atoms near the iron are broadened and shifted in the spectrum of the paramagnetic Fe"-form. The numbered peaks account for 36 of the non-protonated carbons, peak 30, for example, is C-y of Trp 59 (from t871)
No I3C resonance was observable [93] from HI3CO; bound to transferrin (mol. wt. 78000). Evidently, the Fe-C distance is less than about 9 A , and the resonance has broadened out. Transferrin tyrosyl resonances are also broadened by (high-spin) Fe3' in the 'H spectrum of the protein [94], which may indicate the protein-binding groups for iron.
Free radicals Paramagnetic nitroxide radicals (spin labels) have been attached at specific sites on enzymes and inhibitors to perturb 'H NMR spectra by broadening resonances from amino acid residues near the unpaired
176
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
electron of the radical. Distances up to 20 A are probably accessible by this technique. Thus, lysozyme covalently spin-labelled at His-15 broadens the proton resonances of N-acetyl-a-D-glucosamine and di-Nacetyl-D-glucosamine bound at the active site, and these broadenings have been used to estimate the distances from His-15 to the acetamido methyls [95]. Phosphofructokinase (monomer, mol. wt. 90000) can be spin-labelled at a reactive SH site with 4-(2-iodoacetamido) + 2,2,6,6tetramethyl piperidino-oxyl, retaining 60% enzymatic activity [96], but it has little effect on the relaxation rates of bound Mg-ATP protons. A similar labelling (9) of yeast alcohol dehydrogenase at Cys-43 (near active
(9)
site, inactivates enzyme) has suggested that reduced nicotine adenine dinucleotide (NADH) protons are 8-14 A from the radical [97]. The amino acid residues in the hapten combining sites of the Fv (mol. wt. 25000) and Fab (50000) fragments of the myeloma protein MOPC 315 (150000, IgA) have been probed [98] with spin-labelled dinitrophenyl haptens. Nitroxide radicals are bulky and may perturb the structure of the macromolecule. Enzyme inactivation may occur, and accurate information, as with Mn2', again requires reliable estimates of correlation times. They may also flap about on the enzyme causing uncertainty in the location of the radical perturbation. However, ESR can be used to check mobility [95]. Flavodoxins have a natural spin label, but, unlike the nitroxides, the flavin-free radical suffers from electron de-localisation, which interferes with distance calculations. The broadening of protein resonances in the spectra of the semiquinone forms of flavodoxins from Clostridium MP and Peptostreptococcus elsdenii is highly selective [99], and peaks from groups in the vicinity of the radical can be assigned. COUPLING CONSTANTS
The value of the 'H-'H spin-spin coupling constant in fragments of the kind H-C-C-H depends upon the dihedral angle about the C-C bond (Karplus equation). This relationship has been extended [ 1001 with caution to three bond H-N-C-H coupling constants of peptide bonds
P. J. SADLER
177
(Karplus-Bystrov equation). NH Peaks (observable in water or nonaqueous solvents, not in 'H20) must be accurately assigned, and this has caused controversy in the cyclic nonapeptides oxytocin [30] and vasopressin [loll. The complete assignment of the NH resonances of the octapeptide angiotensin I1 (10) in H 2 0 has been accomplished [lo21 by Asn-Arg-Val -Tyr-Val -His-Pro-P he (10)
using an NH-a -CH decoupling technique that carefully avoided saturation of spectrometer amplifiers (a-CH peaks are usually submerged beneath the large water resonance). The data excluded the (Y helix, conventional p turn, and y turn or random coil. In general, a given coupling constant may correspond to 4 angles, and the use of a second 3-bond coupling constant such as I3C-N-C-H or H-C-C-I5N may help to remove such ambiguities. In a novel use of a I3C-N-C-H coupling constant, the time-average conformation of N-acetyl-L-tryptophan in solution was found [lo31 to be different from its complex with chymotrypsin. 13C-'H coupling constants may also be used [lo41 in conjunction with 'H-IH constants to give fractional populations of amino acid side-chain rotamers. Another molecule which distorts upon binding to an enzyme is the tetrasaccharide (NAG-NAM)z*which binds to lysozyme [105]. Since the cleavage reaction is relatively rapid, spectra have to be taken quickly and changes in coupling constant are followed for several minutes. Three-bond couplings to 31Phave been extensively used for conformational analysis of nucleotides [106, 1071. The results suggest a surprisingly rigid nucleotidyl structural unit.
ION AND SMALL MOLECULE BINDING Many binding processes lead to spectral perturbations which are less well understood than those referred to in the previous section, and, as yet, cannot be interpreted to give bond lengths or bond angles. Nevertheless, the thermodynamic binding parameters can usually be derived, and qualitative structural data often obtained.
*
N A G is 2-acetamido-2-deoxy-D-glycopyran0~e. N A M is 2-acetamido-2-deoxy-muramic acid.
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
178
IONS
Protons Proton dissociation produces a shift in the resonance positions of nearby nuclei. The pK, values of ionising groups are readily determined from a graph of chemical shift versus pH. A classic example is ribonuclease. The C-2 'H histidine peaks lie at low field, outside the main spectral envelope, and the pK, values of the four histidines range from 5.8 to 6.7 [108]. Methylation of lysozyme lysines gives 6 new peaks at 270 MHz 11091 and the e-NH2 p K values can be determined. When 'H spectra are complicated, I3CNMR may provide a useful alternative route to ionisation constants. The 0.7 difference in pK, values of cis and trans-N-acetylproline is readily detected [110] by 13CNMR (Figure 3.7), and the trans form predominates at low pH. I3C Protonation shifts aid the elucidation of the structure of the tetra-functional cross-link compound isolated from borohydride-reduced cow skin collagen [ l l l ] , which also has a complex 'H NMR spectrum. The chemical shift of 31Pphosphate resonances are also dependent upon pH. This can be made the basis for the determination of intracellular pH, by monitoring the shift difference between intracellular and serum phosphate [112]. Similarly, in red cells, 2,3-diphosphoglycerate (5 mM in human cells) can be utilised. The I4N resonances of amino acids, e.g. histidine, show large changes 182
-
t
i
-
; c
180
-
E
P
*
E a
178
-
176
-
I
I
I
I
I
1
1
2
3
4
5
6
7
PD Figure 3.7. "C N M R titration curves f o r the COO- resonances of cis ( 0 )and truns (0) N-acetyl-DL-prohe, 0.3 M . The p K values are 3.47 and 4.13 respectively (from [110])
P. J. SADLER
179
at their own respective pK, values C1131. However, the lines are very broad ("N has a quadrupole moment) and only small molecules or those with rapid segmental motion give observable I4N signals ("N is less abundant but gives sharp peaks). The general use of difference spectroscopy to follow pH titrations by NMR should be noted [1141.
Metal ions Diamagnetic metal ions (such as Na', Ca*+, Ga3+) often produce small shifts of the resonances near the binding site and small effects on relaxation times. This is in contrast to the large effects produced by paramagnetic ions, discussed above. Ga3' was preferred [115] as a substitute for Fe3+to study metal binding to enterobactin (1 l), the microbial high-affinity iron transport compound, to retain coupling constant information which is blotted out by the line-broadening, paramagnetic Fe3+.
$=O
r;. 0- C H2-
C -C
( 11 )
The 'H and I3C resonances of the catechol groups shifted when the metal bound, whilst the aliphatic resonances reflected a major conformational change. When the cyclic antibiotic valinomycin forms a complex with K', Rb' or Cs' ions, the carbonyl I3C signals are shifted 4-5 ppm downfield [116]. The shift differences upon complexation with Na' are much less pronounced, which is presumably related to the ability of the antibiotic to distinguish between Na' and K'. The S-Me methionine 'H resonances are shifted and broadened when PtCl:. binds to ribonuclease [117] (Figure 3.8). In its native state, only one residue is accessible, but all four become available for binding at low pH. Cd" and Zn*+binding to metallothionein (mol. wt. 12000) begins at pH 2, and cysteine methylene 'H resonances are broadened [118].
180
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES RNase
Figure 3.8. The effect of chloroplatinite on the 1OOMHz ' H NMR spectrum of 2 m M ribonuclease. The protein is initially partially unfolded, and Pt '+ induces almost complete unfolding by binding to sulphur of methionine residues. Note the disappearance o f the S-CH, resonances at A (from [ I 171)
An alternative way of investigating diamagnetic metal-ion binding is to observe NMR signals from the diamagnetic metal itself. 23Na,39K and 133 C s have been the subject of a few studies [9]; however, quadrupolar nuclei are inherently insensitive to detection. Further advances using high field NMR ihstruments can be expected. SMALL MOLECULES
NMR is an attractive technique for the study of the interaction of small molecules such as drugs with macromolecules because of the possibility of probing the structure, environment, and motional freedom of the bound molecule (see reviews [17, 181).
P. J. SADLER
181
In the presence of human serum albumin, the 'H spectrum of acetylsalicyclic acid is specifically shifted and broadened [119]. The interpretation of changes in TI and T2 require several theoretical assumptions. These have been discussed in detail [1201 for N-acetylsulphanilamide and acetate binding to the active site of carbonic anhydrase. It was concluded that the acetyl groups of these inhibitors have a motion additional to that of the enzyme. It can be shown by NMR that acetate binds to two sites on the enzyme, only one of which is inhibitory to esterase activity (methyls are 4.3 and 4.8 A from the metal in the Mn2+substituted enzyme [121]). Strict care must be taken to avoid paramagnetic impurities when NMR relaxation enhancement by diamagnetic macromolecules is being studied. A preparation of carbonic anhydrase, for example, can contain 0.24 paramagnetic Cu atoms per Zn atom [122]. Applications of the nuclear Overhauser effect (NOE) are promising for studying small molecule binding. For example, NOE effects on the tryptophan aromatic proton signals are observed upon irradiation of upfield resonances of a -chymotrypsin in solution with (+)-tryptophan [123]. The effect is a negative one, the integrated intensity of the tryptophan signals decreasing upon irradiation. The maximum effect is at S = 1 ppm, implicating Val-213 on the edge of the tryptophan ring in the N-formyltryptophan-enzyme complex. NOE studies have also been successfully applied to peptide-neurophysin interactions [ 1241. They indicate that the aromatic ring in position 2 of the bound peptide (e.g. Tyr of Ala-Tyr-Phe-NH,) is in close proximity to a single tyrosine on the protein. It is worth noting here that negative NOES have been observed in protein spectra [27,125] and may be useful for assignment purposes. The resonances of a small molecule are usually broadened when it binds to a macromolecule and this effect, due to decreased freedom of motion, is of general use for molecular mobility studies.
MOLECULAR MOBILITY The most mobile groups are those with nuclei having; (i) the sharpest 'H resonances (smallest linewidths), or (ii) the longest ('H-decoupled) I3C spin-lattice relaxation times. FOLDING AND UNFOLDING
Synthetic polypeptides have been studied in some detail [I261 as model protein systems. The a - C H and NH proton resonances of poly(y-benzyl
182
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
glutamate), for example, are broad in dimethyl sulphoxide indicating a helical conformation [ 1271; on the other hand, poly(p -benzyl aspartate) shows sharp peaks and has the more mobile random coil form. The T I values of the a -carbons of poly(y -benzylglutamate) increase by about 50% when going from helix to coil [128]. Proton resonance spectra of denatured proteins consist of sharp peaks which correspond to a summation of resonances from individual residues [129]. In I3C spectra of denatured proteins, it is possible to distinguish all the carbon resonances of the aromatic side chains of histidine, phenylalanine, tyrosine and tryptophan, and separate resonances from alanine, arginine, glycine, isoleucine, leucine, threonine, valine and occasionally methionine [130]. (A natural abundance I3C spectrum of a 13 mM solution of lysozyme takes only 4 hr accumulation time using 20 mm sample tubes [ 13 11). In their native (folded) conformations, proteins give broader 'H and I3C resonances than in their denatured states, and there are shifts of peak positions too, indicative of the new microenvironments of the nuclei. Unfolding pathways can readily be followed [ 1321. Ribonuclease Speptide-S-protein interactions [ 1331 suggest that a Glu-2 Arg-10 salt bridge stabilises that region of the folded protein. Local mobility of parts of the S-peptide when bound to the S-protein can be deduced with the aid of selective '3C-labelling [134]. This is a general method for monitoring helix formation at individual amino acid residues in proteins; synthetic or biosynthetic selective introduction of 13Cinto protein residues is possible. "C-studies [135] on unstretched elastin in ligamentum nuchae show that it is composed of highly mobile chains. Histones have segments of polypeptide chain which are rich in basic residues and complementary segments rich in apolar, aromatic, acidic and other active residues. 'H NMR studies have shown [136,137] that the former are primary sites of DNA interaction, whilst the latter are sites of histone-histone interaction (Figure 3.9). A model for interactions in chromatin gel has also been proposed from the observed differential broadening of peaks. MEMBRANES
Lecithins, soaps and surfactants can form bilayer structures in water, resembling those in cell membranes, and NMR has been used to determine the degrees of molecular mobility at the interior and surface of the bilayer. The proton spectrum of dipalmitoyl-lecithin (12) shows very
P. J. SADLER HISTONE F2A1
u
0.05 M
9
8
7
6
ppm Figure 3.9. The aromatic region of 270 M H z spectra of calf thymus histone F2Al in '&O. When NaCl is added aggregation occurs, but only residues 33-101 are inuolued. The C-2 and C-4 resonances from His 18therefore remain sharp, whereas those from Tyr 51, 72,88,98 and Phe 61 and 100 are broadened out (from [136])
broad resonances below 40°C, when a phase transition occurs to a liquid crystalline form and resonances sharpen [138] Figure 3.10). The spectra of lipids are often very broad and difficult to interpret [ 1391 but sonication markedly intensifies and sharpens resonances [140]. Both I3C and 'H T , measurements [ 141, 1421 indicate the greater mobility of methylene groups towards the terminal methyl group. "C-Studies [ 1431 indicate that phospholipid molecules bind to the apoprotein of high density lipoproteins with their hydrophobic fatty acid chains, and not with hydrophilic zwitterionic groups. The study of 31P relaxation times provides a specific method for observing dynamic events in the head group region of phospholipid dispersions and membranes [ 1441. Mn2+ions broaden only those resonances from nuclei on the outside of phospholipid vesicles, and therefore the percentages of lipids on the inside and outside surfaces can be calculated [145].
184
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
SMALL VESICLES
Figure 3.10. 220MHz 'HN M R spectra of small vesicles (300A)of dipalmitoyllecithin. A phase (Chapman) transition from crystalline to liquid crystalline phase occurs near 40°C (from t1381)
Sciatic nerves are a popular choice for the NMR study of myelincontaining membranes, as two 10 cm long portions fill a 12 mm diameter NMR tube and yield a "C spectrum in about 30min. The spectrum of canine sciatic nerves is identical with that of canine adipose tissue and similar to liquid triolein [ 1461. No resonances assignable to cholesterol, glycolipids or sphingolipids are detectable, despite their abundance in the myelin sheath; they must therefore be highly immobile. It is often easier to identify the components of such complex materials from I3C rather than 'HNMR, since the I3C chemical shift range is about forty times larger. There will undoubtedly be much future study of intact biological specimens now it is realised that NMR signals can be observed from their mobile components. Most of them at least contain water, and this alone, as the next section will demonstrate, can provide much information.
P. J. SADLER
185
TISSUE STUDIES Gastrocnemius and sartorius muscles from the frog contain about 80% water by weight and can readily be suspended in 8 m m diameter NMR tubes by cotton attached to the bone fragments or tendons at either end. Small weights can be attached to produce slight tensions. In the temperature range 25-5"C, proton water T zdata have been analysed [147] in terms of 3 components: a slowly exchanging fraction (15%), an intermediate fraction (65%) and a fast relaxing component (20%). Twenty per cent of the water cannot take up the geometrical arrangement required for ice formation and remains unfrozen even at -80°C. This fraction may be strongly bound to muscle proteins and is affected by dehydration-hydration cycles [ 1481. When a weight is removed from a muscle under tension, the water TI returns to its unloaded value over a period of several hours [149]. There is now evidence that the water proton TI values in tumour cells are significantly longer than those from the corresponding control cells [150]. Damadian, Zaner, Hor and DiMaio have examined 106 human tumours [8,151] and feel that the technique is now ready for use by pathologists as an additional method for diagnosing malignancy. In fourteen malignant tumours involving lymph nodes, for example, the mean water TI at 100MHz (there is a frequency dependence) was 1.004 5 0.056 sec compared with 0.720 k 0.076 sec for control lymph nodes; in tumours involving bone, 1.027 2 0.152 sec compared with 0.554 ? 0.027 for control bone. The melanomas, however, were an exception to the rule that the TI of malignant tumours is increased. The results are intriguing but the origin of the effects is not understood. Three 31Psignals from adenosine triphosphate are readily observed [152] when chromaffin granules are packed into an NMR tube. The ATP concentration is about 0.1 M in the granule and is released upon addition of Ca2' and X537A (membrane carrier). The NMR study of intact biological specimens is therefore often both feasible and fruitful, and the construction of a 'whole body spectrometer' [ 1531 (for photograph see [ 1541) emphasises the non-destructive nature of the NMR method! Figure 3.1 is a timely indicator of future progress.
CONCLUSION NMR is now a technique with which every biologist should be acquainted. During the thirty years since its discovery, a wealth of information on
I86
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
defined chemical systems has been carefully catalogued and in the past few years, the expertise for handling biochemical systems has been developed. In the future, NMR can confidently be expected to contribute to the solution of many biological problems and to foster much interdisciplinary collaboration. REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24.
J. W. Akitt, NMR and Chemistry (Chapman and Hall, London) (1973). L. M. Jackman and S. Sternhill, Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry (Academic Press, New York, 1969). J. A. Pople, W. G. Schneider and H. J. Bernstein, High Resolution Nuclear Magnetic Resonance (McGraw-Hill, New York, 1959). J. W. Emsley, J. Feeney and L. H. Sutcliffe, High Nuclear Magnetic Resonance Spectroscopy (Pergamon Press, Oxford, 1965). E. Williams, J. A. Hamilton, M. K. Jain, A. Allerhand, E. H. Cordes and S. Ochs, Science, 181 (1973) 869. I. D. Campbell, C. M. Dobson, R. J. P. Williams and A. V. Xavier, Ann. N.Y. Acad. Sci. 222 (1973) 163. D. I. Hoult, S. J. W. Busby, D. G. Gadian, G . K. Radda, R. E. Richards and P. J. Seeley, Nature 252 (1974) 285. R. Damadian, K. Zaner, D. Hor and T. DiMaio, Physiol. Chem. Phys. 5 (1973) 381. R. A. Dwek, Nuclear Magnetic Resonance in Biochemistry (Clarendon Press, Oxford, 1973). F. R. N. Gurd and P. Kein, Methods Enzymol., 27 (1973) 830. A. S. Mildvan and M. Cohn, Advan. Enzymol., 33 (1970) 1. P. J. Quilley and G. A. Webb, Coord. Chem. Rev., 12 (1974) 407. H. A. 0. Hill, in: NMR, eds. P. Diehl, E. Fluck and R. Kosfeld (Springer-Verlag, Berlin) Vol. 4. (1971) 167. G. C. K. Roberts and 0. Jardetzky, Advan. Protein Chem., 24 (1970) 448. W. D. Phillips, Methods Enzymol., 27 (1973) 825. F. A. Bovey, High Resolution NMR of Macromolecules (Academic Press, New York, 1972). B. Sheard and E. M. Bradbury, Progr. Biophys. Mol. Biol., 20 (1970) 187. B. D. Sykes and M. Scott, Annu. Rev. Biophys. Bioeng., 1 (1972) 27. J. S. Cohen, in: Experimental Methods in Biophysical Chemistry, ed. C. Nicolau (J. Wiley, London, 1973) p 521. A. F. Casy, PMR Spectroscopy in Medicinal and Biological Chemistry (Academic Press, London, 1971). N. A. Matwiyoff and D. G. Ott, Science, 181 (1973) 1125. E. F. Elstner, R. J. Suhadolnik and A. Allerhand, J. Biol. Chem., 248 (1973) 5385. H. H. Wasserman, R. J. Sykes, P. Peverada, C. K. Shaw, R. J. Cushley and S. R. Lipsky, J. Amer. Chem. SOC.,95 (1973) 6874. H. Seto, T. Sato and H. Yonehara, J. Amer. Chem. SOC.,95 (1973) 8461.
P. J. SADLER 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.
55. 56.
187
J. M. A. At-Rawi, J. A. Elvidge, D. K. Jaiswal, J. R. Jones and R. Thomas, Chem. Commun., (1974) 220. 1. Putler, A. Barreto, J. L. Markley and 0. Jardetzky, Proc. Nat. Acad. Sci. U.S.A., 64 (1969) 1396. B. D. Sykes, H. I. Weingarten and M. J. Schlesinger, Proc. Nat. Acad. Sci. U.S.A., 71 (1974) 469. W. H. Huestis and M. A. Raftery, Biochem. Biophys. Res. Commun., 49 (1972) 1358. J. A. Sogn, L. C. Craig and W. A. Gibbons, J. Amer. Chem. SOC.,96 (1974) 3306. A. F. Bradbury, A. S. V. Burgen, J. Feeney, G. C. K. Roberts and D. G. Smythe, FEBS Lett., 42 (1974) 179. J. H. Bradbury and B. E. Chapman, Biochem. Biophys. Res. Commun., 49 (1972) 891. J. Dadok and R. F. Sprecher, J. Magnet. Resonance, 13 (1974) 243. J. D. Glickson, W. D. Phillips and J. A. Rupley, J. Amer. Chem. SOC.,93 (1971) 4031. A. Masson and K. Wiithrich, Febs Lett, 31 (1973) 114. S. Karplus, G. H. Snyder and B. D. Sykes, Biochemistry, 12 (1973) 1323. D. J. Patel, L. Kampa, R. G. Shulman, T. Yamane and B. J. Wyluda, Proc. Nat. Acad. Sci. U.S.A., 67 (1970) 1109. D. J. Patel, L . Kampa, R. G. Shulman, T. Yamane and M. Fujiwara, Biochem. Biophys. Res. Commun., 40 (1970) 1224. A. Mayer, S. Ogawa, R. G. Shulman and K. Gersonde, J. Mol. Biol., 81 (1973) 187. J. H. Griffin, J. S. Cohen and A. N. Schechter, Biochemistry, 12 (1973) 2096. G. Robillard and R. G. Shulman, J. Mot. Biol., 71 (1972) 507. D. R. Lightfoot, K. L. Wong, D. R. Kearns, B. R. Reid and R. G. Shulman, J. Mot. Biol., 78 (1973) 71. R. G. Shulman, C. W. Hilbers, Y. P. Wong, K. L. Wong, D. R. Lightfoot, B. R. Reid and D. R. Kearns, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 2042. C. W. Hilbers, R. G . Shulman and S. H. Kim, Biochem. Biophys. Res. Commun., 55 (1973) 953. D. M. Crothers, C. W. Hilbers and R. G. Shulman, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 2899. C. F. Brewer, H. Sternlicht, D. M. Marcus and A. P. Grollman, Biochemistry, 12 (1973) 4448. C. F. Brewer, H. Sternlicht, D. M. Marcus and A. P. Grollman, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 1007. K. D. Hardman and C. F. Ainsworth, Biochemistry, 12 (1973) 4442. J. J. Villafranca and R. E. Viola, Arch. Biochem. Biophys., 160 (1974) 465. S. H. Koenig, R. D. Brown and C. F. Brewer, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 475. E. Meirovitch and A. J. Kalb, Biochim. Biophys. Acta, 303 (1973) 258. B. H. Barber and J. P. Carver, J. Biol. Chem., 248 (1973) 3353. B. H. Barber and J. P. Carver, J. Mol. Biol., in the press. C. F. Brewer, D. M. Marcus, A. P. Grollman and H. Sternlicht, J. Biol. Chem., 249 (1974) 4614. C. H. Fung, A. S. Mildvan, A. Allerhand, R. Komorski and M. C. Scrutton, Biochemistry, 12 (1973) 620. A. S. Mildvan and M. C. Scrutton, Biochemistry, 6 (1967) 2978. G. Navon, Chem. Phys. Lett., 7 (1970) 390.
188
NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES
G. Navon, R. G . Shulman, B. J. Wylinda andT. Yamane, J. Mol. Biol., 51 (1970) 15. T. Nowak and A. S. Mildvan, Biochemistry, 11 (1972) 2813. G . Navon, R. G. Shulrnan, B. J. Wylinda and T. Yamane, Proc. Nat. Acad. Sci. U.S.A., 60 (1968) 86. 60. W. J. Ray and A. S. Mildvan, Biochemistry, 12 (1973) 3733. 61. A. Lanir and G. Navon, Biochemistry, 11 (1972) 3536. 62. A. Lanir, S. Gradsztajn and G. Navon, FEBS Lett., 30 (1973) 351. 63. A. R. Peacocke, R. E. Richards and B. Sheard, Mol. Phys., 16 (1969) 177. 64. T. Nowak, A. S. Mildvan and G . L. Kenyon, Biochemistry, 12 (1973) 1690. 65. C. M. Grisham and A. S. Mildvan, J. Biol. Chem., 249 (1974) 3187. 66. R. A. Dwek, G . K. Radda, R. E. Richards and A. G. Salmon, Eur. J. Biochem., 29 (1972) 509. 67. D. J. Birkett, R. A. Dwek, G. I 50%) of the intensity of the free radical signal from vitamin Q depleted sub-mitochondrial particles suggests that it contributes to the radical signals found in normal particles [72]. Flavin derivatives, particularly lumiflavin, have been widely studied as models for flavoprotein spectra; their much lower molecular weight enables them to rotate sufficiently rapidly in solution at room temperature that their spectra are isotropic and hyperfine splitting can be resolved. Relatively stable radicals from these derivatives can be generated by partial reduction with, for example, dithionite. Metal-containing flavoproteins, in most cases, show ESR signals on reduction with substrate, but their spectra are poorly defined since they rotate slowly. Metal-free flavoproteins rarely give rise to these signals with substrate but can often be studied after reduction with dithionite or light. Since flavoproteins are widely utilised as electron transfer agents (they also catalyse the oxygen linked dehydrogenation of certain compounds) and are able to undergo one- and two-electron reductions, information on the structure of their radicals has a biological relevance. The spectra of lumiflavin and some closely related compounds are now well understood, the structures from the neutral, the anionic and the cationic radicals and the radical chelates in a variety of solvents have been analysed and the spin densities calculated at the ring atoms [73-761. It is found that the highest unpaired spin densities are at N-5 then N-10, C-8 and C-6 and that atoms attached to these will also have appreciable spin density. There appears to be negligible density on the pyrimidine ring (see Figure 4.8). These studies have been confirmed and extended by the use of ENDOR [77]. When
Figure 4.8. The Flauin ring system
D. L. WILLIAMS-SMITH AND S. J. WYARD
22 1
studying frozen solutions with ENDOR, it is often easy to recognise isotropic coupling, i.e. couplings to individual groups, particularly methyl groups, which are able to rotate in the solid phase. Thus, while the ESR signals of flavoproteins are not sufficiently well resolved to be interpreted, good ENDOR spectra have been obtained from a number of them, such a s NADPH dehydrogenase, glucose oxidase, oxynitrilase, azotobacter flavoprotein, D-amino acid oxidase and riboflavin binding protein [78]. These all show isotropic couplings of between 11 and 7 MHz to the methyl group at C-8. The exact values for this coupling fall into two groups, 10-1 1 MHz corresponding to those flavoprotein radicals which have a red colouration and 7-9 MHz to blue flavoprotein radicals. Comparison with values obtained earlier from model compounds led to the important observation that the magnitude of the coupling of the red flavoprotein radicals corresponded to the anionic models, whilst that for the blue was similar to neutral radicals. IRRADIATION STUDIES
A fairly large but rather specialised area of the study of small molecular weight compounds has concerned the possible source of damage to cells by high energy irradiation. Since the primary products of such irradiations will be paramagnetic (either from addition or subtraction of an electron or by homolytic fission of covalent bonds), it has been possible to gain by ESR precise information on the chemical consequences of such processes. Many amino acids and nucleic acids have been irradiated, particularly as single crystals at temperatures from 4.2"K upwards and radical species identified, both with ESR and ENDOR. The major difficulty in this work has been in trying to extrapolate the detailed information gained on simple compounds to systems of such enormous complexity as the whole cell. The interested reader is recommended to consult other work [79]. WHOLE TISSUE ESR
The detection of ESR signals in whole tissues was one of the earliest observations made by those attempting biological ESR work, and a fairly extensive literature has built up concerning conditions under which these signals are found and how they change during various pathological conditions. This work has mainly concerned organic free radicals occurring around g = 2.00 and much of it is difficult to evaluate partly
222
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
because of difficulties in defining the exact conditions under which consistent results may be obtained and partly because of the failure to identify the radicals involved. There are a few exceptions to this. Thus, the ascorbyl radical has been identified in a number of tissues, particularly liver and blood; its concentration is found to alter in neoplastic tissues [80]. The very stable melanin radicals can also be detected in most pigmented tissues. Since the major problem in identifying the species giving rise to the g = 2.00 signal is the lack of resolvable hyperfine structure, it is conceivable that in the future ENDOR may provide us with more precise information, and indeed a radical from bacteriochlorophyll has been recognised by this method in whole bacteria [81]. The ENDOR technique requires an intense ESR signal with signal to noise ratios of at least 100: 1; this is rarely achieved in whole tissues, but should instrumental advances be made, a large new area would become accessible. The problems of identification are not nearly so acute when dealing with metalloproteins as they have a wide range of g values, and a number of these have been observed in whole tissue. Thus, the low spin ferric form of cytochrome P-450 has been observed in liver and its concentration found to be reduced in Morris hepatomas [82]. Adrenal ferredoxin in adrenal glands [83], sulphite oxidase in liver [84], iron-sulphur proteins in pigeon heart (851, methaemoglobin and erythrocyte catalase in whole blood are further examples in mammals. Two other types of signals are likely to be encountered by those contemplating tissue work. Manganese (Mn”) is of fairly ubiquitous biological occurrence and a NO-haemoprotein signal has been reported in liver. The intensity of this latter signal appears to be a function of the amount of nitrate ingested [86]. Under certain conditions a number of nitric oxide complexes may be generated, some of which show pronounced nitrogen hyperfine splitting [87], but as yet the haemoproteins involved have not been identified.
COMPLEX MULTI-COMPONENT ENZYME SYSTEMS RESPIRATION AND PHOTOSYNTHESIS
As mentioned earlier, ESR spectroscopy has made unique contributions to knowledge of iron-sulphur proteins. The areas of greatest relevance to such work are the complex electron transfer chains present in the
D. L. WILLIAMS-SMITH AND S. J. WYARD
223
mitochondria of respiring mammalian cells, in plant chloroplasts, and in photosynthetic bacteria. The exact role of iron-sulphur proteins in these systems is not known, beyond their ability to transfer electrons, but complex I particles depleted of these proteins are unable to carry out phosphorylation, though they still have a functioning electron transfer chain. At present, however, a number of such proteins are still being identified, and ESR is the ideal technique for this as their optical spectra are weak. With the development of methods to study the relationship of the iron-sulphur proteins to the other known components it may be possible to resolve many of the outstanding problems in these systems. Respiration A simplified representation of the mitochondria1 respiratory chain in terms of the oxidation of NADH and succinate by oxygen is illustrated in Figure 4.9 together with Table 4.2 showing the twelve iron sulphur proteins identified, their g values and mid-point redox potentials. The g values found are consistent from a variety of different preparations, though some changes in line share are found. The mid-point potentials are variable, with values from - 20 mV to -265 mV for centre N-2. The first detailed report of the presence of an unusual signal at g = 1.94 in both mitochondria and submitochondrial particles was made i n 1960 [88]. The experimental procedure has changed little since then and consists in principle of incubating the particles anaerobically with substrate or titrating with a suitable reducing agent such as dithionite, quickly freezing the sample and running spectra at low temperatures. Details of the equipment used [89] and the method of determining mid-point potentials from titrations has been published [90]. Both succinate dehydrogenase and NADH dehydrogenase gave a similar signal with a prominent peak at g = 1.94 on incubation with substrate, or with dithionite. Attempted kinetic measurements on NADH dehydrogenase using rapid-freeze techniques [91] showed that the maximum intensity of the g = 1.94 signal was achieved in under 8 m s e c at 1"C, the minimum resolution of the apparatus [92]. This suggested that this component was reacting at a sufficiently fast rate to allow it direct participation in electron transfer. When comparing a number of different yeast systems a close relation has also been observed between the relative signal intensity of the iron-sulphur centres associated with these dehydrogenases and their maximal rate of electron transfer [93]. The g = 1.94 signal came from proteins now designated N-1 and S-1.
224
m“
k
U
3
0
k
u
v)
6
I I
I
al
5rnzz 6
n
. I
U
’tt
Table 4.2. IRON-SULPHUR PROTEINS IN HEART MITOCHONDRIA
Assignment
Mid-point redox potential, mV, (pH) References
g Values
NADH dehydrogenase
N-lal
2.031
1.94
1.93)
- 305
(7.2)
N-lb N-2 N-3 N-4
2.054 2.101 2.103
N-6
2.1 1, 2.07, 1.90, 1.89 (not resolved) -260 (8.0)
Location unclear 5 6
UQ cytochrome c Fe-SR,.,k, HIPIP
- 380 (7’2) - 250 (7.21 ,
1.922 1.886 1.863
-
20 (7.2)/- 13.5- 265 (7.2) -245 (7.2) - 245
1.864
1
2.08 2.1 1
+ 4 0 (7.2) 0
1.89 1.90
2.03 1.90 1.80 +280 2.01 (oxidised) 2.04, 1.99 (at intermediate oxidation levels) 1.867
85, 94-96
I
85, 94-96 85, 94-96 85, 94-96 85,9696 95 98
85, 99 85 85 100
Succinate dehydrogenase
s-1 s-2
2.03 2.03
1.94 1.94
1.92 1.92
0 (7.4) - 260 (7.4)
97 97
N VI N
226
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
With the advent of very low temperature techniques, these early experiments were re-investigated and at below 20°K a new order of complexity was observed. In the NADH dehydrogenase region, for example, seven centres were identified. The original g = 1.94, N-1 signal was probably a mixture of two components of different redox potential and five further proteins were detected of which the signals due to N-5 and N-6 have not yet been resolved [94-961. As might be expected, the spectra are rather complex but full use has been made of reductive titration methods (the higher the redox potential, the sooner the signal should appear) and of differences in relaxation and saturation properties of the proteins. In succinate dehydrogenase another iron-sulphur component, S-2 has been observed below 20°K and it is not reduced by succinate [97]. Centres 5 and 6 are also detectable in submitochondrial particles, and their signals are not produced in the presence of substrate and rotenone. They must therefore be on the oxygen side of rotenone inhibition [95,98]. Some studies have already been made correlating data with tissues showing abnormal respiratory behaviour. Particularly interesting is the recent work on these proteins in mitochondria prepared from control and tumour-bearing rat livers and from Morris hepatomas [98]. These hepatomas showed diminished rates of NAD linked substrate oxidation and succinate oxidation, and in all cases a close relationship was found in their mitochondria between the reduction of oxidative ability in parts of the chain and the intensity of the ESR signals from that part of the chain when the mitochondria were reduced with dithionite. It was not possible to invoke the disappearance of a specific iron-sulphur centre as responsible for the changes, as in each case all centres in the relevant regions were diminished, though differences in the relative changes were found.
Photosynthesis The utiiisation of radiant energy from the sun to reduce COz, thereby generating the simple carbon-, hydrogen- and oxygen-containing molecules that are essential for many species, is a process which in green plants and photosynthetic bacteria involves the utilisation of an electron transfer chain. In plants, there are two primary acceptors of light, pigment system I (which can absorb light 3 700 nm) and pigment system I1 (only absorbs light =s680 nm). Both contain forms of chlorophyll molecules which on photoexcitation stimulate the flow of electrons via a complex system involving cytochromes and iron-sulphur proteins to a number of electron acceptors such as NADP', utilising some of the free energy
D. L. WILLIAMS-SMITH AND S. J . WYARD
221
liberated to phosphorylate ADP. In bacteria, the primary light acceptor is bacteriochlorophyll and pigment system I1 is absent. ESR studies on photosynthesis initially focused attention on two free radical signals, called signals I and 11, g value 2.0025, 2.0047, which were observable when plant chloroplast and subchloroplast particles were irradiated with light [ 1011. Signal I corresponds to the chlorophyll pigment P700, and its optical spectra and the ESR signal behaved in an identical way in samples prepared from red algae [102]. Most of the evidence for the structure of this radical came from ESR and ENDOR measurements on the bacteria Rhodospirillum rubrum and R . spheroides. The in vivo signal had a line width of 9 gauss yet in isolated bacteriochlorophyll it was 13 gauss [103], and the ENDOR couplings in vivo were half those found in vitro [81]. This is confirmation of the proposal that this radical is delocalised over a pair of bacteriochlorophyll molecules, as spin delocalisation should narrow the line width by a factor of l / f l and the couplings should be halved. Signal I1 has not yet been unambiguously identified. It is absent in bacteria, suggesting that it is associated with pigment system 11, and, as its intensity decreases in plastoquinone-depleted chloroplasts, it may be a radical analogous to plastosemiquinone [ 1041. However, the line width of plastosemiquinone in vitro is not identical and it has been proposed that the related a-tocopheroxyl radical might be a more likely candidate [ 1051. Recent work has concerned the presence of iron-sulphur centres in the chain. These were first observed in 1971 and are associated exclusively with pigment system I. The experimental procedure is either to illuminate chloroplasts or subchloroplast particles at various temperatures and redox potentials or to titrate them against dithionite in the presence of redox mediators and record ESR spectra at close to liquid helium temperatures. Thus, on irradiation of the particles at 77"K, peaks at g = 2.05, 1.94, 1.86 appeared, typical of an iron-sulphur protein [ 1061. The signals obtained on reduction were slightly different, occurring at g = 2.04, 1.95, 1.93 and 1.89 and the total number of spins was twice that obtained on illumination. The iron-sulphur proteins were then shown to be part of pigment system I 11071 and double integration revealed a 1 : 1 ratio of radicals formed from W O O to iron-sulphur centres reduced [ 1081. The photochemical behaviour of the signals from subchloroplast particles in the presence and absence of methyl viologen corresponded to optical absorbance changes which had previously been observed at 430 nm [109]. There are small changes in g values when studying different preparations but the signals from photosystem I subchloroplasts have been considered in terms of three
228
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
iron-sulphur components, one with lines at g = 2.05, 1.94 (A), one at g = 2.05, 1.92, 1.89 (B), and one at g = 1.89 (C) (its other lines are not resolvable) [ 1101. At 77°K illumination produces mainly signals (A) and (C); when illuminated at room temperature and during subsequent freezing strong signals from (B) are seen. Much speculation has occurred concerning the possibility that one of these proteins may be the primary electron acceptor. Signal (B) cannot be this, and the kinetics of production of (A) and (C) are rather too slow. Recent data indicate the presence of a fourth broad signal in the same region whose kinetic behaviour is much closer to that expected for the primary acceptor. A further degree of complexity in the ESR spectra of photosynthetic systems is found in Chromatum D, where a high potential iron-sulphur protein and two cytochromes, C555and C553are observable [ I l l ] . By recording spectra at different potentials it has been possible to identify these components and to study their behaviour on illumination [I 121. Ferredoxin-like iron-sulphur proteins can also be seen [ 1131. CYTOCHROME P-450
A large body of evidence has now accumulated suggesting a central role for cytochrome P-450 in the metabolism of foreign compounds and lipids. This haemoprotein, which occurs in a large number of mammalian tissues, (especially the liver and adrenal cortex) and in certain rnicro-organisms is part of an enzyme system responsible for the initial processes (generally hydroxylation or demethylation), in the breakdown of drugs and carcinogens [114]. In the liver, it has been implicated in the metabolism of phenobarbital, benzpyrene, polychlorinated biphenyls and cyclophosphamide; in the adrenal gland, it is responsible for several of the steps in the synthesis of corticosterone from cholesterol. Its presence in certain bacteria enables them to synthesise useful compounds from petroleum waste. As is to be expected from such a versatile and talented system, it also has many unusual physical properties. The currently accepted mechanism of operation is illustrated in Figure 4.10. Cytochrome P-450in its resting state is a low spin ferric haemoprotein; on binding substrate, it becomes high spin. It then undergoes a one-electron reduction, and binds oxygen. After accepting another electron, the substrate is hydroxylated and the cytochrome restored to its resting state. ESR, in combination with optical spectroscopy, is a convenient tool for studying cytochrome P -450 as in principle these spin state and oxidation state changes can be followed [5, 115, 1161. In particular, the spin state change, along with a
D. L. WILLIAMS-SMITH AND S. J. WYARD
229
Hp+SOH
*
# RESTHG STATE9 = 2 h 4 z . z ~ , I ~ z z
e
-
k++ s* 8; * A Z .
F+-S
*
ADRENAL FERREDOXY
+:O \2 -
0-
I
oy. 2022,
0 2
Figure 4.10. The proposed mechanism of action of cytochrome P-450
shift in the P-450 Soret band from 420 to 390nm, may be used as a diagnostic test for the metabolism of compounds by this system. Typical g values for low spin P-450 are g = 2.42, 2.25, 1.92 and high spin g = 8,4,2 and these are in themselves unusual. The low spin values are sufficiently distinctive that it has been possible, after a study of suitable model compounds, to state that one of the fifth and sixth ligands at the haem iron is a thiol [9], and the high spin parameters show a rhombicity of 30%, easily the highest known for any haemoprotein under physiological conditions. One of the most interesting problems concerning the cytochrome P-450 system has been its multiplicity, for it is difficult to conceive of a single enzyme system having the ability to oxidise in a specific way such a wide variety of substrates. Current data suggest that there are at least three forms of the enzymes present in rat liver microsomes. Optical spectra reveal two forms of the CO-bound reduced cytochrome P -450, one with a maximum at 448nm (such as is induced by 3methylcholanthrene) and one at 450 nm (as is induced by phenobarbital), and SDS (sodium dodecyl sulphate) polyacrylamide gel electrophoresis shows three peaks corresponding to molecular weights of 44000 (phenobarbital), 50000, and 53 000 (3-methylcholanthrene) [ 1171. Differences are also seen in the ESR spectra of P-450 in rat liver microsomes induced by different chemicals and there are further differences when
-
230
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
compared with microsomes from other sources. Most marked are the low spin data but in a P-450 system responsible for steps in the synthesis of corticosterone from cholesterol in the rat adrenal, two high spin forms can be observed and under certain conditions both can be resolved in the Same sample. The form at g = 7.8 is the substrate-bound form responsible for 1Ip-hydroxylation of deoxycorticosterone and that at g = 8.1 for side-chain cleavage (SCC)of cholesterol. In individual preparations of liver microsomes and adrenal mitochondria, further perturbations of the g values of the low spin spectra occur on adding substrates; these are changes which cannot be detected by optical spectroscopy and their meaning is not yet clear. The resolution of two high spin forms in adrenal mitochondria has been exploited in studies of the stimulation of steroid biosynthesis by ACTH and the effects of stress. Mitochondria from stressed rats showed increased amounts of high spin P-450, most notably in that corresponding to cholesterol side chain cleavage and similar but not identical effects were observed when hypophysectornised rats were treated with ACTH [118]. This supports the thesis that ACTH stimulation increases the availability of cholesterol for side chain cleavage. In adrenals, ESR can also be used to monitor the presence of reducing equivalents to P-450 as, in this case, the P-450 is linked to NADPH via an iron-sulphur protein, adrenal ferredoxin. This shows typical signals on reduction at g = 2.01, 1.94 [119]. A similar iron-sulphur protein (g = 2.03, 1.94) occurs in mammalian testes connected with a P-450 system [ 1201.
SPIN LABELLING In the previous Sections, results obtained from the use of ESR to study directly the properties of biological molecules which were at least transiently paramagnetic have been discussed. Although these molecules form only a small percentage of all biological materials, a few diamagnetic metalloproteins can be studied after substitution with a paramagnetic metal. In this Section, a far more general way of making diamagnetic systems amenable to ESR techniques will be discussed. The vital property of a spin label is that the shape of its ESR spectrum is dependent upon the rate and type of motion it undergoes. Thus, in a viscous solvent, the spectrum will be anisotropic and more than one component of the g tensor will be observable whilst, in a non-viscous
D. L. WILLIAMS-SMITH AND S. J. WYARD
23 1
solvent, the more rapid molecular rotation may result in an isotropic spectrum and an averaged g value. Furthermore, more complex situations, such as might occur when the spin label is free to move in some directions but restricted in others, may also be interpretable from the shape of its spectrum. When a stable paramagnetic spin label is chemically attached to a biological molecule without interfering with that molecule’s normal function, a ‘spy’ is formed whose spectrum reveals certain properties of the labelled site. Spin labels have found particularly wide application in the study of membranes and enzymes, and measurements can be made on intact biological systems at room temperature. The most commonly used spin labels are nitroxides as they are one of the very few classes of molecules that even closely approximate the behaviour required of spin iabels, namely, that they should be free radicals stable in aqueous solution at pH 7, be easily attached to biological molecules, and have well defined spectra that are motion dependent at the rates of motion likely to occur in the biological molecules in a close to in vivo situation. The spectrum of a typical nitroxide, N-oxyl-2,2,6,6tetramethyl-4-piperidinol is shown in Figure 4.11. Figure 4.1 IA illustrates the spectrum obtained under conditions of rapid motion and shows three peaks due to coupling with the nitrogen nucleus (I = 1). Figure 4.11B and C show the immobilised cases, where the rate of rotation is lower than the frequencies corresponding to the largest differences between the principal components of the hyperfine coupling tensor and that of the g tensor. The rates of motion to which these and intermediate spectra correspond are required and these are normally expressed in terms of the rotational correlation time, yC. This quantity may be regarded as a measure of the time taken for the molecule to reorientate itself and the range of times considered are from 7‘ = lO-’sec (strongly immobilised) to r, = lo-” sec (isotropic). The value of the rotational correlation time may be obtained in two ways. either (in the case of symmetrical rotation) by running a series of calibration spectra in solutions of known viscosity until a spectrum is found which is superimposable on that from the biological sample and then using the Stokes Law formula: 47~77r3 where 77 = viscosity 3kt T = radius of molecule (spherical symmetry assumed)
7,= -
or by a theoretical analysis of the ESR lineshape. At present, the theory is not sufficiently well developed that the latter procedure can be recom-
232
ELECTRON SPIN RESONANCE I N MEDICINAL CHEMISTRY
A
B
C
Figure 4.11. Spin label B , X = OH. (A) In methanol rapid tumbling. (B)In glycerol at 298°K (partly irnrnobilised). (C) In glycerol at 77°K (highly immobilised)
mended with confidence, but it is expected that advances will be made in this area. Interested readers are referred to a number of relevant papers [121-1231. In cases where it is suspected that the molecule is free to rotate rapidly in some directions but not in others (anisotropic motion), it may not be possible to find a useful model and a full theoretical analysis may be necessary. Besides their motion dependance, many spin labels show changes in their g tensor and/or hyperfine coupling tensor in different solvents. These solvent dependent properties have also been utilised in biological systems, particularly for measuring the distribution of spin labelled molecules across aqueous-lipid interfaces.
D. L. WILLIAMS-SMITH AND S. J. WYARD
233
The structures of the nitroxides employed as spin labels fall into a number of simple groups, of which types A and B predominate.
x
(A)
(€3)
X is a functional group that enables the nitroxide to be attached under mild conditions to the biological molecule of interest. For protein binding, it has been found convenient to make derivatives of some of the small compounds known to interact with proteins, such as the iodoacetamide (X = -NH.CO.CHJ), maleimide (XI) and bromoacetate (X = -NH.CO.CH2.CHBr.CO2H) nitroxides. Lipids cannot easily be labelled in this way and so great use has been made of a general procedure for generating nitroxides from ketones [124]. Rozantsev [ 1251 has made a most useful compilation of the synthetic routes to nitroxides. The first experiments concerning the use of nitroxide spin labels, a study of conformational changes in poly-L-lysine and bovine serum albumin [ 1261 were carried out by Stone, Buckman, Nordio and McConnell in 1965, and it is largely due to the impetus provided by this group that such a large literature about them now exists. This has been reviewed in a number of places [127-1301; a few experiments representative of the general areas in which spin labels have found application are given here. ENZYMOLOGY
A large number of isolated enzymes and coenzymes are in principle
amenable to ESR study by spin labelling, and although there is only data on a few dozen at present, this is potentially a large field. In particular, the spectra of labelled haemoglobin and a-chymotrypsin have been the subject of detailed investigations. Interest in haemoglobin stems from the possibility of seeing co-operative interactions between sub-units. It was found that it can conveniently be labelled at cysteine p-93 with nitroxides A, B, X', and in aqueous solution at room temperature the resulting spectra were of the strongly immobilised type. Significant changes were seen on oxygenation due to alterations in local constraints at the spin label rather than differences in rotational behaviour of the haemoglobin molecule as a
234
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
whole [131]. The nitroxides A (X = -NH.CO.CH,I) bound at the same position showed a similar effect but here the amount of change was found to be proportional to the degree of oxygenation and when its spectra were superimposed isobestic points were observed [132]. In this case only two conformational states were being monitored. The related nitroxide B (X = -NHCO.CHzI), however, does not show clear isobestic points and is therefore also sensitive to conformational changes other than those arising from oxygenation or deoxygenation of the p-chains [133, 1341. This was conclusively demonstrated by preparing haemoglobins which contained chains unable to bind oxygen due to prior oxidation in the presence of cyanide (cyanomet a ) , (spin labelled p )* haemoglobin showed a large spectra change on oxygenation, whereas ( ( Y . ) (spin ~ labelled cyanomet p)z showed only a small change. When using label A iodoacetamide, the latter change was not seen. These experiments therefore provide direct evidence for interactions between chains on oxygenation; they also illustrate an important point concerning spin labelling, that the choice of the most suitable spin label is a critical and somewhat random process. It is also possible to spin label protohaem with the nitroxide A, where X = N H z at the propionic acid groups at positions 6 and 7 on the porphyrin ring. By incorporating this into haemoglobin and using a reducing system which will not reduce the spin label (ferredoxin NADP reductase and an NADPH generating system), reduction of methaemoglobin and oxygenation to oxyhaemoglobin can be followed [135]. The modified haemoglobin showed normal oxygen binding properties and visible spectra, and a change in its ESR spectrum was observed on reduction. This conformational change was not predicted from X-ray studies and hence either the spin label method is more sensitive in this case or the effect may be specific to haemoglobin in solution. The oxyhaemoglobin spectra again appeared to show the influence of more than one type of conformational change. Differences in spectra between the carbon monoxy, met-azide and metfluoride derivatives [I361 and a change on interaction with haptoglobins 11371 have also been observed. Alpha-chymotrypsin is an enzyme which hydrolyses peptide bonds (preferentially those formed by aromatic amino acyl residues and a variety of esters and amides forming an acylated intermediate). This property has been exploited for spin labelling, and by reacting the enzyme with a nitroxide substrate analogue, A, Xz, it is possible to record, at low pH, the ESR spectrum of the acyl enzyme 11381. This showed strong
D. L. WILLIAMS-SMITH AND S. J. WYARD
235
immobilisation, and when the pH was raised to 6.8, the kinetics of the appearance of rapidly rotating spin label, (i.e., the rate of deacylation) were measured. The rotational correlation time for the acyl enzyme was 1.2 x lo-" sec at 20°C [139], and little or no motion of the spin label relative to the tertiary structure of the enzyme occurred. A spin labelled derivative of the esterase inhibitor di-isopropylfluorophosphate has also been synthesised; it produces a highly immobilised spectrum when bound to a-chymotrypsin [140]. The free enzyme can be spin labelled with B, X = -NH.CO.CHzI, which reacts at methionine 192. Its ESR spectrum indicates that it is relatively free to rotate; it shows a pH dependency and at alkaline pH it is modified by the presence of indole, an inhibitor of the enzyme 11411. Other isolated enzymes which it has already proved possible to successfully spin label include carbonic anhydrase, creatine kinase, phosphofructokinase, lactate dehydrogenase, liver alcohol dehydrogenase, lysozyme and ribonuclease, as well as the coenzymes NAD' and vitamin BIZ. In principle spin labelling is easily applicable to the study of intact materials, and it has already been used to probe the proteins concerned with muscular contraction. The two major protein components of myofibrils, actin and myosin, have both been labelled. F-actin and G-actin bind the maleimide nitroxide, A, XI, and strongly immobilised spectra are then obtained. The degree of immobilisation increases on polymerisation. When oriented films of F-actin are placed in the ESR cavity in such a way that they are parallel to the applied magnetic field it is found that the two outer lines in the spectrum become much more prominent and that their separation increases. It was concluded that the axis of the N-0 bond is more nearly perpendicular than parallel to the helical axis of F-actin [ 1421. Myosin contains two types of functionally important thiol groups S' and Sz; S' has been labelled with the iodoacetamide derivative B, X = -NH-CO-CHzI and Sz with maleimide nitroxides. S' labelled myosin shows a highly immobilised type of ESR spectrum, and addition of ATP, ADP and PP, increases its mobility. When Mg-ATP is added and the spectrum recorded within two minutes of mixing, a third spectrum is obtained typical of the steady state of ATP hydrolysis. This change was confirmed by adding 5'-adenylyiimidodiphosphate(which combines with myosin and competes with ATP but is not hydrolysed) and the ADP type of spectrum was then observed [ 1431. Further immobilisation is seen when spin labelled myosin and actin are combined; the spectra are found easier to power-saturate. This effect is greatest at a molar ratio for actin: myosin
236
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
of 2 :1. No perturbation of the spectrum corresponding to the steady state of ATP hydrolysis occurs on addition of actin. It has also been possible to spin label glycerinated muscle fibres by incubating them at room temperature with the maieimide or iodoacetamide nitroxides. Most sensitive was the maleirnide derivative; when the fibres were progressively shortened by the addition of ATP, the spectra were composed of decreasing amounts of weakly immobilised label, and increasing amounts of highly immobilised label. This effect continued at lengths well below that at which all cross bridges were interacting with actin sites, and an increase in length beyond that at which the fibers were labelled produced no alteration in the spectrum. The results were not accomodated by the simple sliding filament model [144]. Both actin and myosin appeared to have been labelled in these fibres. MEMBRANE STUDIES
Spin labelling has found application to a wide range of membrane studies concerning both simple model systems and intact biological membranes, living cells and mosses. The procedure is to spin label a lipophilic molecule and incorporate a small amount of it into the system of interest; it is then hoped that alterations in the arrangement of the lipid molecules will be reflected in changes in the ESR spectrum of the nitroxide component. In some cases, it may be possible to demonstrate a specific alignment of the lipid molecules by observing an orientation dependence of the spectrum; in many cases, spectra indicative of anisotropic motion can be obtained. Phospholipid bilayers, liposomes and detergent micelles have often been used as model systems for membranes and have also proved convenient for ESR experiments. Lecithin bilayers, when prepared as films on a glass slide, are known to be formed in such a way that the lecithin molecules have their long axis perpendicular to the membrane surface. When the stearic acid derivatives (C) were incorporated into lecithin bilayers, it was found that they gave ESR spectra characteristic of
D. L. WILLIAMS-SMITH AND S. J. WYARD
237
anisotropic motion around the long axis (i.e. rapid motion around long axis, negligible motion around others). There were pronounced systematic variations in the degree of anisotropy dependent upon the isomer used [145]. Hexagonal phases such as are found in the cardiolipin/Ca2'/water system, also show orientation dependence E1461. Here again, spin labelled stearic acid was used as a probe and the degree of anisotropy was found to decrease as the position of the nitroxide along the hydrocarbon chain moved away from the polar head group. Phase transitions may be a particularly important property of membranes as they occur at around physiological temperatures and may result in large permeability changes in the membrane. The crystallinelliquid crystalline transition in dipalmitoyl lecithin has been examined in detail [1471 using the spin labelled androstane derivative (D). LONG
AXIS
OF
The transition temperature T, was known to be 41°C from previous work and indeed at this temperature a sharp decrease in the linewidth of the spectrum occurred, indicating an increased in the tumbling motion of the label. When the molar ratio spin label :dipalmitoyl lecithin was greater than 0.01:l broadening of the spectra occurred due to spin exchange between neighbouring nitroxides. At certain concentrations, these lines again sharpened above the transition temperature, suggesting that the lateral separation of the spin labels was also different in the two phases. Exchange phenomena have also been studied in oriented multibilayers of dipalmitoyl lecithin, egg lecithin, and dioleoyllecithin. Marked changes in the spectra on addition of increasing amounts of cholesterol were observed and interpreted in terms of variation of lateral diffusion and of clustering of spin labelled molecules [1481. Much of these data on model systems is complementary to information already obtained by other techniques but, as mentioned in other parts of this review, the greatest potential of ESR is in the ability to study complex intact biological systems, and this is true again of the use of spin labels in
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
238
membrane research. Nerve membranes were the subject of some of the first experiments in this area, in particular that of the walking leg nerve of the Maine Lobster (Hornurus Arnericanus). When the simple spin label B, X = H was incorporated, the ESR spectrum was composed of two parts indicating a distribution across hydrophobic and hydrophilic regions [149]. In contrast, androstane derivatives (such as D), which can be incorporated into the nerve membrane by exchange from serum albumin, were entirely in the hydrophobic region. The shape and separation of the hyperfine lines were indicative of rapid anisotropic motion about the long axis of the molecule. The 17-OH group clearly plays a vital role in orientating the molecule within the membrane, since this type of motion was absent in the 17-H analogue [150]. When aliphatic fatty acid derivatives were introduced, their rates of rotation were found to be dependent on the distance between the polar end-group and the nitroxide. Thus the stearic acid derivative C (n = 10) showed effectively isotropic motion, C (n = 3) was much more immobilised and the tricosanoic acid derivative, C (m = 17, n = 3 ) was still more immobilised [151]. This latter spin label was particularly useful in that it proved possible, after orienting the nerve fibres, to analyse its spectrum in terms of spin labels experiencing the applied magnetic field perpendicular and parallel to the axis about which the rapid anisotropic motion was occurring. It was then shown that the preferred orientation of the long axis of the lipid spin label was perpendicular rather than parallel to the axonal axis, the former being about five times more probable. Protein components in nerve tissue from the same source have also been spin labelled using the nitroxide A, X'. Their spectrum, which indicated strong immobilisation, was pH-dependent, mobility being a minimum between pH 3.5 and 5.5, and altered by treatment with urea, guanidine, HCI, and proteases. As the addition of this nitroxide to nerve homogenates reduced ATPase activity giving a strongly immobilised signal, and as ATPase needs to have free S H groups for activity, it was suggested that binding to this protein was at least partly responsible for the spectrum seen from nerve fibres [152]. The mitochondria1 membrane has also been found suitable for ESR studies. When incorporated into such membranes, aliphatic hydrocarbon nitroxides of type E '
& 4
3
(E)
....*,,.-O 4 - O ' length k N 1
1
D. L. WILLIAMS-SMITH AND S. J. WYARD
239
and also some steroid nitroxides have been shown to partition across hydrophobic and hydrophilic phases, and the resolution of their spectral lines suggests a relatively sharp boundary between these phases [153]. Addition of high concentrations of sucrose to the mitochondria suspension, known to inhibit oxygen-dependent hydrogen transport, caused much of this resolution to disappear. This was interpreted either as due to the spin label being in a phase of intermediate polarity or because it was rapidly exchanging between polar and apolar phases. This change in the ordering of the membrane was again observed, but to a lesser degree, in the presence of uncoupling agents such as 2,4dinitrophenol. Carbodi-imide, an inhibitor of ATPase still shows activity when suitably spin labelled, and when interacting with mitochondria1 membranes it becomes highly immobolised ( T = ~ 2 x lo-’ sec) [ 1541. In this membrane-bound form, the nitroxide group is reducible (and hence rendered ESR inactive) by succinate, but not when free, indicating that electrons can be transfered to the ATPase system from the respiratory chain. Its spectrum is not affected by ADP or ATP either alone or as their Mg” complexes, but the paramagnetic MnZt ATP decreased the signal of spin labelled carbodi-imide bound to membrane fragments by 30%. This effect, due to spin exchange interactions, indicates that the binding sites of these two molecules are probably close. The iodoacetamide nitroxide, A, X = -NH.COCH21 binds to horse ferricytochrome c at methionine 65 and shows only slight immobilisation ( T = ~ 9.3 x lo-’’ sec), but when cytochrome c -depleted mitochondria are added the degree of immobilisation increases (7, 3.3 x lO-’sec) [155]. The motion of the nitroxide attached to free cytochrome c becomes progressively reduced as the solvent viscosity is increased on addition of sucrose and, as the motion of the protein as a whole is known to be considerably slower than that observed by ESR, this indicates that the nitroxide is exposed to the solvent. When bound to the mitochondrial membrane, no sucrose effect is seen; thus here the spin label must be facing or interacting with the membrane. Spin labelled yeast cells show ESR spectra whose height is dependent upon the amount of respiratory activity; in cells grown aerobically on a medium containing a suitable spin labelled stearic acid derivative, addition of KCN or flushing with nitrogen caused the signal to decay; when grown anaerobically peak height increased on aeration [ 1561. There have been some attempts to use spin labels to probe for differences in the properties of membranes of normal and tumour cells. Thus mouse embryo fibroblasts have been labelled with C (n = 4, m = 10) and it was found that on transformation with oncogenic DNA or RNA
-
240
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY
viruses or with methylcholanthrene there was an increase in membrane fluidity [157]. The ESR data were correlated with results from freeze-fracture electron microscopy. The method of biosynthetic incorporation of spin label, rather than mechanical addition to isolated material, is a convenient way of ensuring that the results obtained are biologically meaningful and has also been used with such systems as the mould Neurospora crassa [I%], Mycoplasma laidlawii [159], human leucocytes, and mouse L cells [160]. The spectra from these two mammalian cells showed distinct similarities for a variety of spin labels, but different spectra were obtained when the labels were incorporated in human erythrocytes. Fractionation of the cell components showed the stearic acid (C, n = 3) spin label in all the major fractions, but by far the largest concentration was in the nuclear membrane. The ESR spectrum underwent a time and temperature dependent decay and spin labels on the surface membrane were reactivated with K3Fe(CN)6. IMMUNOCHEMISTRY
The spin label technique may also find general application in the field of immunochemistry, for it has been reported in several cases that the interaction of a spin labelled hapten with the corresponding antibody results in immobilisation. When the nitroxide B, X3 was added to antibodies directed against the dinitrophenyl group, the ESR spectrum changed from one typical of rapid rotation to one of high immobilisation ( T est ~ 3.9 x lo-' sec); and titration of such spin labels, monitored by ESR, gave a stoichiometric ratio of two haptens per antibody, in agreement with the results from fluorescence titrations [161, 1621. p-Azobenzoate and p-azophenyltrimethylammonium derivatives gave similar results with their respective antibodies. Variations in the degree of immobilisation were found for antibody preparations from different rabbits [163]. The technique may have considerable potential as an assay when used in a similar way to the well known radioimmunoassay, providing that the haptens can easily be spin labelled. The procedure would be to prepare a spin labelled hapten-antibody complex (spectrum highly immobilised) and add the solution containing the hapten to be assayed. The bound spin labelled hapten should then be competitively displaced by the added unlabelled hapten. From an analysis of the resulting ESR spectrum in terms of highly immobilised bound label and rapidly rotating displaced label, the amount of added hapten could then be calculated.
D. L. WILLIAMS-SMITH AND S. J. WYARD
24 1
Using spin labelled morphine it is possible to use ESR as an assay technique in this way for opium narcotics in urine and saliva, for antibodies derived from morphine are found to be also active against a number of related chemicals such as heroin and cocaine [164-1661. The absence of the possible hazards and inconveniences of radioactivity and the lack of a need to isolate the displaced molecules from those still bound to the antibody favour the spin immunoassay technique. The American army has been using this method of detection since 1972, and the measurement can be made in about 30 seconds.
REFERENCES 1. 2.
3.
4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
19. 20. 21.
G. Palmer and H. Beinert, Anal. Biochem., 8 (1964) 95. J. Peisach, W. E. Blurnberg, S. Ogawa, E. A. Rachrnilewitz and R. Oltzik, J. Biol. Chem., 246 (1971) 3342. J. Peisach and W. E. Blurnberg in: Probes of Structure and Function of Macromolecules and Membranes, ed. B. Chance, T. Yonetani and A. S. Mildvan (Academic Press) Vol. I1 (1971) pp. 231-239. W. E. Blurnberg and J. Peisach, ref. 3, pp. 215-229. J. Peisach, C. A. Appleby and W. E. Blumberg, Arch. Biochern. Biophys. 150 (1972) 725. C. P. Scholes, Proc. Natl. Acad. Sci. U.S.A., 62 (1969) 428. H. Watari, A. Hayashi, H. Morimoto and M. Kotani in: Recent Developments of Magnetic Resonance in Biological Systems, ed. S. Fujiwara and L. H. Piette (Hirokawa Publishing Co. Tokyo, 1968) p. 128. C. R. E. Jefcoate and F. L. Gaylor, Biochemistry, 8 (1969) 3464. A. Roder and A. Bayer, Eur. J. Biochem., 11 (1969) 89. J. S. Griffiths, Nature, 180 (1957) 30. 0. Ristau, H. Rein and F. Hackenberger, FEBS Lett., 9 (1970) 71. H. Brintzinger, G. Palmer and R. H. Sands, Proc. Natl. Acad. Sci. U.S.A., 55 (1966) 397. J. F. Gibson, D. 0. Hall, J. H. M. Thornly and F. R. Whatley, Proc. Natl. Acad. Sci. U.S.A., 56 (1966) 987. W. H. Orme-Johnson, R. E. Hansen, H. Beinert, J. C. M. Tsibris, R. C. Bartholomaus and I. C. Gunsalus, Proc. Natl. Acad. Sci. U.S.A., 60 (1968) 368. W. Dunham, G. Palmer, R. H. Sands and A. Bearden, Biochim. Biophys. Acta, 253 (1971) 373. J. Fritz, R. Anderson, J. A. Fee, G. Palmer, R. H. Sands, W. H. Orme-Johnson, H. Beinert, J. Tsibris and I. C. Gunsalus, Biochim. Biophys. Acta, 253 (1971) 110. E. T. Adman, L. C. Sieker and L. H. Jensen, J . Biol. Chem., 248 (1973) 3987. C. W. Carter Jr., S. T. Freer, Ng. H. Xuong, R. A. Alden and J. Kraut, Cold Spring Harbour Syrnp. Quant. Biol., 36 (1971) 381. K. D. Watenpaugh, L. C. Sieker, J. R. Herriott and L. H. Jensen, ref. 18, p. 359. W. E. Blurnberg and J. Peisach, Arch. Biochem. Biophys., 162 (1974) 502. R. D. Dowsing and J. F. Gibson, J. Chem. Phys., 50 (1969) 294.
242 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
51. 52. 53. 54. 55. 56.
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY R. Aasa, Biochem. Biophys. Res. Commun., 49 (1972) 806. p. Aisen, R. Aasa, B. G. Malmstrom and T. Vanngard, J. Biol. Chem., 242 (1967) 2484. E. M. Price and J. F. Gibson, J. Biol. Chem., 247 (1972) 8031. p. Aisen, R. Aasa and A. G. Redfield, J. Biol. Chem., 244 (1969) 4628. T. Vanngard in: Biological Applications of Electron Spin Resonance, ed. H. M. Swartz, J. R. Bolton and D. C. Borg (Wiley Interscience, New York, 1972) pp. 41 1 - 4 7 , R. Malkin and B. G. Malmstrom, Advan. Enzymol., 33 (1970) 177. W. E. Blumberg in: The Biochemistry of Copper, ed. J. Peisach, P. Aisen and W. E. Blumberg (Academic Press, New York, 1966) p. 49. A. S. Brill and G. F. Bryce, J. Chem. Phys., 48 (1968) 4398. S. Katoh and A. Takamiya, J. Biochem. (Tokyo), 55 (1964) 378. G. H. Rist, J. S. Hyde and T. Vanngard, Proc. Natl. Acad. Sci. U.S.A., 67 (1970)79. J. A. Fee and B. P. Gaber, J. Biol. Chem., 247 (1972) 60. P. H. Haffner and J. E. Coleman, J. Biol. Chem., 248 (1973) 6626. J. A. Fee, Biochim. Biophys. Acta, 295 (1973) 107. R. A. Lieberman and J. A. Fee, J. Biol. Chem., 248 (1973) 7617. S. Lindskog, L. E. Henderson, K. K. Kannan, A. Liljas, P. 0 . Nyman and B. Strandber, in The Enzymes, ed. P. D. Boyer (Academic Press), Vol. 5 (1971) p. 587. J. S. Taylor and J. E. Coleman, J. Biol. Chem., 248 (1973) 749. H. Beinert, ref. 26, pp. 377-388. D. J. E. Ingram: Biological and Biochemical Applications of ESR (Adam Hilger, London, 1969) pp. 190-197. W. G. Zumft, G. Palmer and L. E. Mortenson, Biochim. Biophys. Acta, 292 (1973) 413, 422. B. E. Smith, D. J. Lowe and R. C. Bray, Biochem. J., 135 (1973) 331. W. H. Orme-Johnson, W. D. Hamilton, T. L. Jones, M-Y. W.Tso, R. H. Burris, V. K. Shah and W. J. Brill, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 3142. H. J. Cohen, I. Fridovich and K. V. Rajagopalan, J. Biol. Chern., 246 (1971) 374. D. L. Kessler and K. V. Rajagopalan, J. Biol. Chem., 247 (1972) 6566. J. R. Pilbrow and M. E. Winfield, Mol. Phys., 25 (1973) 1073. B. M. Bahior, T. H. Moss and D. C. Gould, J. Biol. Chem., 247 (1972) 4389. T. H. Finlay, J. Valinsky, A. S. Mildvan and R. H. Abeles, J. Biol. Chem., 248 (1973) 1285. J. A. Hamilton, R. Yamada, R. L. Blakley and H. P. C. Hogenkamp, Biochemistry 10 (1971) 347. J. A. Hamilton, Y. Tamao, R. L. Blakley and R. E. Coffman, Biochemistry, 11 (1972) 4696. S. A. Cockle, H. A. 0. Hill, R. J. P. Williams, S. P. Davies and M. A. Foster, J. Amer. Chem. SOC.,94 (1972) 275. F. s. Kennedy, H. A. 0. Hill, T. A. Kaden and B. L. Vallee, Biochem. Biophys. Res. Commun., 48 (1972) 1533. P. H. Haffner and J. E. Coleman, J. Biol. Chem., 248 (1973) 6630. L. C. Dickinson and J. C. W. Chien, Biochim. Biophys. Res. Commun., 51 (1973) 587. J. C. W. Chien and L. C. Dickinson, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 2783. B. G . Malmstrom, T. Vanngard and M. Larsson, Biochim. Biophys. Acta 30 (1958) 1. A. S. Mildvan and M. Cohn, J. Biol. Chem., 240 (1965) 238.
D. L. WILLIAMS-SMITH AND S. J. WYARD 57. 58. 59. 60. 61. 62.
243
J. P. Slater, I. Tamir, L. A. Loeb and A. S. Mildvan, J. Biol. Chem., 247 (1972) 6784. G . H. Reed and M. Cohn, J. Biol. Chem., 247 (1972) 3073. G . H. Reed and W. J. Ray Jr., Biochemistry, 10 (1971) 3190. G. H. Reed and M. Cohn, J. Biol. Chem., 245 (1970) 662. E. Bayer and H. Kniefel, Naturforsch, 276 (1972) 207.
N. D. Chasteen, R. J. DeKoch, B. L. Rogers and M. W. Hanna, J. Amer. Chem. SOC., 95 (1973) 1301.
63. 64. 65. 66. 67. 68. 69. 79. 71. 7b. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.
86. 87. 88. 89. 90. 91. 92. 93.
I. Yamazaki, H. S . Mason and L. H. Piette, J. Biol. Chem., 235 (1960) 2444. I. Yamazaki and L. H. Piette, Biochim. Biophys. Acta, 50 (1961) 62. I. Yamazaki and L. H. Piette, Biochim. Biophys. Acta, 77 (1963) 47. L. H. Piette, G. Bulow and I. Yamazaki, Biochim. Biophys. Acta, 88 (1964) 120. I. Yamazaki, J. Biol. Chem., 237 (1962) 224. H. Mason, E. Spencer and I. Yamazaki, Biochem. Biophys. Res. Commun., 4 (1961) 236. L. Broman, B. G. Malmstrom, R. Aasa and T. Vanngard, Biochim. Biophys. Acta, 75 (1963) 365. M. Blois Jr. and J. Maling, Biochem. Biophys. Res. Commun., 3 (1960) 132. M. Das, H. Connor, D. Leniart and J. Freed, J. Amer. Chem. SOC.,92 (1970) 2258. D. Backstrom, B. Norling, A. Ehrenberg and L. Ernster, Biochim. Biophys. Acta, 197 (1970) 108. A. Ehrenberg, F. Muller and P. Hemmerich, Eur. J. Biochem., 2 (1967) 286. F. Muller, L. E. G. Eriksson and A. Ehrenberg, Eur. J. Biochem., 12 (1970) 93. F. Muller, P. Hemmerich, A. Ehrenberg, G. Palmer and V. Massey, Eur. J. Biochem., 14 (1970) 185. W. H. Walker and A. Ehrenberg, FEBS Lett., 3 (1969) 315. L. E. G. Eriksson, J. S. Hyde and A. Ehrenberg, Biochim. Biophys. Acta, 192 (1969) 211. L. E. G. Eriksson and A. Ehrenberg, Biochim. Biophys. Acta 295 (1973) 57. S. J. Wyard, Solid State Biophysics (McGraw-Hill, Maidenhead, 1969). N. J. F. Dodd, Brit. J. Cancer, 28 (1973) 257. J. R. Norris, M. E. Druyan and J. J. Katz, J. Amer. Chem. SOC.,95 (1973) 1680. D. Nebert and H. S. Mason, Biochim. Biophys. Acta, 86 (1964) 415. D. L. Williams-Smith, unpublished observations. D. L. Kessler, J. L. Johnson, H. J. Cohen and K. V. Rajagopalan, Biochim. Biophys. Acta, 334 (1974) 86. N. R. Orme-Johnson, R. E. Hansen and H. Beinert, J. Biol. Chem., 249 (1974) 1922, 1928. J. Woolum and B. Commoner, Biochim. Biophys. Acta, 201 (1970) 131. T. Maruyama, N. Kataoka, S. Nagase, H. Sato and H. Sasaki, Cancer Res. 31 (1971) 179. H. Beinert and R. H. Sands, Biochem. Biophys. Res. Commun., 3 (1960) 41, 47. W. H. Orme-Johnson and H. Beinert, Anal. Biochem., 32 (1969) 425. P. L. Dutton, Biochim. Biophys. Acta, 226 (1971) 63. R. C. Bray, Biochem. J., 81 (1961) 189. H. Beinert, G. Palmer, T. Cremona and T. P. Singer, J. Biol. Chem., 240 (1965) 475. T. Ohnishi, R. Katz and B. Chance, Abstr. Commun. Meet. Fed. Eur. Biochem. SOC. 7 (Varna)Abs. no. 655 (1971).
244 94. 95. 96. 97.
ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY N. R. Orme-Johnson, W. H. Orme-Johnson, R. E. Hansen and H. Beinert, Biochem. Biophys. Res. Comrnun., 44 (1971) 446. T. Ohnishi, D. F. Wilson, T. Asakura and B. Chance, Biochem. Biophys. Res. Commun., 46 (1972) 1631. T. Ohnishi, J. S. Leigh, C. I. Ragan and E. Racker, Biochem. Biophys. Res. Commun., 56 (1974) 775. T. Ohnishi, D. B. Winter, J. Lim and T. E. King, Biochem. Biophys. Res. Commun., 53 (1973) 231.
98. 99.
T. Ohnishi, J. G . Hemington, K. F. LaNoue, H. P. Morris and J. R. Williamson, Biochem. Biophys. Res. Commun., 5 5 (1973) 372. J. Rieske, D. H. McLennan and R. Coleman, Biochem. Biophys. Res. Comrnun., 15 (1964) 338.
100. 101. 102. 103.
I. Y. Lee and E. C. Slater, Biochem. Biophys. Acta, 347 (1974) 14. B. Commoner, J. Heise, B. Lippincott, R. Norberg, J. Passoneau and J. Townsend, Science, 126 (1957) 57. H . Beinert, B. Kok and G. Hoch, Biochem. Biophys. Res. Commun., 7 (1962) 209. J. R. Norris, R. A. Uphaus, I. L. Crespi and J. J. Katz, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 625.
104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 1IS. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127.
D. Kohl and P. Wood, Plant Physiol., 44 (1969) 1439. D. Kohl, J. Wright and M. Weissrnan, Biochim. Biophys. Acta, 180 (1969) 536. R. Malkin and A. J. Bearden, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 16. M. C. W. Evans, A. Telfer and A. V. Lord, Biochim. Biophys. Acta, 267 (1967) 530. A. J. Bearden and R. Malkin, Biochim. Biophys. Acta, 283 (1972) 456. B. Ke and H. Beinert, Biochim. Biophys. Acta, 305 (1973) 689. B. Ke, R. E. Hansen and H. Beinert, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 2941. J. S. Leigh and P. L. Dutton, Biochem. Biophys. Res. Cornmun., 46 (1972) 414. P. L. Dutton and J. S. Leigh, Biochim. Biophys. Acta, 314 (1973) 178. M. C. W. Evans, A. V. Lord and S. G. Reeves, Biochem. J., 138 (1974) 177. A. H. Conney, Pharmacol. Rev., 19 (1967) 317. J. 0. Stern, J. Peisach, W. E. Blumberg, A. Y. H . Lu and W. Levin, Arch. Biochem. Biophys., 156 (1973) 404. J. Peisach and W. E. Blumberg, Proc. Natl. Acad. Sci. U.S.A., 67 (1970) 172. A. F. Welton and S. D. Aust, Biochem. Biophys. Res. Commun., 56 (1974) 898. A. C. Brownie, I. Alfano, C. R. E. Jefcoate, W. H. Orme-Johnson, H. Beinert and E. R. Simpson, Ann. N.Y. Acad. Sci., 212 (1973) 344. H. Watari and T. Kimura, Biochem. Biophys. Res. Commun., 24 (1966) 106. J. I. Mason, R. W. Estabrook and .I L. . Purvis, Ann. N.Y. Acad. Sci., 212 (1973) 406. M. Istzkowitz, J. Chem. Phys., 46 (1967) 3048. A. Hudson and G . Luckhurst, Chem. Rev., 69 (1969) 191. s. A. Goldman, G. V. Bruno and J. H. Freed, J. Phys. Chem., 76 (1972) 1858. J. F. W. Keana, S. B. Keanaand D. Beetham, J. Amer. Chem. Sac., 89 (1967) 3055. E. G . Rozantsev, Free Nitroxyl Radicals (Plenum Press, New York, 1970). T. J. Stone, T. Buckman, P. L. Nordio and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 1010. c. L. Hamilton and H. M. McConnell, in: Structural Chemistry and Molecular Biology, ed. A. Rich and N. Davidson (W. H. Freeman, San Francisco, 1968) pp. 115-149.
128.
H. M. McConnell and B. G . McFarland, Quart. Rev. Biophys., 3 (1970) 91.
D. L. WILLIAMS-SMITH AND S. J. WYARD 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166.
245
P. Jost, 0. H. Griffith, Methods in Pharmacology (1972) 223. W. T. Roubal, Progr. Chem. Fats and Lipids, 13 (1972) 63. J. Boeyens and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 56 (1966) 22. S. Ogawa and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 58 (1967) 19. S. Ogawa, H. M. McConnell and A. Horwitz, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 401. H. M. McConnell, S. Ogawa and A. Horwitz, Nature, 220 (1968) 787. T. Asakura, M. amura and M. Shin, J. Biol. Chem., 247 (1972) 3693. H. M. McConn II, W. Deal and R. Ogata, Biochemistry, 8 (1969) 2580. B. Malchy, H. Dugas, F. Ofosu and I. C. P. Smith, Biochemistry, 11 (1972) 1669. L. Berliner and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 55 (1966) 708. E. J. Shimshick and H. M. McConnell, Biochem. Biophys. Res. Commun., 46 (1972) 321. J. C. Hsia, D. Kosman and L. H. Piette, Biochem. Biophys. Res. Commun. 36 (1969) 75. D. Kosman, J. Mol. Biol., 67 (1972) 247. R. W. Burley, J. C. Seidel and J. Gergley,Arch. Biochem. Biophys., 146 (1971) 597. J. C. Seidel and J. Gergely, Cold Spring Harbour Symp. Quant. Biol., 37(1972) 187. R. Cooke and M. F. Morales, Biochemistry, 8 (1969) 3188. P. Jost, L. J. Libertini, V. C. Herbert and 0. H. Griffith, J. Mol. Biol., 59 (1971) 77. J. M. Boggs and J. C. Hsia, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 1406. E. Sackmann and H. Trauble, J. Amer. Chem. SOC.,94 (1972) 4482, 4492, 4499. D. Marsh and I. C. P. Smith, Biochim. Biophys. Acta, 298 (1973) 133. W. L. Hubbell and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 12. W. L. Hubbell and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 63 (1969) 16. W. L. Hubbell and H. M. McConnell, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 20. G. J. Giotta and H. H. Wang, Biochim. Biophys. Acta, 298 (1973) 986. G. Zimrner, A. D. Keith and L. Packer, Arch. Biochern. Biophys., 152 (1972) 105. A. Azzi, M. A. Bragadin, A. M. Tamburro and M. Santato, J. Biol. Chem., 248 (1973) 5520. A. Azzi, A. M. Tamburro, G. Farnia and E. Gobbi, Biochim. Biophys. Acta, 256 (1972) 619. M. Nakarnura and S. Ohnishi, Biochem. Biophys. Res. Commun., 46 (1972) 926. R. E. Barnett, L. T. Furcht and R. E. Scott, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 1992. A. D. Keith, A. Waggoner and 0. H. Griffith, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 819. M. E. Tourtellotte, D. Branton and A. D. Keith, Proc. Natl. Acad. Sci. U.S.A., 66 (1970) 909. J. Kaplan, P. G. Canonico and W. J. Caspary, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 66. J. C. Hsia and L. H. Piette, Arch. Biochem. Biophys., 129 (1969) 296. L. Stryer and 0. H. Griffith, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 1785. L. H. Piette, E. F. Kiefer, A. L. Grossberg and D. Pressman, Immunochemistry 9 (1972) 17. R. Leute, E. F. Ullman and A. Goldstein, J. Amer. Med. Ass., 221 (1972) 1231. R. Leute, Ann. N.Y. Acad. Sci., 222 (1973) 1087. E. S. Copeland, Ann. N.Y. Acad. Sci., 222 (1973) 1097.
2
This Page Intentionally Left Blank
Progress in Medicinal Chemistry-Vol. 12, edited by G. P. Ellis and G. B. West Publishing Company
@ 1975-North-Holland
5 Polarography in Biochemistry, Pharmacology and Toxicology MIROSLAV BREZINA and JIRi VOLKE
J. Heyrovskf Institute of Physical Chemistry and Electrochemistry, Czechoslovak Academy of Sciences, Opletalova 25, 110 00 Prague 1, Czechoslovakia INTRODUCTION
248
APPLICATIONS IN BIOCHEMISTRY AND TOXICOLOGY General remarks The determination of inorganic compounds Determination of metals Oxygen Other inorganic substances Organic substances Introduction Nucleic acids, their components and related compounds Adenine Cytosine Guanine Uracil and thymine Other purines and pyrimidines Desoxyribonucleic acids Polyribonucleotides Ribonucleic acids Conclusions Sulphydryl compounds and proteins Reaction of thiols with mercury and other metals Reduction of disulphide groups Catalytic waves in buffered solutions Catalytic waves in cobalt solutions Oxidation at solid electrodes Polarographic studies of biologically important reactions Hemin enzymes I Proteolysis The Beckman glucose analyzer Various other reactions
25 1 25 1 252 252 254 258 258 258 259 260 260 261 26 1 261 261 26 1 262 262 262 263 264 264 264 269 269 269 270 270 270
APPLICATIONS IN PHARMACY, PHARMACEUTICAL CHEMISTRY AND PHARMACOLOGY Possible applications and recent developments Polarographic activity of pharmaceuticals 241
27 1 27 1 276
248
POL AROGRAPHY
Functionalization of inactive substances Analysis of pharmaceutical formulations Fields with intense development
277 279 282
REFERENCES
286
INTRODUCTION Polarography was invented by Jaroslav Heyrovskg 53 years ago. Since that time, it has spread into many fields of research and practical analysis; simultaneously, it developed as a method and its instrumentation reached a high level [ l , 21. Biochemistry and pharmacy belong to the disciplines where polarography plays a significant role, particularly as an analytical method [3-71. Originally, polarography was defined as a method making use of current-voltage curves obtained in the electrolysis at a dropping mercury electrode of the solutions to be analyzed. Later, the application of solid electrodes [8] which may be stationary, rotated or vibrated was introduced. Polarography with solid electrodes is often called voltammetry. In most cases, polarographic activity of substances results from their ability to be reduced or oxidized on the electrode, i.e., to accept or to donate electrons. The qualitative determination is connected with the oxidation or reduction potential of the substances to be determined. The concentration of the substance is given by the current which is also a function of the number of electrons exchanged between the electrode and polarographically active species which is transported by diffusion to the electrode. Sometimes the mechanism of the electrode reaction and the mass-transport is more complicated. Various other processes can play a limited role for the measured polarographic current, e.g., adsorption, preceding reactions, regeneration of polarographically active substance, and catalysis of reduction of active substrate by polarographically-inactive species. As in many other analytical methods, polarographic quantitative determination is carried out by comparison with a standard of known composition. The absotute measurement of the concentration is possible but often inconvenient. Polarography is not only an analytical method, but also a method for the determination of stability constants of various complexes, and rate constants of many reactions; sometimes, it can assist in determining the structure of an organic molecule. The dropping mercury electrode has two main advantages when used
MIROSLAV BREZINA AND J I R ~VOLKE
249
as a measuring electrode. By dropping, the electrode surface is regularly renewed. This fact increases the precision of the analysis. The high overvoltage of hydrogen evolution on mercury allows negative potentials to reach values as high as -2.5 V (vs. SCE). With solid electrodes (e.g. platinum), positive potentials of about + 1.5 V can be reached but on the negative side, the hydrogen overvoltage is low and is a function of the electrode material and the pH of the solution. Moreover, solid electrodes often exhibit serious drawbacks as regards the background current which can be affected by the formation and/or removal of surface compounds, e.g., oxides. Nevertheless, in biological media only the non-toxic solid electrodes may be used. Platinum, gold and carbon are the most frequently employed materials for solid electrodes. Platinum has a high background current, but it also exhibits the highest degree of reproducibility of the obtained voltammetric curves. Carbon must not be porous and for this reason the pores must be filled, e.g., with paraffin. The so-called Glassy carbon [9] with a very low porosity is recommended. The electrode surface must in all cases be respected, this makes a mechanical or an electrochemical pretreatment necessary before each recording of a voltammetric curve. In classical polarography (sometimes called direct-current polarography), a slow linearly increasing voltage sweep (0.1-1.0 V/min) is applied to the dropping mercury electrode. This polarization speed is often applied in voltammetric measurements, too. With faster voltage-sweeps than 1 V/s, one has to measure the whole current-voltage curve on a single mercury drop. The surface area of the electrode must not increase substantially during the recording of the curve. This is why a hanging mercury drop is suitable for such measurements. Stationary electrodes are often polarised with consecutive triangular sweeps. This method is called cyclic polarography or cyclic voltammetry. A repeated polarisation of a solid electrode in opposite voltage directions can partly renew the electrode surface by an electrochemical process. A special branch of cyclic polarography is the stripping analysis 110-131 which makes possible the determination of very small amounts of substances (subnanograms), i e . , an analysis of lo-'') M solutions. The precision is lower than in clahical polarography (here it is about *2%) and depends on the substance to be determined and on its concentration. The basis of the stripping analysis is an electrolytical accumulation from the solution of the substance to be determined on the stationary mercury electrode. This is effected either as amalgam or as film formation on the mercury electrode. In some special cases, a solid, e.g., carbon electrode,
250
POLAROGRAPHY
can also be used for the electrolytical deposition of a film. The deposited substance is transferred back (redissolved) into the solution by a reverse electrochemical process. The current-voltage curve of this reverse process is a measure of the quantity of the deposited species. During the pre-electrolysis the solution is stirred. The calibration curve is linear if constant pre-electrolysis conditions are ensured. Other recent methods which increase the sensitivity of the determination, are alternating current (a.c.) polarography [ 141 and square-wave polarography [ 151. A.c. polarography applies the superposition of a sinusoidal low frequency alternating potential with a small amplitude (5-50 mV) onto the linear increasing or decreasing potential applied to a dropping mercury electrode. In square-wave polarography, the superimposed alternating potential has the form of a square-wave. Both methods are more useful for determining reversibly behaving systems than for those with irreversible electrode processes. With reversible systems, the lower concentration limit in analysis is as low as 5 x lO-'M. In recent years puke polarography [ 151proved a very successful method in biological studies, in its derivative form especially. As in classic polarography, a slowly increasing voltage is applied to the dropping mercury electrode. At a certain time after the beginning of the drop-life (usually 2sec) a rectangular voltage pulse (usually 30 mV, 1/25 sec) is superimposed on the linear voltage. The analytical evaluation of polarographic or voltammetric currentvoltage curves is based on measuring the height of the waves that result when a dropping-mercury electrode (d.m.e.) or a rotated disc electrode (r.d.e.) with a slow linearly increasing voltage sweep are used. With stationary electrodes, the reduction or oxidation peaks are obtained whose height is a linear function of concentration of the electroactive substance as in case of polarographic (d.m.e.-IlkoviE equation) or voltammetric (r.d.e.-Lev% equation) waves. However, the linear dependence of the peak-height on concentration is subject to the conditions of stationary diffusion, e.g., the relative quiet of the solution, and the constant rate of voltage sweep. A.c. polarography, square-wave and pulse polarography give maxima which are linear functions of concentrations of the analysed electroactive substance. The most characteristic parameter of a compound, when examined polarographically by d.m.e. or r.d.e. is the half-wave potential. It is a potential measured in the half height of the wave on the current-voltage curve. In the case of a thermodynamically reversible redox process, the half-wave potential virtually corresponds to the redox potential of the
MIROSLAV BREZINA A N D JIRI VOLKE
25 1
redox system. In a similar way in polarography with stationary electrodes, the half-peak or peak potential characterize the quality of the electroactive species in the solution. When the solution to be analysed has a high resistance (e.g., in non-aqueous solvents or when the current is too high, e.g., using solid electrodes with a large surface), the controlled potential technique is to be used. A three electrode (working, reference and auxiliary) system with a potentiostat is employed. The supporting (background, indifferent) electrolyte must contain cations or anions, the reduction or oxidation of which occurs at sufficiently negative or positive potentials respectively.
APPLICATIONS IN BIOCHEMISTRY AND TOXICOLOGY GENERAL REMARKS
If necessary, 0.1 ml of the solution to be electrolysed is sufficient for a polarographic analysis. The first step in a biochemical or toxicological analysis is the dissolution of the sample. In several cases, when liquid samples such as some body fluids are investigated, the direct polarographic analysis after dilution of the sample with a suitable supporting electrolyte can be performed. In most cases, however, destruction of the sample and the dissolution of the substances to be determined are necessary. In determining inorganic components in biological materials, ignition, extraction or some other chemical separation method is employed in order to remove interfering compounds from the sample to be analyzed. The biological material can be ignited in a furnace and the ash then dissolved in an acid, or the sample can be destroyed in hot acids under the influence of some oxidizing agents. The most frequently used mixtures for wet treatment are sulphuric, nitric and perchloric acids; sometimes the less aggressive oxidizing mixture of nitric acid and hydrogen peroxide is satisfactory (e.g., for the analysis of tissues). In the determination of organic compounds in biological samples drastic methods such as ignition cannot be used. Physicochemical methods such as extraction, distillation and all types of chromatographic methods are more convenient for separating the substance to be determined from the interfering components. For compounds that will only dissolve in water with difficulty, either mixtures of water and organic solvents (e.g., alcohols, dioxan, acetone) or anhydrous solvents (e.g.,
POLAROGRAPHY
252
acetonitrile, NN-dimethylformamide, dimethylsulphoxide) must be used. An additional advantage of purely anhydrous solutions is the fact that the voltammetric behaviour is rather simplified as compared with aqueous or mixed solution since the effect of protonation is excluded. The choice of a supporting electrolyte depends upon what interfering materials are present. For the polarographic electrolysis or to remove interfering inorganic substances, the complex-forming properties of the supporting electrolyte are very important; the half-wave potential of either the substance to be determined or the interfering substance can be changed substantially. The pH of the solution can play a similar role as a complexing agent, in the analysis of organic substances especially. Hydrogen ions take part in most electrode reactions of organic substances. T H E DETERMINATION OF INORGANIC COMPOUNDS
Determination of metals Almost all metals in various oxidation states can be determined polarographically [3,16]; however, it is not achieved with the same sensitivity and accuracy in all cases. Sometimes it is possible to determine two or more metals in one sample which was prepared for the polarographic electrolysis. The most frequently analyzed biological materials have been blood, urine, human organs, water and gases. A full review of many applications was published in 1958 [32]. Lead and thallium are elements whose polarographic determination causes no trouble. Their half-wave potentials are very favourably situated in the potential range of various electrolytes. Therefore the polarographic waves of these metals are easily measurable. The determination of lead and thallium was often used in toxicology and industrial hygiene (see [31). Other elements which were determined very often in miscellaneous biological materials are copper, cadmium and zinc. Their polarographic behaviour is very typical and the determination is without problems. Blood, urine and tissues are the most analyzed biological samples. The polarographic determination of manganese, iron, cobalt and nickel is not so often used as that of the above mentioned elements. Probably, competition with other analytical methods is the main reason. However, the polarography of these metals has been thoroughly investigated and it is frequently used for studies of equilibrium constants of their complexes.
MIROSLAV BREZINA AND J I R ~VOLKE
253
Special attention is to be paid to alkali metals. Their reduction potentials are very negative (above - 2 V vs. SCE) and the electrolyte components must be reducible at very high negative potentials. The tetra-alkylammonium bases or salts are very convenient for such purposes; these must be pure. The small variability of the half-wave potential of the reduction of sodium, potassium, rubidium and caesium ions makes impossible their polarographic discrimination. The half-wave potential of lithium is about 200 mV more negative than that of sodium or potassium and therefore it can be determined in the presence of up to a 10-fold excess of sodium or potassium. Some methods for separate determination of sodium and potassium have been described [17]. This procedure is based on the preliminary separation of the potassium by the perchlorate precipitation. Polarography of alkaline metals and ammonium ion has proved successful for studying complexes of alkali metals with biologically important macrocyclic compounds such as valinomycin [ 181, macrotetrolides [19,201 and polyethers [20,21]. In the rare metal group, one application must be mentioned here, i.e., the exact determination of a small amount of gold [22,231 (up to 0.5 pg in 5 ml of solution with an accuracy of 2 2%) in urine. The polarographic determination of tin has been carried out in various foods; the content of bismuth, antimony and arsenic has been measured mainly in blood, urine and tissues (see [3]). The methods of metal analysis described in this chapter can be employed with the highest sensitivity and accuracy of classical polarography (about 50 pg 2%, at higher concentrations & 1%). For trace metal analysis the anodic stripping voltammetry is convenient [lo-131. Anodic stripping analysers [24J are capable of performing rapid trace analysis for Pb, T1, Zn, Hg, Sb, As, Bi, Cd, Cu, Au, Ag, in the 10-6-10-10M concentration; 50 to 150 routine analyses per day can be carried out on samples of 5-100mg. The accuracy of an analysis depends on the metal and its concentration, e.g., for 100 ng Cd it is 24%, for 0.1 ng 2 12%. Stripping voltammetry can be successfully applied in clinical chemistry. In the literature of the firm Esa [25,26] trace metal (Zn, Cd, Pb, Cu, Bi and T1) analysis of blood and tissues is described. Using very sensitive analytical methods, the need for cleaner reagents becomes a necessity. The purest reagents contain about 10-7-10-8 M of various metals. Electrochemical cleaning systems [27Jenable electrolytes suitable for trace analysis to be prepared.
*
254
POLAROGRAPHY
Polarography of metals not mentioned here has been only seldomly applied to the analysis of biological materials. Oxygen The most useful application of polarography in biochemistry is the determination of oxygen. The reduction of oxygen proceeds in two steps at a mercury dropping electrode. In the first wave oxygen is reduced to hydrogen peroxide and in the second, more negative wave, the peroxide formed is reduced to water. Although the reduction of oxygen on the d.m.e. is very typical, this electrode, however, is not convenient for the determination of oxygen directly in biological media. The reduction of oxygen at solid electrodes [8, 281 is not so simple as on the d.m.e. When platinum or silver electrodes are in an active state, e.g., they have been freshly polished and are free of surface-active substances, the reduction of oxygen to water proceeds in one 4-electron wave. When the platinum or silver electrodes are inactivated, as e.g., by the presence of surface active substances the 4-electron reduction wave of oxygen is divided into two parts; mostly the first part is higher than the second one. It is necessary to know this fact, especially in the cases of continuous measuring of oxygen concentration at a constant potential with instruments without the registration of the whole polarographic curve. This problem could play an important role when using commercial p 0 2 analyzers. Therefore it is to be recommended that the potential used in measuring the whole polarographic curve be checked. At gold and carbon electrodes the oxygen is reduced in two waves, which differ very often in their height. The first wave increases with the activity of the electrode surface at the cost of the second one, especially with a carbon electrode. A dropping mercury electrode was used for the determination of oxygen in blood [29-321 in lymph [31]during various enzymatic processes [33-351 and in various gases [36-381. In order to exclude any toxic effect of mercury and other components of the electrode system on the activity of biological material a special arrangement for estimating the oxygen consumption of various animal and human tissues has been described [39,401. A saline solution flows slowly at a constant rate through an analyzer and enters first a small flat chamber with the tissue under investigation resting upon a cellophane membrane to prevent its unwanted leaching. After leaving the contact chamber in which some oxygen has been consumed, the saline comes into
MIROSLAV BREZINA AND J I R ~VOLKE
255
the electrode compartment from which it is drained away. The method described was used for measurement in various kinds of sliced tissues in vitro [41-431. An analyzer utilizing the same principle was used to estimate the oxygen uptake of human skin in vivo [44-461. The first important paper about the use of solid electrodes was published by Davies and Brink [47]. These authors described two types of platinum microelectrodes for determining oxygen tension in various body fluids and living tissues in vitro and in vivo. The so-called recessed electrode is made by sealing a platinum wire of about 0.2 mm diameter with a soft glass tube in such a way that the glass extends beyond the end of the wire and forms a sleeve about 1 mm long. After a definite period, which is necessary for establishing an equilibrium between the oxygen in the sleeve and the solution being studied, the current of an oxygen reduction at suitable potential is measured. To use the recessed electrode for analysis of blood, the opening of the tube was then covered with a semipermeable membrane in order to prevent the erythrocytes from penetrating and interfering in the analysis. The so-called open type of platinum microelectrode is made by a simple platinum contact with the whole investigated medium. The results obtained with this type of electrode are not always quite reproducible, as the surface active substances can inactivate the electrode. Such an electrode can serve as an oxygen monitor measuring the decrease or increase of oxygen content in biological medium only. In 1953 Clark, Wolf, Granger and Taylor [48] found that a shiny platinum cathode covered with a layer of cellophane is suitable for direct measurement of oxygen tension in whole blood. The cellophane membrane prevents the undesirable effects of the red cells on the electrode. However, the great electrical resistance of the membrane and the possibility of contamination of the reference electrode with surfaceactive biological materials does not allow the use of this electrode for analysis in all media. This is why a new model of oxygen electrode has been constructed [49]. Both the platinum cathode and the silver anode were covered with a plastic membrane, and between membrane and electrodes the electrolyte is situated. Since its invention, Clark’s oxygen electrode has been used in many laboratories all over the world. The Nijmegen group of Kreuzer merits special mention [50-531. The so-called catheter oxygen electrode constructed in Nijmegen has the following characteristics. The calibration curve is linear up to 100% O2 and is reproducible within 1% (relative). The current output is of the order
256
POLAROGRAPHY
of 4 p A for an electrode of 300 p n diameter with 100% O2 at 37°C. The output depends on the temperature with a temperature coefficient of 2.9% (relative) at 20°C and 3.1% at 38°C. For all measurements in gases the response time is 0.2-0.3 sec for 95% deflection at 37°C. This catheter electrode was used for recording the oxygen pressure in respiratory air and for the measurement in various biological fluids in vitro and in vivo. A special catheter PO, electrode of 2 mm diameter with low flow dependency and fast response was used for continuous recording of oxygen pressure in blood in vivo [54]. The cathode is a platinum foil about 3 pm thick which is sealed between a tube and a cylinder of polyvinyl chloride or glass to form a ring-shaped electrode. The anode is a silver ring which also carries the membrane held by a silver cap. The current output of 0.6 pA for 100% O2at 37°C with a 6 pm Teflon membrane is equivalent to that of a single wire cathode of 100 p m diameter. Laboratory oxygen analyzers are produced by several firms, e.g., Beckman 1551 and Radiometer [56]. The Beckman Model 777 Oxygen Analyzer measures both gaseous oxygen and dissolved oxygen in aqueous or non-aqueous solutions. Oxygen is reduced at a potential - 0.75 V vs. a silver-silver chloride reference electrode on a gold electrode. Both electrodes are protected from the sample being measured by a Teflon membrane. A cellulose-base KCI gel serves as an electrolyte agent in place of the membrane. To counteract temperature errors a thermistor is built into the sensor. The thermistor reduces temperature errors to less than 5 5% of the reading over the 15 to 45°C range. Accuracy of the sensor at a constant temperature is 21% full scale. The instrument can be calibrated for the p 0 2 range 0-50 mm Hg, the analyzer drift is k 1% full scale/24 hr, and the speed of response 90% in 10-20 sec, net weight approx. 3.6 kg. Radiometer pOz electrode, type E 5046 consists of a platinum cathode (20 pm diameter) and silver-silver chloride reference electrode placed in an electrochemical solution behind a 20 p n thick polypropylene membrane. A polarizing voltage of about 650 mV is applied. The polarographic current is about 10-"A per m m H g of oxygen tension at 38°C. Zero current is lower than lo-'' A, response time less than 60 sec at 38°C 99% of full deflection. The pOz electrode is used with the pH-Meter 27 GM or the Astrup Micro-Equipment, in conjunction with the Oxygen Monitor. The scale can be calibrated to the range 0-100 mm Hg pOz. Thermostated cells provide measurements at constant temperature of volumes down to 70pl. The small volume makes this cell useful to measure the pOz of capillary blood. The cell is supplied with accessories for blood sampling.
MIROSLAV BREZINA AND JIRI VOLKE
257
The polarographic steady state current measured with the pOz electrode is a linear function not only of the partial pressure of oxygen in the analyzed media and the cathode area but also of the diffusion coefficient and the solubility of oxygen in the membrane. The membrane must not be too thick or must not have low oxygen permeability. An oxygen depletion in the electrolyte between membrane and cathode surface may arise and this results in a decrease of the polarographic current. In this case the method has a low sensitivity and a long response time. When the membrane is too thin, the convection of the solution along the membrane can cause the oxygen transport to the electrode to become irregular and the polarographic current does not correspond to the oxygen concentration in the analyzed media. The constant thickness of the electrolyte layer between the electrode surface and the membrane can be secured by inserting a disc of filter paper. The temperature dependence of the polarographic current is deduced from that of the oxygen diffusion and permeation coefficients and of the oxygen solubility in the membrane and media. With increasing temperature the polarographic current rises and the response time becomes shorter. Some theory concerning the characteristics of membrane-covered polarographic oxygen electrodes has been presented [53]. Polarographic oxygen electrodes covered with a membrane were very often used for the analysis of biological media. In the proceedings of the International Symposium on Oxygen Pressure Recording in Nijmegen 1968, e.g. [53], some interesting applications of oxygen electrode are to be found [57-631. These papers contain many other references. Venous oxygen fluctuation under various influences were measured by Yokota and Kreuzer [64,65]. These authors have used polarographic oxygen electrode to the study of alveolar oxygen tension in alveolar gas and blood of anesthetized dogs [66,67]. A pOz electrode was frequently employed for the measurement of oxygen concentration in bacterial cultures e.g. [68-701. With the same method May and Jacob [71] measured continuously the respiration activity of mitochondria fractions from Polystictus versicolor. The respiration of various cells (e.g., lymphocytes, macrophages) was studied [72]. All applications of the polarographic pOz electrode cannot be described here. Several examples which were reported may serve as an information source about a useful method for the determination of oxygen in biological samples.
POLAROGRAPHY
258
Other inorganic substances Solutions of certain anions such as bromate and iodate or their corresponding acids undergo reduction at the dropping mercury electrode and can be analyzed easily. Iodine and bromine can be determined after oxidation to iodate or bromate respectively. During the anodic polarization of a dropping mercury electrode the chloride, bromide, iodide, cyanide, thiocyanide and sulphide ions form slightly soluble salts or stable complexes with mercury. In the presence of these substances so-called anodic oxidation waves appear on polarographic curves. The polarographic determination of chloride has received most attention. In many cases the dilution of the sample with 0.1 N H2S04 is satisfactory and the solution can be polarographed directly [3]. Free halogens in acid media can be reduced to the corresponding halide ions at the mercury electrode or a t various solid electrodes. However, this electrode reaction is not convenient for analytical purposes. Therefore halogens are very often determined indirectly after reaction with some reducing agents [3, 161. Iodine can be oxidized to iodate which gives a well defined cathodic wave. The amount of some gases, such as SO, [73], CO [74], can be automatically continuously measured by polarographic analyzers [75]. Measuring electrodes with a constant surface, e.g. carbon, platinum, gold disks or rods are most often used. Present research is concentrated on the so-called coulometric analyzers [73]. The analysed gas is brought by the shortest route directly to the measuring electrode. The substance to be determined is completely reduced or oxidized electrochemically at a large area electrode. For determining SOz in the atmosphere, the last type of an analyzer according NovAk is to be recommended. Due to its simplicity, it is constructed in a light portable form. The accuracy is for 0.3ppm SO, k 5%, the minimum measurable amount is 0.005 ppm SO,. The automatisation is so satisfactory, that the analyzer can be used for several months without any maintenance. ORGANIC SUBSTANCES
Introduction The polarographic analysis of biological material for determining organic compounds differs from that of inorganic components (p. 252). The
MIROSLAV BREZINA AND J I R ~VOLKE
259
descriptions of procedures cannot be generalized, as each problem is to be solved individually. The survey of the analysis of organic compounds in biological materials and in pharmaceutical preparations has been reviewed [3]. The compounds are arranged here partly according to the group which 'is reducible or oxidizable, and partly according to their biochemical significance. So alkaloids, vitamins, hormones, enzymes and proteins have been collected into separate chapters-regardless of the group undergoing the electrochemical change. Organic compounds containing groups whose electrode reaction is reversible, yield well-defined, easily measurable polarographic waves, e.g., quinones, p -aminophenols, indophenols, thiazines, phenazines. Besides these few reversible systems, a great number of organic substances are irreversibly reduced or oxidized, in this case, the good measurement of polarographic waves depends on a suitable situation of their half-wave potentials on the potential axis (see also [76]). The half-wave potential and the character of the polarographic wave is mostly dependent on pH of the supporting electrolyte. Therefore, it is to be recommended that organic substances be electrolysed in sufficiently buffered solutions. In this short review, we can only pay attention to the most important and progressive applications of polarography in this field. Substances which are relevant to pharmacy or pharmacology will be mentioned in another part of this review [3-71.
Nucleic acids, their components and related compounds For understanding the complicated function of nucleic acids, it is desirable to gain information about the electrochemical properties of these polymers or their components. The first study about the polarography of purine derivatives was published by Heath [77] in 1946. Most new ideas for the electrochemical study of this attractive problem [78-831 were presented by Elving, (University of Michigan, Ann Arbor) and PaleEek (Institute of Biophysics, Czechoslovak Academy of Sciences, Brno). Polarography and related methods can yield valuable knowledge, not only about the electron transfer between a substance being studied and the electrode but also about other properties, e.g., adsorbability of the molecule and changes of the molecular structure. First of all, our attention will be devoted to simple components of nucleic acids. D-Ribose is reducible at the dropping mercury electrode in its free aldehydic form only, i.e., in neutral media El,*= - 1.8 V (vs. SCE) [84,851.
260
POLAROGRAPHY
Sugars bound in nucleosides or nucleotides are polarographically not reducible, but they can influence the electron and mass transport and the adsorbability of the molecule, which consequently may change the polarographic behaviour somewhat (e.g., shift of the half-wave potential or decrease of the wave-height). Adenine. The polarographic reducibility of adenine was discovered by Heath [77] and studied by a number of authors, especially by Smith and Elving [86]. Adenine is reducible in an acid solution, probably in its protonated form only. At about pH 5, its polarographic wave diminishes and at pH 6.5 it disappears. E,12of the 4-electron reduction of adenine is - 0.97 V - 0.090 pH [78]. At a stationary hanging mercury drop electrode (HME) the reduction of adenine occurs in the pH range 0-5 in a single peak with the peak-potential E , = - 1.06-0.086 pH. At a pyrolytic graphite electrode (PGE) adenine shows no cathodic reduction activity over the available potential range from pH 1 to I2 [83]. However, a large anodic peak appears between pH 3.6 and 10, with E , = 1.4 V-0.05 pH. Over the potential range from 0.60 to - 1.80 V, usually only a single well-defined a.c. peak is observed, at higher concentrations of adenine, three other peaks appear. The Faradayic peak which can be observed in the pH range 1-6 corresponds to the reduction wave of adenine. The reducibility of adenine is not substantially influenced when it is bound in dinucleotides [80]. The half-wave potential becomes more negative in the series deoxyadenosine monophosphate (dAMP) G adenine < adenosine < deoxyadenosine < adenosine monophosphate < adenosine triphosphate (ATP). The difference in El,, between AMP and adenine is only 25 mV. The adsorbability of these substances, measured by the differential capacity method [87,88] increases in the series adenine < dAMP < deoxyadenosine < ATP < AMP. The pulse polarography of adenine and its nucleosides and nucleotides agree with those obtained with the classical polarographical method. However, at higher pH the pulse-polarographic waves can be better measured than the d.c. polarographic waves [Sl]. Polarographic methods have been employed in the determination of adenine in blood [89-911, in various tissues 189,921 and in DNA- and RNA-hydrolysates [78,89,931. Cytosine. The polarographic reducibility of cytosine was described in 1962 [86,94]. A four-electron reduction wave with E l i z = - 1.170-0.084 pH becomes lower in alkaline solutions and disappears at pH about 10 [SO, 951. This behaviour is given as in the case of adenine by
MIROSLAV BREZINA AND JIRi VOLKE
26 1
the dissociation of the polarographically active protonated form in the alkaline solutions. Below pH 3, the wave merges with the discharge of the supporting electrolyte. Sugar and phosphate components exert a relatively great influence on the half-wave potential of the related compounds. Elit becomes more negative in the order cytidine (-1.58V)< dCMP < CMP < cytosine (- 1.70 V). This fact can be explained by the electron-withdrawing effect of the pentose ring. Using HME cytosine shows a single reduction peak of almost constant height between pH 3.7 and 7. E , is - 1.14-0.084 pH [83]. Cytosine yields a small single a.c. peak. Guanine. Guanine is polarographically non-reducible [78]. On the basis of oscillopolarographic studies [81] it can be said that the polarographic reduction of guanine proceeds at a very negative potential. The reduction wave of guanine coalesces with the discharge of the supporting electrolyte. Guanine and guanosine can be electrochemically oxidized at a pyrolytic graphite electrode [86,96,97] with Ep/2= + 1.02 V in 2 M H,SO,. Uracil and thymine. Uracil and thymine are not reducible polarographically under normal conditions [78,86,95], but in alkaline media they yield an anodic wave caused by the formation of a mercury salt [98]. Other purines and pyrimidines. Xanthine is not reducible polarographically [86], and hypoxanthine can be reduced only in an acidic medium [86,99]. The polarographic behaviour of many other purine and pyrimidine derivatives is described in papers [78,80,83, 100, 1011. Desoxyribonucleic acids. Among nucleotides which are components of nucleic acids, only CMP and AMP yield well-defined polarographic waves [80,81,95]. Under favourable conditions denaturated DNA can be reduced in a polarographic wave, the height of which roughly corresponds to the theoretically calculated value with respect to the lower diffusion coefficient in the relation to this parameter of a simple nucleotide. The DNA is reduced in an adsorbed state only [81, 1021. Native DNA is polarographically not reducible 1103, 1041. In native DNA the polarographically reducible bases in Watson-Crick double helix are not available for electron transport. Better results than with classical polarography can be obtained with the pulse-polarographic technique [ 105,1061. The lowest concentration of denaturated DNA still yielding a measurable wave is about 1 pg/ml. Besides the well-defined reduction wave another pulsepolarographic wave can be observed. This wave does not have a Faradayic character and can also be obtained with native DNA. Polyribonucleotides. Synthetic single-stranded polyribonucleotides con-
262
P OLAROGRAPHY
taining bases that are polarographically reducible in a monomeric form, e.g., polycytidylic and polyadenylic acid behave like denatured DNA, (i.e., they yield polarographic reduction waves). While for classical polarographic measurements relatively concentrated solutions of polynucleotides are necessary, analysis carried out with pulsepolarographic technique can be performed with M solution (referred to the monomer content). Polynucleotides with polarographically irreducible bases give no polarographic waves. Polyuridylic acid, copolymer polyuridyloguanylic acid, and polyribothymidyl acid yield pulsepolarographic waves at a high sensitivity of the instrument only [81,107, 1081. Ribonucleic acids. Information about the polarographic activity of RNA is much poorer than in the case of DNA [107]. Recently it has been shown that the double-stranded RNA and its thermally denatured form behave in a similar manner to native and denaturated DNA [ 1091. Pulse-polarographic analysis in connection with a gel electrophoresis can be used for testing double-stranded RNAsamples prepared for biological experiments (interferon-inducing and antiviral activities). Conclusions. From modern polarographic techniques, pulse-polarography has been found best suited for studying nucleic acids. It is possible to determine single-stranded polynucleotides, e.g., denaturated DNA, polyadenylic acid and polycytidylic acid in concentrations about 1 pglml. Polynucleotides having a double-helix structure in which reducible groups are blocked show very small pulse-polarographic signals. Therefore it is possible to determine traces (less than 1%) of single-stranded polynucleotides in samples containing double-helix polynucleotides. The analysis obtained with classical polarographic methods corresponds roughly with those reached by the pulse-polarographic technique, but the sensitivity is much lower. The difference between the polarographic behaviour of single-stranded and double-helical form of polynucleotides makes possible the study of the conformation of nucleic acids [81,82, 108-1 131. Polarography can be utilized also in the study of structural changes of polynucleotides under the influence of the temperature El 12, 114, 1151 or irradiation [116]. The photodynamic destabilisation of DNA has been described [117].
Sulphydryl compounds and proteins Sulphydryl compounds belong to the most important among biologically active substances. The polarographic behaviour of sulphydryl compounds
MIROSLAV BREZINA AND JIRI VOLKE
263
has been studied intensely in past years. Their polarographic activity can be divided into five classes; a) The formation of insoluble salts or polarographically inactive complexes with mercury ions or with ions of some other metals. b) Reduction of disulfide groups. c) Catalysis of hydrogen ion reduction in buffered solutions. d) Catalysis of hydrogen ion reduction in buffered solutions containing cobalt salts. e) Oxidation at solid electrodes. Reaction of thiols with mercury and other metals. Similarly as chloride (see p. 258) sulphydryl groups can yield anodic waves which are caused by the formation slightly soluble salts with mercury during the anodic polarisation of a dropping mercury electrode [3]. At concentrations higher than 10-4M, two or more waves can be observed. The sum of the wave-heights which should be measured in quantitative measurements, corresponds to a one-electron exchange. Since the oxidation of the thiol groups on a platinum electrode occurs at about 1.0V more positive potentials and since the electrolysis on a large mercury electrode forms mercury salts, the results suggest, that the following reaction is taking place: RSH + Hg+ RSHg + H'
+e
In some cases various mercury salts can be formed. Higher concentrations cannot be analysed because above the limiting concentration the anodic wave of thiol does not increase with concentration. This limiting concentration (10-4-10-3M) varies with the species and can be influenced by many factors such as pH and the presence of some other substances. The analysis of thiol compounds is simple and in some cases advantageous, e.g., by means of different anodic waves, glutathion or similar substances can be determined together with ascorbic acid [3, 1181. Sulphydryl compounds form with ions of metals such as silver, copper, lead and cadmium insoluble salts or polarographically inactive complexes 131. These reactions are important not only for the study of the binding of heavy metals in biological systems but they can also be used for the determination of thiols by the so-called amperometric (polarometric) titrations [2] (For more details see [2,3, 161). In this method, the dependence of the current passing through the cell on the volume of the added titrating agent is noted. The current is measured at a constant potential at which either the substance to be determined or the titrant or both are reduced or oxidized. This method of analysis is very simple, rapid and precise (about 0.2%), however, in some cases it can be unspecific. Often
264
POLAROGRAPHY
the rotating or vibrating platinum electrode is employed as an indicator electrode. The use of argentometric, mercurimetric or iodometric amperometric titration is common 13, 1181. The reactions of thions and disulphide groups with mercuric salts have been described [ 118, 1191. Amperometric titration of sulphydryl groups is often applied to the analysis of proteins or body fluids and tissues, respectively [3]. In particular the papers of Benesch, and Lardy and Benesch [121,122] and of others [123-1261 are to be mentioned here. Reduction of disulphide groups. Sulphydryl compounds can be polarographically determined mostly after their oxidation to disulphides. Cystine and oxidized glutathione are reduced irreversibly in a wide range of pH at a relative positive potential with the addition of 2 electrons. The electrochemistry of some other disulphides has been described [3]. Catalytic waves in bugered solutions. Some sulphydryl compounds, especially proteins, yield in buffered solutions, preferably between pH 6 and 10, even at very high dilution, polarographic waves at potentials closely preceding the discharge of the supporting electrolyte cation [3, 1271. This type of polarographic effect is called praenatrium wave and is caused by the evolution of hydrogen at the surface of the dropping mercury electrode. The hydrogen overvoltage is decreased by the catalytically active groups. However, these catalytic waves are also produced by other organic substances, especially by those which contain nitrogen. Therefore, this polarographic effect is very unspecific and it is influenced by many factors. The height of this catalytic wave is not a linear function of the concentration of the active substance; it approaches a limiting value. The praenatrium wave can therefore be recommended for an analysis of pure substances only in some special cases. Catalytic waves in cobalt solutions. Compounds containing sulphydryl or disulphide groups give two different types of catalytic waves in buffered ammoniacal solutions of cobalt, which are very often named BrdiEka catalytic waves. The simple compounds of a low molecular weight (e.g., cystine, cysteine) produce a characteristic round maximum, whereas more complicated compounds such as proteins give a typical double-wave [3, 127-1311. These catalytic effects result from the reduction of hydrogen ions supplied to the electrode by the acidic component of the buffered solutions; the presence of ammonium ions is not necessary, as some suitable amino acid as a proton-donor is satisfactory. The decrease of hydrogen overvoltage caused by cystine or proteins is about 400mV.
MIROSLAV BREZINA AND JIRI VOLKE
265
Although the mechanism of the electrode process has not been explained in detail, there is no doubt that it is a complex of cobalt(I1)ions with the sulphydryl compound which plays a decisive role in this polarographic catalytic effect. Proteins not containing sulphydryl or disulphide groups are catalytically inactive and among natural amino acids only cystine and cysteine show such a characteristic wave in cobalt solutions. Cobalt complexes with dissociated sulphydryl group of cysteine. Therefore it is to be expected that after the electrodeposition of metallic cobalt from the complex, the dissociated sulphydryl groups will take up protons from the acid component of the solution. The protons supplied to the electrode surface through the sulphydryl groups are discharged at a decreased hydrogen overvoltage. Disulphide groups can be reduced at a more positive potential to sulphydryl groups, the trivalent cobalt, if used, to the divalent form. Similar effects of thiols and proteins as in cobalt solutions, with much smaller sensitivity however, can be observed in buffered electrolytes containing nickel(I1). The nature of the bonding and structure of the complexes of proteins with cobalt is not fully understood. It follows at least from the analogous polarographic effects due to proteins and to their free cystine and cysteine nuclei. The shape of the polarographic double-wave of proteins differs somewhat from that of cystine and cysteine. A two-humped shape of the protein catalytic effect can be explained by a different activity of some other amino acids in the protein molecule or by the globular and linear structure of the polypeptides. Another possibility is, that this shape results from the adsorption of protein molecules at the electrode surface where they promote, in the adsorbed state presumably, the evolution of hydrogen from the sulphydryl groups [127]. This last theory [132] assumes, that the second part of the protein double-wave is caused by sulphydryl groups located in charged, hydrophillic regions of the films of the flattened protein, while the first part is given by sulphydryl groups present in uncharged, hydrophobic regions. Although its theory is not quite clear, the BrdiCka catalytic effect of cysteine or proteins is so typical and sensitive, that it is suitable for use in the analysis. It is not possible to discuss here the whole theory of catalytic currents of sulphydryl compounds (see also [I 33-1411) but some characteristics must be mentioned. At a suitable concentration of ammonium ions and ammonia, providing the electrolyte with an adequate buffer capacity, the height of the protein double-wave increases with the increasing concentration of the cobaIt in the shape of a parabola. In solutions containing cobalt(II1) ions the
266
POLAROGRAPHY
protein wave is usually slightly higher than in solutions of cobalt(I1) ions at the same concentration. Cystine and SH- or -S-S- substances of a low molecular weight have similar dependence of the wave-height on the cobalt concentration as proteins. While the catalytic effect of cystine in Co(I1) solutions has about the same sensitivity as that of proteins, the height of the cystine catalytic wave in Co(II1) solutions is approximately 1000 times smaller . BrdiCka catalytic processes are strongly influenced by the concentration of ammonia and ammonium ions. Any change in their concentration ratio brings a shift in the dissociation equilibria of SH-compounds and their complexes. In excess of ammonia, the coalescence of the protein double-wave occurs, giving a single wave and a considerable increase in the single wave appears as compared to the double wave. For analytical purposes the composition of the supporting electrolyte is to be kept constant. For the determination of cystine and similar SH- or -S-S- substances Co(I1) solution is necessary (e.g., 0.001 M CoCl,, 0.1 M NH,, 0.1 M NH4CI). For the analysis of proteins Co(II1) electrolyte (0.001 M Co(NH&Cl,, 1 M or 0.1 M NH,, 0.1 M NH4Cl) is to be recommended. The BrdiCka catalytic effect is very sensitive and the sensitivity depends not only on the character of the species to be determined, but also on the composition of the supporting electrolyte. Some sulphydryl compounds can be determined in concentrations as low as 1 Fg%. It is important to know that the calibration curve of Brdieka catalytic waves is n o t a straight line, but ascends towards a limiting value. Therefore, a sufficient dilution of the sample is to be recommended. In the polarographic analysis it is impossible to distinguish different types of proteins. Thus its application to quantitative analysis is mainly limited to samples containing either a single type of protein or a protein mixture of a similar composition. Analytical applications of the polarographic protein test cover a broad field and cannot be described here. They involve the estimation of proteins in various body fluids and tissues [3]. A very interesting use of the BrdiCka protein wave is the controlling of the purity of the tobacco mosaic virus [142,143]. This virus in the pure state shows the first part of the double-wave only. However, the non-virus protein present as impurities, yields the whole double-wave, the second hump of which disappears if the virus is purified. The polarographic studies of TM-virus at low temperature led Rutkay to the opinion, that p,!arographic effect of protein is given by three maxima, which can under special circumstances coalesce. He suggests, that the maxima
MIROSLAV BREZINA AND J I R ~VOLKE
267
(waves) can be caused by various groups of cystein which can be bound on cobalt, =C=O, =NH and -SH (from positive to negative potential). Although it could not be quite confirmed, this was used to study the re-formation of tobacco mosaic virus capsid from its disordered polypeptide chains [144,145]. As it has been shown, the structural changes of proteins can be followed polarographically. The stability of polypeptides after the break down of certain intramolecular bonds is accompanied by the liberation of a part of polarographically active groups, which are masked inside the native molecule [3,127, 1301. For example if serum proteins are denaturated in a slightly alkaline solution (e.g., 0.2 M KOH), a distinct increase of the protein double-wave with time is observed. After reaching the maximum value, the height of the protein double-wave begins to decrease and corresponds to a slow coagulation of proteins. A similar effect takes place if native proteins are exposed to irradiation. The alkaline denaturation of serum proteins proved to be of practical use in detecting greater or lesser differences between normal and various pathological sera. For example, the proteins of sera from patients suffering from cancer cannot be denatured with alkali as can those of normal people. A similar difference is found if the sera are tested with pepsin in dilute solutions of hydrochloric acid. The higher protein waves in the normal sera, in comparison with carcinomatic sera, indicate higher content of polarographically active groups liberated through denaturation or peptic cleavage. In such a manner, the activity of different proteolytic enzymes has been estimated. Since the products of proteolytic cleavage can exhibit higher catalytic waves than those of the native proteins, the proteolytic effect can be investigated polarographically. Larger protein molecules can be precipitated with sulphosalicyclic acid. The polarographic analysis of these filtrates corresponds to the content of proteolytic products mainly. The fact that various proteolytic systems are active at different pH regions enabled a distinction to be made between some enzymes present in blood sera (146,1471 and those in spinal fluid [147,148]. The polarographic examination of sulphosalicylic filtrate has been applied by BrdiEka and later on by many others to the determination of some degradation products or fractions of the serum proteins in blood of ill humans [3,127,130,147-1531. The increase of BrdiEka protein wave in sulphosalicylic filtrate is characteristic of sera of patients suffering from cancer or some inflammatory disease. The BrdiEka test is simple, quick and reproducible. Sulphydryl compounds which cause the increase of the
268
POLAROGRAPHY
polarographic activity of sulphosalicylic filtrate of serum proteins could not be the degradation products only but also proteins containing a sugar component (e.g., mucoproteins). For diagnostic purposes, the following procedure of the BrdiEka filtrate test is to be recommended. Add 0.4ml of fresh blood serum to 1 ml 0.1 M KOH and allow to stand for 45 min. Precipitate the proteins at room temperature by the addition of 1 ml of 20% sulphosalicylic acid. After 10 min, filter the precipitate through a hard filter paper and add 0.5 ml of clear filtrate to 5 ml of buffered hexamino-cobalt(II1) solution M in 0.1 M NH4CI, 1 M NH,). Compare the increase in the height of the wave of the serum filtrate from the patients with the average height of that obtained from 10 or 20 normal humans. Disregarding the non-specifity of the BrdiEka filtrate test, the main interest in clinical practice is directed towards its application in the diagnosis of cancer. The extensive statistical material, collected during the last 30 years, shows that the height of the filtrate wave increases appreciably during the progress of the malignant disease. A surgical or successful therapeutic intervention is followed by a decrease in the filtrate wave, which returns to its normal value. The recurrence of the disease is always accompanied by a new increase in the value of the BrdiEka test. In contrast to cancer and inflammatory diseases, the decrease of the filtrate wave under the normal value takes place with patients suffering from infectious hepatitis or cirrhosis of the liver E154, 1551. Urine can be analysed in the same manner as serum, but the quantitative changes in the content of pathological proteins or smaller polypeptides fluctuate over wide limits [156]. A considerable increase of the filtrate wave in urine was observed with patients suffering from burns. In order to obtain information about the polarographic properties of the individual protein components in blood sera, the polarography was combined with paper electrophoresis. After electrophoretic separation, the cut strips of paper with separated fractions of albumins and globulins are eluted in physiological sodium chloride solution and each sample is analyzed polarographically. These combined methods were applied for study of various pathological cases [147]. Many applications of polarography in the determination of proteins or simple sulphydryl or disulphide compounds in various biological materials have been described [3]. For the estimation of traces of proteins in nucleic acid preparations, a pulse polarographic method is more suitable than classical polarography
MIROSLAV BREZINA AND J I R ~VOLKE
269
[157]. It enables the detection of proteins in concentrations of approximately 0.1 pg/ml, and a few micrograms of the sample suffice for the estimation of 1% protein in a nucleic acid preparation. Oxidation at solid electrodes. Oxidation of cysteine, cystine and both forms of glutathione at a platinum electrode was studied extensively by PradBE, Koryta and Ossendorfovh 1158-1601. The oxidation of these substances occurs by reaction of the adsorbed radical with a surface oxide. Therefore, the calibration curve approaches a limiting value at higher concentrations. Reproducible results are obtained at pH 7.2 using cyclic voltammetry. This method makes it impossible to estimate these substances in blood serum 1161,1621 and in some body organs [161,163]. Cysteine as electrochemical indicator can also be determined in vivo after intravenous injection, as for example in the assessment of the blood supply to the kidney [164-1661.
Polarographic studies of biologically important reactions Polarography enables us to gain a great amount of information about various reactions which play an important role in the living organism, and much interest has been shown in the following enzymatic reactions [3]. Hemin enzymes. The catalytic function of hemin enzymes is due to the reactivity towards oxygen and hydrogen peroxide of the iron bound in a complex with porphyrin. Besides measuring the polarographic waves of the enzyme redox systems, it is also possible to study polarographically the nature of the catalytic activity of many enzymes and to evaluate their reaction rates with the substrate. The scheme for the reaction of hemin or catalase with hydrogen peroxide was proposed and verified by calculating rate constants according the derived equations 13,167-1691. Hemin forms an addition compound with hydrogen peroxide and peroxide can be then reduced with a smaller overvoltage (see p. 254). In the reaction of catalase with hydrogen peroxide, oxygen is regenerated and so the polarographic reduction wave of oxygen grows at the expense of the peroxide wave in the presence of catalase. The polarographic catalytic effect of hemin compounds is sensitive as it could be registered at concentrations as low as lO-’M. The polarographic catalytic activity of cytochrome c and peroxidase was studied too [3,170-1731. Conformation of methemoglobin, metmyoglobin, cytochrome c, together with poly-L-lysine and glycogen phosphorylase adsorbed at the mercury electrode, was studied by various polarographic techniques in
270
POLAROGRAPHY
the Institute of Molecular Biology, Academy of Sciences of GDR, Berlin [174-1771. Proteolysis. The polarographic proof of proteolysis and the estimation of Some proteolytic enzymes by means of the increase of the polarographic activity of hydrolysed proteins are described on p. 267. In this part, we should mention another method of following the proteolytic activity. Some polypeptides yield high polarographic catalytic waves in the BrdiEka cobalt(II1) solution (see p. 265). However, the products of their hydrolysis are polarographically much less active in cobalt (111) solution (see p. 265). In such a manner, the influence of serum of pregnant women on the hydrolysis of protein pituitary extract and on oxytocin and vasopressin was measured [178,179]. A curious method for determining the enzymatic activity of trypsin and of the duodenal content has been demonstrated on the cleavage of p -nitroadides of some aminoacids [ 1801. The half-wave potential of p -nitroaniline reduction is more negative than that of the substituted derivatives and so the hydrolysis of the nitroanilides can be measured by the decrease of its reduction wave. The Beckman glucose analyzer. This analyzer introduces to biochemical laboratories a convenient method for determining glucose in centrifugated blood plasma or serum, or in urine. Protein precipitation from the sample is not required. Up to 60 samples per hour can be handled. Glucose is determined on the basis of measuring the rate of oxygen consumption by means of the polarographic oxygen sensor (see p. 256). A digital-meter provides direct readout in milligrams of glucose per 100 ml of the sample. The rate of oxygen consumption is directly proportional to the glucose concentration. Glucose reacts with oxygen in the presence of glucose oxidase, and the hydrogen peroxide produced must be destroyed by catalase or some other catalysts. Various other reactions. Some other enzymatic reactions, such as the catalysis of the oxidation of aldehydes and of xanthine [181], and oxidation of catechol and hydroquinone catalyzed by tyrosinose [ 1821 have been described in the book [3]. The activity of cholinesterase is proportional to the increase of the anodic wave of thiocholine, which is produced by the hydrolysis of acetylcholine [ 1831. Reactions of deoxyribonucleic acids with actinomycin [184], daumomycin [185] and with methylene blue [186] were followed by measuring the height of polarographic wave of reactants studied.
MIROSLAV BREZINA AND J I R ~VOLKE
271
APPLICATIONS IN PHARMACY, PHARMACEUTICAL CHEMISTRY AND PHARMACOLOGY In this chapter on polarography in pharmacy we intend to stress only the fundamental ideas of the application and to describe and interpret the recent trends and developments in this field which, at least in our opinion, seem promising. The reader will find a systematic treatment based on older literature in the book by BEezina and Zuman [3] or in a shorter version (in German) in a review [187]. More recent reviews present later development in a brief form [188]. If one is interested in the problems of polarographic activity of organic compounds, in particular in mechanisms, Perrin’s review [ 1981 should be consulted. More details about polarography of heterocyclics are to be found in a specialised review chapter [6]. A vast quantity of material about organic electrochemistry are contained in 2 recent book [188a]. POSSIBLE APPLICATIONS AND RECENT DEVELOPMENTS
Pharmaceutical analysis has been a characteristic field in which polarography, in particular in its classical d.c. form is often used, mostly of course as an analytical tool [3,187,188]. The term analysis involves here not only qualitative and quantitative determination of the substances to be investigated, but also the interpretation or determination of their structures based on their polarographic or electrochemical behaviour. Accordingly, there are a number of publications dealing with the relationship between the structure and electrochemical parameters, e.g., in alkaloids and vitamins [3]. In polarographic pharmaceutical analysis proper, one uses this method for the determination of pharmaceuticals as pure substances, their determination in pharmaceutical formulations, determination of intermediates in the synthesis or in the production (e.g., determination of codeinone in dihydrocodeinone, the former being reduced at less negative potentials [189]), and detection of impurities and decomposition products, such as the determination of quinazoline carboxaldehyde, formed from formulations containing oxazepam after shelf storage [190]. On the other hand (very often in theoretical studies of the stability of drugs), one can carry out kinetic studies (mostly by measuring the current as a function of time at a constant applied potential), e.g., in following the hydrolysis of esters under different conditions or the
272
POLAROGRAPHY
oxidation of ascorbic acid. This possibility enables a study to be made of the stability of pharmaceuticals in formulations, and, in contrast to most spectral methods, one can usually easily decide whether an electroactive group disappears from the molecule or is formed. The above applications have been well-known among specialists for about 20 or 30 years. However, three other fields should be mentioned in this context. In the first place, the determination of drugs in biological materials such as blood, urine and saliva etc. becomes still more important. In such a determination, the procedure is made more difficult by two additional factors: the low concentration of the substance to be determined and the presence of surface-active substances which deform the shape of the d.c. polarographic curves. Both problems must be overcome because this type of determination is of the utmost importance for present pharmacology and toxicology. The toxicological analyses are usually easier to perform because the overdose causing the poisoning necessarily results in a higher concentration level in the body fluids. The problem of low concentration is often solved by working with a pulse or differential-pulse mode, the presence of surface-active substances can be shown by extracting the substances to be determined and, if possible, their metabolites with various solvents and separating them, mostly by thin-layer chromatography [ 1911. A more detailed discussion of these techniques will1 be given in the paragraph on polarography of psychotropic drugs since recent advances have been achieved especially in this field. A further important complex of techniques must be mentioned which was not covered in earlier reviews because it has been developed only in the course of the last 15 or 20 years. This is the automated polarographic analysis and in most cases continuous. It is usually based on measuring the limiting (diffusion-controlled) current with a polarised indicator electrode at a constant potential, corresponding to the region of the limiting current. The values obtained are used for controlling or monitoring chemical processes. Special flow-cells had to be developed. The fundamentals of this branch of practical polarography were laid by the Prague polarographic school [75] and can be followed in a considerable number of methods published or protected by patents. Moreover, the research in this field led to commercial production of automatic polarographic analysers used in industry. In pharmaceutical analysis the situation is somewhat different. The increasing demand for large numbers of analyses both in pharmaceutical control and in production, have led to the development of an
MIROSLAV BREZINA AND J I R ~VOLKE
273
automated polarographic method of analysis for tablet and capsule formulations. In contradistinction to the normal polarographic automated techniques using polarized electrodes in continuous-flow systems [75], complete polarograms are needed; this is due to the presence of decomposition products and components interfering in the constant potential techniques. The problem was solved by combining a polarographic analyser with a continuous-flow system [192]. The suitability of the new method was demonstrated by analysing tablets and capsules containing Lorazepam (7 - chloro - 5 - (0 - chlorophenyl) - 1,3 - dihydro - 3 - hydroxy 2H - 1,4 - benzodiazepin - 2 - one), one of the modern psychotropic drugs. In performing the analyses, standards and samples of intact and powdered tablets or capsules are placed on the sample plate. This series is followed by further standards on the same plate. The samples are introduced into the so-called SOLIDprep unit and dispersed in 0.1 M sodium acetate-acetic acid in methanol-water, serving as supporting electrolyte. After a complete dissolution of the drug the sample is aspirated into a flow system and automatically filtered. A decantation trap is placed between the unit ensuring the dissolution and the proportioning pump. The filtrate is then segmented with nitrogen and pumped into the so-called ‘steady-state extension unit’. By means of changing the flowrate and of valve control the duration of the sample’s steady state is prolonged. In the continuous deaerator, a stream of methanol-saturated nitrogen transforms the solution into a turbulent liquid film. The purging gas is thermostated in order to prevent evaporation. The sample is then transported to the polarographic flow cell. The polarographic analyser and recorder are synchronised to initiate a scan at the moment the sample attains steady state at the flow cell. The recording is usually made in the d.c. polarographic mode. The development of such an automatic analyser is connected with a difficult problem; the measurement can be only carried out while the sample concentration at the detector is kept constant; consequently steady state of relatively long duration must be achieved. In this particular case, the so-called steady-state extension unit had to be constructed in order to overcome the limitations in rate determination. The description of this unit has not been given in full. The last major development is not of a purely analytical nature: it is based on the idea of applying polarographic results for working out electrochemical routes of organic synthesis. The experience obtained from d.c. polarographic experiments can be utilized. By preparative electrolysis in extremely small volumes (about 1 ml) with a dropping mercury electrode in connection with the identification of products thus
POLAROGRAPHY
274
formed, one obtains sufficient information about the role of the potential of the working electrode in the particular electrode process. The next step is a transfer of the results with the dropping mercury electrode to a large-area mercury electrode. This is not always possible and the situation must be checked by means of experiments with a stationary hanging mercury drop electrode the conditions which correspond more closely to those at a mercury pool, even if the latter is stirred. A suitable reduction potential (mercury electrodes are not convenient in oxidations because of their dissolution at less negative positive potentials; consequently oxidations should be carried out with platinum or gold anodes) is then chosen according to the required product, particularly in multistep processes. A simple example is the electrolytical preparation of pyridoxamine [193] from pyridoxal: the first step is a chemical one (Scheme 1). The
6
HO Me
HO
CH=NOH \ CHPH
- )I
\ c w H
+NH20H -H20
Me
SCHEME 1
resulting aldoxime is reduced at a potential which has been found on a d.c. polarogram (Scheme 2). The reduction potential corresponds to the H+
+
4e
+
4H+
__ - HzO
Me
SCHEME 2
limiting current of the first wave of the aldoxime. If a more cathodic potential were chosen, e.g. that corresponding to the limiting current of the second wave, an undesirable product would result according to Scheme 3. HO M
e
1
H+
\
T
-
+
2~ + 2H'
:@ SCHEME 3
] H + . + NH,
MIROSLAV BREZINA AND JIRS VOLKE
275
In more recent applications, use is made of chemical reactions following an electrochemical initial step. A typical example of this procedure is the so-called electrocyclization [ 1941. Here an electroactive group is partly reduced at the electrode and in a follow-up reaction reacts with another (electroinactive) group in a sterically suitable position, e.g. ortha in a benzene nucleus, forming a new, closed ring; the resulting ring itself or substituents on it may be further reduced at more negative potentials; consequently, the composition of the reduction products is a function of the electrode potential as has been found by means of polarography and related methods (Scheme 4 ) . From the analogous 2-nitro-2’-thiocyanato-
SCHEME 4
substituted biphenyls seven-membered sulphur and nitrogen containing heterocyclic rings have been obtained [ 1951 which are similar to modern psychotropic drugs. Further synthetic work is in progress. The recent electrochemical literature reports anodic oxidations at platinum electrodes leading to a synthesis of some alkaloids of the morphine group [196], e.g. laudanosine. Originally, even the polarographic investigations of organic substances were carried out in aqueous solutions. Owing to the low solubility of many organic compounds in this medium, the possibility of analysing them was rather limited. This is why mixtures of water with non-aqueous solvents, such as methanol, ethanol or acetone were introduced. In addition to the increased solubility one could observe that in such mixtures (with e.g. 20 or 30% non-aqueous solvent) adsorption phenomena which complicated the shape of the current potential curves almost completely disappeared. Later, new non-aqueous solvents [208], such as dioxan, tetrahydrofuran, acetonitrile, dimethyl sulphoxide and, in particular, dimethylformamide were introduced which dissolve organic substances much more easily than alcohols; moreover, a completely new experimental technique developed, i.e. polarography in aprotic solvents with total exclusion of water ([H201 < M) which makes the interpre-
276
POLAROGRAPHY
tation of polarographic results much easier and simplifies them by the fact that no protonation reactions occur. This results also in finding that new, interesting products are obtained in electrosynthetic work which usually differ from those in water or in protic solvents. Free radicals are more stable than in water and undergo other types of follow-up reactions. The non-aqueous solution, usually with a tetraalkyl ammonium salt (e.g., 0.1 M NBu4C104)as supporting electrolyte, has a rather low conductivity. This led to the development of special three-electrode circuits [2], built into modern polarographs. Such an apparatus compensates for the IR-drop in the solution and applies the required potential to the indicator electrode. POLAROGRAPHIC ACTIVITY O F PHARMACEUTICALS
The determination or investigation of inorganic components is relatively seldom carried out in pharmaceutical chemistry. It occurs, for example, in the determination of heavy-metal impurities in chemicals or pharmaceuticals, originally by d.c. polarographic methods, but anodic stripping analysis as described at the beginning of this chapter would be more convenient, or the determination of zinc present in zinc-protamine-insulin as prescribed by the Czechoslovak pharmacopeia [ 1971. Much more important is the polarographic behaviour of organic substances. This is based on the presence of electroactive groups, reducible or oxidizable at the dropping mercury electrode. The electroactive groups with examples from pharmaceutical chemistry are shown in Table 5.1. It should be emphasized that the aldehydic and ketonic groups, Table 5.1. POLAROGRAPHICALLY ACTIVE GROUPS IN ORGANIC PHARMACEUTICAL CHEMISTRY Active group
Examples ~~
-CHO
-CHO in conjunction with a double bond )C=N-
~~~
~
~
Formaldehyde, benzaldehyde, vanillin, pyridoxal. Citral, citronellal, cinnamaldehyde, myrtenal, some steroids. Heterocyclic rings in alkaloids, benzodiazepines, local anaesthetics, riboflavine etc.
%-Br
Bromisoval.
$C-NO,
Chloroamphenicol.
MIROSLAV BREZINA AND J I R ~VOLKE
211
as well as >C=N- are most frequent among polarographically active pharmaceuticals. In addition to these, further electroactive groups can appear in \ pharmaceuticals, such as -NO, -C=C-, ,C=S, -N=N-, -0-0-, -S-S- etc. All these are reducible and the reduction mechanisms with the corresponding electron consumptions can be found in the pertinent literature [ 1981. Sulphydryl groups, cyclic derivatives without sulphur (barbituric acid derivatives, hydantoins) and with sulphur give anodic waves caused by reaction with mercury ions in the anodic dissolution of the d.m.e. In this group, too, a number of pharmaceutically important substances are to be found. As regards qualitative analysis, the above-mentioned active groups reduce in characteristic potential regions so that from the value of the half-wave potential if the molecule or the remaining part of the skeleton to which the group is attached (benzene, pyridine etc.) is known, one can conclude what the reducible grouping is. On the other hand, the half-wave potential of the same group is not a constant since it is affected by the whole molecule in which it is present. This phenomenon is shown e.g. with an aldehydic group attached to different heterocyclic nuclei. Of course, these phenomena are often utilized in studying the relationships between structure and electrochemical properties of drugs. The quantity of the substance is determined from the concentration in the solution, the linear function of which is the limiting current which should be, in this case, diffusion-controlled. It follows from this requirement that the electrode process corresponds either to reduction or oxidation of the substance. In connection with this, we would like to warn the reader against using the so-called catalytic reduction waves [199] which are caused by catalytic reduction of hydrogen ions (compare e.g. with p. 258) in presence of some nitrogen-containing compounds, such as alkaloids or pyridine derivatives (e.g. nicotinamide). Such waves were sometimes recommended [200] as an extremely sensitive tool (they may be several orders larger than normal reduction waves) for determining these substances, in particular alkaloids. However, they are not specific and, in particular, are extremely sensitive towards the presence of other substances in the solution. FUNCTIONALIZATION OF INACTIVE SUBSTANCES
Even substances which are neither reduced or oxidized at the dropping mercury electrode can be determined by means of polarography unless an
278
POLAROGRAPHY
amperometric (in Czech literature more often polarometric) titration is preferred. The polarometric titration of such a compound is carried out with the solution of an electroactive reagent reacting with the substance to be determined stoichiometrically and with sufficient speed. A potential corresponding to the limiting current of the reagent is applied to the indicator electrode. Only at the point of equivalence does a current increase begin. A typical example is the titration of phenolic substances such as morphine with diazotized sulphanilic acid [203]. However, in pharmaceutical polarography, the so-called functionalization is popular, i.e. the introduction of a polarographically active group, or in a more general way a chemical transformation into an electroactive derivative. Typical examples are shown in Table 5.2.
Table 5.2. FUNCTIONALIZATION IN ORGANIC POLAROGRAPHY
Group introduced Method
Substance to be determined
-NO2
Either by direct nitration or by reacting phenols with HNO,
Phenazone [201], morphine [202], cephaeline [203].
-NO
Nitrosation with HNO,
Secondary amines [204], local anaesthetics [204]
-Br
H,SO,
Introduction of oxygen into the molecule
+ KBr + KMn04
Citric acid [205]; CBr,COCHBr, is formed.
Reaction with ammonia, primary amines, hydrazine, hydroxylamine, sernicarbazide, Girard D reagent
Ketones [206], aldehydes [206] irreducible under given conditions; better developed waves with some reducible compounds; simultaneous determination of pyridoxal and pyridoxal-5-phosphate [ 1931, 17-ketosteroids [238].
Oxidation with bromine, ceriurn(IV), H20,, HNO,, NaN02.
Menadiol to the quinone [239], lidocaine to its N-oxide [207], chloropromazine to its sulphoxide [212].
The accuracy of these indirect methods is somewhat lower than in normal polarography, i.e. -c 3-5%.
MIROSLAV BREZINA AND J I R ~VOLKE
279
ANALYSIS OF PHARMACEUTICAL FORMULATIONS
Why are polarography or related polarographic methods so often used in practical pharmaceutical analysis’ The main reason is that the solutions to be analysed, (such as injections or tinctures) are mostly simple mixtures whose composition is at least approximately known. This is in contrast to the usual problems of quantitative organic analysis on the one hand, and to the analysis of biological systems on the other hand. For this reason, the analytical procedure is often simple, the solution being diluted with the solution of the supporting electrolyte and the polarographic curve being recorded after deaereating with a stream of nitrogen or argon. This prerequisite is important although it is often claimed [209] that in a.c. oscillopolarography, in current sampled polarography and in differential pulse polarography, it is not necessary to remove dissolved oxygen from the solution because of the slowness of its reduction. However, one must not forget that it can chemically react with the intermediates of the electrode process and thus adversely influence the shapes of the current-voltage curves. The same dilution technique is usually applied in the analysis of ethereal oils [210]. It can be used in the determination of aldehydes such as citral and citronella1 in lemon oil (Oleum citri) and in Oleum verbenae, of anisaldehyde in Oleum anisi stellati, Oleum anisi and Oleum foeniculi, of cinnamaldehyde in Oleum cinnamomi, and of ketones, such as carvone, in Oleum anethi, of pulegone and diosphenol. In all these cases, the oil is first diluted with ethanol and to this solution an aqueous solution of the supporting electrolyte is added, usually only 0.1 M LiCl. The preparation of the polarographic sample becomes more complicated in the analysis of drugs [211], e.g., of seeds or barks. A typical procedure can be exemplified with the determination of cinnamaldehyde in cinnamon bark. A quantity of about 2-5 g of this bark is pulverised and extracted with chloroform for 10 hr in a Soxhlet apparatus. The solvent is then distilled off and the residue dissolved in ethanol. An aliquot of this is mixed with an aqueous solution of the supporting electrolyte and polarographed. A similar procedure proved successful in the determination of carvone in Semen carvi; it differs only in the application of ethanol for the extraction. This extraction in the determination of cuminaldehyde in the Roman caraway seeds was a failure because electroactive impurities were extracted simultaneously. For this reason, it was necessary to make use of steam distillation.
280
POLAROGRAPHY
Probably the most useful application is the analysis of tablets: first they must be pulverised and then digested either with hot water in the case of water-soluble substances (e.g., dihydrocodeinone hydrochloride) or with some other, usually non-aqueous solvent, the resulting suspension is mixed with the base solution. In purely aqueous solutions, the analysis should be carried out with larger volumes (higher dilution) whereas with non-aqueous solvents or their mixtures with water, practically no readsorption of the substance to be determined onto the particles of the insoluble components of the tablet occurs. With regard to the possibility of readsorption, the determination is made by standard addition (internal standard) or with the help of a calibration curve obtained in the presence of the insoluble particles. An important advantage of the method is that usually even a single tablet can be analysed [213]. It is an interesting finding that the indirect method of functionalization of an inactive substance for polarographic analysis can also be used with tablets. Thus chloropromazine [212] (Scheme 5) coated tablets are pulverised and mixed with sodium nitrite; after adding water, chloropromazine is oxidized with 65% nitric acid; the oxidizing agent proper is nitrous acid.
After 15 min of oxidation, dimethylformamide is added and insoluble particles are filtered off at decreased pressure. An aliquot of the solution is mixed with a Britton-Robinson buffer pH 1.8. The final solution gives a two-electron cathodic wave of the sulphoxide. The precision amounts to 2 1.42% as compared with -+ 1.48% in ultraviolet spectroscopy. Surprisingly enough in the analysis of tablets by direct polarography, an accuracy of about ? 2-3% has been obtained on average [213,226,227]. The procedures for the polarographic determination of tablets, coated tablets and capsules are practically the same. The polarographic (and not only the polarographic) analysis of ointments is one of the most difficult tasks. The active component which is to be determined must be first extracted, usually with non-aqueous solvents, often at a raised temperature. Some authors recommend glacial acetic acid for this purpose, but satisfactory results are obtained by extracting a benzene solution of the ointment with 2 M hydrochloric acid [214].
MIROSLAV BREZINA AND J l R i VOLKE
28 1
If still more complicated systems have to be analysed which contain interfering substances or mixtures of compounds, e.g., isomers, separation processes must be performed before the polarographic analysis proper. In most cases, chromatographic methods are applied: adsorption chromatography (on aluminium oxide in the case of ipecacuanha alkaloids [203]), paper chromatography (morphine in blood [216]), ion exchangers and the so-called chromatopolarography [217,218]. In this method, the polarographic limiting current is measured at constant potential in a solution leaving the chromatographic column. In this way, a number of peaks result as a function of time. The areas under these peaks correspond to the amount of the active component in the solution. The authors of this procedure succeeded in separating closely related compounds, such as isomers with identical half-wave potentials (e.g., nitrophenols) but from the pharmaceutical point of view more interesting is the separation of strychnine and brucine [219]. However, the polarographic determination is based on their catalytic hydrogen waves and is not specific. More promising is a thin-layer separation of isomers, for example, followed by extraction from the thin-layer and polarographic determination after mixing the extract with the solution of the supporting electrolyte. The presence of the adsorbent during electrolysis does not interfere. The extraction from the adsorbent is best carried out with dimethylformamide. The first published combination of TLC with d.c. polarography [191] is based on the separation of a mixture of 2- and 3nitro-4-acetaminophenetole which results after nitration in the indrect determination of phenacetin after nitration. Later, the method was used with other adsorbents (i.e., silica gel and magnesium silicate) and proved successful in the separation and determination of small quantities (10-100 p g ) of psychotropic substances (see also below). In all the above-mentioned cases, the active component itself had to be determined. However, polarography often serves to determine toxic decomposition products or reaction intermediates in pharmaceutical synthesis. Besides the presence of codeinone in dihydrocodeinone [ 1891, one can detect the presence and amount of toxic ketones in chloramphenicol [221] or of papaveraldine in papaverine [222]. Although polarography was introduced into pharmacopoeias in Czechoslovakia as early as in 1954, in the later edition from 1970 the number of articles prescribing a polarographic determination has been reduced (niacinamide, ascorbic acid, aureomycin and insulin). In the present edition, the method is only used for the determination of zinc in zinc-protamin insulin. More articles with a polarographic determination
282
POLAROGRAPHY
are prescribed by USP XV, and, by the latest edition of the pharmacopoeia of the German Democratic Republic. The U.S. Pharmacopoeia uses this method for the determination of some sulphonamides. The reason why polarography is not frequently used in pharmacopoeias is based on the necessity of working with standard samples (i.e., in practical analysis it is never used as an absolute method); consequently, it is more suitable for the determination of low percentages of a substance in a solution or a mixture rather than for the quantitative assay of pure substances. FIELDS WITH INTENSE DEVELOPMENT
The present situation in the application of polarographic techniques in pharmaceutical analysis and in pharmacology can be best exemplified with the important and rapidly expanding group of psychotropic drugs. In particular, this holds true with all benzodiazepine derivatives [223-2371. All those which are used in medicine as tranquillizers have been investigated and found to be polarographically active. In this article, only the properties of the five most important benzodiazepines will be dealt with in some detail although the recently introduced substances of this class have been also studied polarographically and analytical methods have been suggested for them by the Frankfurt school [223-2281. At present classical benzodiazepines are chlorodiazepoxide (1, Librium), diazepam (2, Valium), oxazepam (3, Serenid D), nitrazepam (4, Mogadon) and dipotassiumchlorazepate (5, Tranxillum).
MIROSLAV BREZINA AND JIRi VOLKE
283
The active grouping which all of them have in common is the conjugated double bond C=N in the seven-membered ring; this double bond is reduced in a two-electron process [224]. In compound (1) the polarographic reduction starts with the most positive wave corresponding to the two-electron reduction of the N + O group and ends with the reduction of the -N=C-NHMe grouping [2231. In oxazepam (3), protonation of the hydroxyl group results in its electrochemical splitting off in acid media; in such a solution a 4-electron wave is obtained which corresponds to the electrode process associated with a -C=N- and that of C-OH in a single wave [226]. In compound (4), the aromatic nitro group is reduced [225] with the uptake of 4 + 2 or 4 electrons, depending on pH; this occurs in addition to the reduction of the -C=N- group. The polarographic determination of the salt of (5) is in essence based on the polarographic reduction of an artifact, i.e., it can be looked upon as an indirect method [228]. This reaction proceeds very rapidly in solution and it is (7) (Scheme 6), the so-called desmethyldiazepam which is then determined. All these compounds are easily determined by d.c. polarography because the waves are diffusion-controlled and a linear function of
M in buffered solutions containing concentration between 5 * lO-’-2 . 5-20% dimethylformamide (pH values mostly about 4-5; for details see the individual papers). Consequently, about 15 pg of the substance can be determined in 1 ml. The values differ somewhat owing to different wave-heights (different electron consumptions) of the substances. Several different techniques are recommended for assaying the benzodiazepines in single tablets, but according to the authors’ experience, the simplest and most reliable method, [225,226] is when powdered tablets are extracted with dimethylformamide by shaking. Without filtering, this extract is mixed with the aqueous supporting electrolyte and polarographed. On the other hand [1901, if extraction of (3) is carried out with methanol, 30 min of shaking are necessary, followed by centrifuging. Only the clear supernatant is mixed with the solution of the supporting electrolyte. Experiments by the same authors showed that a rapid extraction with dimethylsulphoxide was not sufficient.
284
POLAROGRAPHY
The polarographic investigation of the decomposition products of some benzodiazepines is also possible: in acid solutions, the benzodiazepine ring is opened with formation of the corresponding substituted benzophenones, the polarographic behaviour of which is characteristic [224] (Scheme 7). The decomposition product of oxazepam (3) on
Ph SCHEME 7
shelf storage [ 1901 is quinazoline carboxaldehyde which is reduced at about 200 mV less negative potentials. A stronger degree of decornposition can be followed by d.c. polarography; if only 1% of the decornposition product is present, differential pulse polarography must be applied. In a considerable number of publications, attempts to determine the tranquillizers of the benzodiazepine group in body fluids have been described. By giving the individual procedure, we also intend to show the way in which generally the polarographic determinations in body fluids are carried out. The simplest method of determining chlorodiazepoxide is based on mixing the human serum with 0.1 M sulphuric acid in a ratio 1 : 1. In the authors’ opinion [230], the drug is more strongly adsorbed on the electrode surface than the serum proteins and this makes the direct analysis possible. In a similar manner, nitrazepam (4) and diazepam (2) are determined in blood serum [230,2311 or in cadaver blood [237] without prior solvent extraction. The sensitivity of these methods lies in the vicinity of l-lOpg/ml. A higher sensitivity can be reached if the substance is extracted, e.g., in case of (2) [2331 with 30 ml of benzene from 5 ml of serum. In such a determination, 0.05-1.0 pgg/ml can be detected. Extraction into light petroleum from human plasma [238] made alkaline enables a cathode-ray determination of therapeutic doses (2) on the 0.02 pglml level. Another group of authors [239] have suggested a cathode-ray or pulse polarographic determination of nitrazepam (4) in the presence of its metabolites containing an NHz or NHCOMe group instead of the original nitro group. However, the most detailed and evidently most reliable method has been described for chlorodiazepoxide (1) in human plasma [236]. In essence (1) or its metabolites are extracted into diethyl ether from plasma buffered to pH 9.0. This operation is followed by a TLC separation on silica gel since the peak potentials E , lie close to each other. The substances are eluted from the thin layer with methanol, and the
MIROSLAV BREZINA AND J I R ~VOLKE
285
residue after distillation is dissolved in 0.1 N sulphuric acid. The compounds shown in Scheme 8 are determined here.
(')
- c,a=N? --C/&)
Ph
0
Ph
'0
SCHEME 8
Since the overall recovery is about 62%, a method utilizing internal standard has been used; 0.05 pglml or 0.10 pgglml can be determined following either a single 30mg dose by intravenous or oral routes, or following chronic administration. An increased output signal has been gained by the current sampling method in differential pulse polarography. The method is thus about 20 times more sensitive than normal d.c. polarography. The thin-layer chromatographic separation of the individual components of a mixture, followed by a polarographic determination in the benzodiazepine series, had been first introduced [219] for the analysis of a mixture of nitrazepam (4) and its metabolites containing NH2 and NHCOMe groups. In this chapter, the application of polarography in the determination of benzodiazepines is emphasized in which this method has played an outstanding role but it can be also useful with some other psychotherapeutics such as fluorine-substituted butyrophenones [240] or those with seven-membered, possibly heterocyclic rings [241] such as in imipramine. In the latter case, anodic oxidation waves are obtained with rotated noble metal electrodes. The analytical applications of polarography in the benzodiazepine series are based on thorough theoretical foundations [223-2281. The mechanisms of the electrode processes are known, the electron consumptions have been determined coulometrically, and the products of the electrode reaction have been isolated in most cases. This not only allows a reliable choice of the analytical conditions but it also enables us to draw important conclusions as regards preparative electrochemistry. Besides the group of psychotropic compounds, we must quote here the antibiotics as drugs where a rapid recent development of techniques can be observed. D.c. polarographic techniques for the determination of chloramphenicol [242], tetracycline [2431 and streptomycin [244] are available. The polarographic determination of penicillin G potassium salt is only indirect and is preceded by the introduction of a nitroso group [245]. The synthesis of chloramphenicol comprises the chemical reduc-
286
POLAROGRAPHY
tion followed by hydrolysis of p - nitro - 2 - acetamido - 3 - hydroxypropiophenone to 1 - p - nitrophenyl - 2 - amino - 1,3 - propanediol. A polarographic control of this process is possible 12461 because their simultaneous determination is possible in mixtures. The difference in half-wave potentials AEl,z of both nitro compounds in mixtures is much greater than that obtained by subtracting the half-wave potentials in pure solutions. There is a mutual influence of both compounds in the reduction; evidently, the adsorption of reactants plays a role. With the differential pulse polarography [245], the antibiotics can be determined at low concentration, if necessary, at the ppm or even sub-ppm level. Tetracycline hydrochloride is determined in aqueous acetate buffer pH 4 (detection limit 0.1 ppm), but for the analysis of chlortetracycline hydrochloride, oxytetracycline hydrochloride and free tetracycline, a non-aqueous medium must be used. Streptomycin sulphate is analysed in alkaline solution, trace quantities of zinc being masked by Na2EDTA, and the detection limit is 1 ppm. A determination in blood serum or urine is also possible but the peak potentials are shifted here to more negative values. The polarographic determination is preceded by ultrafiltration. Penicillin G potassium and ampicillin must be first functionalised by nitrosation. The authors also recommend an analysis of mixtures which is however demonstrated only with chloramphenicol and tetracycline, at 2.4 and 4.2 ppm, respectively. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11
J. Heyrovskf and J. Kdta, Principles of Polarography (Publishing House of Czechoslovak Academy of Sciences, Prague, 1965) p. 581. L. Meites, Polarographic Techniques (Interscience, New York) 2nd edn. (1965) p. 752. M. Biezina and P. Zuman, Polarography in Medicine, Biochemistry and Pharmacy (Interscience, New York, 1958) p. 862. M. Biezina, Progr. Polarogr. 2 (1962) 667. P . Zuman and M. Biezina, Progr. Polarogr. 2 (1962) 687. J. Volke, Physical Methods in Heterocyclic Chemistry, ed. A. R. Katritzky (Academic Press, New York) Vol. I , (1963) pp. 217-323. J. Volke, Talanta, 12 (1965) 1081. R. N. Adarns, Electrochemistry at Solids Electrodes (Marcel Dekker, New York, 1969) p. 402, Prospectus, Tokai Electrode Mfg. Co, Ltd., Tokyo, Japan. F. Vydra, K. Stulik and E. Juliikovii, Electrochemical Stripping Analysis (Stsitnf nakladatelstvi technick6 literatury, Prague), in the press. J. W. Rose, R. D. de Mars and I. Shain, Anal. Chem., 28 (1956) 1768.
MIROSLAV BREZINA AND J I R ~VOLKE
12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
287
J. G. Nickelby and W. D. Cooke, Anal. Chem., 29 (1957) 933. R. D. de Mars and I. Shain, Anal. Chern., (29 (1957) 1825. B. Breyer and H. H. Bauer, Alternating Current Polarography and Tensammetry (Interscience, New York, 1963) p. 288. G. C. Barker, Square-Wave and Pulse Polarography, in: Progr. Polarogr. 2 (1962) 411. I. M. Kolthoff and J. J. Lingane, Polarography (Interscience, New York) 2nd edn. (1952) p. 990. K. Abresch, Angew. Chem., 47 (1935) 683. M. Biezina, A. Hofmanovfi-Mattjkovi and J. Koryta, Biophys. Chern., in the press. K. Angelis, M. Bfezina and J. Koryta, J. Electroanal. Chem., 45 (1973) 504. T. Ryan, J. Koryta and A. Hofmanovi-Mat6jkovi and M. Biezina, Anal. Lett., 7 (1974) 335. J. Koryta and M. L. Mittal, J. Electroanal. Chern., 36 (1972) App. 14. F. Linhart, cas. LCk. Ces., 92 (1953) 1298. F. Linhart, Collect. Czech. Chem. Comrnun., 18 (1953) 302. Prospectus Esa, Inc., 175, Bedford Street, Burlington, Mass., USA. Trace Metal Analysis of Blood, Esa, Inc., 175, Bedford Street, Burlington, Mass., USA. Trace Metal Analysis of Tissue, Esa, Inc., 175, Bedford Street, Burlington, Mass., USA. The Model 2014P Reagent Cleaning System, Esa, Inc., 175, Bedford Street, Burlington, Mass., USA. M. Bfezina, Ber. Bunsengesellsch., 77 (1973) 849. K. Wiesinger, Die Polarographische Messung der Sauerstoff spannung des Blutes (Schwabe and Co., Basel, 1950) p. 80. S. M. Berggren, Acta Physiol. Scand., 4 (1942) Suppl. 11, p. 92. K. Beecher, R. Follanshee, A. J. Murphy and F. N. Craig, J. Biol. Chem., 146 (1942) 197. Ref. 3, pp. 138-147. J. P. Baumberger, Amer. J. Physiol., 129 (1940) 308. L. Selzer and J. P. Baurnberger, J. Cellular Cornp. Physiol., 19 (1942) 281. D. H . Miiller, The Polarographic Method of Analysis (Publ. J. Chem. Education, 1951) p. 209. V. Vitek, Collect. Czech. Chem. Comrnun., 7 (1933) 537. B. Mosinger, Pracovni Ltkaistvi, 6 (1954) 287. B. Mosinger, Physiol. Bohemoslov., 6 (1957) 126. L. Serik, Advances in Polarography, Proc. 2nd Intern. Congr. Cambridge 1959 (Pergamon Press, London, 1960) p. 1057. L. Serfik, Abhandl. der Deutsch. Akad. d. Wissensch. zu Berlin, Klasse fur Chern., Geol., Biol., (1964) p. 305. A. KrCilek, L. Serak and V. JanouSek, Giorn. dell Arteriosclerosi, 4 (1966) 127. 0. ZajiCek and M. Kindlova, J. Periodont. Res., 7 (1972) 242. 0. Zajitek, M. Skach and M. Kindlovi, c s . Stomatol., 74 (1974) No. 5 , in the press. G. Lejhanec, L. SerBk and P. HybfiSek, Acta Univ. Palackianae Olomucensis, 30 (1962) 67. G. Lejhanec, P. HybfiGek and L. Serik, Ann. Ital. Dermatol. Clin. Sperim., 19 (1965) 398.
288 46. 47. 48. 49. SO.
51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87.
POLAROGRAPHY
p. HybBSek, G. Lejhanec and L. SerBk, Acta Fac. Med. Univ. Brunensis, 41 (1971) 259. p. W. Davies and F. Brink Jr., Rev. Sci. Instr., 13 (1942) 524. L. C. Clark, R. Wolf, D. Granger and Z. Taylor, J. Appl. Physiol., 6 (1953) 189. L. C. Clark, Trans. Amer. SOC.Art. Int. Org., 2 (1956) 41. F. Kreuzer and C. G. Nessler, Science, 128 (1958) 1005. F. Kreuzer, Bull. SOC. Fribourg. Sci. Naturell., 48 (1958) 231. R. Schuler and F. Kreuzer, Respir. Physiol., 3 (1967) 90. R. Schuler and F. Kreuzer, Progress in: Respiration Research, Vol. 3, Oxygen Pressure Recording in Gases, Fluids and Tissues (S. Karger, Basel, 1969) pp. 64-78. H. P. Kimmich and F. Kreuzer, Ref. 53, p. 100. Prospectus, Beckman, Fulleton, California, USA. Prospectus, Radiometer, Copenhagen, Denmark. H. H. Beneken and F. Kreuzer, Ref. 53, p. 13. I. A. Silver, Ref. 53, p. 124. D. W. Lubbers, H. Baumgartl, H. Fabel, A. Huch, M. Kessler, K. Kunze, H. Riemann, D. Seiler and S. Schuchhardt, Ref. 53, p. 136. K. Kessler and W. Grunewald, Ref. 53, p. 147. K. Kunze, Ref. 53, p. 153. W. J. Whalen, Ref. 53, p. 158. N. T. S. Evans and P. F. D. Naylor, Ref. 53, p. 161. H. Yokota and F. Kreuzer, Respiration Physiol., 15 (1972) 350. H. Yokota and F. Kreuzer, Pflugers Arch., 340 (1973) 307. H. Yokota, L. J. C. Hoofd and F. Kreuzer, Pfliigers Arch., 340 (1973) 273. H. Yokota and F. Kreuzer, Pfliigers Arch., 340 (1973) 291. H. E. Jacob, Z. Allg. Mikrobiol., 11 (1971) 691. H. E. Hacob, Path. Microbiol., 36 (1970) 57. H. E. Jacob, Abhandl. der Deutsch. Akad. der Wissensch., Berlin, KI. Medizin, (1966) 87, 93. R. May and H. E. Jacob, Z. Allg. Mikrobiol., 10 (1970) 275. P. G. Munder and M. Modolell, Z. Anal. Chem., 212 (1965) 177. J. V. A. Novik, Collect. Czech. Chem. Commun., 30 (1965) 2703. J. V. A. NovBk, Collect. Czech. Chem. Commun., 20 (1955) 1076. J. V. A. Novbk, Continuous Polarographic Analyzers, in: Progr. Polarogr. 2 (1962) 569. P. Zuman, Organic Polarographic Analysis (Pergamon Press, Oxford, 1964) p. 313. J. C. Heath, Nature, 158 (1946) 23. P. J. Elving, Ann. N.Y. Acad. Sci., 158 (1969) 124. P. J. Elving and J. W. Webb, in: The Purines-Theory and Experiment (The Israel Academy of Sci. and Human., Jerusalem, 1972) pp. 371-391. B. Jan& and P. J. Elving, Chem. Rev., 68 (1968) 295. E. Paletek, Progr. Nucleic Acid Res. Mol. Biol. 9 (1969) 31. E. Paletek, Methods Enzymol. 21 (1971) 3. G. Dryhurst and P. J. Elving, Talanta, 16 (1969) 855. S. M. Cantor and Q. P. Peniston, J. Amer. Chem. SOC.,62 (1940) 2113. K. Wiesner, Collect. Czech. Chem. Commun., 12 (1947) 64. D. L. Smith and P. J. Elving, J. Amer. Chem. SOC.,84 (1962) 1412, 2741. V. Vetterl, Collect. Czech. Chem. Commun., 31 (1966) 2105.
MIROSLAV BREZINA AND J I R ~VOLKE 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132.
289
V. Veterl, J. Electroanal. Chem., 19 (1968) 169. B. Filipowicz and W. Leyko, Bull. SOC.Sci. Lettres Lodz, C1. 111, 4 (1953) 1. W. Leyko and H. Panusz, Bull. SOC.Sci. Lettres Lodz, C1. 111, 5 (1954) 1. S. Eguchi, Nichidai Igaku Zasshi, 20 (1961) 2368, Chem. Abstr., 61 (1964) 3496a. S. Fiala and H. E. Kasinski, J. Nat. Cancer Inst., 26 (1961) 1059. V. Bulant, M. Urks and H. PaiizkovB, Antibiotiki, 9 (1964) 545. E. PaleCek and B. Janil, Arch. Biochem. Biophys., 98 (1962) 527. B. Jan& and E. PaleCek, Arch. Biochem. Biophys., 105 (1964) 225. G . Dryhurst and G. F. Pace, J. Electrochem. SOC.,117 (1970) 1259. G. Dryhurst, Anal. Chim. Acta, 57 (1971) 137. 0. ManouSek and P. Zuman, Collect. Czech. Chem. Commun., 20 (1950) 1340. D. Hamer, D. M. Waldron and D. L. Woodhouse, Arch. Biochem. Biophys., 47 (1953) 272. P. J. Elving, S. J. Pace and J. E. O’Reilly, J. Amer. Chem. SOC.,95 (1973) 647. G . Dryhurst, in: Bioelectrochemistry and Bioenergetics, Vol. 1, Papers presented at Mousson i 1973) pp. 271-288. the 2nd Int. Symp. in Bioelectrochem., (Pont ? V. Brabec, J. Electroanal. Chem., 50 (1974) 235. E. PaceCek, J. Mol. Biol., 20 (1966) 263. E. PaleCek and V. Vetterl, Biopolyrners, 6 (1968) 917. E. PaleEek and B. D. Frazy, Arch. Biochem. Biophys., 115 (1966) 431. E. Paletek, Arch. Biochem. Biophys., 125 (1968) 142. E. PaleEek, J. Electroanal. Chem., 22 (1969) 347. E. PaleEek, Collect. Czech. Chem. Commun., 37 (1972) 3198. E. PaleCek and J. DoskoCil, Anal. Biochem., 60 (1974) 518. E. Palefek and V. Brabec, Biochem. Biophys. Acta, 262 (1972) 125. V. Brabec and E. PaleEek, Biophysik, 6 (1970) 290. V. Brabec and E. PaleCek, Biopolymers, 11 (1972) 2577. E. PaleCek, V. Vetterl and J. Sponar, Nucl. Acids Res., 1 (1974) 427. A. BezdEkovB and E. PaleCek, Studia Biophys., 34 (1972) 141. E. PaleCek and I. FriC, Biochem. Biophys. Res. Commun., 47 (1972) 1262. E. LukBSovi and E. PaleEek, Radiat. Res., 47 (1971) 51. H. Berg and H. Bar, Studia Biophys., 3 (1967) 133. P. Zurnan, Collect. Czech. Chem. Commun., 16 (1955) 876. S. J. Leach, Aust. J. Chern., 13 (1960) 520, 547. C. D. Proctor, Illinois Acad. Sci. Trans., 49 (1956) 80. R. Benesch and R. E. Benesch, Arch. Biochem., 19 (1948) 35. R. Benesch, H. A. Lardy and R. E. Benesch, J. Biol. Chem., 216 (1955) 663. S. Rosenberg, J. C. Perrone and P. L. Kirk, Anal. Chem., 22 (1950) 1186. E. B. Schoenbach, E. B. Armistead and N. Weissman, Proc. SOC.Exp. Biol. Med., 73 (1950) 44. S. K. Bhattacharya, Nature, 183 (1959) 1327. H. B. Collier and S. C. McRae, Can. J. Biochem. Physiol., 33 (1955) 404. R. BrdiCka, M. Biezina and V. Kalous, Talanta, 12 (1965) 1149. R. Brditka, Collect. Czech. Chem. Commun., 5 (1933) 112. R. BrdiCka, Collect. Czech. Chem. Commun., 5 (1933) 148. R. BrdiCka, Research, 1 (1947) 25. R. Brditka, Z. Phys. Chem., (Leipzig), Sonderheft (1958) 165. B. A. Kuznetsov, Experientia Suppl., 18 (1972) 381.
290 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168.
POLAROGRAPHY 0. H. Miiller, Methods of Biochemical Analysis, ed. D. GIick (Interscience, New York) Vol. 11 (1963) p. 329. S. G . Mairanovskii, Catalytic and Cinetic Waves in Polarography (Pergamon Press, New York, 1968). W. Lamprecht, S. Gudbjarnason and H. Katzlmeier, Z. Anal. Chem., 181 (1961) 201, 216. P. Mader, Collect. Czech. Chem. Commun., 36 (1971) 1035. A. Calusaru, J. Electroanal. Chem., 15 (1967) 269. M. Ito, Mie Med. J., 13 (1964) 149, 14 (1964) 95, 16 (1966) 105, 16 (1967) 173. I. M. Kolthoff and P. Mader, Anal. Chem., 41 (1969) 924, 42 (1970) 1762. I. D. Ivanov and E. E. Rachleeva, Polarography of Structure and Function of Biopolymers (In Russian) (Izdat. Nauka, Moskva, 1968) p. 344. V. Kalous, Experientia Suppl., 18 (1971) 349. G. Rutkay-Nedeckq, Biochim. Biophys. Acta, 26 (1957) 455. G. Rutkay-NedeckL, Collect. Czech. Chem. Commun., 25 (1960) 3363,27 (1962) 2744, 28 (1963) 585, 29 (1964) 1809. G. Rutkay-Nedeckq and B. Bezuch, J. Mol. Biol., 55 (1971) 101. G. Rutkay-Nedeckq and B. Bezuch, Experientia Suppl., 18 (1971) 553. S. JanouSek, K. Herfort and J. Skachova, c s . Gastroenterol., 9 (1955) 113, 11,(1957) 167. J. Homolka, Polarography of Proteins and its Chemical Applications (In Czech) (Statni ZdravotnickC Nakladatelstvf, Praha, 1964) p. 143. J. Homolka and F. Hanzal, Cas. U k . ces., 101 (1962) 340. R. BrdiEka, Acta Intern. Ver. Krebshekampf., 3 (1938) 13. M. Biichner, Moderne Chemische Methoden in der Klinik (G. Thieme, Leipzig) 2nd edn. (1961) p. 568. V. Kalous, Z . Phys. Chem., (Leipzig), Sonderheft (1958) 187. A. Kotent, Neoplasmn, (Bratislava), 5 (1958) 396. A. Kotent, Z. Brada and I. BoSkovB, Clin. Chim. Acta, 2 (1957) 508. J. Berman, Cas. LCk. Ces., 87 (1948) 226. I. D. Mansurova, Exchange Processes at Diffusive Diseases of Liver (In Russian) (Izd. Donis, Dushambe, 1967) p. 288. M. Biezina, Rev. Polarogr. (Japan), 11 (1963) 26. M. VorliCkovii and E. Paletek, Biochim. Biophys. Acta, 331 (1973) 276. J. PradBt and J. Koryta, J. Electroanal. Chem., 17 (1968) 167, 177. J. Koryta and J. PradiE, J. Electroanal. Chem., 17 (1968) 185. N. Ossendorfovh, J. P r a d i t and J. Koryta, J. Electroanal. Chem., 18 (1970) 311. J. Prad6t, J. Pradatovi and J. Koryta, Biochim. Biophys. Acta, 237 (1971) 450. N. Ossendorfova, J. Pradit, J. PradBtova and J. Koryta, Biochim. Biophys. Acta, in the press. J. Pradht, J. Pradatovi, J. Koryta and J . Vrubel, Folia Biol. (Prague) 17 (1971) 322. J. P r a d i t , N. Ossendorfovi and J. Koryta, Croat. Chem. Acta, 45 (1973) 87. J. Koryta, J. P r a d i t , J. PradhCovh and N. Ossendorfovh, Experientia Suppl., 18 (1971) 367. J. PradiE and J . Koryta, Ber. Bunsenges., 77 (1973) 808. R. BrdiEka and K. Wiesner, Collect. Czech. Chem. Commun., 12 (1947) 39. J. KouteckL, R. Brditka and V. HanuS, Collect Czech. Chem. Commun., 18 (1953) 61 I .
MIROSLAV BREZINA AND J I R ~VOLKE
169. 170. 171. 172. 173. 174. 175. 176. 177.
29 1
V. HanuS, Thesis, Czechoslov. Acad. Of Sciences, Prague (1955). G. L. Brown, Arch. Biochem. Biophys., 49 (1954) 303. B. Chance, Arch. Biochem., 24 (1949) 389, 410. B. Swedin, Acta Chem. Scand., 1 (1947) 500. B. S. Walker, J. Amer. Chem. SOC.,63 (1941) 2015. M. Janchen and F. Scheller, Studia Biophys., 39 (1973) 1. F. Scheller, H. Jehring and N. Retter, Studia Biophys., 39 (1973) 47. F. Scheller and H. Jehring, Studia Biophys., 39 (1973) 21 1. F. Scheller, M. Janchen, G. Etzold and H. Will, Bioelectrochem. and Bioenerget., Vol. 1, Papers Present at the 2nd Int. Symp. in Bioelectrochemistry, (Pont B Mousson) (1973) pp. 23-31. 178. M. Bartfk and E. Michnovi, Veterin. Med. (CSSR), 11 (1966) 645. 179. M. Bartfk, M. Teleha, V. Cunderlikovi and K. Zwick, Endocrinol. Exp., 5 (1971) 143. 180. M. Chavko, M. Bart11 and E. Kasafirek, Collect. Czech. Chem. Commun., 37 (1972) 3956. 181. E. Knobloch, Collect. Czech. Chern. Commun., 12 (1947) 407. 182. J. Doskotil, Collect. Czech. Chem. Commun., 15 (1950) 614, 718. 183. V. FiSerova-Bergerovi, Collect. Czech. Chem. Commun., 28 (1963) 331 1. 184. V. Luzatti, F. Masson and L. S. Lerman, J. Mol. Biol., 3 (1961) 634. 185. E. Calendi, A. di Marco, M. Reggiani, B. Scarpinato and L. Valentini, Biochim. Biophys. Acta, 103 (1965) 25. 186. H. Berg and F. A. Gollmick, Abhandl. Deutsch. Akad. Wiss. Berlin, (1966) 533. 187. J. Volke, Leybold Polarogr. Ber. 2 (1954) 175. 188. M. Biezina and J. Volke, Polarographische Methoden in der Medizin und Pharmazie, Beckman Report (1968) No. 1: 2, pp. 11-18. 188a. M. M. Baizer, ed. Organic Electrochemistry (M. Dekker, New York) (1972). 189. Ref. 3, p. 366. 190. J. A. Goldsmith, H. A. Jenkins, J. Grant and W. Franklin Smyth, Anal. Chim. Acta 66 (1973) 427. 191. H. Oelschlager, J. Volke and G. T. Lim, Arch. Pharm. (Weinheim), 298 (1965) 213. 192. L. F. Cullen, M. P. Brindle and G. J. Papariello, J. Pharm. Sc. 62 (1973) 1708. 193. 0. ManouSek, Thesis, 1961, Czech. Acad. of Science, Prague (1961). 194. J. Hlavatg, J. Volke and 0. ManouSek, Collect. Czech. Chem. Commun., in the press (1975). 195. J. Hlavatg and J. Volke, unpublished results. 196. L. Miller, Lecture at Eurochem. Conference on Organic Electrochemistry, Schloss Elnau, April 1974. 197. Czechoslovak Pharmacopoeia, 3rd edn., Vol. 2 (Aventinum, Prague, 1970). 198. C. L. Perrin, Mechanisms of Organic Polarography in Progress in Physical Organic Chem. (S. G. Cohen et al. ed) (Interscience, New York) Vol. 3. (1965) p. 166. 199. E. Knobloch, Collect. Czech. Chem. Commun. 12 (1947) 581. 200. H. F. W. Kirkpatrick, Quart. J. Pharm. Pharmacol. 18 (1945) 338; 19 (1946) 8, 127, 526; 20 (1947) 87. 201. D. M. Hamel and H. Oelschlager, J. Electroanal. Chem. 28 (1970) 197. 202. H. Baggesgaard-Rasmussen, C. Hahn and K. Ilver, Dansk Tidsskr. Farm. 19 (1945) 41. 203. A. Jindra, V. Jungr and J. Zqka, Ceskoslov. Farm. 1 (1952) 177, 185. 204. B. Novotng, Cesk. Farm. 3 (1954) 12.
292 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246.
POLAROGRAPHY P. J. Elving, R. E. Van Atta, Anal. Chem. 26 (1954) 295. P. Zuman, Nature 165 (1950) 485. H. Oelschlager and H. Hoffmann, Arch. Pharm. (Weinheim) 299 (1966) 1025. C. K. Mann and K. Barnes, Electrochemistry in Nonaqueous Solvents (M. Dekker, New York, 1971). A. M. Bond, Talanta 20 (1973) 1139. Ref. 3, pp. 242, 252. Ref. 3, p. 245. H. Oelschlager and K. Binge, Arch. Pharm. (Weinheim) 307 (1974) 410. D. M. Hamel and H. Oelschlager, Arch. Pharm. (Weinheim) 304 (1971) 148. V. Volkovi, Acta Chim. Acad. Sci. Hung. 9 (1956) 247. J. Fidelus, M. Zietek, A. Mikolajek and 2. Grochowska, Mikrochim. Acta (1972) 84. J. Erben, unpublished results. W. Kemula, Progr. Polarogr. 2 (1962) 397, 409. W. Kemula and Z. Stachurski, Rocz. Chem. 30 (1956) 1285. J. Volke and H. Oelschlager, Proc. 25th Congr. Pharmacol. Prague, (Butterworths, London) Vol. 11, (1967) p. 105. H. Oelschlager, J. Volke and G. T. Lim, Arzneim.-Forsch. 17 (1967) 637. E. Knobloch and E. Svitek, Chem. Listy 49 (1955) 37. J. Kiepinskg, Cesk. Farm. 7 (1958) 13. H. Oelschlager, Arch. Pharm. (Weinheim) 296 (1963) 396. H. Oelschlager, J. Volke and E. Kurek, Arch. Pharm. (Weinheim) 297 (1964) 431. H. Oelschlager, J. Volke, G. T. Lim and U. Frank, Arzneim.-Forsch. 16 (1966) 82. H. Oelschlager, J. Volke, G. T. Lim and R. Spang, Arch. Pharm. (Weinheim) 302 (1969) 946. H. Oelschlager and H. P. Oehr, Pharm. Acta Helv. 45 (1970) 708. H. Oelschlager and F. I. Sengiin, Arch. Pharm (Weinheim) 307 (1974) 401. J. Barrett, W. F. Smyth and J . P. Hart, J. Pharm. Pharmacol. 26 (1974) 9. E. Jacobsen and T. V. Jacobsen, Anal. Chim. Acta 55 (1971) 293. S. Halvorsen and E . Jacobsen, Anal. Chim. Acta 59 (1972) 127. E. Jacobsen, T. V. Jacobsen and T. Rojahn, Anal. Chim. Acta 64 (1973) 473. J. M. Clifford and W. F. Smyth, Z. Anal. Chemie 264 (1972) 149. E. Jacobsen and T. V. Jacobsen, Anal. Chirn. Acta 60 (1972) 472. D. J. Berry, Clinica Chim. Acta 32 (1971) 235. M. R. Hackmann, M. A. Brooks, J. A. F. de Silva and T. S. Ma, Anal. Chem. 46 (1974) 1075. M. Clifford and W. F. Smyth, Analyst 99 (1974) 241. M. Biezina, V. Volkovi and J. Volke, Collect. Czech. Chem. Commun. 19 (1954) 894. 0. Hrdy, Cesk. Farm. 3 (1954) 196. J. Volke, L. Wasilewska and A. Kejharovi-Ryvolovi, Pharmazie 26 (1971) 399. J. Volke, M. M. El-laithy and V. Volkovi, J. Electroanal. Chem., 60 (1975) 239. J . Doskoeil and M. VondriEek, Chem. Listy 46 (1952) 564. 0. Krestynovi-TElupilova, V. Macik and F. Santavq, Collect. Czech. Chem. Commun. 19 (1954) 234. G. B. Levy, P. Schwed and J. W. Sackett, J. Arner. Chem. SOC.68 (1946) 528. Princeton Applied Research, Application Note AN-111. D. DumanoviE, J. Volke and R. JovanoviE, Ass. Offic. Anal. Chem., 54 (1971) 884.
Progress in Medicinal Chemistry-Vol. 12, edited by G . P. Ellis and G . B. West @ 1975-North-Holland Publishing Company
6 Methods Related to Cyclic AMP and Adenylate Cyclase B. G. BENFEY, M.D., Dipl. Chem. Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada INTRODUCTION Adenylate cyclase Guanylate cyclase Cyclic nucleotide phosphodiesterase Cyclic AMP Cyclic GMP Protein kinase Phosphorylase kinase Phosphorylase Glycogen synthase Triacylglycerol lipase
294 294 297 297 299 301 301 303 3 04 306 306
EXPERIMENTAL APPROACHES USED IN THE STUDY OF CYCLIC NUCLEOTIDES Broken cell preparations Intact tissues and cells Body fluids Cytochemical studies Tissue fixation
306 306 310 311 311 312
ASSAYS OF CYCLIC NUCLEOTIDES Assay of cyclic AMP by activation of phosphorylase Enzymatic assay of cyclic GMP Assay of cyclic nucleotides by enzymatic cycling techniques Protein binding assay Protein kinase assay Radioimmunoassay Determination of relative levels of cyclic AMP by prelabelling of tissues or cells
313 313 314 315 316 318 318 319
ASSAYS OF OTHER ENZYME ACTIVITIES Protein kinase Phosphodiesterase Phosphorylase Phosphorylase kinase Phosphorylase phosphatase Glycogen synthase Triacylglycerol lipase
320 320 320 32 1 32 1 322 322 323
293
CYCLIC AMP AND ADENYLATE CYCLASE
294 ATPase Pyrophosphatase 5’-Nucleotidase
324 324 325
CONCLUSIONS
325
ACKNOWLEDGMENT
325
REFERENCES
326
INTRODUCTION Earl W. Sutherland, who died in 1974 at the age of 58, described his discovery of cyclic AMP (cyclic adenosine 3’ 3’-monophosphate) in an article baser! on the lecture he delivered in Stockholm in 1971 when he received the Nobel Prize in Physiology or Medicine [1]. Cyclic AMP was found in 1956 in the course of studies of the effect of adrenaline and glucagon on carbohydrate metabolism. It was found as a heat-stable factor, formed by particulate fractions of liver homogenate in the presence of ATP, Mg”, and adrenaline or glucagon, which is required for the activation of phosphorylase in the supernatant fraction of the homogenate [2]. The factor was isolated by ion exchange chromatography and its proposed structure proved to be identical with that suggested for a product isolated from the barium hydroxide digest of ATP [3,4]. In the early reports, it was thought that cyclic AMP was a dinucleotide but subsequent work showed it to be a mononucleotide [51. Robison, Butcher and Sutherland, in a monograph on cyclic AMP in 1971 [6], considered it to be a versatile regulatory agent acting to control the rate of many cellular processes, particularly as it occurs in all animal species, unicellular organisms, bacteria, and many plants [7,81. The knowledge that cyclic AMP is involved in hormone effects stimulated interest in other cyclic nucleotides. Yet only one of these, cyclic GMP (cyclic guanosine 3’,5‘-monophosphate), is known to occur in nature, having been discovered first in rat urine in 1963 [9] and then in all mammalian tissues examined in other organisms and in bacteria, but whether it too plays a regulatory role in cell activity is not known [61. ADENYLATE CYCLASE (EC 4.6.1.1.; ATP PYROPHOSPHATELYASE (CYCLISING))[lo, 1 1 1
The enzymatic activity responsible for the production of cyclic AMP was initially called adenyl cyclase, but it was realised that this name does not
B. G . BENFEY
295
refer to an adenine nucleotide, and two trivial names were proposed in 1970-adenylate cyclase and adenylyl cyclase-where the terms adenylate and adenylyl refer to the ester character of the substrate. Adenylate cyclase has been obtained in soluble form from bacteria [ 12,131, and can be prepared free of contaminating phosphodiesterase, ATPase (adenosine triphosphatase) and pyrophosphatase activities, but the relationship of the bacterial enzyme to the enzyme found in higher forms of life is not yet clear. The bacterial enzyme, for example, does not respond to mammalian hormones or sodium fluoride. In higher forms, adenylate cyclase is bound to membranes and, to a greater or lesser degree, is found in other cell structures such as microsomes and mitochondria. Attempts to obtain adenylate cyclase of multicellular organisms in a highly purified form have so far been unsuccessful. Increases in specific activity on a protein basis have been relatively small, as, for example, 10to 20-fold for plasma membranes from skeletal muscle [141 and 25-fold for plasma membranes from liver [15]. Adenylate cyclase of frog erythrocyte ghosts has been purified 150-fold, and it had no phosphodiesterase activity but a high ATPase activity [16]. Adenylate cyclase from bovine thyroid membranes has been purified 100-fold, but ATPase, p -nitrophenyl phosphatase and 5’-nucleotidase were co-purified with it [17]. Much of the adenylate cyclase activity of mammalian tissues can be solubilised or dispersed in Triton solution [IS]. Particulate preparations can be stored at -70°C for long periods without loss of activity, but at higher temperatures the activity from cells of multicellular organisms is rapidly lost [6]. The reaction catalysed by adenylate cyclase is shown in the first reaction (on the left) in Figure 6.1. The mechanism has not been established, but it is known that a divalent cation, Mg2+or Mnz+,is required and that pyrophosphate is formed stoichiometrically with cyclic AMP [ 191. The reaction requirements for adenylate cyclase from different mammalian tissues are probably similar. In a representative study of adenylate cyclase from mammalian heart muscle [20,21], the apparent K , for ATP was O.OSmM, and ATP concentrations in excess of those of Mg” inhibited the enzyme. Concentrations of Mg2’ as high as 10mM were required, however, to saturate the enzyme. It was concluded that Mg2+ binds to a second and possibly allosteric site and that the consequence of this binding is significantly increased reactivity of the catalytic site for substrate. Mn2’ is more effective than Mg2+in increasing the activity of adenylate cyclase (apparent K , for Mg” is 2-3 mM and for Mn2’ 0.7 mM).
2%
CYCLIC AMP AND ADENYLATE CYCLASE NH,
0-
y-
1
0I
0--P-0--P-on
y-
-0-P-0-P-0
8
6
/g\
-0-P
4
-0--P-0-cn
im bn
Figure 6.1. Adenylate cyclase catalyses the formation of cyclic A M P and inorganic phosphate (centre) from A T P (left); cyclic nucleotide phosphodiesterase catalyses the formation of 5’-AMP (right) f r o m cyclic AMP (centre)
Ca2+ is usually inhibitory in mammalian preparations. The ACTHstimulated adenylate cyclases of bovine adrenal cortex and of rat fat cell ghosts are exceptions [ 2 2 ] . In the mammalian myocardium, Caz+ is inhibitory and competitive with respect to Mg” ( K ,of about 0.3 mM) [20]. Thus, MgZ’, by binding to an allosteric site, might serve in the physiological regulation of adenylate cyclase, and Ca2+,by binding to this site, may have a modulating action. Like Mg2+, adrenaline and sodium fluoride increase the V,,, of adenylate cyclase in mammalian myocardium without changing the apparent K,,, for ATP or K , for Mg2+ [21]. Sodium fluoride stimulates adenylate cyclase activity in most preparations from multicellular organisms [23], whereas the majority of ATPases are inhibited. Sodium fluoride, however, does not stimulate adenylate cyclase activity in intact cells, although it probably enters most cells readily. The mechanism by which the action of a hormone on its tissue receptor leads to stimulation of adenylate cyclase is not known. Studies concerning the relationship of hormone binding to enzyme stimulation are in their infancy, and there may not be a direct relation between the number of receptors occupied and the extent of enzyme stimulation. The problems involved in the identification of the glucagon [24] and catecholamine receptors 125-271 have been discussed elsewhere.
B. G . BENFEY
297
GUANYLATE CYCLASE
The enzyme catalysing the formation of cyclic GMP (Figure 6.2) from GTP (guanosine 5'-triphosphate), namely guanylate cyclase, has been found in both particulate and high-speed supernatant fractions of many tissues. Guanylate cyclase has not been consistently affected in cell-free systems by compounds that cause an increase in cyclic GMP levels in intact cells [28,29]. Guanylate cyclase exhibits nearly complete dependence on Mn2+.The apparent K , for Mn2+is near 0.5 mM and the activity is barely detectable when Mg2' or Ca2' is substituted for Mn2+.The K,,, for GTP is between 0.1 and 0.4mM. Sodium fluoride has no effect on guanylate cyclase activity [29].
,;s-x:> 0
II
1
-0-P-0 1 I 0
Figure 6.2. Cyclic G M P
3',5'-CYCLIC NUCLEOTIDE PHOSPHODIESTERASE (EC 3.1.4.17; 3',5'-CYCLIC-AMP 5'-NUCLEOTIDOHYDROLASE) [30]
An Mg2'-dependent enzyme capable of destroying the biological activity of cyclic AMP was found by Sutherland and Rall in extracts of heart, brain and liver [31]. About 60% of this activity in beef heart homogenates was associated with particulate fractions after low-speed centrifugation. The soluble enzyme from beef heart was later purified and freed from interfering enzymes, and this preparation has been used in the identification of cyclic AMP in test solutions [32]. The reaction catalysed by phosphodiesterase is the second one (on the right) in Figure 6.1. Phosphodiesterase is now known to occur in several forms [33,34], and most of the earlier studies on effects of compounds on phosphodiesterase activity are therefore of limited value, particularly as they were carried out in the presence of high concentrations of cyclic AMP. Studies on phosphodiesterase are now performed at physiological concentrations
298
CYCLIC AMP AND ADENYLATE CYCLASE
of cyclic AMP (0.1 to 5 pM), and many compounds which inhibit the low-K, phosphodiesterase have little effect when measured at high substrate concentrations, as under these conditions activity due to the high-K,, enzyme is primarily being measured. Cyclic GMP is metabolised by some phosphodiesterases which also attack cyclic AMP, and these usually possess a higher affinity for cyclic GMP. A specific cyclic GMP phosphodiesterase has recently been identified in rat liver [35]. In mouse epidermis, the enzymatic hydrolysis of cyclic AMP is much more strongly stimulated by low concentrations of Mn2’ than by those of Mg2+;in contrast, the hydrolysis of cyclic GMP is exclusively stimulated by Mgz+ [36]. Methylxanthines inhibit phosphodiesterase in many tissues, and theophylline is about six times as potent as caffeine [6]. The potentiation of drug effects by methylxanthines has often been interpreted as accumulation of cyclic AMP, but methylxanthines also promote the accumulation of cyclic GMP [37]. Methylxanthines also inhibit adenylate cyclase activity in some tissues, as, for example, the noradrenaline-stimulated adenylate cyclase activity in rat erythrocyte ghosts [38], the vasopressinstimulated adenylate cyclase activity in toad bladder epithelium [39], the increase in cyclic AMP produced in brain slices by depolarising stimuli and by adenosine [40], and the adenylate cyclase activity in guinea-pig lung particles [41]. Both basal and glucagon-stimulated phosphorylase activity in rat liver slices are inhibited by theophylline [42]. However, methylxanthines also have pharmacological effects which are not related to inhibition of phosphodiesterase [43]. Catecholamines inhibit phosphodiesterase from beef heart [44] and guinea-pig lung [45], and so does N6,2’-O-dibutyryl cyclic AMP [47] which has been used to mimic cyclic AMP effects as it is more lipid-soluble and, therefore, assumed to penetrate cell membranes more readily than cyclic AMP [6,46]. A large number of other drugs inhibit the phosphodiesterase activity of rat brain and cat heart [48]. Ca2+( K , 2.3 p M) stimulates beef heart phosphodiesterase [49], and a Ca2+- and Mg2’-dependent phosphodiesterase has been isolated from brain [50,51]. This suggests that increased Ca” concentrations in cells may lead to a suppression of the increased rate of cyclic AMP accumulation, thus preventing a prolonged increase in cyclic AMP. Although phosphodiesterase is generally not found extracellularly, its location at the external surface of skeletal muscle was demonstrated by incubating frog muscles with labelled cyclic AMP [52].
B. G. BENFEY
299
CYCLIC AMP
The chemistry of cyclic nucleotides has been recently reviewed [53]. Cyclic AMP (Figure 6.1, middle formula) is a relatively stable compound; at 92°C in N HCI its half-life is 55 min and at 98°C in N NaOH it is 36 min [ 5 ] . Acid or alkaline hydrolysis preferentially breaks the 5’-bond, which is in contrast to the action of phosphodiesterase which attacks only the 3’-bond. The 3‘-bond of cyclic AMP has a high energy content, the free energy of hydrolysis of this bond being -11.9 kcal/mol [54]. Thus, the 3’phosphodiester bond of cyclic AMP is about 3 kcal/mol more ‘energyrich’ than the P,y-pyrophosphate bond of ATP (free energy of hydrolysis: 8.8 kcal/mol). The enthalpy of hydrolysis of cyclic GMP is lower than that of cyclic AMP but is still indicative of a high-energy bond [ 5 5 ] . Nevertheless, there is no evidence that the cyclic nucleotides serve as adenylating or guanylating agents. Cyclic AMP has been proposed to be the second messenger in hormone effects, with the hormones themselves acting as first messengers [6]. However, in most instances we do not know how cyclic AMP affects physiological functions. This is a reflection of our ignorance of the nature of basic cell processes in general. Progress in understanding how cyclic AMP acts is limited by a lack of knowledge of the fundamental biochemistry and biophysics of processes such as contractility, rhythmicity, permeability, transport, and secretion, and Sutherland’s second messenger concept has been critically discussed [56]. The basal concentration of cyclic AMP in various tissues is of the order of 0.1 to 1 nmol/g of tissue (wet weight) or about 0.5 to 5 pmol/mg of protein [6]. Assuming an even distribution throughout the intracellular water, the concentration would thus be in the range of 0.25 to 2.5 pM. This is much less than the concentration of other adenine nucleotides. The concentration of ATP is usually on the order of 1 to 5 pmllg of tissue (wet weight) or about 5mM. The concentration of 5‘-AMP is usually about one tenth of this, with the concentration of ADP most often falling somewhere in between. These nucleotides, therefore, exist in cells in concentrations which are 1000 to 10000 times greater than that of cyclic AMP 161. The subcellular distribution of cyclic AMP shows that the actual concentration of free cyclic AMP may be very small due to protein binding, but in certain parts of some cells it may be very high as a result of
300
CYCLIC AMP AND ADENYLATE CYCLASE
compartmentalisation. In rat liver, the concentration of cyclic AMP is about 1 p M , at least one order of magnitude greater than the concentration which fully activates the liver phosphoryiase system in extracts [23], yet phosphorylase and other cyclic AMP-sensitive systems in rat liver are not activated [571. Likewise, the cyclic AMP content of rat fat cells under basal conditions is about 0.5 nmol/g of tissue [58],although half-maximal stimulation of adipose tissue protein kinase occurs with 0.05 p M cyclic AMP [59]. Most of the cyclic AMP in fat cells is either inactive or insensitive to protein kinase. The concentration of cyclic AMP is subject to large fluctuations under the influence of hormones. Thus, in the isolated perfused rat liver preparation, high concentrations of glucagon increased the concentration of cyclic AMP to a level which was more than 60 times that of normal [6]. This result illustrates the enormous capacity which cells have for changes in their content of cyclic AMP. The changes in the level of cyclic AMP which are physiologically significant are, however, often small compared with those occurring under experimental conditions. For example, physiological concentrations of glucagon produce only a two-fold increase in cyclic AMP in the liver. This is, nevertheless, associated with a maximal stimulation of glucose output, and increasing the concentration of glucagon, and hence of cyclic AMP, beyond this point has no further effect [6]. There have been frequent difficulties relating physiological effects to cyclic AMP levels. For example, adenosine inhibits the effect of noradrenaline on cyclic AMP accumulation in rat fat cells but does not interfere with the lipolytic effect of the catecholamine [58]. Both ACTH and its o-nitrophenyl sulphenyl derivative stimulate corticosterone synthesis in isolated rat adrenal cells to the same maximal rate, but the analogue has a 30- to 100-fold smaller effect than ACTH on cyclic AMP accumulation [601. The multiple drug effects on smooth muscles are also little understood. Thus, isoprenaline relaxes and adrenaline and noradrenaline contract the isolated rat aorta, but all three catecholamines increase cyclic AMP formation [61]. Adrenaline induces relaxation and prostaglandin E, contraction in the estrogen-treated rat isolated myometrium, but both adrenaline and prostaglandin El elevate the cyclic AMP level in the tissue [62]. An example of a critical evaluation of the evidence linking cyclic AMP to a physiological effect (cardiac contractility) is that of Sobel and Mayer [63]. Another problem is the lack of homogeneity of the tissue employed in
B. G . BENFEY
301
many studies. Thus noradrenaline stimulated cyclic AMP accumulation in both brain slices and glial cells [64]. Accordingly changes in brain cyclic AMP may take place in glial cells and not in nerve cells. Cyclic AMP accumulation by catecholamines is generally associated with P-adrenoceptor stimulation, but there is recent evidence that (Y adrenoceptor stimulation also can lead to cyclic AMP accumulation. For example, in slices of rat cerebral cortex, the effect of isoprenaline on the cyclic AMP level was inhibited by a P-adrenoceptor blocking drug and yet inhibition of the effect of noradrenaline required the combination of a j? and an a-adrenoceptor blocking drug 165,661. CYCLIC GMP (CYCLIC GUANOSINE 3',5'-MONOPHOSPHATE) [67]
The tissue levels of cyclic GMP (0.01 to 0.1 nmollg) are generally about 10% of those of cyclic AMP. The physiological role of cyclic GMP is unknown and a control function for cyclic GMP in tissues has not been established. Parasympathomimetic agents increase cyclic GMP in many tissues, providing extracellular Ca2' is present [37]. This suggests that the increase in cyclic GMP is a secondary event brought about by an increased cytoplasmic Ca2+concentration. In frog heart, cyclic AMP levels rise and cyclic GMP levels fall during early systole but return to normal during early diastole 1681. The situation is complex in smooth muscles. Both acetylcholine and the phosphodiesterase inhibitor, l-methyl-3-iso-butylxanthine, elevate cyclic GMP levels, but acetylcholine causes contraction of the muscle preparation whereas the phosphodiesterase inhibitor produces relaxation [69]. In the guinea-pig ileum, the increase in cyclic GMP brought about by a parasympathomimetic drug can be prevented by the inclusion of isoprenaline in the incubation medium; however, isoprenaline causes an increase in the concentration of cyclic AMP that can be inhibited by a parasympathomimetic drug [70]. In guinea-pig lung slices, acetylcholine and bradykinin increase both cyclic GMP and cyclic AMP yet indomethacin and aspirin inhibit the effect on cyclic AMP but not that on cyclic GMP [71]. PROTEIN KINASE (EC 2.7.1.37; ATP: PROTEIN PHOSPHOTRANSFERASE) [72-741
In the course of studies designed to elucidate the mechanism by which cyclic AMP regulates phosphorylase activation and glycogenolysis in
302
CYCLIC AMP AND ADENYLATE CYCLASE
skeletal muscle (Figure 6 . 3 ) ,a cyclic AMP-activated protein kinase was found which catalysed the phosphorylation by ATP of phosphorylase kinase and other protein substrates, such as casein, protamine, and histone [75]. Both cyclic AMP and cyclic GMP-dependent protein kinases have been found in numerous animal tissues [76]. Catecholamine
1
Adenylate cyclase
n
ATP
‘“1“’
Protein kinase
n
Nonactivated phosphorylase kinase Phosphoryiase
Activated phosphorylase
Q
kinase
Phosphorylase 2
n
GlycogeniPi
Glucose-l-phosphate
Figure 6.3. Catechofarnineactivation of phosphorylase
In animal cells, activation of protein kinase is the only well-understood action of cyclic AMP. The concentration of cyclic AMP needed for half-maximal stimulation of purified protein kinase from rabbit skeletal muscle was 15-30 nM [77]. The apparent K , for ATP in the presence of 10 mM Mg” was approximately 15 pM. The binding of cyclic AMP to protein kinase was reversed by mild chemical treatment and by gel filtration on Sephadex [77]. A partially purified cyclic GMP-activated protein kinase from lobster muscle was later found to have a K , for cyclic GMP of about 0.08 pM and for cyclic AMP of about 4 pM. A cyclic AMP-activated protein kinase isolated from the same tissue had an apparent K , for cyclic AMP of about 0.02 p M and for cyclic GMP of about 1.2 p M [76]. Cyclic GMPdependent phosphorylation of endogenous protein has been demonstrated in membranes of mammalian smooth muscle [78]. In the presence of 10 mM Mn2+,a half-maximal increase in the phosphorylation of these proteins occurred with 20-30 nM cyclic GMP, but ten-fold higher concentrations of cyclic AMP were required to produce the same increase in phosphorylation. It is now assumed that cyclic AMP increases the catalytic efficiency of protein kinase by dissociating a holoenzyrne into the regulatory and catalytic subunits. In the absence of the cyclic nucleotide, the regulatory
B. G . BENFEY
303
or cyclic AMP-binding subunit binds to the catalytic subunit, thereby reducing its activity. A protein inhibitor of cyclic AMP-activated protein kinase has been isolated from mammalian tissue and this promotes a five-fold increase in the binding constant of cyclic AMP to protein kinase [79]. The inhibitor is assumed to interact with the catalytic subunit at the regulatory subunit binding site. Thus, inhibitor protein and regulatory subunit modulate the catalytic subunit activity by an identical mechanism. However, it may be more complex, as a protein kinase modulator protein has been isolated which alters the substrate specificity both of cyclic AMP and of cyclic GMP-activated protein kinases, increasing the phosphorylation of some protein substrates and decreasing that of others [go]. In most cases, the effect of cyclic AMP has been to increase the activity of protein kinase, but instances have been found in which the cyclic nucleotide decreased the protein kinase activity. In these cases the level of substrate phosphorylation was lower in the presence than in the absence of cyclic AMP. Such a net decrease in the level of phosphorylation of a protein substrate may result either from a decrease in the activity of a protein kinase or from an increase in the activity of a protein phosphatase, or both [81]. Cyclic AMP-inhibited protein kinases have been described [82]. Protein binding can contribute to the high basal level of cyclic AMP in many tissues, and its intracellular concentration may be regulated not only by the activities of the two enzymes-adenylate cyclase (which mediates the synthesis of cyclic AMP) and phosphodiesterase (which mediates its degradation)-but also by binding proteins which protect cyclic AMP from degradation and limit its access to its sites of action. Protein kinase does not bind either N6,2’-0-dibutyryl cyclic AMP [74], which has been employed to mimic cyclic AMP effects [6], or I-succinyl cyclic nucleotide tyrosine methylester [83], which is used in the radioimmunoassay of cyclic nucleotides [84]. It should be added that cyclic nucleotide-stimulated protein phosphorylation represents only a fraction of the total amount of protein phosphorylation that occurs in the body. PHOSPHORYLASE KINASE (EC 2.7.1.38; ATP: PHOSPHORYLASE PHOSPHOTRANSFERASE) [74]
The activation of phosphorylase was the first effect of cyclic AMP to be discovered. Of all the systems which are affected by cyclic AMP, the
304
CYCLIC AMP AND ADENYLATE CYCLASE
phosphorylase system is the one which has been most carefully studied and about which most is known. The sequence of events according to which the activation of one enzyme leads to the activation of another to stimulate glycogenolysis (Figure 6.3) has been likened to the cascade of reactions involved in blood clotting. Although some amplification may take place at an earlier stage (i.e., one molecule of hormone may stimulate the formation of more than one molecule of cyclic AMP), most of the amplification involved in hormonal responses mediated by cyclic AMP probably occurs after the formation of cyclic AMP. The first enzyme in this sequence and the one which is affected by cyclic AMP is a protein kinase. It catalyses the transfer of the terminal phosphate of ATP to phosphorylase b kinase, thereby activating the enzyme. Phosphorylase kinase has been known for a longer time than protein kinase. It exists in resting muscle in a form which has little activity at or below the normal intracellular pH and exhibits less than maximal activity even at higher pH values. In the presence of ATP and Mg2+, it is converted to a form (activated phosphorylase kinase) having 25-50 times as much activity at pH 6.8 and about twice as much activity at pH 8.2. The ratio of kinase activity at pH 6.8 to that at pH 8.2 has been widely used as an index of kinase activation. Activation of phosphorylase kinase is exceedingly rapid in response to an increase in the level of cyclic AMP. Phosphorylase b kinase of muscle is relatively specific for its normal substrate, phosphorylase b, and Caz+ is a cofactor. The apparent K , values for Ca2+for the activated kinase were 0.2 p M at pH 8.2 and 0.5 p M at pH 6.8 and for the non-activated kinase 3 p M at pH 8.2 and indeterminate at pH 6.8 [85].Thus activation of phosphorylase kinase reduces the Ca2' requirement of the enzyme. Activated phosphorylase kinase is inactivated by a phosphatase, but the specificity of this phosphatase has not yet been worked out. GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1; a-1,4-GLUCAN :ORTHOPHOSPHATE GLUCOSYLTRANSFERASE)
Phosphorylase activity has been found in a large number of tissues and animal species. Muscle phosphorylase b is converted to phosphorylase a under the catalytic influence of phosphorylase kinase by a reaction requiring both Ca2+ and Mg" (Figure 6.3). Both forms of muscle phosphorylase require 5'-AMP, although the K , for phosphorylase Q is so low that for physiological processes the assumption of independence is
B. G . BENFEY
305
probably correct. In other words, the intracellular concentration of 5'-AMP is always high enough to ensure maximal (or close to maximal) activity of phosphorylase a. On the other hand, the intracellular concentration of 5'-AMP may at times greatly exceed the K , of skeletal muscle phosphorylase so that, for example, in severe anoxia, phosphorylase b is fully as active as phosphorylase a. The effect of 5'-AMP on muscle phosphorylase b is to increase the affinity of the enzyme for its substrates, glycogen and inorganic phosphate. Liver phosphorylase b is not stimulated to full activity by 5'-AMP, but it serves as a substrate for phosphorylase kinase from heart muscle, and phosphorylase b from heart muscle is activated by liver phosphorylase kinase. Phosphorylase kinase from skeletal muscle is also effective on liver phosphorylase. The apparent K , of active liver phosphorylase for glycogen and inorganic phosphate are similar to those reported for muscle phosphorylase a. The conversion of phosphorylase b to a involves the addition of the terminal phosphate of ATP. The phosphate is then removed as the enzyme is converted back to phosphorylase b under the catalytic influence of phosphorylase phosphatase (EC 3.1.3.17; phosphorylase phosphohydrolase). Phosphorylase phosphatase from rabbit skeletal muscle has been purified 700-fold [86]. Terminology to deal with this type of situation has not yet been developed or has not come into widespread use [87]. Conversion of one molecular form of an enzyme to another more active form is referred to as activation of that enzyme. Effects such as those of 5'-AMP on phosphorylase b where the activator must be present continuously for activity to be maintained and where the formation of a new molecular species is not involved, is referred to as allosteric stimulation or, where less is known about the mechanism, simply as stimulation of the enzyme. Tnterconversion allows metabolic reactions to be switched on and off without changes in the intracellular concentration of metabolites. The regulation of phosphorylase activity in muscle has been reviewed [88,89], and cyclic AMP is only one of several regulatory influences which impinge on the phosphorylase system. It has been proposed that in skeletal muscle contraction is coupled to glycogenolysis by Ca2+ [891. When Caz+ is released from the sarcoplasmic reticulum in response to nerve stimulation, it triggers off muscle contraction and at the same time stimulates phosphorylase kinase. This leads to the conversion of phosphorylase b to phosphorylase a and subsequent glycogenolysis. An energy-requiring process (contraction) is thus linked to an energy-
306
CYCLIC AMP AND ADENYLATE CYCLASE
producing metabolic sequence. Electrical stimulation of skeletal muscle, however, leads to a very rapid increase in phosphorylase a with no increase in cyclic AMP or activated phosphorylase kinase. A similar effect occurs in perfused mammalian heart following an increase in the concentration of Ca2+ in the medium [MI.Also, anoxia stimulates phosphorylase b to a conversion in heart muscle without activation of phosphorylase kinase. It has been suggested that, in heart muscle, Ca” is an immediate means of increasing glycogenolysis, whereas cyclic AMP which requires several enzymatic steps and, hence, needs time, provides a relatively slower effect [90]. GLYCOGEN SYNTHASE (EC 2.4.1.11; UDP GLUC0SE:GLYCOGEN [91] LY-~-GLUCOSYLTRANSFERASE)
Glycogen synthase exists in two forms: the D (glucose-6-phosphate dependent) or b (less active) and the I (glucose-6-phosphate independent) or a (active) forms. Muscle glycogen synthase has been obtained in a high state of purity and found to incorporate one phosphate group per subunit during the I to D conversion. The phosphorylation of both glycogen synthase and phosphorylase kinase is catalysed by the same protein kinase. Thus the cyclic AMP-activated protein kinase turns on glycogen degradation by way of the phosphorylase reaction and turns off glycogen synthase-catalysed glycogen synthesis. It is not known what is the relationship among the phosphatases that activate glycogen synthase and inactivate phosphorylase and thus reverse the metabolic flow. TRIACYLGLYCEROL LIPASE (TRIGLYCERIDE LIPASE, EC 3.1.1.3; GLYCEROL ESTER HYDROLASE)
Triacylglycerol lipase from adipose tissue is directly activated by a cyclic AMP-dependent protein kinase [92]. Adipose tissue lipase is also activated by skeletal muscle cyclic AMP-dependent protein kinase 1931. EXPERIMENTAL APPROACHES USED IN THE STUDY OF CYCLIC NUCLEOTIDES BROKEN CELL PREPARATIONS
These are useful as the environment in the vicinity of the cyclases can be greatly simplified and, therefore, better controlled than in most organised
B. G . BENFEY
307
systems. In most mammalian tissues, adenylate cyclase has been located in the low-speed fraction of homogenates which contains fragments of the cell membrane. Often these preparations can be washed repeatedly, thus eliminating many metabolites and soluble enzymes, and in some cases can be taken through additional purification steps with the retention of hormonal reactivity. The ultimate goal is to obtain a preparation containing only adenylate cyclase, but it has not yet been possible to purify the enzyme from mammalian sources beyond a certain point without destroying its sensitivity to hormonal stimulation. The great lability of adenylate cyclase is the chief limitation during studies with broken cell preparations. Most can be quick-frozen and stored at -70°C for long periods (and can be thawed at least once) without altering the properties of the enzyme. Aliquots of a given preparation treated and stored in this manner yield results which are highly reproducible. Often the data cannot be related quantitatively to that obtained from studies with more highly organised systems, not only because of the altered circumstances of the adenylate cyclase (e.g. dilution effects, absence of endogenous inhibitors or activators), but also because of possible differences in the way the hormone is handled (e.g. metabolism, tissue uptake). As a rule, adenylate cyclase activity in broken cell preparations is less sensitive to hormonal stimulation than activity in intact cells [94], and guanylate cyclase activity does not respond to agents which increase the cyclic GMP level in intact cells [29]. As adenylate cyclase is probably oriented within the membrane with receptors exposed on the outside and catalytic components on the inside, the formation of vesicles on homogenisation result in the expression of artifactual properties. Thus inside-out vesicles reduce or prevent hormone interaction and right-side-out vesicles effectively reduce total enzyme activity if the vesicles were impervious to the exogenous substrate. The non-stimulated or basal adenylate cyclase activity observed in homogenates varies considerably from tissue to tissue [18], and there is a lack of correlation between such a measure of basal activity and the tissue content of cyclic AMP. A major factor complicating the quantitative interpretation of most adenylate cyclase measurements is contamination by other enzymes including ATPase, inorganic pyrophosphatase, cyclic nucleotide phosphodiesterase, and various deaminases. Some degree of inhibition of phosphodiesterase is necessary in most preparations, and a methylxan-
308
CYCLZC AMP AND ADENYLATE CYCLASE
thine is usually included in the incubation medium for this purpose. In many preparations, cyclic AMP does not accumulate at all in the absence of a phosphodiesterase inhibitor. For example, in the absence of theophylline, only 15% of the cyclic AMP formed by washed heart particles was found to survive, while in the presence of 8 mM theophylline, the recovery was 68% [20]. For the assay of adenylate cyclase activity of washed dog heart particles, a medium has been employed which contained 40 mM Tris or 20 mM potassium phosphate buffer (pH 7.5) with 4 mM ATP, 6.6 mM magnesium sulphate, 13 mM caffeine, and 2-5 pglml of bovine serum albumin in a volume of 1.8 ml 1951. The reaction was stopped by heating the vessels for 3 min in a boiling water bath. After centrifugation, the supernatant fluid was assayed for cyclic AMP by the dog liver phosphorylase-activation method. While formation of cyclic AMP from ATP did not occur, the nonenzymatic formation of significant amounts of cyclic GMP from GTP in solutions heated for 3 min at 100°C at a pH near neutrality has been observed 1961. Guanylate cyclase reactions can be terminated by decreasing the pH of the mixture with or without heating. For the assay of guanylate cyclase, a medium has been employed containing 40 mM Tris buffer (pH 7.4), 2 mM GTP, 3 mM manganous chloride, and 10 mM theophylline [28]. A more recent method uses 0.2 M Tris buffer (pH 7.7) with 0.4 mM GTP, 2 mM manganous chloride and 2 mM cyclic GMP in a volume of 0.15 ml [97]. It has become more common to measure the rate of conversion of radioactively labelled ATP to cyclic AMP [98,99], taking care to avoid contamination of the counting fluid with ATP, as the concentration of ATP in the reaction mixture exceeds that of cyclic AMP by several orders of magnitude. Passage of the reaction mixture through a Dowex 50 column followed by precipitation of all nucleotides other than cyclic AMP by ZnS04-Ba(OH)2 treatment has been recommended [98,100]. Precipitation with Ba(OH)2 and ZnS04 should not be carried out in the presence of high concentrations of ATP, as formation of cyclic AMP by a chemical reaction can occur [3]. Barium sulphate cannot be used for the purification of cyclic GMP solutions because it removes the nucleotide. Addition of carrier cyclic AMP aids in the recovery of labelled cyclic AMP, as in the presence of 2 m M unlabelled cyclic AMP and 8 m M theophylline, recovery of cyclic AMP formed by washed heart particles was 100% [20]. Paper chromatography has been employed for the isolation of cyclic
B. G . BENFEY
309
AMP 1201. Paper chromatography offers the advantage that the content of ATP degradation products can be monitored. The spots are made visible under ultraviolet light, cut out, and placed in vials containing scintillation fluid for counting. Also, thin-layer chromatography has been used for the isolation of labelled cyclic nucleotides [ 101, 1021. Cyclic nucleotides have been isolated by column chromatography on aluminum oxide [ 103-1051, and aluminum oxide chromatography has been combined with ion exchange chromatography [ 102,106,107]. [cx-~*P]ATPor GTP has been used for the assay of cyclase activity, and the cyclic nucleotides were then isolated by column chromatography on aluminum oxide [ 103, 1041. Cyclic nucleotides, nucleosides, and purines pass through the column and all other nucleotides are adsorbed. As the only phosphorylated compounds passing are the cyclic nucleotides, an a 32 P-labelled nucleoside triphosphate becomes a specific precursor for use in this assay. The high ATPase activity of broken cell preparations from many tissues makes the use of an ATP-regenerating system mandatory. This may consist of 20mM phosphoenol pyruvate and 130pg/0.15ml of pyruvate kinase 1201, which serves to recycle the ADP formed by ATPase back to ATP. ATPase activity has been determined during the assay of adenylate cyclase by thin-layer chromatography of a sample of the eluate containing ATP and ADP collected from the Dowex 50 column prior to the fraction containing the bulk of the cyclic AMP [108]. Samples were cochromatographed with an aliquot of unlabelled marker nucleotides. The ATP and ADP spots were made visible with ultraviolet light, scraped from the plates into scintillation fluid and counted. During 2min of incubation, guinea-pig heart preparations degraded 250000 pmol/min/mg protein of ATP and formed 73 pmollminlmg protein of cyclic AMP. The respective data for guinea-pig lung were 60000 and 144 pmol/min/mg protein, and for bronchial smooth muscle, 340000 and 245 pmol/min/mg protein [108]. The ATP-regenerating system may complicate the analysis because of its possible influence on the adenylate cyclase activity. For example, 5’-adenylyl imidodiphosphate (AMP-PNP) labelled with ( Y - ~ ~ an P, analogue of ATP containing Nitrogen substituted for Oxygen between the terminal phosphates, has been used to circumvent the problems encountered in maintaining the substrate concentration during kinetic studies of adenylate cyclase activity [109]. AMP-PNP is a substrate for adenylate cyclase and is only slowly hydrolysed by ATPase. Unlabelled AMP-PNP
3 10
CYCLIC AMP AND ADENYLATE CYCLASE
has been used and the cyclic AMP formed in the presence of adenylate cyclase has been determined by the protein-binding method [1101. INTACT TISSUES AND CELLS
Studies with intact tissues can often be carried out under conditions where the physiological response and the change in cyclic AMP can be measured simultaneously. The information obtained may, therefore, be more directly relevant to the physiological situation. The change in cyclic AMP and the physiological response can be compared as functions of the dose of the hormone needed to elicit the response and the time required for the response to be manifested. The isolated perfused heart was first used to measure cyclic AMP levels while simultaneously monitoring a mechanical or functional response [ l l l ] . However, studies on temporal and dose relationship only suggest, but in no way prove, an involvement of cyclic AMP in the observed response. A decisive answer will come only when the exact function of cyclic AMP (if any) in the response is discovered. A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [I 121. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP; the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114, 1151. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. A method has also been described for isolating [3H]GMPfrom piatelets incubated with r3H1guanine [117].
B. G . BENFEY
311
BODY FLUIDS [118-120]
cyclic AMP and cyclic GMP are the only organic phosphate compounds which have been detected in urine. Urinary cyclic AMP is derived from two sources: plasma, by glomerular filtration and tubular secretion, and kidney, by synthesis and diffusion from kidney cells into the tubular lumen. The daily urinary excretion of cyclic AMP in man ranges from 1 to 9 pmol and that of cyclic GMP from 0.4 to 3 pmol, whereas cyclic AMP levels in human plasma range from 10 to 26nM and cyclic GMP levels from 2 to 16nM. Cyclic nucleotide degradative activity occurs in mammalian whole blood, but it is not known whether phosphodiesterase exists in plasma in vivo. The enzyme can be released from damaged platelets and granulocytes in vitro. However, degradation of cyclic AMP in whole blood is slowed by reduction in temperature, immediate centrifugation and addition of methylxanthines, and plasma can be pipetted directly into HClO,. It is usually necessary to process several milliliters of plasma owing to the low cyclic nucleotide levels and losses with purification. Little is known of the origin of plasma cyclic nucleotides under basal conditions. The liver may be a source of cyclic AMP, as the isolated perfused rat liver releases cyclic AMP into the perfusate, being a highly sensitive index of glucagon and adrenaline effects [121]. Release of cyclic AMP has also been reported from the perfused adrenal gland [122] and the superior cervical ganglion [ 1231, and lung fibroblasts incubated with prostaglandin El released cyclic AMP into the medium [124]. In man, the volume of distribution of the cyclic nucleotides exceeds the extracellular fluid space 11251, and only about 15% of injected labelled cyclic AMP is excreted by the kidney. Extrarenal cyclic nucleotide clearance probably involves hepatic metabolism and/or biliary excretion, but cyclic AMP is also destroyed by renal phosphodiesterase. The isolated perfused rat kidney rapidly removes cyclic AMP from the perfusion medium [126]. CYTOCHEMICAL STUDIES
The interpretation of results of studies with cyclic AMP in tissue homogenates, slices, and intact tissues is difficult because the material is not homogeneous. Cytochemical studies in liver [I271 and heart [128,1291 have also shown that cells may respond differently to various stimulants
312
CYCLIC AMP AND ADENYLATE CYCLASE
of adenylate cyclase activity. The basis of the cytochemical reaction is the precipitation of a heavy metal salt of the pyrophosphate produced by the action of adenylate cyclase on ATP. PbZ' precipitates pyrophosphate from the medium with the final product easily seen under the electron microscope. In the liver, isoprenaline-stimulated adenylate cyclase has been found to be located almost exclusively on the surface of the parenchymal cells, with little or no deposit on the surface of the reticulo-endothelial cells. In contrast, the predominant effect of glucagon resulted in the deposition of reaction product on the reticulo-endothelial cell surface, although deposits were also present on the parenchymal cells. In the presence of F-, there were substantial deposits on the surface of both types of cells. These results demonstrate that distinct enzyme systems are present in parenchymal and reticulo-endothelial cells. No theories concerning the function of adenylate cyclase in the liver have considered its role in the endothelial cells which act mainly on phagocytes. These cells contribute 35% of the cells in the liver and can be expected to contribute substantially to biochemical measurements of adenylate cyclase activity and cyclic AMP concentrations when liver slices, homogenates, or cell fractions constitute the enzyme source. In the heart, the lead salt has been found to be deposited along the plasma membrane and sarcotubular system of muscle cells, and precipitation was enhanced by adrenaline, glucagon and F-. In contrast, histamine caused particularly intense staining at the plasma membrane and the membrane of the pinocytotic vesicles of endothelial cell capillaries. This raises the possibility that cyclic AMP formed in endothelial cells of the coronary capillaries under the influence of histamine may affect cardiac cells, thereby acting as a local hormone. The localisation of cyclic AMP in lymphocytes and neurons by immunocytochemical methods has also been described [ 1301, and immunofluorescence has been used for the localisation of cyclic nucleotides in canine thyroid gland [131]. TISSUE FIXATlON
The most critical step in the measurement of cyclic nucleotides and associated enzyme activities in intact tissues is the fixation of the tissue, which must be rapid and thorough, for the rate of turnover of cyclic nucleotides and activated enzymes in tissues is high and increased levels are evanescent.
B. G . BENFEY
313
In general, it appears that fixation by fast freezing, (for example, by clamping between aluminum blocks chilled in liquid nitrogen), followed by pulverisation in impaction mortars chilled to the temperature of liquid nitrogen, and finally rapid homogenisation of the frozen, powdered tissue in 0.1 N HCl, containing radioactively labelled cyclic nucleotides to determine recovery, is the most reliable method of fixation. Certain tissues, such as the rat epididymal fat pad [132] and rat adrenal gland [133] have been homogenised directly in 0.1 N HCI with good results. However, comparison of the more complicated method involving freezing and the easier direct homogenisation method is recommended before the latter is employed routinely. The method of determining cyclic nucleotides can influence the choice of the method of tissue fixation. With the protein kinase method for the determination of cyclic AMP, dissection of samples from the heart of open-chest dogs and rapidly immersing them in liquid dichlorodifluoromethane cooled to -150°C in liquid nitrogen gave the same results as freezing the tissue in situ with clamps cooled in liquid nitrogen 11341. However, the biopsy method could not be used when cyclic AMP was assayed by the phosphorylase activation method [135]. Changes in cyclic nucleotide levels in the brain have been difficult to measure. A microwave oven has been used to kill small animals, such as rats, within seconds [136-1391, the brain then being removed and frozen. The basal levels of cyclic nucleotides found in tissues vary widely depending on the method of killing the animal, as has been shown by the analysis of eight different tihues of the rat [140]. For example, cerebellum cyclic AMP levels (pmol/mg of protein) were 10.59 after immersion in liquid nitrogen, 161.19 after decapitation, 106.20 after ether anaesthesia, and 63.98 after pentobarbital anaesthesia.
ASSAYS O F CYCLIC NUCLEOTIDES ASSAY OF CYCLIC AMP BY ACTIVATION OF PHOSPHORYLASE
The first assay procedure to be reported [23] and which is still being used with minor modifications [6] employs dog liver phosphorylase. The preparation of the enzyme entails the isolation of active phosphorylase and subsequent inactivation with phosphorylase phosphatase. Inactive dog liver phosphorylase is stable for many years when frozen at - 20°C.
314
CYCLIC AMP AND ADENYLATE CYCLASE
In the first stage of the assay, inactive phosphorylase is incubated for 20 min at 25°C with an 11000 g supernatant fraction of dog liver homogenate, ATP, MgS04, caffeine, together with controls, cyclic AMP standards (0.1 to 6 pmol), or suitably diluted unknown samples. In the second stage, the amount of active phosphorylase at the end of the first stage is determined by measuring the rate of glycogen formation from glucose-lphosphate during incubation at 37°C for 30 min. The assay may be complicated by the presence of interfering materials such as ADP, glucose-1-phosphate and glucosed-phosphate, UDPglucose and Ca2+.Cation and anion exchange chromatography has been used to purify cyclic AMP from complex mixtures. The presence of inhibitors of the effects of cyclic AMP on the assay system is detected by adding known amounts of cyclic AMP along with the experimental extracts in the assay system. The specificity of the assay system in terms of activators is greatly enhanced by using purified phosphodiesterase. Thus, any cyclic AMP-like activity found in extracts after ion exchange chromatography which is destroyed by incubation with phosphodiesterase is probably cyclic AMP. The preparation of beef heart phosphodiesterase has been described [6]. The most serious problem encountered with the assay system is a quantitative variability; that is, a particular set of extracts assayed under identical conditions in separate assays may give figures which are quantitatively different. The reason for this is unknown. The relative cyclic AMP activity in the extracts is the same; that is, when sample B contains twice as much cyclic AMP as sample A on day 1, it will also contain twice as much on day 2. However, the absolute values of cyclic AMP may vary. Skeletal muscle phosphorylase has also been used for the assay of cyclic AMP [135]. The muscle system is even more tedious than that with liver phosphorylase because of the high concentrations of phosphorylase b which must be used. This is necessitated by the stimulation of muscle phosphorylase b activity by 5'-AMP, the major catabolite of cyclic AMP. ENZYMATIC ASSAY OF CYCLIC GMP [69]
Cyclic GMP is first converted to 5'-GMP with the aid of phosphodiesterase, 32Pis then transferred from [y-32P]ATPto GMP by the action of GMP kinase (EC 2.7.4.8; ATP :GMP phosphotransferase), and after degradation of the remaining [32P]ATPto ADP and 32P9with the aid of myosin (EC 3.6.1.3; ATP phosphohydrolase), the inorganic phosphate is
B. G . BENFEY
3 15
precipitated and the amount of f3*P1GDPin the supernatant determined. As little as 0.1 pmol of cyclic GMP per tube can be determined. For the isolation of cyclic GMP from tissue extracts, 5’-nucleotides are coprecipitated with nascent ZnC03, and the cyclic GMP is purified and separated from cyclic AMP on a Sephadex column. Only two other assays allow the determination of amounts as small as 0.1 pmol of cyclic GMP per tube: the cycling system [141, 1421 and the radioimmunoassay [84,143]. The assay described above is faster and less laborious than these methods involving enzymatic cycling. ASSAY OF CYCLIC NUCLEOTIDES BY ENZYMATIC CYCLING TECHNIQUES
The following method has been described for the assay of cyclic AMP [144, 1451. Cyclic AMP is converted to 5‘-AMP with the aid of phosphodiesterase and then to ATP with myokinase (EC 2.7.4.3; ATP :AMP phosphotransferase; adenylate kinase) and pyruvate kinase (EC 2.7.1.40; ATP :pyruvate phosphotransferase). ATP is measured by determining the orthophosphate which accumulates during incubation of ATP with a cycling system containing myosin, pyruvate kinase, and phosphoenol pyruvate. Alternately, the ATP is determined by its luminescent reaction with firefly luciferin and luciferase [ 145-1471. With a sensitivity to about 1 pmolltube of cyclic AMP, this assay is almost as sensitive as the phosphorylase method, but with a linearity over three orders of magnitude, it is linear over a much wider range than the phosphorylase method. An extremely sensitive method has been developed which permits the detection of as little as 0.05pmol of cyclic AMP and cyclic GMP [141, 1421. However, the cyclic nucleotides must be purified from interfering materials in tissues, and meticulous attention in the use of enzymes as analytic reagents is required. The cyclic nucleotides are isolated by thin-layer chromatography and hydrolysed with the aid of phosphodiesterase. 5’-AMP is converted to ATP by the combined actions of myokinase and pyruvate kinase. The ATP generated from cyclic AMP then serves as the catalyst in an enzymatic magnification system in which equivalent amounts of glucose-6-phosphate, proportional to several thousand times the ATP present, are generated as a result of the dismutation between hexokinase and pyruvate kinase. The glucose-6phosphate is determined fluorometrically. 5’-GMP is converted to GDP by the action of GMP kinase with the aid of creatine phosphokinase. The amount of GDP generated from cyclic GMP is magnified with the aid
3 16
CYCLIC AMP AND ADENYLATE CYCLASE
of an enzymatic cycling system composed of succinate thiokinase and pyruvate kinase. In this system, pyruvate equivalent to about 2000 times the GDP concentration is generated. The pyruvate is converted to lactate with the 2.id of lactate dehydrogenase and an equivalent amount of NAD' is produced and this is measured fluorometrically. PROTEIN BINDING ASSAY
This is now the most widely used method of assay for cyclic AMP. Kits are commercially available which make the assay a fast and relatively simple procedure [148, 1491. For cyclic AMP, a binding protein from muscle is utilised which presumably is the regulatory subunit of a cyclic AMP-activated protein kinase. The addition of a heat-stable inhibitor of the protein kinase [79] increases the affinity for the enzyme. The cyclic AMP binding activity is quantitatively adsorbed on cellulose ester (Millipore) filters, a simple means of separating bound from free ligand and so forming the basis for estimation of the extent of competition for binding sites between ['Hlcyclic AMP and unlabelled cyclic AMP to be assayed. Tissue extracts do not require purification, and the assay is sensitive to 0.05-0.1 pmol of cyclic AMP. The preparation of the cyclic AMP binding protein from bovine muscle has been described [ 1501. It involves homogenisation, centrifugation, pH 4.8 precipitation and ammonium sulphate precipitation, followed by fractionation on DEAE cellulose. The preparation binds 0.3 pmol of cyclic AMP per p g of protein and has an enzymatic activity of 24 pmol of 32 P per p g of protein per 10min. Over 200pg of the protein can be quantitatively adsorbed on a single Millipore filter. The yield of binding protein from 500 to 1OOOg of muscle is sufficient for more than 100000 assay tubes. The binding activity is stable for 18 months at -20°C. The protein kinase inhibitor is prepared from bovine muscle by homogenisation, boiling, filtration, and precipitation with trichloroacetic acid 1793. The cyclic AMP binding reaction is conducted in a volume of 50 p l of 50 mM sodium acetate buffer, pH 4.0. The reaction components include [3H]cyclic AMP, standard solutions or unknown samples, sufficient binding protein to bind less than 30% of cyclic AMP, and a maximally effective concentration of the protein kinase inhibitor preparation. At 0°C binding equilibrium is established within 60 min, and in the presence of the inhibitor preparation the equilibrium plateau is stable for at least 4 hr. Without the inhibitor preparation, a slow decline in bound counts occurs.
B. G. BENFEY
317
At equilibrium the mixtures are diluted and passed through a 0.45 p m Millipore filter. The filter is then washed and dissolved in a counting vial. At pH 4 the binding constant K , of cyclic AMP is 2-3 nM; in the presence of the inhibitor preparation the binding constant approaches 1 nM. [3H] cyclic AMP is utilised at 0.5-1 pmol/50 pl in the presence of the inhibitor or 2 pmol/50 pl in its absence. As these are saturating concentrations of [3H]cyclic AMP, the effect of added unknowns or standard solutions can be determined from a nearly theoretical decrease in the total bound ['Hlcyclic AMP which is linear when plotted logarithmically. A protein binding assay for cyclic AMP using rabbit skeletal muscle has been described [151]. A similar method uses a binding protein from bovine adrenal cortex whose preparation is simple and can be accomplished in 2-3 hr [152-1541. The preparation can be stored at -20°C for at least 3 months. Unbound cyclic AMP is separated from bound cyclic AMP by adsorption on charcoal and as little as 0.01 pmol of cyclic AMP per tube can be identified. For cyclic GMP, a cyclic GMP-activated protein kinase from lobster muscle is utilised. The cyclic GMP assay is somewhat less sensitive than the radioimmunoassay but has the advantage of the short time needed for the preparation of the binding protein; 0.5 to 1 pmol of cyclic GMP can be estimated [l55]. The cyclic GMP binding protein is prepared from lobster tail through the dialysed ammonium sulphate precipitation step described by Kuo and Greengard [76]. The binding activity is stable for months at -7O"C, but activity is lost with repeated thawing and freezing. The cyclic GMP assay is carried out in a volume of 100 p l of 50 mM sodium acetate buffer, pH 4.0. The other reaction components are 6-10 pmol [3H]cyclic GMP, standard cyclic GMP solutions or unknown samples, and cyclic GMP binding protein. Tubes are incubated at 0°C for 75 min and the reaction mixtures are then diluted, filtered, and counted. Cyclic AMP interferes with cyclic GMP binding 15-20% only when present in a ten-fold excess; 5'-AMP interferes 15% at 0.1 mM and 30% at 1 mM concentrations. Samples from tissues containing very low concentrations of cyclic GMP need purification prior to the assay. Small columns of Dowex 1 separate cyclic GMP from cyclic AMP with nearly quantitative recovery of both nucleotides. ATP is retained on the column and 5'-AMP is separated from cyclic GMP. Desalting procedures are essential when incubation media containing large amounts of salt must be concentrated prior to the assay. Binding is decreased 35% by 145 mM NaCl.
3 18
CYCLIC AMP A N D ADENYLATE CYCLASE PROTEIN KINASE ASSAY
The method is based upon the ability of low concentrations of cyclic nucleotides to activate protein kinases which catalyse the phosphorylation of protein substrates, such as histone, by ATP [156, 1571. The extent of phosphorylation is proportional to the amount of the cyclic nucleotides. The limits of sensitivity of the method are about 0.3 pmol for cyclic AMP and 0.5pmol for cyclic GMP. Purification on a Dowex 50 column separates the two cyclic nucleotides from each other and removes any substances, such as ATP, which might interfere with the assay. Cyclic GMP is further purified by column chromatography on aluminum oxide and Dowex 1. Cyclic AMP-activated protein kinase is prepared from bovine heart and cyclic GMP-activated protein kinase from lobster tail. For the cyclic AMP assay [y-32P]ATP (10pM) is incubated with 10 mM magnesium acetate, cyclic AMP standard (0-10 pmol) or water, histone mixture, cyclic AMP-activated protein kinase, and 0.1 M sodium acetate buffer, p H 6.0, for 5 min at 30°C. The reaction is terminated by the addition of trichloroacetic-acid-tungstate-sulfuricacid, the precipitate is dissolved in N NaOH, and the radioactivity counted in scintillation fluid. The conditions for the cyclic GMP assay are the same, except that cyclic GMP samples or standards up to 20 pmol are used with the cyclic GMP-activated protein kinase preparation. Using casein as substrate, cyclic AMP has been measured directly in crude tissue extracts by the stimulation of the rate of phosphorylation of casein catalysed by skeletal muscle protein kinase [158]. By using high concentrations of casein and [Y-~’P]ATP,the interference with the protein kinase activity by materials present in the tissue extracts is minimised and preliminary purification is not necessary. The phosphorylated casein is isolated on filter paper discs. As little as 0.5 pmol of cyclic AMP can be measured. The assay is rapid and simple. RADIOIMMUNOASSAY
This is the most comnonly used method of assay for cyclic GMP. Assay kits for cyclic GMP and cyclic AMP are commercially available. The method is based upon competition of the cyclic nucleotides with isotopically labelled cyclic nucleotide derivatives for binding sites on specific antibodies [84,143]. The cyclic nucleotides are rendered immunogenic by conjugating a succinylated derivative to protein. Succinyla-
B. G . BENFEY
319
tion occurs at the 2'-O-position, and the free carboxyl group of the derivative is conjugated to protein [1591. Rabbits are immunised by the injection of the cyclic nucleotide protein conjugate. Anti-sera can be obtained that cross-react minimally with other nucleotides so that chromatographic preparation of tissue samples is minimised or avoided. While [3H]cyclic nucleotides can be used as markers in the radioimmunoassay, greater sensitivity is achieved by using iodinated derivatives with greater specific activity and which can be assayed with greater counting efficiency. High specific activity derivatives of the cyclic nucleotides are prepared by iodinating ('"I or '"I) the tyrosine methyl-ester derivative of the succinylated cyclic nucleotides. Free and antibodybound I-labeled cyclic nucleotides are separated by ammonium sulphate precipitation and the precipitate is counted in a gamma spectrometer or a scintillation counter [160]. Binding equilibrium at pH 6.2 is reached in 24 hr, but sensitive and reproducible assays are obtained in 2-18 hours. The sensitivity and assay ranges are 0.01-2 pmol per tube for cyclic AMP and 0.01-1 pmol per tube for cyclic GMP. This permits the measurement of cyclic AMP in less than 0.5 mg and of cyclic GMP in less than 5 mg of most tissues (wet weight). By using specific cyclic AMP and cyclic GMP antibodies and ['*'I]- and ['311]succinylcyclic nucleotide tyrosine methylesters, the cyclic nucleotides can be assayed simultaneously, the precipitate being counted in a dual channel spectrometer. DETERMINATION OF RELATIVE LEVELS OF CYCLIC NUCLEOTIDES BY PRELABELLING OF TISSUES OR CELLS
Various mammalian cells and tissues, when incubated in a solution containing adenine, rapidly concentrate this purine and convert it into 5'-AMP and subsequently into ATP. The basis of the prelabelling technique [112] is that, in tissues and cells prelabelled with radioactive adenine, the relative amounts of cyclic AMP newly formed from ATP can be determined by isolating the cyclic nucleotide and measuring the amount of radioisotope which it contains [161]. The limitation on the measurement of small amounts of cyclic AMP with an acceptable precision, i.e., the limit of sensitivity of the assay, is a function of the specific activity of the radioactive adenine utilised for the prelabelling procedure. Thus, by increasing the specific activity of the radioactive adenine proportionally smaller amounts of cyclic AMP can be measured with the same precision.
320
CYCLIC AMP AND ADENYLATE CYCLASE
For any given tissue or cell preparation, it is desirable to compare the amount of radioisotope which the cyclic AMP contains with the absolute levels of the cyclic nucleotide. The result of such a comparison for heart tissue slices demonstrates that the radioactive cyclic AMP formed reflects the changes of the absolute levels of the cyclic nucleotide [113]. A similar result has been obtained in brain slices [114,115]. Thus, the prelabelling method provides a convenient means of studying dynamic changes in cyclic AMP levels in tissues and cells. It is essential that cyclic AMP be isolated free of other labelled adenine products, and this involves fractionation on Dowex 50 columns and/or paper chromatography of the tissue extract.
ASSAYS OF OTHER ENZYME ACTIVITIES PROTEIN KINASE
The activity of protein kinase is readily assayed by measuring the rate of toPa protein substrate, such as histone, transfer of 32Pfrom [ Y - ~ ~ P I A T casein, or protamine [77,80,82]. The phosphorylated substrate is collected on a filter, eluted, and counted. CYCLIC NUCLEOTIDE PHOSPHODIESTERASE
By using highly specific radioactive cyclic nucleotides and the snake venom nucleotidase, the most sensitive, specific and convenient method of assay for phosphodiesterase has been evolved. The assay is carried out both at 1 p M and 1 mM substrate concentrations [ 1621. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg2+,the reaction is stopped by brief heating in a water bath, the 5’-nucleotide formed is converted to the nucleoside by snake venom 5’-nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. The entire assay can conveniently be carried out in a glass scintillation vial [821. The reaction volume contains Tris buffer (pH 7 . 9 , Mg2+, [3H]cyclic nucleotide, and snake venom 5’-nucleotidase. After a I0-min incubation at 37”C, the reaction is stopped by the addition of a slurry of AGl-X2 anion exchange resin. The resin binds substrate cyclic nucleotides but does not bind nucleosides. Scintillation fluid is added and the amount
B. G . BENFEY
321
of [3H]nucleoside present in the vial is counted. Unreacted cyclic nucleotide which is adsorbed by the resin does not interact with the scintillation fluid and, therefore, does not interfere with the detection of unbound ['H]nucleoside. PHOSPHORYLASE
A convenient method of assay of phosphorylase activity is based on the rate of liberation of inorganic phosphate from glucose-1-phosphate, i.e., phosphorylase is measured in the direction of glycogen synthesis [6]. After a 10-min incubation at 37°C followed by precipitation with trichloroacetic acid, the supernatant fraction is analysed for inorganic phosphate by the method of Fiske and SubbaRow [163]. This involves the formation of phosphomolybdic acid from the phosphate present and the reduction of the phosphomolybdic acid to produce a blue colour whose intensity is proportional to the amount of phosphate present. The preparation of liver phosphorylase has been described [6,164]. Phosphorylase activity has also been determined in the direction of glucose-1-phosphate production [ 1651. The velocity is only one third of that measured in the direction of glycogen synthesis, but glucose-lphosphate can be determined with a lower blank and a higher sensitivity than when inorganic phosphate is measured. Glucose-1-phosphate is determined by conversion to 6-phosphogluconic acid with the simultaneous reduction of NADP by phosphoglucomutase and glucose-6deh ydrogenase and the fluorometric measurement of NADPH. Incubation is carried out in the absence and presence of 5'-AMP. The ratio of the activity in the absence of 5'-AMP to the activity in the presence of 5'-AMP is termed phosphorylase activity ratio [165]. Increases in this ratio reflect relative increases in the percentage of phosphorylase in the activated form. Activity assayed in the presence of 5'-AMP is designated total phosphorylase activity. +
PHOSPHORYLASE KINASE
The rate of activation of phosphorylase is measured at pH 6.0 or 6.8 and pH 8.2, and the result is expressed as the ratio of phosphorylase kinase activity measured at the low pH to that measured at the high pH. An increase in the activity ratio of phosphorylase kinase indicates transformation to the activated (phosphorylated) form of the enzyme. Activated phosphorylase kinase is 25 to 50 times more active than inactive phos-
322
CYCLIC AMP AND ADENYLATE CYCLASE
phorylase kinase at the low pH and has about twice the activity at the high pH. Thus activation of the enzyme results in a large increase in activity at the low pH and some increase at the high pH. Phosphorylase kinase activity in dog heart homogenate has been measured by incubation at pH 6.0 and 8.2 with ATP, Mg2+,and crystalline phosphorylase b [135]. The b to a conversion is stopped by dilution and the amount of phosphorylase a is then determined as described before [165]. In the rat heart, the assay has been carried out at pH 6.8 and 8.2 [166]. A blank containing heart extract, but not phosphorylase b, is run through the b to a conversion step and phosphorylase is added afterwards. This is necessary to correct for the formation from rat heart extracts of an activator of phosphorylase (possibly ADP) during the conversion from b to a. 5’-AMP formed by contaminating enzymes from ATP during the conversion of b to a is destroyed by adding adenylic deaminase after the b to a conversion step and incubating for 5 min. Formation of 5‘-AMP or other activators of phosphorylase b during incubation was not sufficient to interfere with the assay of phosphorylase kinase in the dog heart [135]. PHOSPHORYLASE PHOSPHATASE
Phosphorylase phosphatase (EC 3.1.3.17; phosphorylase phosphohydrolase) catalyses the conversion of phosphorylase a to phosphorylase b with the release of inorganic phosphate The reaction can be followed by the disappearance of phosphorylase a activity or by the release of radioactive phosphate from 32P-labeHedphosphorylase a [167]. In the indirect method, crystalline phosphorylase a is incubated with the enzyme preparation to be assayed, aliquots are withdrawn, diluted (which stops the phosphatase reaction), and the diluted solutions are assayed in the absence of 5’-AMP for remaining phosphorylase a activity by incubation with glucose-1-phosphate and glycogen and determining the inorganic phosphate released. In the direct method, the trichloroacetic acid-soluble 32Piis measured which is released from ”P-labelled phosphorylase a, which is prepared from phosphorylase b with [y-”P]ATP. GLYCOGEN SYNTHASE
The rate of incorporation of UDP[’4C]glucose into glycogen is measured in the absence and presence of glucose-6-phosphate [ 1681. The ratio of the
B. G . BENFEY
323
former to the latter is termed synthase activity ratio. A decrease in this ratio reflects a relative increase in the fraction of the enzyme in the glucose-6-dependent (D) form. The activity assayed in the presence of glucose-6-phosphate represents total synthase activity. Glycogen is separated from unreacted UDP['4Clglucose by spotting an aliquot of the reaction mixture on a square of filter paper and eluting the UDPglucose with 66% ethanol. The filter paper is then placed in a scintillation vial and the radioactivity of the ['4C]glycogen counted. The method can lead to an underestimate of the activity of glycogen synthetase in crude tissue extracts, as the presence of phosphorylase and (Y -amylase may cause the degradation of the radioactive glycogen [91]. TRIACYLGLYCEROL LIPASE
Rat adipose tissue homogenate has been incubated with cyclic AMP, ATP, and magnesium chloride in potassium phosphate buffer, pH 6.7, and the reaction terminated by pipetting aliquots of the mixture into chloroform plus potassium phosphate buffer, pH 6.8, for extraction of free fatty acids and assay by the method of Duncombe [93,169]. The latter is a colorimetric procedure for the determination of long-chain fatty acids in the range of 0.05 to 0.5 pmol. Partially purified lipase from rat adipose tissue has been incubated with ['4C]triolein as a substrate in sodium phosphate buffer, pH 6.8, in the presence of 1 mM NaCi and 20 mglml of bovine serum albumin 11701. Fatty acids were isolated by adsorption on Amberlite IRA 400 in a scintillation vial, excess solvent was removed, the resin washed, and the fatty acids were displaced from the resin with NCS solubiliser and counted. The activity of the lipase has also been assayed with the ultramicro method of Novak [171] to determine net free fatty acid release from endogenous substrate [ 1721. Incubation of rat adipose tissue homogenate was carried out in 40mM phosphate buffer, pH 6.8, in the presence of 30mM EDTA and 2% bovine serum albumin. Lipolysis in isolated fat cells has been measured by determining free fatty acids and glycerol [173]. Free fatty acids were determined after extraction with a two-phase heptane-isopropyl alcohol-water system [174], whereas glycerol was determined by a coupled enzymatic assay which involves formation of glycerol-3-phosphate by ATP, oxidation of glycerol-3-phosphate with NAD', and measuring the native fluorescence of NADH [175].
324
CYCLIC AMP AND ADENYLATE CYCLASE ATPase (EC 3.6.13 ; ATP PHOSPHOHYDROLASE)
In liver plasma membranes, ATPase activities were determined as follows [176]. (a) Total ATPase (Mg"ATPase plus Na'-K'-Mg''ATPase, Na' plus K'-activated ATPase): The medium contained 5 mM ATP, with or without [y-"P]ATP, 5 mM MgC12,66 mM NaCI, 33 mM KCI, and 25 mM Tris or histidine buffer, pH 7.4. (b) Mg*'ATPase: The same conditions were used except that 1 mM ouabain was added. (c) Na'-K'-Mg'"ATPase was estimated by subtracting the activity found under the conditions (b) from the activity found in (a). (d) Ca"-activated Mg2'ATPase: CaC12 was added in varying concentrations (0.1 p M to 10mM) to the medium used in (a) and (b). In these experiments 0.5 mM EGTA was added in the control assay. (e) Ca2'ATPase was estimated by using CaCL instead of MgCh under the conditions (a) and (b). In all cases the reaction, initiated by the addition of ATP, was run at 37°C for 10 min and was stopped by adding ice-cold trichloroacetic acid. Protein was removed by centrifugation and inorganic phosphate was estimated in the supernatant either by the method of Fiske and SubbaRow or by counting the radioactivity which was isolated as follows: To I ml of the supernatant were added 0.33 ml 7.5% ammonium molybdate and 1 ml isobutanol. After extraction, an aliquot of the organic phase was counted. By a sensitive method, ATPase activity in chromafine granule membranes has been measured by incubating 1 mM [y-3ZP]ATPin 10mM Tris-HCI buffer (pH 6.5) with 160 mM KCl, 5 mM NaCI, and 1 mM MgC12 11771. lncubation was stopped by adding ice-cold trichloroacetic acid, the tubes were centrifuged, the supernatant was treated with charcoal to remove [y-"P]ATP, and the radioactivity of the 32P,in the supernatant determined after centrifugation.
INORGANIC PYROPHOSPHATASE (EC 3.6.1.1; PYROPHOSPHATE PHOSPHOHYDROLASE [I781
The hydrolysis of inorganic pyrophosphate has been carried out in cacodylate buffer, pH 5.5 [179], trichloroacetic acid was then added to terminate the reaction, denatured protein was sedimented by centrifugation, and suitable aliquots of the supernatant solution were assayed for inorganic phosphate by the method of Fiske and SubbaRow [1631.
B. G. BENFEY
325
5‘-NUCLEOTIDASE (EC 3.1.3.5; 5’-RIBONUCLEOTIDE PHOSPHOHYDROLASE [180]
5’-[3H]AMP has been incubated in the presence of MgC12 in Tris-HC] buffer, pH 7.4, and the [3H]adenosine formed isolated for radioassay by fractionation on an ion exchange column of QAE Sephadex [181,182]. Also, 5‘-AMP has been incubated with MgSO, [14], the reaction terminated by trichloroacetic acid precipitation and inorganic phosphate determined in the supernatant by the method of Fiske and SubbaRow 11631. Tartrate has been included in the assay medium in order to inhibit acid phosphatase [183]. Inorganic phosphate has been assayed by the method of King [184].
CONCLUSIONS With the availability of commercial kits, the determination of cyclic nucleotides has become relatively easy and much less time-consuming than in early times. Nevertheless, the exact role played by these cyclic nucleotides in the body is still unknown, although by correlating effects on physiological and pathological functions with changes in cyclic nucleotide levels and probing with analogues of cyclic nucleotides, a large amount of information is gradually being gathered together. Possibilities for drug development based on the cyclic AMP system have been discussed [185]. One promising approach is to manipulate phosphodiesterase which can be selectively inhibited by drugs [ 1861. The relation of cyclic AMP to clinical chemistry and medicine has also been discussed [120, 187, 1881. The literature on cyclic nucleotides continues to be produced at a fast rate.
ACKNOWLEDGMENT Support by the Medical Research Council of Canada is acknowledged.
326
CYCLIC AMP A N D ADENYLATE CYCLASE
REFERENCES 1.
2. 3. 4. 5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
E. W. Sutherland, Science, 177 (1972) 401. T. W. Rall, E. W. Sutherland and J. Berthet, J. Biol. Chem., 224 (1957) 463. W. H. Cook, D. Lipkin and R. Markham, J. Amer. Chem. Soc., 79 (1957) 3607. E. W. Sutherland and T. W. Rall, J. Amer. Chem. Soc., 79 (1957) 3608. D. Lipkin, W. H. Cook and R. Markham, J. Amer. Chem. Soc., 81 (1959) 6198. G . A. Robison, R. W. Butcher and E. W. Sutherland, Cyclic AMP (Academic Press, New York, 1971). K. A. Drlica, J. M. Gardner, C. I. Kado, I. K. Vijay and F. A. Troy, Biochem. Biophys. Res. Commun., 56 (1974) 753. B. Kessler and R. Levinstein, Biochim. Biophys. Acta, 343 (1974) 156. D. F. Ashman, R. Lipton, M. M. Melicow and T. D. Price, Biochem. Biophys. Res. Commun., 11 (1963) 330. J. P. Perkins, in: Advances in Cyclic Nucleotide Research, ed. P. Greengard and G. A. Robison (Raven Press, New York) Vol. TI1 (1973) pp. 1-64. E. R. Stadtman, in: The Enzymes, ed. P. D. Boyer (Academic Press, New York) Vol. VIII (1973) pp. 1-49. M. Hirata and 0. Hayaishi, Riochim. Biophys. Acta, 149 (1967) 1 . M. Tao and F. Lipmann, Proc. Natl. Acad. Sci., U.S.A., 63 (1969) 86. D. Id. Severson, G. 1. Drummond and P. V. Sulakhe, J. Biol. Chem., 247 (1972) 2949. S. I
= Thallium(II1j nitrate = -CO.O.CH,CCI,
STRUCTURAL CONSIDERATIONS The naturally-occurring penicillins and cephalosporins, and thousands of their semisynthetic derivatives, are based on two fundamental structural units, the penam (1) [2] and cepham (2) [3] systems. The numbering of these bicyclic systems is different from that usual for heterocycles [4]. The numbering system for penicillin is also used in the case of cephalosporins and other derivatives, including new members of the group of p-lactam antibiotics [3].
SUBSTITUTIO 1 IN T H E T H I ZOLIDINE AND THIAZINE RINGS The classification of structural variations is not easy: in every arrangement there are many overlaps because of multiple substitution. Strictly speaking, only three types of ring substitution can occur in penams,
398
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
namely at positions 1 , 2 or 3, and five in cephems, at positions 1 , 2 , 3 , 4 or 3 and 4, connected with double bond shift or disappearance. POSITION 1
In the presence of oxidizing agents (e.g. sodium metaperiodate, hydrogen peroxide, m-chloroperbenzoic acid, t-butyl-hypochlorite, iodobenzene dichloride, etc.) both penicillins and cephalosporins undergo facile oxidation to either sulphoxides or sulphones. The esters of penicillin sulphoxides (4) were described in 1949 [l]. It was found later [5] that the free acids could be oxidized by periodate. When a 6(7)-p-acylamino side-chain is present in the molecule the resulting reagent-approach control due to the N-H proton promotes the formation of the (S)-sulphoxide (4a) [6,7].
The oxidized derivatives possess a very much lower antibacterial activity. The activity retained by the (R)-sulphoxides (4b) and the sulphones ( 5 ) is about five times higher than that of the corresponding (S)-sulphoxides [8]. Other ]-substituted derivatives, such as sulphonium-ylides (6), have been described as intermediates during exposure to diazo compounds, but none of these has been prepared [9,10].
SUBSTITUTIONS F 4INLY
T POSIT10 1 2
Some new penicillins were obtained by the reaction of &substituted cysteine derivatives with an oxazolone [l 11. The antibiotic activities of these penicillins (71, prepared only in crude form, were similar to that of benzylpenicillin.
J. Cs. JASZBERENYI AND T. E. GUNDA
3 99
( 7) 79
Y=Y'=Me
7b 7c
Y=Y'=Et Y-Me YlEt
7d
Y-H
q-Me
A large number of these compounds have emerged during the search for new cephalosporin syntheses. The first derivatives were the 2acyloxymethylpenicillins (8) and (9), which are usually by-products in the
I
I
(12)
(9)
(Pr'C0)20
1
oxid
2 Ac,O
ring expansion of penicillin sulphoxides [ 12-16]. The activities of the corresponding acids are lower than those of the parent penicillins (Table 8.1).
The 2P-halogenomethylpenicillins (lo), which are easily convertible into cephems, were obtained by Japanese scientists [ 171. Unfortunately, no mention was made of the biological activity. Starting from penicillin, the 2-carboxypenam derivatives ( I 1a and b) were synthesized [ 181. Since their structures are not so closely related to those of normal penicillins as in the previous example, the decrease in antibacterial activity is more drastic. With the aid of an interesting cephalosporin ring-contraction, the penam (13) was obtained [ 9 ] . This compound did not exhibit significant activity.
400
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS Table 8.1. ANTIBACTERIAL ACTIVITY OF 2-ACYLOXYMETHYLPENICILLINS MEASURED AS THE MINIMAL INHIBITORY CONCENTRATION
Compound
8 a , R = G, R'=H 8b, R = G , R'=H Penicillin G t121 8a, R = V , R'=H 8a, R = G , R'=H 8a, R = T , R'=H 12, R = V , R'=H Penicillin V t531
Strain S. aureus 663 sensitive (dml)
S. aureus 11,127 resistant (1*s/ml)
S . aureus 209P
0.3
125
-
0.16
31
-
0.005
125
-
-
-
135
-
-
100
-
-
140
-
-
128
-
-
1800
(units/rng)
J. CS. JASZBERENYI AND T. E. GUNDA
401
G N H ~ J - ~ ~ ~ ~ ~ G~N H~f l s Y C o 2 H +
0 Bacterium
0 (ria)
H. influenzae Staph. aureus
MIC hg/rnl)
(11 b)
MIC (pg/ml) 250 16-125
250 16-31
Penam derivatives with different C-2 and C-3 substituents were synthesized by the group at Astra Lakemedel Co. [19,20] (14) using a new p-lactam ring-closure, but they do not report any activity data either. 1. N2CHCO2H
GN 0 H a J M eCO2R
2. Zn/H+
G N H F S G E t 0
C02 H
In the cephalosporin sulphoxides the 2-methylene group contains moderately labile protons. The 2-methylene group has some allylic character, but the 2-halogeno derivatives can not be obtained from the A’-sulphides; however, the corresponding sulphoxides do undergo this
ref lux
I R1 R2andR3
are H, Me,Et or Ph
X : Br, N3 (14
402
MODIFICATIONS AND ANALOGUES OF p-LACTAM ANTIBIOTICS
reaction. This problem is discussed in detail in the monograph Cephalosporins and penicillins [21]. The different methods for introducing halogen or -OR substituents into position 2 are summarized in Table 8.2. The activity of these derivatives is reduced, but is better than that of the penicillins if penicillinase-producing strains are used as test organisms (Table 8.3). The effect of the bulkiness of the 2a-substituents is well illustrated by the data of Table 8.4. The 2-exomethylene derivatives [60] (24, 25) have opened a new route for the synthesis of further compounds, because the 2-exomethylene group can easily be transformed into other derivatives. The Mannich reaction providing (24) through the action of formaldehyde and amine salts on cephalosporin-(S)-sulphoxides is highly stereospecific: no reaction has been observed in the case of cephalosporin-(R)-sulphoxides [63]. Scheme 8.1 shows the derivatives obtained from the exomethylene parent compound (24). For the bioassay results, see Tables 8.5, 8.6 and 8.7. As far as compounds of type (32) are concerned, their activities against Staphylococcus aureus lie in the range 0.4-13 pglml, but they have no significant activity against gram-negative strains. In the presence of 25% human serum their activity is highly reduced (MIC > 50 Fg/ml) [25]. Table 8.2. METHODS FOR INTRODUCING ALKOXY AND ACYLOXY SUBSTITUENTS INTO POSITION 2 O F CEPHALOSPORINS
N R $H c“
phd :Jo
0 0
C02TCE
0 (151
”Me
R=H
(18)
R=H
C02R2 (201
R% H
A3-Cephem
Reagent
Product
Reference
(15) R = H (16) (18) R = H (20) R4 = H
NBS, AIBN MeOH, NEt, SOZCI*,py 1. CI,, R ~ O H 2. Zn, H’ if R2 = TCE 1 . Ac20, reflux 2. Zn, H’ 1. Pb(OCOR’)), 2. Zn,H’
(16)=(15, R = B r )
22 22 23, 24 23, 24
(18) R = H (20) R’ = V, RZ= TCE, R’ = H, OAc
(17) = (15, R = OMe) (19) = (18, R = C1) (21) = (20, RZ= H, R4 = OR’)
(22) = (20, R ’ = V , R2 = R’ = H, R4 = OAc) (23) = (20, R ’ = V , R2 = H, R4= OCOR’)
39, 59 *
39, 59
Table 8.3. IN VITRO ANTIBACTERIAL ACTIVITY OF 2 a - O SUBSTITUTED CEPHALOSPORINS [53], MEASURED AS THE MINIMAL INHIBITORY CONCENTRATION (pglml) Compound
Strain
S. aureus penicillin res. V-41 (20) R' = T, R2= H, R3 = OAc, R4= OMe (20) R' = T, R2 = H, R3 = OAc, R4= OEt (22) Keflin Penicillin V
S. aureus rnethicillin res. X-400
E. coli N-10
Klebsiella pneumoniae X-26 9.8
0.5
> 20
116
3.0
> 20
> 200
180
16.0 0.3 > 20
> 20 > 20 > 20
82 11.4 >loo
100
0.7 >loo
Table 8.4. IN VITRO ANTIBACTERIAL ACTIVITY* OF 2-ALKOXYCEPHALOSPORINS [53] Compound
Strain
S. aureus (SA) 3055
B. subtilis X-12
Sarcina lutea X-186
Proteus vulgaris X-45
7-P henoxyacet32 amido-DCA (20, R'=V R2=R3=R4=H)
36
33
22
(20), R' = V, R2 = R' = H,
23
28
21
-
(20), R' = V, R2= R3 = H, R4= OEt
17
23
17
-
(20), R' = V, R2= R' R4= OPr'
trace
16
trace
-
30
45
45
-
19
30
22
14
R4= OMe
= H,
Keflin (20), R' = T, R2 = R' R' = OMe
*
= H,
These data are given as zone diameters in millimeters, determined by disc-plate procedure with 6 m m discs [295]. These and other data [53] are personal communications of Dr Douglas 0. Spry, Lilly Research Laboratories, Eli Lilly and Co., t o whom the authors are greatly indebted.
404
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
On the basis of the work of CIBA-Geigy A. G. and the Woodward Forschungsinstitut, the original total synthesis of cephalosporin has been extended with the incorporation of many fascinating details to the preparation of new derivatives [64-731. Some features of this extensive work are shown in Scheme 8.2. Although an analysis of antibiotic activities toward different strains has not been published so far, it was found that (39) ('Cephalocillin') and (40) are able to inhibit S. aureus, Proteus vulgaris and Bacillus megaterium [26]. 0
r
uJ::R2 C02ti
a ,;*. 0
CH~R~ c02Rl
axH2 C02H
J. CS. JASZBERENYI AND T. E. GUNDA
405
Table 8.5. IN VITRO ANTIBACTERIAL ACTIVITY O F DIFFERENT 2-SUBSTITUTED CEPHALOSPORINS AND TRICYCLIC ANALOGUES [53] MEASURED AS ZONE DIAMETERS IN MILLIMETERS*
Compound
Strain
36a, R = V, R2= H, R3= C02Me, R ' = Me ' = Me 36b, R = T, R2 = OAc, R3= C02Me, R 36c, R = T, R2= OAc, R' = Ac, R' = Et P henoxyacetamido-3-cephem-4-acid 38a, R = V, R' = C02Me, R' = Me 38b, R = V, R3 = C02H, R" = H 35, R = V, R' = CO,H, R' = H Keflin
S. aureus ( S A ) 3055
B. subtilis X-12
Sarcina lutea X-186
19 28 30 29 26 15 21 28
23 28 31 36 24 17 21 44
13 28 29 26 23 10 13 40
~~~~
*
~
Data taken from Dr D. 0. Spry, The Lilly Research Laboratories, Eli Lilly and Co., to whom the authors are greatly indebted. Solution (1 mg/ml) in buffer at p H 6.5. Determined by 6 mm discs.
The key compounds of Sheehan's new total synthesis are (41a) and (41b). These provide not only the construction of classical P-lactam, but the basis of new variations not accessible from natural sources [27-321. The crucial step is the condensation of (41a) and (41b) with an appropriate Table 8.6. ANTIMICROBIAL ACTIVITY OF 2-SPIROCYCLOPROPYL DERIVATIVES [53] MEASURED AS T H E MINIMAL INHIBITORY CONCENTRATION (pglml)
(28b) (28a)
:I
OAc
0 COZH
Compound
Strain S . aureus penicillin res. V-41
0.2 28a 28b 0.5 Keflin 0.3 Penicillin G >20
S. aureus methicillin res. X-400
E. coli N-10
P. aeruginosa X-528
K . pneumoniae X-26
136 150 20 > 20
>200 180 11.4
>200 >200 > 200 > 100
64.7 6.2 0.7 63.0
-
406
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
Table 8.7. IN VITRO ANTIBACTERIAL ACTIVITY OF 2-ALKYLCEPHEMS AND EXOMETHYLENE DERIVATIVES [60]* MEASURED AS THE MINIMAL INHIBITORY CONCENTRATION (pglml)
Substituents
Strain Gram-positive?
R' R 2 V H V H V H V H T OAc T OAc T OAc Penicillin
*
R3
R'
H H M e H H M e exo-CH2 H H§ exo-CH2 XI' H G
V-32 1.211.0 12.8/> 20 15.0/> 20 0.611.0 0.611.O 2.1/> 20 0.69 > 20
Gram -negative
N-IOS
X-26
0.711 .O 10.4/> 20 12.2/> 20
> 50 > 50
> 50
> 50 > 50
> 50
> 50
> 50
0.511.0 0.5/1 .O 1.8/> 20
> 50
0.6
-
V-84
> 20
40.0
X-68
19.5 29.0
18.8 4.8 23.6
>50 4.4 7.6
41.0
63.0
40.0
Test by gradient plate procedure.
t Benzylpenicillin-resistant strains of S. aureus, MIC's in absence/presence of human serum. $ N-10: E. coli, X-26: Klebsiella pneumoniae, X-68: Aerobacter aerogenes.
8 Keflin. 'I X = CH,.S.Ph-p-Br [25]. T [251. a
No data.
ketocarboxylic acid derivative, giving rise to the desired bicyclic product [33-35]. Similar derivatives (42) were reported [36], and a new cephalocillin synthesis shown in (43-46) has been developed recently [37,38]. COMPOUNDS WITH SHIFTED DOUBLE-BOND
3-Cephems (or A3-cephalosporins) can be converted into two isomers, with the double bond in the 2-position (A2-cephalosporin) or 3exomethylene cephalosporins. A convenient means of obtaining 2-
J. CS.JASZBERENYI AND T. E. GUNDA
407
cephems is the alkaline hydrolysis of 3-cephem esters (48) [13], but the esterification of A3-acids via the acid chloride [40] or double-bond isomerization with an amine base offer alternative routes to (47) [41,42]. 4-Substitution may also lead to shifting of the double-bond. These compounds together with their reported antimicrobial activity are listed in Table 8.8. They are clearly much less active relative to 3-cephems or penicillins, due to the altered conformation of the whole system. This will be discussed later. Neither of the interesting 3-exomethylene derivatives (48a) and (48b) [43-49] exhibits notable activity. The anhydropenicillin (49) [50-52] may also be classified as a derivative containing an exocyclic double-bond. This compound is often cited as a typical instance contradicting the ‘lability’ of p-lactams, a misbelief descending from the early period of penicillin chemistry. Penicillin G is indeed labile in aqueous solutions, but there is also much evidence as to the stability of other p-lactam compounds. The anhydropenicillin can undergo melting without decomposition. It is ineffective as an antimicrobial agent. It is worth noting that the anhydro-a-phenoxyethylpenicillene(50), which can be obtained from (49) by oxidation with mercuric acetate, and which does not contain a p-lactam ring, has significant activity (e.g. the MIC values toward S. aureus Smith and Kleb. pneumoniae are 0.5 and 3.13 pg/ml, respectively). In contrast, the corresponding penicillenic acid is characterized by a complete lack of activity [till. The A3-doublebond makes a considerable contribution to the bioactivity of cephems. Further, cephams having no double-bond exhibit no activity. These compounds can be obtained by catalytic hydrogenation [74], or as intermediates during the ring expansion of penicillin sulphoxides. The ratio of (51) and (20a) and the nature of the group R2depend on the conditions [12-14, 75-771. Recently, the formation of 3-halogenocephams (52) was observed [ 171 when 2p-halogenopenicillins (10) were kept in dimethylformamide at room temperature. In spite of their inactivity, they are useful intermediates. Mention must be made of two further compounds, (53) [lo] and (54) [36], where X =halogen. SYSTEMS CONTAINING THREE OR MORE RINGS
Strictly speaking, the 2-spirocyclopropyl derivative (28) also belongs to this group, but only compounds bearing extra rings at positions 3 and 4 are
408
MODIFICATIONS AND ANALOGUES OF 6-LACTAM ANTIBIOTICS
I
LF3C02H
CF3C02H
R'-NHa> 0
HO
C02TCE 140)
0
C02TCE CF3COp
TC-NHa70
ICF3COJ20
I
Scheme 8.2
J.
hS
N
CS.
JASZBERENYI AND T. E. GUNDA
P = TCE
H2Nay"
R - N H ~ ' ~ ; :
D H F I H 4 , CrCl
0B
H
409
NHC02Q
0
o=me
X
0
Y = -NCO
XZ-NHC02C
X: -OH
5
R:-H R'; -R
I
1I I R-NH-P
R-NHaS-CH7X 0
1
&,&+I CHO
I
I I.
..
1
C02But
I x 2
-OH
X = -OH
x=
-Cl
x=
-
COZH CF3C02H. -20'
R-M-7-yH20Troc
CHO
Y = -H. -Me
I
Scheme 8.2 (contd.)
-CI
I-;
1
410
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS
PMNn
discussed here. Some of these compounds were prepared via nucleophilic displacement of the 3'-acetoxy group. Using this nucleophilic displacement, a vast number of compounds substituted on 3' (or C-10) have been synthesized; some of them are of pharmacological interest (see Table 8.16),but this rapidly developing field of 'semisynthesis' is not treated in this review. This topic has been surveyed [84a]. The bioactivities of lactones (59) are comparable with those of the 3'-acetoxy parent compounds but in the presence of serum protein, their
Y, Y'= H,Me
J. C S . JASZBERENYI AND T. E. GUNDA
H H
1.
RN
NaNdDMSo
j 2 1.. P LiN(Pri), hMp
B @
2. H ~ l c a t .
C 02H
3. acylation 6. hydrol.
CO&
411
Table 8.8. ANTIBACTERIAL ACTIVITY O F 2-CEPHEM ACIDS
> z R’
R2
T
R3
R4
R3
Activity
(CHz),-COzEt Me
H CO,H
H Me
?
R
COzH H
V
H
H
CF,
OH
CHSR Me H H Me H
CH2R’ Me Me Me Me Me
COzH CO,H CO,H C02H CO,H CO,H
H H OAc Me Me H
~~
Reference ~
1
‘Actitre against S. aureus and Str. pyogenes’ ‘Deutliche wenn auch geringe antibakterielle wirkung gegen Staphylococcen ’. ‘No activity’ ‘No activity’ 20 pg/mI against gram-positive and 200 pg/ml against gram-negative strains. 15 units/mg
58 54
55
56
25 53 13
U
J. CS. JASZBERENYI AND T. E. GUNDA R
-
413
V, G,T
R' = OAc
HLcJcH2
RN0
electroreduction
CO2H
I
R'
- 0-N 5 -S Bz
0
CuCIZ/MeOH I
t
x=
-CI
- OCOzEt
(L)
acid catalyst.
' " I B0? O R 2 COzR1 Me
+
RNHfiJ 0
C02R'Me
414
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
activities toward gram-positive test micro-organisms are vastly diminished. Gram-negative activity even disappears in the presence of a phenylglycine side-chain [85]. As the lactones are resistant to chemical hydrolysis, their interaction with the bacterial enzyme system in acid form is not probable. Table 8.9 shows the comparison of (55) and (59) with the parent compound (20c) [86]. The derivatives (60), (61) and (62) containing a 3-spiro function [87] possess only a slight activity, which may be due to some reversion to uncyclized forms. The 3-cephem double-bond is very unreactive and does not undergo most of the reactions characteristic of a double-bond. As a dipolarophile, on long exposure to diazomethane it yields pyrazolinocephams (63) and (64), which lack antibacterial activity [88-901. The dispiro compound (65) [91] and also (66) and (67) [92] are of interest, even if they are practically ineffective (Table 8.10). In fact, these and the previously-described compounds well emphasize the role of the A’-double-bond.
J.
CS.
JASZBERENYI AND T. E. GUNDA
415
Table 8.9. IN VITRO ANTIBACTERIAL ACTIVITY OF 3,4TRICYCLIC CEPHALOSPORINS [86] (MINIMUM INHIBITORY CONCENTRATION, pglml)
Compound
S. aureus
S. aureus +5% human serum albumin
Deactivation coeficient
(20) R = T (59) R = T (55) R = T
0.05-0.1 0.1-0.25 0.15-0.2
0.1-0.25 100 1-2
2-2.5 400-1000 5-10
DERIVATIVES O F THE 3(4)-CARBOXYL GROUP
For an excellent review of this field up to 1966, see Hamilton-Miller [93]. The different amide derivatives (68) of penicillin were investigated in the very early period. Their syntheses started from penicillanic acid anhydride, but later different mixed anhydrides were applied for this purpose [94-981. Moreover, other procedures described in peptide chemistry are also useful, e.g. the DCC method [99], or synthesis through active esters [loo]. Their characteristic feature is that the retained in vitro activity, which is only 5-15% of that of the parent free acid, nearly disappears in vivo or in vitro if serum is present. For example, the amide of penicillin V has an activity of 219unitslmg toward S. aureus, which decreases to 3.3-23 unitslmg in the presence of serum. The activity of N,N-bis-
416
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS
Me
benzylpenicillanylhydrazineis only 22 unitslmg [94]. In the case of mouse protection tests the effectiveness of penicillin G or V amide and phenethylpenicillin amide is practically zero [ 1011. However, if the substituted amide group can easily undergo cleavage in vivo, then the activity returns. The 0-hydroxylamine derivatives (69) [1021 exhibit high inhibition effects toward penicillinase-producing S. aureus. Similarly, the a-alkoxy- and a-alkylmercapto-alkyl amides of penicillin Table 8.10. IN VITRO ANTIBACTERIAL ACTIVITY OF 3,4TRICYCLIC CEPHALOSPORINS (66) AND (67) [92]; MINIMAL INHIBITORY CONCENTRATION (pg/ml) Compound (66)
Strain
S. aureus Oxford Str. faecalis Str. pneumoniae C N33 E. coli NCTC 10418 P . rettgeri ~
*
~~
62.5 (125)* 125 62.5
>250 250 ~
Compound (67)
> 250 (> 250)
> 250 > 125 > 125
> I25 ~~-
Serial dilution in 5% blood agar; figures in brackets are MIC values in nutrient broth. These data are taken from the personal communication of Dr R. J. Stoodley, (The University of Newcastle upon Tyne), whom we thank for these in vitro data.
J. Cs. JASZBERENYI AND T. E. GUNDA
J
\
CICOZEt
417
RNHa2 0
CON R i
have proved to be effective in mouse protection tests [loll. The antimicrobial activities of these types cannot be explained in terms of a simple hydrolytic process, as proved by the fact that the activity is retained in the presence of staphylococcal peniciliinase. Presumably enzymatic hydrolysis takes places after the binding to transpeptidase and neither before nor after this step can the p-lactamase interact with the antibiotic molecule having a transformed carboxy group. a-Amino acids attached to the carboxyl group of penicillin and cephalosporin are also of interest. The biological data of different authors are not always in agreement with each other, due to the lack of systematic investigations. On the other hand, bioassays have been carried out on the protected amino acid derivative in some cases. Benzyl esters of glycine and phenylalanine amides of penicillin V (69c and d) [991 were found to be 1/40 and 1/10 times as active as penicillin V. The inhibiting properties of glycine amide of penicillin V (69e) appear more promising [loo]; activity data related to penicillin V, as determined by the paper-disc method are given in Table 8.11. Chauvette and Flynn [42] found the alanine derivative of cephalothin to have only slight activity.
418
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS Table 8.11. RELATIVE IN VITRO ANTIBACTERIAL ACTIVITY DATA OF THE GLYCINEAMIDE OF PENICILLIN V [lo01
Activity in %*
Strain Proteus sp. S. aureus Str. pyogenes Str. viridans Str. faecalis
*
38 88 100 100
96
Antibacterial activity data are given related to that of penicillin V against the above mentioned strains, as determined by the paper-disc method.
The dipenicillins (70) [lo31 can also be included in this group. It was believed that the penicillin moiety would play the roIe of a side-chain capable of inhibiting sterically the approach of penicillinase, and thereby an enhanced stability toward the enzyme would occur. According to the bioassay, the more sensitive the test strain was to the parent compound, the more activity was retained. Usually the esters proved poorer than the free acids. A functional derivative of penicillin amide is the nitrile (71) [104], the activity of which is even lower. The different anhydride and thioanhydride derivatives may be regarded merely as intermediates useful for obtaining amides and esters. The penicillin anhydrides were synthesized long ago [IOS,1061. Some cephalosporin anhydrides too were described recently [ 1061. Cephalothin anhydride (72a) was reported to be effective against S. uureus Smith in a dose of 0.24 pglml. Assuming quantitative hydrolysis, the antimicrobial
(70)
R = G,M,O,C!O R'= H or BZ
J. C S . JASZBERENYI AND T. E. GUNDA
419
RNHf12coNH2 Ts CI
0
-H20
RNF3CN
(71) R Activity
v
10units/mg
G 70units/mg
effect of benzylpenicillin thioanhydride (73) should be identical with that of the corresponding penicillin. In the majority of tests this assumption proved to be valid, but when B. subtilis was used as test organism an anomalously high activity ( - 4000 units per mg) was observed [107]. Again, this fact could be explained only on the basis of hydrolysis in the cell itself. One comes up against a similar problem in the case of (74), described in 1965 [lot?]. It was found that strains sensitive to penicillins showed the same MIC values toward both penicillin and (74); this was not observed in tests with resistant strains (Table 8.12).
420
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS Table 8.12. IN VITRO ANTIBACTERIAL ACTIVITY OF DIFFERENT PENICILLIN DISULPHIDES [lo81 (MINIMAL INHIBITORY CONCENTRATION IN pglml)
Compound
S. aureus (sensitive) S. aureus (resistant)
(74a), R = V (74b), R = G (74c), R = Oxa (74d), R = M
0.016 (0.04)* 0.012 (0.012) 0.25 (0.2) 1.0 (1.25)
*
75 (6.25) 37 (> 100) 0.6 (0.4) 3.1 (2.5)
The figures in parentheses are the MIC values of the corresponding penicillins.
Among the functional derivatives a great number of penicillin and cephalosporin esters have been synthesized to date. With the exception of the ‘active’ esters, these derivatives have served only as protected intermediates for further chemical transformations. The 3(4)-carboxy group can usually be functionalized by more or less standard procedures, and instead of details, therefore, their preparations are summarized in Table 8.13. The different neutral esters are nearly or wholly ineffective, but in vivo they show activity when administered other than orally to mouse, rat or guinea-pig. In vivo, in man, monkey or dog these esters are not active. In the case of alkyl and aralkyl esters a dosage for complete inhibition against Strept. hemolyticus in mouse is 0.01-0.05 glrnouse (intraperitoneal) vs. 0.1 g/mouse for the sodium salt of benzylpenicillin [109]. These data can be explained by the fact that the serum of these animals contained an esterase capable of hydrolyzing the esters to the free acid [ 124-1 261. The first really active ester was reported in 1948 [105], namely the diethylaminoethyl ester of penicillin G (75a), which showed full activity in the plate bioassay due to its ready conversion to the free penicillin. The hydroiodide of this ester (Estopen, Lactopen, Leocillin) was further examined [127]. The difference of its activity in relation to the free penicillin might lie in the fact that the ester group is functionalized in cationic form at physiologic pH values, while the free penicillins and their salts are in anionic form [128]. More recently reported active esters are the different acyloxymethyl esters [129]. The pharmacology of penicillin G acetoxymethyl ester (Penamecillin) was investigated in 1966 [130]. A further derivative is the acetoxymethyl ester, or preferably the pivaloyloxymethyl ester, of am-
J . c s . JASZBERENYI AND T. E. GUNDA
Table 8.13. METHODS FOR PREPARATION OF PENICILLIN AND CEPHALOSPORIN ESTERS Compound
Method
Reference
With diazoalkanes
109, 13
Via mixed anhydrides
42, 97, 110-113
With dicyclohexyl carbodiimide
42
With halogen compounds
12, 114, 115
BY silylation
116-118
With trialkyltin derivatives
119, 120
With diazoalkanes
14,19, 121
With dicyclohexyl carbodiimide
42, 122
Via acid chloride
40, 60
With isobutylene
123
Penicillin esters
Cephalosporin esters
a,) R'= -CH$H2NEt2 R- G b.) R'= CH20C0 Me R- G
-
C.]
-
R'= CHZOCOC M e 3 R- A
42 1
422
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
picillin [114] (7.52). The serum-level vs. time curves attained after administration of these drugs (Figure 8.1) clearly show that these derivatives are more efficiently absorbed than ampicillin from the gastrointestinal tract. Moreover, the amount of ampicillin excreted in the urine is more than twice as high as after administration of free ampicillin.
0
1
2
3
L
5
6 Hours
Figure 8.1. Mean serum levels of ampicillin in normal volunteers following oral administration of250 mg of ampicillin (-0-0-1 and 358 mg of piuampicillin .HCI ( 250 mg ampicillin) (- x - x -) immediately after breakfast [ I 141
-
Many other active esters have been described, primarily in the patent literature, e.g. quinoline esters [131], and a series of others [132-1341. Other 3(4)-substituents derived from the carboxyl group are the reduced forms, first of all the aldehyde [lo81 (76), but nothing was reported about their activity. The sodium borohydride reduction of penicillin mixed anhydrides led to the penicillanyl alcohol [28] (77), which was further converted into nitrogen derivatives [ 1351 (78a-d). According to the bioassay carried out on S. aureus Smith, the reduction of the carboxyl group brought about a decrease in antimicrobial activity, e.g. the MIC values are 3-6 pglml for (77al-a3) vs. 0.031 pglml for ampicillin. The azide derivative (7th) proved to be effective only against S. uureus 209 (12 pglml) [135], whereas (78c and d) were ineffective in vitro. In vivo, (78b) in a dose as high as 50 mglkg was able to protect mouse infected with S. uureus Smith when administered subcutaneously, but was ineffective orally.
J . CS. JASZBERENYI AND T. E. GUNDA
HLc2
RNHa2
cox
RN0
423
0
cyx
2. M s C l
Compound R 770,
L’
77Q.2
G
7703
A
2. Py
It is worthwhile to make a comparison between cephalosporins and penicillins subjected to the elongation of the carboxyl group. Similarly to the previous case the penicillins (79) [136-138] were found to be more effective (2 and 0.5 pglml for (79dl) and (79d2), respectively, and the same values for (79el) and (79e2), against S. aureus 209P [I391 and [140]), than the corresponding cephem (MIC 25-100 pglml for R’= hydrogen in (80~3)against S. aureus, and MIC>200pg/ml against a variety of gram-negative strains) [141,193]. A number of diff erently-substituted penam derivatives (79f, g) have been obtained as intermediates in the total syntheses developed mainly by the Woodward-Heusler and Sheehan groups, but no exact biological data are known. For the corresponding hydroxy penams and cephems, see [551 and 11421. The 3-hydroxypenam is in equilibrium with the open form ((81)=(82)), but its methyl ether (83) and acetyl derivative (84) can be prepared pure. It is most surprising that the derivative (85) possesses an appreciable biological effect: MIC values, S. aureus 6.3 pglml, Strept.
424
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
pyogenes 1.6 pglml and Diplococcus pneumoniae 0.4 pglml [143]. New 4-substituted derivatives (86H91) have been reported recently [38]. Cephalosporins without a 4-carboxy function are known, but these, (92) [131 and (93) [1441, are not biologically interesting.
0) X=-CO?K
b) X - - COCl C)
X=- COCHN?
d) X = - CH*.CO?H e ) X--
CH2.CH2.C02H
f ) X--NCO g)
x--
NHCO~R’
C
C
I
1) R = V
3 R=G 3lR=T
c ) X=-C+CO,H AcOH
d ) X--COCi?.OAc
1
425
J . C S . JASZBERENYI AND T. E. GUNDA
OEt
OH Z,Y=H~Me
(86)
SOCl2
(901
i
1
LiN(SiMej\
2. C0CIz
? M~
xylene
SUBSTITUTION AT POSITION 5(6) In spite of the fact that these compounds are not so widely known as the classical semisynthetic derivatives, a large number of 5-substituted penams and 6-substituted cephems and cephams have already been produced. These compounds are usually inactive, due to the lack of peptide character and other essential substituents (e.g. the 3(4)-carboxyl
426
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
group). In other cases the p-lactam moiety is stable enough to be resistant to hydrolysis leading to the decrease or disappearance of bioactivity. Among many others, the groups of Sheehan and of Bose have done thorough and fundamental work in this field. Unfortunately, few biological data are accessible from this set of derivatives, but for the previouslymentioned reasons, and because of permeability-solubility problems and the steric hindrance caused by the 5(6)-substituent, the majority of these compounds may be regarded as ineffective. These purely chemical investigations (not detailed here) are of great value in facilitating the synthesis of new penicillins and cephalosporins and their analogues, with absolute stereochemical requirements as well (Table 8.14).
Table 8.14. PENAMS AND CEPHAMS (CEPHEMS) SUBSTITUTED AT POSITION 5(6) Structure
Substituent ( R )
Reference I45 146 147
I46
148
PhZCH'COMe-
149
Ph,CH.CO-
I49
SR
150
J . CS.JASZBERENYI AND T. E. GUNDA
421
Table 8.14. (contd.)
Structure
Substituent ( R )
Reference
151 152
.dJ
hi--+
153-7
0
159
160 0
161
GNd&0
161
162
163
428
MODIFICATIONS AND ANALOGUES OF /3-LACTAM ANTIBIOTICS Table8.14. (contd.)
Structure
Substituent ( R )
NH2 VNH GNH
I
Reference
pNOzPh Ph Ph
160
pN0,Ph
165
n=oii
R=MeiH
166
167
R = -0C CH,NH, R' R2 ~~
RNHfls+ 0
0
G G V V V H H
pNO2Ph Ph pN0,Ph Ph PNH2Ph Ph pNOzPh
167
165
168
J. Cs. JASZBERENYI AND T. E. GUNDA
429
SUBSTITUTION AT POSITION 6(7) AMIDES
There are thousands of P-lactam antibiotics belonging to the 6(7)substituted derivatives: the natural and semisynthetic penicillins and cephalosporins. As there are already several reviews [ 169-1791 dealing with these compounds, we shall not touch upon them here, in spite of their unique position in chemotherapy. The most important ones are to be found in Tables 8.15 and 8.16.
Table 8.15. PENICILLINS OF CLINICAL IMPORTANCE
R
Name
PhCH, PhOCH2
Benzylpenicillin (Penicillin G ) Phenoxymethylpenicillin (Penicillin V)
H&.cH(cH,),
I co; PhOCH
I
Et PhCH
Penicillin N (Synnematin B, Cephalosporin) Propicillin
I
Ampicillin (a-Aminobenzylpenicillin)
I
Carbenicillin (a-Carboxybenzylpenicillin)
NR PhCH CO2H PhO*CH
I
Ph PhCH I
$OIH PhOCH
I
Phenbenicillin Sulbenicillin (n-Sulfobenzylpenicillin) Phenethicillin
Me
Ce 0 Me
Methicillin
430
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS Table 8.15. (contd.)
R
Name
Oxacillin
Cloxacillin
*
Dicloxacillin
Flucloxacillin
Me
Amoxycillin
Epicillin
Nafcillin
Quinacillin
J. C S . JASZBERENYI AND T. E. GUNDA
431
A structural feature common to all previously-described and widelyused compounds is that the acylamino substituents on C-6(7) next to the lactam carbonyl are in the P-configuration. Bioassays reveal that the epipenicillins (94) and epicephalosporins (95) having an acylarnino sidechain in the a-configuration exhibit negligible antibacterial properties [180,1811. The first example of such compounds was epihetacillin, obtained from hetacillin by treatment with triethylamine [ 1821. The lack of activity can be explained in accordance with the mode of action of p-lactam antibiotics [183]; in the case of the epi-derivatives the structural similarity between the terminal D-alanyl-D-alanine moiety and the antibiotic molecule no longer exists. Details will be discussed in the section on the mode of action. As these epi-derivatives are practically inactive, they will not be treated in detail here. Kaiser and Kukolja [184] give an excellent review. NON-AMIDE TYPE SUBSTITUENTS AT C-6(7)
From 6-APA and 7-A(D)CA numerous compounds have been synthesized with non-amide type substituents on C-6(7) (Table 8.17). 6(7)a-METHOXY DERIVATIVES
Besides the derivatives possessing a reversed 6(7)-configuration or nonamide type substituents, the 6(7)~-acylamino-6(7)a-substitutedp-lactam antibiotics are of considerable interest. Intensive investigations began in this field after the cephamycin group had been isolated and found to show a greater antimicrobial activity than cephalosporin C against gramnegative organisms [ 196-2001 (Table 8. I S ) ; moreover, the cephamycins exhibited a higher resistance toward p-lactamase [201]. A procedure has been reported for the removal of the aminoadipoyl side-chain of cephamycin C (96c) [202] (utilizable for cephalosporin C as well), thereby enabling the easy refunctionalization of the 7-amino group to yield semisynthetic cephamycins (98). The preferred route, which involves diazotization of the C-6(7) amino group is useful to functionalize C-6(7) by the introduction of methoxy and other groups [ 1891. The 7a-methoxycephalothin (R=T) (99) end-product exhibits cephalothin-like activity and an enhanced resistance against cephalosporinase. At the same time, it is curious that the inhibiting-ability of 6a-methoxypenicillin G (100) is 15% that of penicillin G [189], while the activity of 6p-methoxypenicillin G (101) is at most only 1% of that of
432
Table 8.16. SOME IMPORTANT CEPHALOSPORIN COMPOUNDS
R
R' OAc
Cephalosporin C
-+g)
Cephalosporin CA
OAc
Cephalothin (Keflin)
-hi
Cephaloridine
OAc
Cephaloglycin
H
Cephalexin
H
Cephradine
sf-7
Cefamandole
N-N
he
Cefazolin
SiI;XMe
OAc
Cephanone
OAc
Cephapirin
OAc
Cephacetrile
0 Ac
Cephaloram
S
'
5C.NN \,
,Me Me
Cephachlomezin
J. CS. JASZBERENYI AND T. E. GUNDA
433
penicillin G. Further, 7/3-methoxycephalosporins (102) did not show activity at as high a concentration as 100 pglml [203]. The synthetic availability of 7 a -methoxycephalosporanic acid (103) has allowed the preparation of not only 7a-methoxycephalosporin C (104) [204,205] but also the semisynthetic cephamycins and thus, similarly to semisynthetic penicillins they are now easily obtainable. Cama and Christensen [206] reported that 6(7)-Schiff bases (105) may also serve as starting materials for a stereospecific route enabling the introduction of different groups, including azido, fluoro and isonitrile. Firestone and Christensen [207] obtained 6a-hydroxypenicillin (107a) and its derivatives through an N-acylimine intermediate (106), but these had a markedly lower activity compared to the 6a-methoxy derivative (100). There is a direct one-step route to 6a-methoxypenicillin sulphoxides (108) [208], which were converted into cephalosporins via a subse-
quent ring expansion [209]. Other procedures [210,211] led primarily to the 6(7)a-mercaptomethyl derivatives, which gave the desired methoxy derivative on further reaction. The 7a-methoxycephems are more active in several cases than the corresponding parent compounds. With other substituents, such as methyl, mercaptomethyl dr higher alkoxy groups, a rapid drop in activity is observed [211]. As a result of research on cephamycins a new synthetic analogue, Cefoxitin, has appeared from the total synthesis developed by Ratcliffe and Christ ensen [2 12-2 1 41.
434
MODIFICATIONS AND ANALOGUES OF 6-LACTAM ANTIBIOTICS
Figure 8.2. The action of p-lactarn antibiotics on the growing cell cross wall is illustrated by the efect of rnethicillin (0.5 pglrnl) in cultures of Staphylococcus aureus 58148 (rnethicillin-sensitive strain) [164]. (a) Control (14 hr, 2 x 17600) (b) Cells treated with (14 hr, OSpg/ml methicillin 1.1 X 17600). (Electron-microscope pictures provided rne~hici~lin by Dr F. Rozgonyi, Institute of Microbiology, University Medical Schooi, Debrecen, Hungary, to whom the authors are indebted)
Table 8.17. NON-AMIDE SUBSTITUTION ON C-6(7) Rz
H
R’
R2
Activity
Reference
H H H
Br
Inactive against M. pyogenes aureus 209P
185
I
H
H
No significant activity
186
H
H
N o significant activity
187
C1
J . CS. JASZBERENYI AND T. E. GUNDA
435
Table 8.17. (contd.)
R'
R2
Activity
Reference
H H H
HO AcO
I88
vo
Inactive against several gram-negative and gram. positive microorganisms
Br I
Br
No significant activity
187
HO
H H
Little activity or inactive against different organisms
190, 192
Little activity
182
vo
I
Ph
H Me
H
GNH
No significant activity
180
Me0
GNH
Less active than benzylpenicillin (1% of the 6aM e 0 penicillin G )
189
VCHz
H
Appreciable antibacterial activity
191
MeNH
H
No interesting bioactivity after acylation
191a
(CH,),N.CH=N
H
Ample activity, especially against gram-negative strains
R2
191b
H
CO, H
H H H H PhCH=N Me,N
CI
c1 H NHz PhCH=N CHz.CH=CHz
OAc H H H H H
194 195 38 38 38 195
436
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS Table 8.18.7a-METHOXYCEPHALOSPORINS OF NATURAL ORIGIN
CO2H (96 a-d)
R'
R2
Producing strain
Name
Me0
p-HOS02C6H4.CH=C(OMe)-
Streptomyces griseus NRRL 3851
Cephamycin A
Me0
p-HO.C,Ha.CH=C(OMe)-
Streptomyces
Cephamycin B
griseus
NRRL 3851
Me0
NH2
Strep tomyces clavuligerus NRRL 3585 and S. lactamdurans NRRL 3802
Cephamycin C
Me0
Me
Streptomyces lipmanii NRRL 3584
7a-methoxycephalosporin C
J. CS.JASZBERENYI AND T. E. GUNDA
1 esterification
CH
2
mc 2
N
Y
diazotization
CH,OAc
CO2H
C02R'
(7051
X = Li
R'=R hydrol
X=Br
X = OMe
431
438
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
These efforts to find a satisfactory route to 7a-methoxycephalosporins and 6a-methoxypenicillins proved to be reasonable, in so far as the cephamycin family (A, B, C) turned out to possess an advantageous antimicrobial spectrum and activity both in vivo and in vitro, with appreciable p-lactamase stability. The latter feature is especially conspicuous in the case of cephamycin C (1 1lc) [196-2001 (Tables 8.19-8.22).
6(7)-ALKYL DERIVATIVES, AND DIFFERENT OTHER COMPOUNDS SUBSTITUTED ON C-6(7)
A number of investigations have been based on the conclusions of Strominger and Tipper [183,215]. It was estimated that by structural analogy between the terminal D-alanyl-D-alanine moiety of Nacetylmuramylpentapeptide and 6cy-methylpenicillin the latter should possess an enhanced bioactivity. The first experiments aimed at obtaining this compound were unsuccessful [216,217], as the copper complex (112) could not be split after alkylation. Starting from 6-APA, Reiner and Zeller 12161were able to introduce the hydroxymethyl function into C-6, but the low activity of the end-product suggested the presence of (113) in the epi-conformation. The use of formaldehyde yielded the spiro derivative (1 14). The syntheses of (115) and its cephem analogue (116) were finally solved [218]. At the same time 6a-alkylpenicillins were obtained via
J. C s . JASZBERENYI AND T. E. GUNDA
I
439
C02Et
CO2Et
5
RCHO
R-CH=N
R-cH=NaJ ,,
Me5
P h L i 678'c)
CH2R' 2' MeSCl
0
C02R
0
__
CH2R'
CO2R
2,h- D N P H ,TS OH
C02R
R'= O N H Z Cefoxitin R'=
TT N
Meg H
MeOH
OAC TN 7d-MeO-Cepha-
lothin CO2R
COZR
stereospecific alkylation, but instead of the natural 6P-acylamino sidechain their product had a 6P-dialkylamino group 1561. The Schiff-base intermediate (1 17) was used in another method and, after functionalization of C-6 with phenyl lithium, treatment with alkyl halides gave the desired 6a-alkyl derivative (1 18) [219].
440
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS Table 8.19. RESISTANCE O F CEPHAMYCIN A TO ENZYMATIC DEGRADATION [197] Antibiotic
Cephamycin A Cephalosporin C Cephalothin Cephaloridine
Inactioation (%) by Alcaligenes faecalis, cells *
Aerobacter cloacae, broth**
0 >99 56 -
16 96 66 96
* Exposure of 4 mglml solution for 3 hr to washed cells in 0.1 M, pH
**
7.5 phosphate buffer at 10 times the cell concentration of an 18-hr nutrient broth culture. Residual activity determined by bioassay. Table 8.20. RESISTANCE OF CEPHAMYCIN C TO ENZYMATIC DEGRADATION [I971 Enzyme source (washed cells)
Substrate inactivated (%)* Cephamycin C
Alcaligenes faecalis MB-9 A . oiscosus MB-12 E. coli 236 Proteus morganii 251 P. morgani 356 P. mirabilis 241
*
Cephalosporin C
0
> 99
54
> 99
38 80 69 5
> 99 > 99 > 99 72
Exposure of 4 mg/rnl for 4 hr to washed bacterial cells in 0.1 M, pH 7.5 phosphate buffer at 10 times the cell concentration of an 18-hr nutrient broth culture. Residual activity determined by bioassay.
Surprisingly, these derivatives did not come up to expectations, as seen in Table 8.23 12181 for the esters (1 15). Nevertheless, the corresponding sodium salts did exhibit some inhibiting properties when tested against 79
J.
CS.
JASZBERENYI AND T. E. GUNDA
44 1
Table 8.21. IN VIVO ACTIVITY AGAINST CLINICALLY ISOLATED STRAINS O F PROTEUS [200]
P. mirabilis 3344f P. mirabilis 3343 P. mirabilis 3347$ Proteus sp. 3348 P. morganii 3376$ P. morganii 334%
Cephamycin C
Cephalothin
Cephaloridine
approx. 310 350 186 957 517 273
391 2575 72 1 2505 > 20000 > 20000
179 3270 3020 11350 10000 9270
*
Infection given intraperitoneally with 45 to 300 LD5, suspended in 5% hog gastric mucin. Therapy by the subcutaneous route as two doses 0 and 6 hr after infection. t Cephalosporin-susceptible by agar diffusion disc test. $ Produces a p-lactamase more active against cephalothin than against cephamycin C.
Table 8.22. IN VITRO ACTIVITY O F CEPHAMYCIN C AGAINST SOME CEPHALOSPORIN-RESISTANT CLINICALLY ISOLATED CULTURES [I991 End point* ( p g / m l ) Test organism
Cephamycin C
Cephalothin
Cep haloridine
MIC
MBC
MIC
MBC
MIC
47 63 63 >I000 63 16
63 156 63 4000 >I000
63 190 63 >8000 >I000 140
750 1000 1000 2000 1000 140
Proteus s p . 3348 8 P. mirabilis 3343 16 P. mirabilis 3347 47 P. morganii 3376 94 P. morganii 3345 63 E. coli 3349 16
*
110
MBC 1000 I000
1000
> 8000 1000 291
Minimal inhibitory concentrations (MIC) determined in brain-heart infusion broth containing lo' cells/ml. Minimal bactericidal concentrations (MBC) determined by subculture into broth. Figures given are averages of two tubes except that the readings from four tubes were averaged for the E. coli data.
442
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS
(115)
a) R'= Me b) R'= But Cl R'=
H
d) R'= No
Table
8.23.
ANTIBACTERIAL ACTIVITY DATA OF 6-METHYLPENICILLINS AND METHYLCEPHALOSP0RIN S [2181
Compound
~
Concentration (wglml) Penicillin G methyl ester 6a-methylpenicillin G methyl ester 6P-methylpenicillin G methyl ester
Str. pyogenes t
S . aureus 209P* ~~~
8 500 500
~
~~
Activity
Concentration (mglkg)
Activity
+5
25
+
- II -
up to 325
-
no data
S. aureus SC 2399 7-phenoxy acetamidodesacetoxycephalosporanic acid
15
7-
+
Str. pyogenes SC 3862
MICS 1
1
no data
no data
50
50
7a-methyl-7-phenox y-
acetamidodesacetoxycephalosporanic acid methyl ester
7a-methyl-7-phenoxyacetamido desacetoxycephalosporanicacid
inactive
z 1250
+
*
In vitro disc assay on agar plates. In vivo, in the mouse, parenteral administration. $ MIC values, determined in a tube dilution assay. 5 +=Active. 11 - = Inactive.
t
P
W P
444
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
different organisms. Sodium salt (116d) was found to have 0.2-16% activity relative to that of penicillin G, which is an ‘appreciable antimicrobial activity’ [219]. It is worth emphasizing that (115d) was effective against Pasturella multicoda 1590 and Diplococcus pneumoniae 3377 in a 2.5 pg/mI dose. In fact, the compound has a narrow, not penicillin G-like spectrum and its activity is further decreased if deuteromethyl or ethyl groups are present instead of the 6a-methyl group. The decrease in activity was as high as 80% when the protons of the methyl group were exchanged for deuterium. Although their investigations were aimed at obtaining the 6 a hydroxymethylpenicillin, Firestone, Schelechow and Christensen solved the preparation of the 6,6’-dimer of penicillin G (121) [220]. On exposure of the intermediate (120) to oxygen the 6a-dimer (121) was isolated, together with a small amount of the desired derivative (122a). Compound (121) was found to have a markedly reduced bioactivity. Its stereochemistry is correct, but the examples furnished by other derivatives (e.g. 6 a -ethylpenicillin) provide the explanation in the steric hindrance due to the bulky substituents.
The 6a-(substituted-methyl) penicillins and the analogous cephem derivatives were prepared by the hydroxymethylation of the appropriate Schiff-bases (122 and 123) [221]. There have recently been obtained penicillins (1 22f-k) having 6a-substituents in higher oxidation state [222]. The relative order of activity when tested against B. subtilis by the agar diffusion method was found to be g i > f a > j k > h. On the addition of acrylonitrile to the Schiff -base two isomeric spiro compounds resulted. One was hydrogenolyzed to the corresponding free acid (124), but was found to be inactive against B. subtilis. The 7a-formyl- and 7a-
-
-
-
J . CS. JASZBERENYI AND T. E. GUNDA
R=
a] -CHZOH
f ) - CH2CH2CN
b) -CH?NH2 C) -CH,Cl
- CHO - CH(0H)Me I ) - C 0 Me 11 - C O 2 M e
d) -CH3
e ) -CH2F
445
9) h)
k] - C 0 2 N a
(1 24)
acetylcephalothin derivatives possessed activities a little higher than those of the corresponding penicillins. In addition, the 6a-hydroxy, formyloxy and benzyloxy derivatives [207] were also markedly less active than the methoxy compound. None of the 6(7)a-alkyl and acetylpenicillins and cephalosporins were more effective than the unsubstituted parent compound [223]. Against gram-positive bacteria, the ratio (activity of parent compound/activity of substituted compound) was 3 5, while against gram-negative bacteria no inhibiting properties were shown up to 200pg/ml. An analysis of the electron-withdrawing properties of the 6a-substituents, further supported by hydrolysis data, has shown that the decreased bioactivity of the penicillins is caused by the lactam-stabilizing effect of the 6a-methyl group [223]. At the same time, the 7a-acetyl derivative, which is particularly sensitive to base hydrolysis, showed a negligible activity again [223,92] (Tables 8.24 and 8.25). A subsequent reaction between the 6(7)a- and p-substituents may also take place, as indicated by (125) and (126) [223]. The facts and data gained so far seem to confirm the assumption, in accordance with the conclusions [223], that any new 6(7)a-substitution of either electron-withdrawing or donating groups, leaving the 6(7)p-amido side-chain intact, leads generally to a decrease in the bioactivity and a narrowing in the spectrum. With the 6a- or 7a-methoxy group, however,
446
MODIFICATIONS AND ANALOGUES OF 8-LACTAM ANTIBIOTICS
in contrast with the other substituents, only a slight activity decrease, or activity increase [224-2261 is observed. On the other hand, in the majority of cases an enhanced stability toward p-lactamase was found. Extended chemical investigations have made possible the syntheses of the 6amethyl derivatives suggested by Strominger and Tipper [183,215]. In contrast with their assumption, these compounds show decreased activity, but in our opinion this fact does not undermine their hypothesis. There is a more definite steric similarity between the terminal D-Ala-DAla moiety of the mucopeptide in the cell wall before cross-linking and the 6a-methylpenicillins. However, as can be seen from the hydrolysis data [223], the methyl group stabilizes the p-lactam carbonyl-nitrogen bond: this means a reduction in acylating ability, and thus a diminished bioactivity results. In the case of electron-withdrawing groups, which are usually more bulky than the methyl group, the resulting steric hindrance restrains the interaction of the more reactive p-lactam bond with the enzyme (see [223] and p. 459). The 7-spiro compound (128) obtained from D-mandelamido-3carbamoylmethylcephem (127) [2271 is worth noting because it was reported to have some antibacterial activity. Novel compounds of interest which have no amide-like substituents have been synthesized [228,229], namely derivatives of type (129)-( 135). Table 8.24. IN VITRO ANTIBACTERIAL ACTIVITY OF 6a-SUBSTITUTED PENICILLINS (1 18,
R' = H)[259]* Substituent
MIC in p,g/rnl t
R
R2
Str. pyogenes SC 3862
B. megathenurn SC 9509
V V V G G
H Me SMe H
0.004 0.18 37.5 0.004 25.0
0.6 23 -i-
~
*
SMe
-
J . E. Dolfini (E. R . Squibb and Sons, Inc., Princeton, New Jersey) personal communication. Our thanks are due to Dr J. E. Dolfini for these in vitro data. t Determined by two-fold tube dilution assay. i- N o data.
J. Cs. JASZBERENYI AND T. E. GUNDA
Table 8.25. IN VITRO ANTIBACTERIAL ACTIVITY OF 7a-SUBSTITUTED CEPHALOSPORINS (110) [2591* Substituent
MIC in pg/ml t
R
Y
S . aureus SC 2400
G G G G A A V V V
H Me Ac SMe H Me H Me Ac
23. I 50 >lo0
* J.
>lo0 37.5 >lo0 1.4
150 >200
B. rnegatherium SC 9509 0.09 62.5
> 100 15 0.09 31.5 $
10.9 75
E. Dolfini (E. R. Squibb and Sons, Inc., Princeton, New Jersey), personal communication. Our thanks are due to Dr J. E. Dolfini for these in vitro data. t Determined by two-fold tube dilution assay. $ No data.
448
MODIFICATIONS AND ANALOGUES OF O-LACTAM ANTIBIOTICS
J. C s . JASZBERENYI AND T. E. GUNDA
449
NUCLEAR ANALOGUES WITH ENLARGED RING SYSTEMS Only a few such compounds have been described to date. Heusler [721 reported compound (1 36), containing a tetrahydrothiazepine ring system, that showed no antimicrobial activity up to 100 pg/ml. Other compounds of this type, e.g. (137) [230], (138) [231] and (139) [92], are also practically inactive. The cycloaddition of ketenes to thiazepines led to (140) [232]. Although no report has appeared about the bioassay, it is highly probable that these compounds have no activity.
('38)
R = -C02Me
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
450
In fact, when the substituents of these compounds are more or less the same as the normal cephalosporins (e.g. (136) or (138)), the enlarged parent thiazolidine ring nitrogen atom of the p-lactam moiety is freed from the stereochemical 'yoke' causing the non-planarity of the nitrogen bonding in the penam and A3-cephem systems.
NUCLEAR ANALOGUES WITH SHIFTED SULPHUR ATOM With the aid of a fascinating photochemical ring-closure procedure, such compounds (141) (142) have been synthesized [233], [234], but they are reported to exhibit no bioactivity against S. aureus Oxford and A. faecalis at 1 mglml. x
N2/'\,
LN,/
0
hv
-
xF") N,/'
0
GN G N0H f i > C 0 2 H
Me Me (141)
NUCLEAR ANALOGUES WITH TWO SULPHUR ATOMS These compounds have been mentioned independently by Woodward (143) (144) [26], Kukolja (143b) [2351 and a patent [236]. The cepham compounds (143a, b) are inactive, but compounds of type (144) possess antimicrobial activity [26].
C02H
I
R"SH 2. CF$O2H
1.
t (143)
a)
R=R'=H
b) R = R ' = M e
J.
CS.
JASZBERENYI AND T. E. GUNDA
45 1
'C0,B"f
This must conclude our description of the analogues of penicillins and cephalosporins, although derivatives with nitrogen and oxygen heteroatoms, without sulphur, other fused p-lactam ring systems, monocyclic and other analogues have not been discussed.
T H E MODE OF ACTION OF B-LACTAM ANTIBIOTICS INTRODUCTION
There have been a number of hypotheses regarding the mode of action of p-lactam antibiotics [237,238]. That most widely used at present is the 'structural analogue' hypothesis [ 183,2151, based on the structural similarity between penicillins and a possible conformation of the acy1-Dalanyl-D-alanine end of the N-acetylmurarnyl-pentapeptide strand of the bacterial cell wall. A number of experimental data support this hypothesis (for recent reviews see [237] and [238]) and the model attractively explains the mechanism of action of p-lactam antibiotics, especially penicillins. Nevertheless, certain more recent data contradict the 'structural analogue' model [239-2411 and a 'conformational response' model has been set up [242,243]. It must be emphasized, however, that the contradictions are sometimes only apparent, and a one-sided interpretation of the structure-activity data may lead to false conclusions, e.g. the problem of the 6(7)cy-substitution in penicillins and cephalosporins. MEMBERS O F THE P-LACTAM GROUP OF ANTIBIOTICS
A similar mode of action is characteristic of different penam and cepham-like compounds, which mostly possess common moieties but may exhibit marked differences in antimicrobial spectra and in inhibiting, binding and permeability properties [244-2461.
452
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS
COZ H
Penicillins : R' R2
variable H in biosynthetic penams; R2 # H in synthetic derivatives. ,Me J\Me
\
X
Cephalosporins : R' variable
X R2
R6
H or OMe in biosynthetic cephems; R2 # H or OMe in synthetic derivatives H (mostly) CH2.0Ac in biosynthetic cephalosporins CH2R6in synthetic derivatives and the naturally occurring Cephamycin family H in total synthetic derivatives variable
Cephalocillins : R3 = R'
as in cephalosporins, but Me
R' = R' R'
THE ROLE OF P-LACTAM ANTIBIOTICS IN THE INHIBITION OF ENZYMES
P-Lactam antibiotics are capable of inhibiting the biosynthesis of the cell wall (Figure 8.2) [247]. This property is due primarily to their selective inhibition of the transpeptidase and DD-carboxypeptidase involved in the cross-linking reaction of bacterial cell-wall synthesis. Entering the final reactions of the cell-wall construction, they prevent the enzymes from building up the cross-linked peptide chains (Figure 8.3). The enzymatic cross-links between the pentapeptide chains is of basic importance as regards the rigidity of the wall of the bacterial cell. This cross-linking is not the same in the different bacteria: in some cases it is achieved by peptide chains [248,250] or amino acids [249], while in other cases it is carried out without such cross-linking peptides or amino acids [250],
J.
CS.
JASZBERENYI AND T. E. GUNDA
453
2 L-ala
D-ala-D-ala
UDP-MurNac-QI-D-glu-Q,
UMP
"1 p-c,,-
UDP PP-c,,
E.
2 D-ala
-t-
c-UDP-GlcNac
P-c,,
E, 'cell wall' C-E, QI:L-alanine (mostly) or L-serine or glycine A: Qz:L-lysine or Dap or L-ornithine B: or other amino acids C: E,:alanine racernase D: E.:D-ala-D-ala svnthetase E,.:UDP-MurNac-pentapeptide ligase P-C,,: E,,:peptidoglycan transpeptidase E,:DD-carboxypeptidase PP-C,,:
+ D-ala
D-Cycloserine O-carbamoyl-D-serine Penicillins and cephalosporins Bacitracin C,,-isoprenoid alcohol monophosphate C,,-isoprenoid alcohol pyrophosphate
Figure 8.3. Final reactions of the cell wall synthesis, and the action of some antibiotics in this process. (For details and references see [237], Chapter 3)
between an amino and a carboxyl group of the peptide chains (Figure 8.4). In the case of p-lactam antibiotics not only transpeptidase and carboxypeptidase, but also glycosidase inhibition is observed [251I. Although this inhibition requires higher concentrations and in contrast to the inhibition of the above-mentioned enzymes is not complete, the investigation of this effect may result in interesting new data.
454
MODIFICATIONS AND ANALOGUES OF 8-LACTAM ANTIBIOTICS
R'
R'
I
I I
L-ala
L-ala
D-glu H2NR3
D-gh
I
I
R ~ HN- Q I D-ala
+
D-ala
I
+
-+ -+-+ RZH N ~ Q
E
D-ala-NHR3
I
E
D-ala R': -GlcNac-MurNac-
I
R2: cross-linking peptide chain, or an amino acid, or a carbonyl group of another peptide chain in the bacterial cell wall E: transpeptidase
H,NR3: cross-linking peptide chain, amino acid or another peptide chain in the cell wall H,NQ: L-lysine or other amino acid (with more than two functional groups)
Figure 8.4. Cross -linking reaction in the bacterial cell wall, catalysed by transpeptidase
THE MODE OF ACTION
The 'structural analogue' model The role of p-lactam antibiotics in these reactions has been pointed out by several workers [183,215,252-255], and supported by many experimental data. It was assumed that the structural similarity between penicillins and the terminal acyl-D-Ala-D-Ala moiety of the pentapeptide chain plays an important role [ 1831, as exemplified by the Dreiding stereomodels of acylD-Ala-D-Ala and a 6a-methylpenicillin shown in Figure 8.5. This structural similarity is not so close in 3-cephems as in penicillins, but 2-cephems, although practically inactive derivatives as antibiotics [41,79], are better structural analogues of acyl-D-Ala-D-Ala. As emphasized by the Strominger group [183,215,252] and depicted in Figure 8.5, the amide bonds in acyl-D-Ala-D-Ala and penicillins assume the same spatial position. This is considered to be very important in the inhibition of transpeptidase and DD-carboxypeptidase. This similarity has been subjected to critical analysis, considering the numerical data relating to the amide bonds [256]. First of all, the peptide chain in the pentapeptide has an amide linkage between the two Dalanines which is 25% longer than the exocyclic amide linkage in the
Figure83 Dreidingstereornodelsof acyl-D-alanyl -D-alanine(1eft)and a 6a-methylpenicillin (right). (Photograph providedby Joseph Hapak, Debrecen, Hungary)
455
456
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS
0
It
penam system; on the other hand, in p-lactams the C-C-N bond angle is 90.5" instead of the normal 117",and in penicillins the normal dihedral angle (180O) is distorted to 135.7'. These data d o not support the obvious similarity, even if the possible template effect of the enzyme surface fixing the acyl-D-Ala-D-Ala terminal in a 'penicillin-like' conformation is considered [257]. Transpeptidation reactions are considered to be achieved by reversible acylation of peptidoglycan transpeptidase (Figure 8.6). According to the 'structural analogue' model, the same acylation would take place in the
-
acyl-D-ala-D-ala + E acyl-D-ala-E + D-ala acyl-D-ala-E RNH, +acyl-D-ala-NHR E
+
+
RNH,: an amino acid or peptide chain for cross-linking or another peptide chain of the wall E: transpeptidase Figure 8.6. Reaction of the transpeptidation [ZlS]
case of p-lactam antibiotics (penicillins, cephalocillins, cephalosporins, generally 'A'), but the covalent acyl-enzyme compound (A-E) would be an irreversibly acylized and thus deactivated derivative of transpeptidase (Figure 8.7). Deactivated transpeptidase (A-E) is a penicilloyl-enzyme A + E zi==
*I
k-1
EA
k2
A-E
k , : forward reaction constant of complex formation k - l : constant of the reverse reaction k2: reaction constant of acylation by penicillins and cephalosporins Figure 8.7. The two-step reaction of inhibition of transpeptidase and carboxypeptidase (E) by p-lactam antibiotics (A), according to the 'structural analogue' model
(Figure 8.8), an irreversibly inactivated derivative [245], probably a thioester [215,254]. In this deactivation reaction the first step is the formation of an enzyme-antibiotic complex (EA). Because of the irreversibility, the second step gives a stable, covalent compound, but no further
Figure 8.8. Proposed structure of penicilloylenzyme [ZlS]
J.
cs. JASZBERENYI AND
T. E. GUNDA
457
steps exist which would liberate the enzyme for the cycle. This model supports another possibility: the DD-carboxypeptidase might form a hydrolyzable acyl-enzyme (A-E) compound, and this could be a plactamase function of the modified enzyme [183,258]. In the equation (Figure 8.7) the formation of the complex is not hindered in the case of classical penam and cephem systems, nor (probably) for the 6(7)a-methyl derivatives, because of the close structural similarity between these antibiotics and acyl-D-Ala-D-Ala (Figure 8.5). It is disadvantageous to substitute the hydrogens of the lactam moiety by bulky substituents because this causes steric hindrance and may inhibit to some extent the formation of the EA complex, i.e. it lowers the stability constants of the EA complexes. In this case a reduced antibacterial activity is probable, even when the substituent at C-6(7) is an electron-withdrawing one and activates the antibiotics as acylating agents ( k r becomes greater). It is easy to observe this steric hindrance (Figures 8.9 and 8.10). The decreased stability of the lactam bond (or its increased acylating ability) does not imply an increased antimicrobial activity. On the contrary, the activity is greatly reduced [223,259,260]. However, this example does not disprove the ‘structural analogue’ model of the mode of action. This directs attention to the importance of the joint investigations of structure-stability-antimicrobial activity. If the substituent of the amidebearing carbon (C-6 in penicillins and C-7 in cephalosporins) does not cause steric hindrance in the case of 6a-methylpenicillins and the ‘normal’ 6a-H penicillins, the constant kl is the determining factor (if such a covalent acylized enzyme compound exists at all). It is easy to see from hydrolysis data [223] that the 6a-methylpenicillin derivative has an increased stability (or decreased ability to acylize) compared with classical penicillins. This seems to be responsible for the reduced antibacterial activity of the 6a-methyl compound (Table 8.26). Other important data relating to this problem result from the comparison of MIC values and transpeptidase-inhibiting concentrations [260] (Table 8.27). The concentrations for the inhibition of transpeptidase are markedly lower than the corresponding MIC values. The difference is considered to be determined by the transport of the antibiotic molecule [260]. Explanations given for the reduced antibacterial activity of 6amethylpenicillins suggest either that the 6a-methyl group does not influence the interaction between the enzyme and the antibiotic molecule, or that the increased stability of the lactam bond reduces the acylating ability, as mentioned above. If the lactam is substituted at C-6 in the penicillin by alkyl, 0-alkyl or S-alkyl groups, an enhanced p-lactam
Figure 8.10. Dreiding stereomodels ojacyl-D-alanyl -D-alanine and a 7a-acetyl-cephem-3-carboxylic acid (right). The a-position at C7 is substituted with a relatively bulky group, which causes steric hindrance for transpeptidase and carboxypeptidase enzymes. (Photograph provided by Joseph Hapdk, Debrecen, Hungary)
460
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS Table 8.26. RELATIVE HYDROLYSIS CONSTANTS O F DIFFERENT6a-SUBSTITUTED PENICILLINS ( I 18). R = V, R’ = H 12231
R2
k (relative)
H Me Ac
1 0.26 2.36
k : relative hydrolysis constants in basic solutions Table 8.27. INHIBITION OF GROWTH AND TRANSPEPTIDASE BY 6-SUBSTITUTED PENICILLINS [260] Compounds
6a-substituent
Penicillin v derivatives
H Me OMe SMe
Penicillin G derivatives
H Me OMe OEt
MIC ( p g l m l )
I00
> 200 100
> 200 13.5
> 200 200
> 200
Concentrations required for 50% inhibition ( p g / m f ) t 0. I 100.0 1 .0 100.0
0. I 100.0 1 .o 10.0
*
The MIC values were determined by the two-fold tube dilution method with E. cofi Y10. t Transpeptidase was prepared from E. coli Y10 and assayed according to the method, described in ref. 12961.
stability is observed (Table 8.28). This effect is regarded as primarily steric rather than polar.
The ‘conformational response’ model In different Streptomyces strains enzymes have recently been found which have both carboxypeptidase and transpeptidase activity 1242, 261-2661 (Figure 8.11 ). Fluorescence and CD investigations of the interaction between the enzyme R61 from Streptomyces and penicillin G [242], indicate that the binding of penicillins gives rise to a change in the
J.
CS.
JASZBERENYI AND T. E. GUNDA
461
Table 8.28. THE RELATIVE RATES OF BASE HYDROLYSIS OF 6-SUBSTITUTED PENICILLINS AT pH 10 [260] Penicillin V
Penicillin G 6cr-substituent
H OMe OEt
Hydrolysis constant (rel.)*
6a-substituent
Hydrolysis const. (rel.)
1 .0
H Me OMe SMe
1 .o 0.1 0.3 0.1
0.2 0.1
* Rate constants determined by titration at constant pH of the penicilloic acid product.
R-D-ala-D-ala + E, + H,O + +
R-D-ala + D-ala + El
R-D-ala-D-ala + R’-NH, + E, + + + R-D-ala-NHR’ + D-ala + E2
E,: carboxypeptidase enzyme or function E2: transpeptidase enzyme or transpeptidase function (in this case E l and E2 are different functions of an E transpeptidase-DD-carboxypeptidase) R’-NH,: peptide chain, amino acid or another peptide of the bacterial cell wall involved in the ttanspeptidation reaction Figure 8.11. Reactions of DD-carboxypeptidase-transpeptidase ( E )
conformation of the enzyme-protein rather than linkage to the enzyme through a covalent bond. This was concluded from the ready reversibility of the process. The effect of penicillins on the enzyme conformation is exemplified by the influence of penicillin G on the heat-denaturation of carboxypeptidase-transpeptidase in 3.6 M guanidinium chloride solution. The fact that the conformational change is not reflected in the far-u.v. dichroism of the enzyme-protein suggests that it is not an extensive change [242]. From these investigations the binding of penicillin to the acceptor site of carboxypeptidase-transpeptidase seems unlikely, because good acceptors for transpeptidation do not affect the interaction of penicillin and enzyme. This binding is believed to proceed elsewhere than at the site of binding of the substrate to the enzyme. On the basis of various experimental data, a ‘conformational response’ model has been suggested [242,243]. This model of the mode of action of p-lactam antibiotics postulates that the inhibiting effect of penicillins arises through
462
MODIFICATIONS AND ANALOGUES OF 8-LACTAM ANTIBIOTICS
the ‘freezing’ of the conformation of the enzyme, which, although bound to the substrate, loses its catalytic activity in the transpeptidation and carboxypeptidation reaction. In the case of a Streptornyces DDcarboxypeptidase it was found [242] that in inhibition experiments on this enzyme with various synthetic peptides the enzyme was bound to those peptides which have a cis-amide bond in the terminal dipeptide. Just as in the pentapeptide of the bacterial cell wall, the amino acid prior to the terminal dipeptide is an L-amino acid, which causes a conformational change in the enzyme. This change induces the distortion of the cis-amide bond, and the terminal dipeptide has a conformation between cis and trans in the enzyme-bound peptide. Accordingly, the bond has a higher energy, of about 30-40 kJ [242], and this energy-rich amide bond is of very great importance as regards the opening reaction. In the case of the penicillins, interaction of enzyme and antibiotic would cause a change of about 45 kJ in the Gibbs free energy at 25°C. After this change, even if the enzyme is able to bind the substrate, in order to form an active state such as in the absence of penicillins or cephalosporins, the enzyme requires the energy of the Gibbs free energy change and also that (30-40 kJ) which would come from the distortion of the amide bond by binding to the enzyme. This is probably an energy barrier for the enzyme, which prevents it from catalyzing the hydrolysis reaction even if the binding is not hindered. Unanswered questions in the ‘conformational response’ hypothesis concern the existence, necessity and role of the ‘penicillin binding site’ in the enzymes. SUMMARY
There are no fundamental differences in the ‘structural analogue’ and the ‘conformational response’ hypotheses, but the latter is nevertheless regarded as a denial of the former. There are basic structural requirements for p-lactam antibiotics, which are of great importance in the formation of the EA complex. The first is to have the necessary steric requirements, independently of whether the binding of penicillins and cephalosporins is realized because of the conformational similarity to the acyl-D-Ala-D-Ala terminus, or because of the existence of a ‘penicillin binding site’. After the formation of this EA complex there are two possibilities, acylation or conformational exchange. The hypotheses, however, were formulated from studies of enzymes of different origins. and thus the data cannot be freely compared. To clarify the question of the mode of action, further work is needed; this will be of great
J.
CS.
JASZBERENYI AND T. E. GUNDA
463
significance since it may direct chemical research in this field. For this reason it would be important to explain the fundamental structural requirements for p-lactam antibiotics from experimental data.
STRUCTURE-ACTIVITY RELATIONSHIPS In addition to the effect of the 6(7)-acylamino side-chain on microbial activity, attention must also be paid to structural variations at other positions. This is a difficult task and a number of errors may arise in the interpretations as a result of the low number of such variations in comparison to the tens of thousands of semisynthetic penicillins and cephalosporins. The most interesting problem is that of the 2-substitution, since the differences in electronic effects on the p-lactam ring of a given alkyl or alkoxy substituent in the a or p configuration are negligible compared to the differences, due to possible steric hindrances, in the interaction between antibiotic molecule and enzyme(s). Unfortunately, the number of known and tested compounds is enough to allow only the mere assumption that the 2a-substituted derivatives retain more activity. Further, as seen from Table 8.4, bulky 2a-alkoxy groups are undesirable in cephalosporins. A large number of data concerning the corresponding p-derivatives would be of great assistance. The activity of the parent compound is more or less retained in 2-methylenecephalosporins ( Table 8.7) and 2-spirocyclopropylcephalosporins (Table 8.6). These facts can be explained on the basis of the conjugation effect; the exocyclic double bond tends to decrease the amide-like resonance in the p-lactam ring, thereby facilitating the pyramidal hybridization of the bridgehead nitrogen atom. The electronic effect of the cyclopropyl ring in unsaturated systems is well known to be close to that of a double bond. It would be worthwhile to compare the cephalocillin, penicillin and the still unknown 2-spiro analogue of penicillin in the same screening. The differences in conformation of penicillin, A*- and A3-cephems and cephams have already been studied by different methods [39,267-2701. The steric position of the 3-carboxy group in penicillins is closer to that in A’-cephems than to that in A3-cephems. On the other hand, the bridgehead nitrogen atom of penicillins and A3-cephalosporins is definitely pyramidal, in contrast to its planar structure in A*-cephems and cephams (Table 8.29) [269]. p-lactams fused with 7 or 8 membered rings (compounds
464
MODIFICATIONS AND ANALOGUES OF S-LACTAM ANTIBIOTICS Table 8.29. CHARACTERISTIC DATA OF DIFFERENT &LACTAMS
Compound
Q*
A3-Cephalosporins Penicillins A’-Cephalospofins Cephams Unfused p-lactams Free amides
+ 0.82 +0.80 +0.76 + 0.75
R N
t
R c=o$
0.23 0.40 0.065
1.21 1.20 1.21
0.0
1.21 1.24
*
Positive net charge on the p-lactam carbonyl C atom [271]. t Distance of N atom from plane [269]. $ C=O bond length in Angstroms [270].
(136)-(140) have no activity, further supporting the necessity of a non-planar p-lactam nitrogen. In the latter cases an amide-type resonance might rather be formed. The extent of this charge delocalization (in which the unshared electron pair of the nitrogen atom also takes part) is decreased by the non-planarity of the nitrogen atom and, in A3-cephems, by the enamine resonance as well. The charge delocalization in the p-lactam C(7)-N bond has been interpreted via the EHMO and CND0/2 calculations of Boyd [271] and Hermann [272]. From the MO calculations it can be seen that the positive net charge on the carbonyl carbon atom is greater in active compounds (Table 8.29),and thus the acylating ability of the molecule is higher. As far as the unsettled problem of anhydropenicillin is concerned, the p-lactam ring of this compound seems electronically ‘overcompensated’, similarly maybe to the penicillin sulphoxides, disregarding the differences in the thiazolidine rings, which is a factor of great uncertainty. The next problem to be discussed is that of the carboxyl group. It is often stated that the presence of a free 3(4)-carboxy function is essential for the bioactivity of p-lactam antibiotics. The diminished activity of compounds with a functionalized carboxyl group is beyond question. From the comparison of activities towards both gram-positive sensitive and resistant strains it was assumed that if a compound exhibits full activity, then in vivo hydrolysis occurs. This is not likely in the case of ceph-
0
J.
CS.
JASZBERENYI AND T. E. GUNDA
465
alosporin lactones (59) and substances with diminished activity, but here and in the former case the compounds are resistant to p-lactamase. We therefore assume that enzymatic hydrolysis takes place after the binding to transpeptidase, and neither before nor after this step can the plactamase interact with the antibiotic molecule containing a transformed carboxyl group. In other words, the presence of a free 3(4)-carboxyl group is not essential for bioactivity, although it is necessary (a) to obtain a molecule possessing excellent antimicrobial properties, and (b) for the ability to interact with p-lactamase. This is further supported by the facts that the conformation of the 3(4)-carboxyl group seems to be not very relevant and that compounds such as (85) also have considerable antimicrobial activity. The penetration of p-lactam compounds through biological membranes and their binding to serum have been investigated mainly by Biagi and Hansch, in terms of the lipophilicities. The lipophilic character is represented by the constant T , introduced by Hansch [273,274], defined as log(Kx/Kp) where K , is the partition coefficient between an organic solvent (usually n-octanol) and water for the parent compound and K , is that of the derivative. The correlation between bioactivity and T is expressed in the form [275,276]: l o g ( 1 / C ) = - k T 2 + k ' T +crp
+ k"
where C is the drug concentration giving a fixed biological response (MIC values in vitro and CDso values in vivo) and cr is a measure of the electronic effect of substituent X (e.g. the Hammett constant). If the T term in the equation is associated with a negative k', this means that increase of the hydrophilic character of the compound increases its activity. The validity of these formulae is characterized by the correlation coefficient r, the value of which is 1 in an ideal case. On the other hand, there is a very good parallelism between the chromatographic R , values and the constants T.The R , value is defined as: RM = log (1 /Rr - 1)
Rr is determined by reversed-phase chromatography for each compound, and thus relative lipophilicities can be established by a very simple and convenient method [277]. It has been found [275,276] that there is a good correlation between hydrophobic character and the results of in vivo mouse protection tests ( r 0.9), even if the cr factor is not taken into consideration. This
-
466
MODIFICATIONS AND ANALOGUES OF P-LACTAM ANTIBIOTICS
emphasizes the role of lipophilicity above all. In in vitro tests a poor correlation was found, r = 0.3 - 0.4, but if these tests were carried out in the presence of serum the correlation again proved a good one, especially if the total penicillin was regarded as bound to the serum. This can be justified on a kinetic basis. From the chromatographic RM values it was found [278-2801 that the lipophilicity requirements are not the same when gram-positive and gram-negative microorganisms are tested (Figure 8.12). There is a parabolic dependence of log (1/C) on the RM values of cephalosporins for E. coli, Staph. aureus and T. pallidum. This is in agreement with the
1 -1,0
-0,s
0
0,5
1,0
1,5
RM
Figure 8.12. Relation of lipophilic character. R,, of 14 difere’erent cephalosporins to their antibacterial activity, log l / c [294]
postulate of a parabolic relationship between the rate of penetration of substances through biological membranes and their lipophilic character [281,282]. The maxima of the curves differ for gram-negative and gram-positive strains, which means that the high lipid content of the cell membrane of E. coli can retain the most lipophilic molecules, which thus do not reach their site of action 12831. Accordingly, only the most hydrophilic compounds are able to cross the membrane. In the case of gram-positive micro-organisms (and T. pallidum, which also lacks a high lipid content of its cell wall) the membrane permits the high activity of compounds more lipophilic in nature. It was suggested, that compounds with intermediate RM values might possess broad-spectrum antimicrobial activity.
J . C S . JASZBERENYI AND T. E. GUNDA
467
The following linear relation of lipid solubility to serum binding [284-2861 has been found to hold: log (penicillin bound to serumlfree penicillin) = k.rr - k‘ ( Y = 0.8 - 0.9)
Nevertheless, it was necessary to give three equations with different constants for penicillins with an a-amino or a-ether group in the side-chain, or with an ortho aromatic ether group (methicillin analogues). It was suggested that the mechanism involves hydrophobic binding and p-lactam antibiotics are able to bind to serum when ionic sites for this are not available, e.g. the case of penicillin amides, cephalosporin lactones (Table 8.9) and certain 2-substituted cephalosporins (Table 8.7), when the increased lipophilicity leads to increased serum binding. To summarize: (1) For reasonable activity a p-lactam should have the following characteristics: (a) An intact p-lactam ring (cf. penicilloic acid). (b) A pyramidal-like hybridization of the p-lactam nitrogen (cf. A’-cephems, cephams, and larger or open thiazolidine rings). (c) The absence of bulky groups at the 6(7)a-position (cf. 6(7)cu-substituted derivatives) and the presence of the ‘natural’ 6(7)p-configuration of the acylamino side-chain. (d) The presence of an S atom at its ‘natural’ place (cf. sulphur-free synthetic derivatives [287-2921 and disulphide analogues).
(2) The appropriate energy content and reactivity of the p-lactam O=C-N bond (cf. the problem of 6(7)a-methyl and methoxy substitution and 2-substituted derivatives). Although the molecular orbital calculations on penicillins and cephalosporins [271,272,293] are not enough to yield exact conclusions, it is likely that a parabolic relationship exists between the electron population of the O=C-N bond and the antimicrobial activity. (3) The rate of penetration of compounds through cell membrane exhibits a parabolic dependence on the hydrophilicity.
468
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS
(4) The extent of serum binding is in an approximately linear relation with lipophilicity. To establish an exact structure-activity relationship all of these factors have to be taken into consideration. The steric problems present the greatest difficulty. Structure-activity relationships have been carried out mainly on the 6(7)-acylamino and cephem-3-methyl structure variations, which is far from enough, and it is the task of chemists to synthesize other derivatives for the biologists.
ACKNOWLEDGEMENTS The authors wish to express their appreciation to Professor R. Bogniir and Dr F. Sztaricskai (L. Kossuth University, Debrecen), as well as to Professor F. Hernhdi and Dr Gy. Barabhs (University of Medical Sciences, Debrecen) for their valuable comments and helpful discussions. Thanks are also due to Mrs M. Punyiczki and Miss I. Petrikovits for technical help and Dr David Durham for linguistic advice.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
11. 12.
H. T. Clarke, J. R. Johnson and R. Robinson, eds. The Chemistry of Penicillin (Princeton University Press, Princeton, New Jersey, 1949) pp. 156,927,946,1008. J. C. Sheehan, K. R. Henery-Logan and D. A. Johnson, J. Amer. Chem. SOC., 75 (1953) 3292. R. B. Morin, B. G. Jackson, E. H. Flynn and R. W. Roeske, J. Amer. Chem. SOC.,84 (1962) 3400. A. M. Patterson, L. T. Capell and D. F. Walker, eds. The Ring Index (American Chemical Society, Washington) 2nd edn. (1960). J. M. Essery, K. Dadabo, W. J. Gottstein, A. Hallstrand and L. C. Cheney, J. Org. Chern., 30 (1965) 4388. D. H. R. Barton, F. Comer, P. G. Sammes, J. Amer. Chern. SOC.,91 (1969) 1529. R. D. G. Cooper, P. V. Demarco, J. C. Cheng, J. Amer. Chem. Soc.,91(1969) 1528. M. Gorman and C. W. Ryan, in: Cephalosporins and Penicillins Chemistry and Biology, ed. E. H. Flynn (Academic Press, New York, 1972) p. 540. M. Yoshirnoto, S. Ishihara, G. Nakayarna, N. Soma, Tetrahedron Lett., (1972) 2923. M. Yoshimoto, S . Ishihara, E. Nakayama, E. Shoji, H. Kuwano and N. Soma, Tetrahedron Lett., (1972) 4387. F. H. Carpenter, G. W. Stany, D. F. Genghof, A. H. Liverrnore and V. du Vignaud, J. Biol. Chem., 76 (1948) 915. D. H. R. Barton, F. Corner, D. G . T. Greig, P. G. Sammes, C. M. Cooper, G. Hewitt and W. G. E. Underwood, J. Chern. SOC. (C), (1971) 3540.
J. CS. JASZBERENYI AND T. E. GUNDA 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.
469
R. B. Morin, B. G. Jackson, R. A. Mueller, E. R. Lavagnino, W. B. Scanlon and S. L. Andrews, J. Amer. Chem. SOC., 91 (1969) 1401. D. 0. Spry, J. Amer. Chem. SOC.,92 (1970) 5006. R. D. G. Cooper and D. 0. Spry, ref. 8, pp. 194-199. D. 0. Spry, Chem. Commun., (1973) 3001. T. Karniya, T. Teraji, Y. Saito, M. Hoshimoto, 0. Nakaguchi and T. Oku, Tetrahedron Lett., (1973) 3001. D. H. R. Barton, P. G. Sammes, G. Hewitt, B. E. Looker and W. G. E. Underwood, Belg. Patent 770,730; Farmdoc Complete Spec. Book, 11047T. N. G. Johansson and B. Akermark, Tetrahedron Lett., (1971) 4784. K. Johanssen, B. 0. H. Sjoberg and B. E. 0. Akermark, Ger. Patent 2,219,601; Farmdoc Complete Spec. Book 76051T. C. F. Murphy and J. A. Webber, pp. 156, 167 in Ref. 8. E. H. W. Bohrne and J. E. Dolfini, Chem. Commun., (1972) 941. D. 0. Spry, Tetrahedron Lett., (1973) 3717. D. 0. Spry, Tetrahedron Lett., (1973) 2413. G. W. Kaiser, C. W. Ashbrook, T. Goodson, I. G. Wright and E. M. Van Heyningen, J. Med. Chem., 14 (1971) 426. R. B. Woodward, Pharm. J., 205 (1970) 562. J. C. Sheehan and K. G. Brandt, J. Amer. Chem. Soc., 87 (1965) 5468. Y. G. Perron, L. B. Crast, J. M. Essery, R. R. Fraser, J. C. Godfrey, C. T. Holdrege, W. F. Minor, M. E. Neubert, R. A. Partyka and L. C. Cheney, J. Med. Chem., 7 (1964) 483. J. C. Sheehan, Brit. Patent 1,273,243; Chem. Abstr., 77 (1972) 48204m. J. C. Sheehan, U.S. Patent 3,487,070; Chem. Abstr., 72 (1970) 66793d. J. C. Sheehan, US. Patent 3,487,071; Chem. Abstr., 72 (1970) 66791b. J. C. Sheehan, U.S. Patent 3,487,072; Chem. Abstr., 72 (1970) 66792~. J. C. Sheehan, Ger. Patent 1,943,159; Chem. Abstr., 75 (1971) 491042. J. C. Sheehan, Ger. Patent 1,943,161; Chem. Abstr., 75 (1971) 49103~. J. C. Sheehan, Brit. Patent 1,273,241; Chem. Abstr., 77 (1972) 48787d. Astra Lakemedel Co., Ger. Patent 2,219,601. H. W. Schnabel, D. Grimm and H. Jensen, Liebigs Ann. Chem., (1974) 477. K. Kiihlein and H. Jensen, Liebigs Ann. Chem., (1974) 369. R. D. G. Cooper, P. V. Demarco, C. F. Murphy and L. A. Spangle, J. Chem. SOC.(C), (1970) 340., and U.S. Patent 3,578,660. C. F. Murphy and R. E. Koehler, J. Org. Chem., 35 (1970) 2429. J. D. Cocker, S . Eardley, G. I. Gregory, M. E. Hall and A. G. Long, J. Chem. SOC., (1966) 1142. R. R. Chauvette and E. H. Flynn, J. Med. Chem., 9 (1966) 741. M. Ochiai, 0. Aki, A. Morimoto, T. Okada, K. Shinozaki and Y. Asahi, Tetrahedron Lett., (1972) 2341. M. Ochiai, 0. Aki, A. Morirnoto, T. Okada and T. Kaneko, Tetrahedron Lett., (1972) 2345. M. Ochiai, 0. Aki, A. Morimoto and T. Okada, Tetrahedron Lett., (1972) 3241. M. Ochiai, E. Mizuta, 0. Aki, A. Morimoto and T. Okada, Tetrahedron Lett., (1972) 3245. M. Ochiai, 0. Aki, A. Morimoto, T. Okada and H. Shimadzu, Chem. Commun., (1972) 800.
470 48. 49. 50. 51.
52. 53. 54. 5s. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83.
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS D. 0. Spry, Tetrahedron Lett., (1973) 165. R. R. Chauvette and P. A. Pennington, J. Org. Chem., 38 (1973) 2994. S. Wolfe, J. C. Godfrey and C. T. Holdrege, J. Amer. Chem. Soc., 85 (1963) 643. S. Wolfe, Can. J. Chem., 46 (1968) 459. S. Wolfe, J. C. Godfrey and C. T. Holdrege, Can. J. Chem., 46 (1968) 2549. Unpublished data, for which the authors are greatly indebted to Dr. D. 0. Spry, The Lilly Research Laboratories, Eli Lilly and Company. J. E. Dolfini, Ger. Patent 2,221,361; Chem. Abstr., 78 (1973) 43492q. K. Heusler, Helv. Chim. Acta, 55 (1972) 388. G. V. Kaiser, C. W. Ashbrook and J. E. Baldwin, J. Amer. Chem. Soc., 93 (1971) 2342. C. F. Murphy and J. A. Webber, pp. 151-166 in Ref. 8. N. G. Weir, Ger. Patent 2,139,289; Chem. Abstr., 77 (1972) 3 4 5 0 3 ~ . R. D. G. Cooper, Ger. Patent 1,950,390; Chem. Abstr. 74 (1971) 22860k. I. G. Wright, C. W. Ashbrook, T. Goodson, G. W. Kaiser and E. M. Van Heyningen, J . Med. Chem., 14 (1971) 420. S. Wolfe, C. Ferrari and W. S. Lee, Tetrahedron Lett., (1969) 3385. D. 0. Spry, Chem. Commun., (1973) 671. J. Cs. JhszberCnyi, Unpublished results (1973). K. Heusler and R. Barns, Ger. Patent 2,032,827; Chem. Abstr., 74 (1971) 100032h. K. Heusler and B. Fechtig, Ger. Patent 2,057,380; Chem. Abstr. 75 (1971) 88628t. K. Heusler and R. Barns, Ger. Patent 2,057,379; Farmdoc Complete Spec. Book 40750B. R. Scartazzini and H. Bickel, Ger. Patent 2,151,559; Farmdoc Complete Spec. Book 31168T. R. Scartazzini and H . Bickel, Ger. Patent 2,151,566; Farmdoc Complete Spec. Book 31167T. Belg. Patent 753,091; Farmdoc Complete Spec. Book 04171s. R. Scartazzini, H. Peter, H. Bickel, K. Heusler and R. B. Woodward, Helv. Chim. Acta, 55 (1972) 408. R. Scartazzini and H . Bickel, Helv. Chim. Acta, 55 (1972) 423. K. Heusler, Helv. Chim. Acta, 55 (1972) 2567. R. B. Woodward, K. Heusler, J. Gosteli, P. Naegeli, W. Oppozler, R. Ramage, S. Ranganathan and H. Vorbruggen, J. Amer. Chem. Soc., 88 (1966) 852. E. Van Heyningen and L . K. Ahern, J. Med. Chem., 11 (1968). D. H. R. Barton, F. Comer, D. G. T. Greig, G. Lucente, P. G. Sammes, and W. G. E. Wedgewood, Chem. Commun., (1970) 1059. G. E. Gutowski, B. J. Foster, C. J. Daniels, L. D. Hatfield and J. W. Fisher, Tetrahedron Lett., (1971) 3433. Neth. Patent 68,00751. J . Cs. JBszberCnyi, T. E. Gunda, E. R. Farkas, unpublished results (manuscript in preparation). J. D. Cocker, B. R. Cowley, J. S. G. Cox, S. Eardley, G. I. Gregory, J. K. Lazenby, A. G. Long, J. C. P. Sly and G. A. Somerfield, J. Chem. Soc., (1965) 5015. U.S. Patent 3,278,531. Brit. Patent 1,155,493; Chem. Abstr., 71 (1969) 81387~. J. Bradshaw, S. Eardlcy and A. G. Long, J . Chem. Soc. (C), (1968) 801. R. Hcymes, G. Amiard and G. Nomink, Fr. Patent 1,524,172; Chem. Abstr., 71 (1969) 81386w.
J. Cs. JASZBERENYI AND T. E. GUNDA
471
84. J. D’A. Jeffery, E. P. Abraham and G. G. F. Newton, Biochem. J., 81 (1961)591,and Brit. Patent 1,155,493. 84a. C. F. Murphy and J. A. Webber in: ref. 8, pp. 151-166. 85. S. Kukolja, J. Med. Chem., 11 (1968) 1067. 86. G. Nornink, Chim. Therap., 6 (1971) 53. 87. H.Fazakerley, D. A. Gilbert, G. I. Gregory, J. K. Lazenby and A. G. Long, J. Chem. SOC.(C), (1967) 1959. 88. R. A. Archer and B. S. Kitchell, J. Org. Chem., 31 (1966)3409. 89. E. R. Farkas,T. E. GundaandJ. Cs. Jaszbertnyi.TetrahedronLett.,(1973)5127. 90. T. E. Gunda, J. Cs. Jhszberknyi and E. R. Farkas, Acta Chim. (Budapest), 83 (1974) 213. 91. R. J. Stoodley, N. S. Watson, J. Chem. SOC.Perkin I, (1973)2105. 92. R. J. Stoodley, (1974),private communication. 93. J. M. T. Hamilton-Miller, Chemotherapia, 12 (1967)73. 94. R. P. Holysz and H. E. Stavely, J. Amer. Chem. SOC.,72 (1950)4760. 95. D. E. Cooper, and S. B. Binkley, J. Amer. Chem. SOC., 70 (1948)3966. 96. D. A. Johnson, J. Amer. Chem. SOC., 15 (1953) 3936. 97. N. R. Barnden, R. M. Evans, J. C. Hamlet, B. A. Henis, A. B. A. Jansen, M. E. Trevett and G. B. Webb, J. Chem. SOC.,(1953)3733. 98. B. K. Koe, Nature, 195 (1962) 1200. 99. M. A. Panina, I. T. Strukov, and A. S. Khokhlov, Antibiotiki, 9 (1964)685,Chem. Abstr., 61 (1964) 11983~. 100. Cs. Nagy, personal communication, for which the authors express their thanks. 101. H. T. Huang, T. A. Seto, J. M. Weaver, A. R. English, T. J. McBride and G. M. Schull, Antimicrob. Agents Chemother., (1964)493. 102. C. Coronelli, G. C. tancini, R. Pallanza, G. Tamoni and P. Sensi, I1 Farmaco, (Sci. Ed.), 21 (1966)450. 103. A. M. Felix, J . Unowsky, J. Bontempo, and I. R. Frier, J. Med. Chem., 11 (1968)929. 104. S. Khokhlov, A. M. Panina, A. V. Uvarov, Doklady Akad. Nauk SSSR, 135 (1960) 875,Chem. Abstr., 55 (1961) 11394. 105. F. H. Carpenter, J. Amer. Chem. SOC.,70 (1948)2964. 106. M. Wolf, W. Chester and J. H. Sellstedt, U.S. Patent 3,763,152; Farmdoc Complete Spec. Book 61380U.
107. R. M. Evans and A. B. A. Jansen, J. Chem. SOC., (1954)4037. 108. W. J. Gottstejn, G. E. Bocian, L. B. Crast, K. Dadabo, J. M. Essery, J. C. Godfrey and L . C. Cheney, J. Org. Chem., 31 (1966) 1922. 109. F. K. Kirchner, J. R. McCormick, C. J. Cavallito and L. C. Miller, J. Org. Chem., 14 (1949)388. 110. P. Gomis, M.Izquierdo and A. Jurado, Bull. SOC.Chim. France (1968)420. 111. G. R. Foster, K. R. Hardy and J. H. C. Nayler, J. Org. Chem., (1971) 1917. 112. U.S. Patent 3,536,698; Chem. Abstr., 74 (1971) 22861111. 113. Ger. Patent 2,024,359; Chem. Abstr., 74 (1971)22826d. 114. W. V.Daehne, E. Frederiksen, E. Gundersen, F. Lund, P. Mprch, H. J. Petersen, K. Roholt, L. Tybring and W. 0. Godtfredsen, J. Med. Chem., 13 (1970) 607. 115. U.S. Patent 3,488,729; Chem. Abstr., 72 (1970) 100728~. 116. K. W. Glombitza, Liebigs Ann. Chem., 673 (1964) 166. 117. Ger. Patent 2.118,693;Chem. Abstr., 76 (1972)85828t. 118. Ger. Patent 2,068,268; Chem. Abstr., 75 (1971) 110327t. 119. P. Bamberg, P. Ekstrom and B. Sjoberg, Acta Chim. Scand., 22 (1968)367.
472
MODIFICATIONS AND ANALOGUES OF B-LACTAM ANTIBIOTICS
120. 121. 122.
U.S. Patent 3,621,029; Chem. Abstr., 76 (1972) 252812. U.S. Patent 3,665,003; Chem. Abstr., 77 (1972) 620142. G. V. Kaiser, R. D. G. Cooper, R. E. Koehler, C. F. Murphy, J. A. Webber, I. G. Wright and E. M. Van Heyningen, J. Org. Chem., 35 (1970) 2430. R. J. Stedman, J. Med. Chem., 9 (1966) 444. C. J. Cavallito, F. K. Kirchner, L. C. Miller, J. H. Bailey, J. W. Klimek, W. C. Warner, C. M. Suter and M. L. Tainter, Science, 102 (1945) 150. J. Ungar, Brit. J. Exp. Path., 28 (1947) 88. G. A. Snow, Biochem. J., 82 (1962) 6. K. A. Jensen, P. J. Dragsted, I. Kiaer, E. J. Nielsen and E. Frederiksen, Acta Path. Microbiol. Scand., 28 (1951) 407. E. T. Parmele, R. E. Rhodes and R. J. Ferlauto, Antibiot. Chemotherap., 3 (1953) 971. A. B. A. Jansen, and T. J. Russell, J. Chem. SOC.,(1965) 2127. H. P. K. Agersborg, A. Batchelor, G. W. Cambridge and A. W. Rule, Brit. J. Pharmacol., 26 (1966) 649. Brit. Patent 1,255,034; Chem. Abstr., 76 (1972) 25286e. L. C. Cheney, J. C. Godfrey, B. L. Crast and J. L. Luttinger, Fr. Patent 1,491,583; Chem. Abstr., 69 (1968) 52131n. H. F. McDuffie, Jr. and D. E. Cooper, U.S. Patent, 2,650,218; Chem. Abstr., 50 (1956) 41Of. H. F. McDuffie, Jr. and D. E. Cooper, U.S. Patent, 2,578,570; Chem. Abstr., 46 (1952) 7127d. M. R. Bell, S. D. Clemans, L. D. Oesterlin, J. Med. Chem., 13 (1970) 389. E. M. Kleiner, L. B. Senyavina and A. S. Khokhlov, Khim. Geterotsikl. Soedin. (1966) 702. E. M. Kleiner, Synthesis of (79e), unpublished results. E. M. Kleiner, A. S. Guseva, N. S. Ovchinnikova and A. S. Khokhlov, Antibiotiki, 13 (1968) 581. S. M. Chaikovskaya, R. A. Makarova, A. E. Tebyakina, E. M. Kleiner, A. S. Guseva and A. S. Khokhlov, Antibiotiki, 13 (1968) 155. E. M. Kleiner, Antibacterial activity data for (79e), unpublished results. For these unpublished data the authors are indebted to Dr E. M. Kleiner. T. Jen, B. Daniel, J. Frazee and J. Weisbach, J. Med. Chem., 15 (1972) 1172. Brit. Patent 1,281,548; Chem. Abstr., 77 (1972) 114398f. J. C. Sheehan, H. C. Dalzell, J. M. Greenwood, D. R. Ponzi, J. Org. Chem., 39 (1974) 277. M. L. Sassiver and R. G. Shepherd, Tetrahedron Lett., (1969) 3993. J. C. Sheehan and J. J. Ryan, J. Amer. Chem. SOC.,73 (1951) 1204, and 4367. J. Sheehan and G. D. Laubach, J. Amer. Chem. SOC.,73 (1951) 4376. J. C. Sheehan and E. J. Corey, J. Amer. Chem. SOC.,73 (1951) 4756. J. A. Erickson, Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, Massachusetts (1953). R. Pfleger and A. Jager, Chem. Ber., 90 (1957) 2460. I. Ugi and E. Wischhoffer, Chem. Ber., 95 (1962) 136. J. C. Martin, V. A. Hoyle, Jr., and K. C. Brannock, Tetrahedron Lett., (1965) 3589. R. Pratt, G. A. Taylor and S. A. Proctor, J. Chem. SOC.(C),(1967) 1569. J. C. Sheehan and J. A. Schneider, J. Org. Chem., 31 (1966) 1635. L. PauI, P. Polczynski and G. Hilgetag, Chem. Ber., 100 (1967) 2761.
123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154.
J. CS. JASZBERENYI AND T. E. GUNDA 155. 156. 157. 158. 159. 160. 161. 162. 163. 164.
165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190.
413
L. Paul, P. Polczynski and G. Hilgetag, Z. Chem., 8 (1968) 417. A. K. Bose and B. Anjaneyulu, Chem. and Ind., (1966) 903. S. M. Deshpande and A. K. Mukerjee, J. Chem. SOC.(C), (1966) 1241. Ref. 1, Chapter 26. A. K. Bose, G . Spiegelman and M. S. Manhas, Chem. Commun., (1968) 321. A. K. Bose, V. Sudarsanam, B. Anjaneyulu and M. S. Manhas, Tetrahedron, 25 (1969) 1191. G. Amiard, R. Heymes and G. Nomink, Fr. Patent 1,494,067; Chern. Abstr., 71 (1969) 124461rn. A. K. Mukerjee and N. N. Suvorov, Khim. Geterotsikl. Soedin. (1972) 74. A. K. Bose, G. Spiegelrnan and M. S. Manhas, J. Chem. SOC.(C) (1971) 2468. F. Rozgonyi, L . Viczi, Gy. Lustyik, P. Jkkel, Z . Nagy and L. Rtdai, Lecture presented at the Annual Meeting of the Hungarian SOC. Microbiol., Koszeg, Hungary, 1972. Abstract: Acta Microbiol. Acad. Sci. Hung., 20 (1973) 27. A. K. Bose, H. P . S. Chawla, B. Dayal and M. S. Manhas, Tetrahedron Lett., (1973) 2503. A. K. Bose, J. L. Fahey and M. S. Manhas, J. Heterocycl. Chem., 10 (1973) 791. M. S. Manhas, J. S. Chib and A. K. Bose, J. Org. Chem., 38 (1973) 1238. S. Kukolja, J. Amer. Chern. SOC.,93 (1971) 6269. F. P. Doyle and J. H . C. Nayler, Advan. Drug Res., 1 (1964) 1. J. C. Sheehan, in: Molecular Modification in Drug Design, Advances in Chemistry Series, No. 45, (Amer. Chem. SOC.,Washington, 1964) pp. 15-24. J. C. Sheehan, Ann. N.Y. Acad. Sci., 145 (1967) 216. E. Van Heyningen, Advan. Drug Res. 4 (1967) 1. M. S. Manhas and A. K. Bose, in: Synthesis of Penicillin, Cephalosporin C and Analogs, ed. A. K. Bose, (Marcel Dekker, Inc., New York, 1969) pp. 36-105. J. 0. Klein and M. Finland, New England J. Med., 269 (1963) 1019, 1074, 1129. E. P. Abraham, Amer. J. Med., 39 (1965) 692. G. V. Kaiser and S. Kukolja, in: ref. 8, pp. 74-95. M. L. Sassiver, A. Lewis and R. G . Shepherd, Antirnicrob. Ag. Chernother., 1968 (1969) 101. A. N. Klimov, in: Penicilliny i Cefalosporiny, ed. A. D. Katschanov (Medicina, Leningrad, 1973). S. M. Navashin and I. P. Fornina, in: Polusynthetitseskiye Penicilliny (Semisynthetic penicillins), ed. V. R. Sobolyev (Medicina, Moscow, 1974). D. A. Johnson and D. Mania, Tetrahedron Lett., (1969) 267. T. Sawai, T. Saito and S. Mitsuhashi, J. Antibiot., 23 (1970) 488. D. A. Johnson, D. Mania, C. A. Panetta and H. H. Silvestri, Tetrahedron Lett., (1968) 1903. J. L. Strominger and D. J. Tipper, Amer. J. Med., 39 (1965) 708. G. V. Kaiser and S . Kukolja, in: ref. 8, pp. 105-120. G. Cignarella, G. Pifferi and E. Testa, J. Org. Chem., 27 (1962) 2668. E. Evrard, M. Claesen and H.Vanderhaeghe, Nature, 201 (1964) 1124. J. P. Clayton, J. Chem. SOC.(C), (1969) 2123. D. Hauser and H. P. Sigg, Helv. Chim. Acta, 50 (1967) 1327. L. D. Cama, W. J. Leanza, T. R. Beattie and B. G. Christensen, J. Amer. Chem. SOC., 94 (1972) 1408. Y. S. Lo and J. C. Sheehan, J. Amer. Chem. SOC.,94 (1972) 8253.
474
MODIFICATIONS AND ANALOGUES O F P-LACTAM ANTIBIOTICS
191. 191a. 191b. 192. 193. 194.
J. C. Sheehan and Y. S. Lo, J. Org. Chem., 38 (1973) 3227. W. Durckheimer and M. Schorr, Liebigs Ann. Chem., 702 (1967) 163. F. Lund and L. Tybring, Nature, New Bio., 236 (1972) 135. Y . S. Lo and J. C. Sheehan, J. Amer. Chem. SOC.,94 (1972) 8253. U.S. Patent 3,799,922; Farmdoc Complete Spec. Book 26908V. R. B. Morin, B. G . Jackson, E. H. Flynn, R. W. Roeske and S. L. Andrews, J. Amer. Chem. SOC.,91 (1969) 1396. G. V. Kaiser and C. W. Ashbrook, unpublished results quoted in: ref. 8, p. 105. R. Nagarajan, L. D. Boeck, M. Gorrnan, R. L. Hamill, C. E. Higgens, M. M. Hoehn, W. M. Stark and J. G . Whitney, J. Amer. Chem. Soc., 93 (1971) 2308. E. 0. Stapley, M. Jackson, S . Hernandez, S. B. Zimmerman, S. A. Currie, S. Mochales, J. M. Mata, H. B. Woodruff and D. Hendlin, Antimicrob. Agents Chernother., 2 (1972) 122. T. W. Miller, R. T. Goegelrnan, R. G . Weston, I. Putter and F. J. Wolf, Antimicrob. Agents Chemother., 2 (1972) 132. A. K. Miller, E. Celozzi, B. A. Pelak, E. 0. Stapley and D. Hendlin, Antimicrob. Agents Chemother., 2 (1972) 281. A. K. Miller, E. Celozzi, Y. Kong, B. A. Pelak, H. Kropp, E. 0. Stapley and D. Hendlin, Antimicrob. Agents Chemother., 2 (1972) 287. D. D. Daoust, H . R. Onishi, H. Wallick, D. Hendlin and E. 0. Stapley, 3 (1973) 254. S. Karady, S. H. Pines, L. M. Weinstock, F. E. Roberts, G . S. Brenner, A. M. Hoinowski, T. Y . Cheng and M. Sletzinger, J. Amer. Chem. Soc., 94 (1972) 1410. W. A. Slusarchyk, H. E. Applegate, P. Funke, W. Koster, M. S. Puar, M. Young and J. E. Dolfini, J. Org. Chem., 38 (1973) 943. R. W. Ratcliffe and B. G. Christensen, Tetrahedron Lett., (1972) 2907. G . A. Koppel and R. E. Koehler, J. Amer. Chem. Soc., 95 (1973) 2403. L. D. Cama and B. G. Christensen, Tetrahedron Lett., (1973) 3505. R. A. Firestone and B. G . Christensen, J. Org. Chem., 38 (1973) 1436. J. E. Baldwin, F. J. Urban, R. D. G . Cooper and F. L. JosB, J. Amer. Chem. SOC.,95 (1973) 2401. Belg. Patent 747,119; Farmdoc Complete Spec. Book 67316R. T. Jen, J. Frazee and J. R. E. Hoover, J. Org. Chem., 38 (1973) 2857. W. A. Spitzer and T. Goodson, Tetrahedron Lett., (1973) 273. R. W. Ratcliffe and B. G. Christensen, Tetrahedron Lett., (1973) 4645. R. W. Ratcliffe and B. G. Christensen, Tetrahedron Lett., (1973) 4649. R. W. Ratcliffe and B. G. Christensen, Tetrahedron Lett., (1973) 4653. D. J. Tipper and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 1133. R. Reiner and P. Zeller, Helv. Chim. Acta, 51 (1968) 1905. S. Wolfe and W. S. Lee, Chem. Commun., (1968) 242. E. H. W. Bohme, H. E. Applegate, B. Toeplitz, J. E. Dolfini and J. Z. Gougoutas, J. Amer. Chem. Soc., 93 (1971) 4324. R. A. Firestone, N. Schelechow, D. B. R. Johnston and B. G. Christensen, Tetrahedron Lett., (1972) 375. R. A. Firestone, N. Schelechow and B. G . Christensen, Chem. Commun., (1972) 1106. D. B. R. Johnston, S. M. Schmitt, R. A. Firestone and B. G . Christensen, Tetrahedron Lett., (1972) 4917. G . H. Rasmusson, G. F Reynolds and G. E. Arth, Tetrahedron Lett., (1973) 145.
195. 196. 197.
198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222.
J. CS. JASZBERENYI AND T. E. GUNDA
223. 224.
225.
226. 227. 228. 229. 230. 231.
232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243.
244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254.
475
E. H. W. Bohme, H. E. Applegate, J. B. Ewing, P. T. Funke, M. S. Puar and J. E. Dolfini, J. Org. Chem., 38 (1973) 230. H. R. Onishi, D. R. Daoust, S. B. Zimmerman, D. Hendlin and E. 0. Stapley, Abstracts of XIIth Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlantic City, N.J., 1972, p. 77 (and preceding two abstracts). W. Brumfitt, J. Kosrnidis, J. M. T. Hamilton-Miller and J. N. G. Gilchrist, Abstracts of XIIIth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 1973, Abstract No. 52 (and preceding two abstracts). C. H. O’Callaghan, Lecture, presented at the Meeting of the Society of Drug Research, London (1973). G. A. Koppel and R. E. Koehler, Tetrahedron Lett., (1973) 1943. D. M. Brunwin and G. Lowe, Chem. Commun. (1972) 192. G. Lowe, Ger. Patent 2,305,972; Farmdoc Complete Spec. Book, 50819U, Chem. Abstr., 79 (1973) 1 1 5 6 0 2 ~ . A. K. Bose, J. L. Fahey, and M. S. Manhas, J. Heterocycl. Chem., 10 (1973) 791. I. Ager, D. H. R. Barton, D. G. T. Greig, G. Lucente, P. G. Sammes, M. V. Tayler, G. H. Hewitt, B. E. Looker, A. Mowatt, C. A. Robson and W. G. E. Underwood, J. Chem. SOC.Perkin I. (1973) 1187. D. N. Reinhoudt, Rec. Trav. Chim., 92 (1973) 20. G. Lowe and M. V. J. Ramsay, J. Chern. SOC.Perkin I. (1973) 479. D. M. Brunwin and G. Lowe, J. Chem. Soc. Perkin I. (1973) 1321. S. Kukolja, J. Amer. Chem. SOC.,94 (1972) 7590. R. B. Woodward, Ger. Patent 2,153,554; Chem. Abstr., 77 (1972) 126700m. E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond and W. J. Waring, The Molecular Basis of Antibiotic Action (John Wiley, London, 1972) p. 71. C. H. O’Callaghan and P. W. Muggleton, in: ref. 8., pp. 472473. M. Leyh-Bouille, M. Nakel, J-M. FrBre, K. Johnson, J-M. Ghuysen, M. Nieto and H. R. Perkins, Biochemistry, 1 1 (1972) 1290. G. G. Wickus and J. L. Strominger, J. Biol. Chem., 247 (1972) 5307. M. R. Pollock and H. R. Perkins, unpublished observations quoted in ref. 242. M. Nieto, H. R. Perkins, J-M. Fr%reand J-M. Ghuysen, Biochem. J., 135 (1973) 493. J-M. Ghuysen, M. Leyh-Bouille, M. Nakel, J. Dusart, K. Johnson, J-M. Frkre, J. Coyette, H. R. Perkins and M. Nieto, Proceedings of the Symposium on Molecular Mechanisms of Antibiotic Action on Protein Biosynthesis and Membranes, Granada (Elsevier, Amsterdam, 1972). Ref. 237, p. 94. K. Izaki, M. Matsuhashi and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A., 55 (1966) 656. K. Izaki, M. Matsuhashi and J. L. Strominger, J. Biol. Chem., 243 (1968) 3193. J. P. Duguid, Edinburgh Med, J., 53 (1946) 401. J-M. Ghuysen, D. J. Tipper, C. H. Birge and J. L. Strominger, Biochemistry, 4 (1965) 2245. Ref. 237, p. 82. J-M. Ghuysen, Bacteriol. Rev., 32 (1968) 425. R. Hartmann, J. V. Holtje and V. Schwartz, Nature, 235 (1972) 426. J. L. Strominger, K. Izaki, M. Matsuhashi and D. J. Tipper, Fed. Proc., 26 (1967) 9. E. M. Wise, Jr., and J. T. Park, Proc. Natl. Acad. Sci. U.S.A., 54 (1965) 75. D. J. Tipper and J. L. Strominger, J. Biol. Chem., 243 (1968) 3169.
476
MODIFICATIONS A N D ANALOGUES O F P-LACTAM ANTIBIOTICS
255. 256. 257. 258. 259. 260.
H. H. Martin, J. Gen Microbiol., 36 (1964) 441. Ref. 237, p. 75. B. Lee, I. Mol. Biol., 61 (1971) 463. Ref. 237, p. 87. J. E. Dolfini, personal communication. P. R. K. Ho, R. D. Towner, J . M. Indelicato, W. A. Spitzer and G. A. Koppel, J. Antibiot. (Tokyo), 25 (1972) 627. M. Leyh-Bouille, J-M. Ghuysen, M. Nieto, H. R. Perkins, K. H. Schleifer and 0 . Kandler, Biochemistry, 9 (1970) 2961. M. Leyh-Bouille, J-M. Ghuysen, M. Nieto, H. R. Perkins, K. H . Schleifer and 0 . Kandler, Biochemistry, 9 (1970) 2971. M. Leyh-Bouille, J. Coyette, J-M. Ghuysen, J. Idczak, H. R. Perkins and M. Nieto, Biochemistry 10 (1971) 2163. M. Nieto, H. R. Perkins, M. Leyh-Bouille, J-M. Frtre and J-M. Ghuysen, Biochem. J., 131 (1973) 163. J. J. Pollock, J-M. Ghuysen, R. Linder, M. R. J. Salton, H. R. Perkins, M. Nieto, M. Leyh-Bouille, M. F r t r e and K. Johnson, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 662. J-M. FrCre, J-M. Ghuysen, H. R. Perkins and M. Nieto, Biochem. J., 135 (1973) 463. G. H. F. Green, J. E. Page, J. E. Staniforth, J. Chem. Soc., (1965) 1595. R. D. G . Cooper, P . V. DeMarco, J. C. Cheng and N. D. Jones, J. Amer. Chem. Soc., 91 (1969) 1408. R. M. Sweet and L . F. Dahl, J. Amer. Chem. SOC.,92 (1970) 5489. R. M. Sweet, Ref. 8, pp. 281-283. D. B. Boyd, J. Med. Chem., 16 (1973) 1195. R. B. Hermann, J. Antibiot. (Tokyo), 26 (1973) 223. C. Hansch and T. Fujita, J. Amer. Chem. SOC., 86 (1964) 1616. J. A. Singer and W. P. Purcell, J. Med. Chem., 10 (1967) 1000. C. Hansch and A. R. Stewart, J. Med. Chem., 7 (1964) 691. C. Hansch and E. W. Deutsch, J. Med. Chem., 8 (1965) 705. C. B. C. Boyce and B. V. Milborrow, Nature, 208 (1965) 537. G. L. Biagi, A. M. Barbaro, M. C. Guerra and M. F. Gamba, J. Chromatogr., 44 (1969) 195, and 371. G. L. Biagi and M. L. Guerra, J. Med. Chem., 13 (1970) 511. G. L. Biagi, A. M. Barbaro and M. C. Guerra, Advan. Chem. Ser., (1972) 61. J . T. Penniston, L . Beckett and D. L. Bentley, and G. Hansch, Mol. Pharmacol., 5 (1969) 333. J. W. McFarland, J. Med. Chem., 13 (1970) I 192. E. J. Lien, G. Hansch and S . M. Anderson, J . Med. Chem., 1 1 (1968) 430. G. L. Biagi, Antibiotica, 5 (1967) 198. A. E. Bird and A. C. Marshall, Biochem. Pharmacol., 16 (1967) 2275. H. Bundgaard, Dansk. Tidsskr. Farm., 45 (1971) 283. D. M. Brunwin, G. Lowe and J. Parker, Chem. Commun. (1971) 865. D. M. Brunwin, G. Lowe and J. Parker, J. Chem. SOC.(C) (1971) 3756. D. M. Brunwin and G. Lowe, Chem. Commun. (1972) 589. F. Moll, 2. Naturforsch., b 21 (1966) 297, and F. Moll, H. Thoma, Z. Naturforsch., b 24 (1969) 942. F. Moll and P . Kastenmeier, 2. Naturforsch., b 27 (1972) 727. G. Lowe and D. D. Ridley, Chem. Commun. (1973) 328, and refs. 233 and 234.
261. 262. 263. 264. 265. 266. 267. 268. 269. 270. 271. 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. 288. 289. 290. 291. 292.
J . C S . JASZBERENYI AND T. E. GUNDA 293. 294. 295. 296.
477
D. B. Boyd, J. Amer. Chem. SOC.,94 (1972) 6513. T. Berti and G. L. Biagi, G . Ital. Chemioter., 17 (1970) 93. Ref. 8, pp. 542-544. K. Izaki, M. Matsuhashi and J. L. Strominger, J. Biol. Chem., 243 (1968) 3180.
This Page Intentionally Left Blank
Index Acetylcholinesterase, purification, 119 Acetylsalicylic acid, NMR, 181 Acidurias, diagnosis, 65-68 Adenine in polarography, 260 Adenylate cyclase, 294-296 assay, 308 in frog erythrocytes, 295 in mammals, 295 Affinity chromatography, 106 applications, 118 experimental aspects, 116, 143 gel, 136, 140 immunochemical uses, 129 Agarose, 108, 110, 126, 128-131, 134, 135, 141 Aldoses and GLC-MS, 58 Amino acids, purification by chromatography, 179 GLC-MS, 37 Aminoglycosides, chemistry, 372, 373 resistance, 370 Amoxycillin, 430 Amphetamine, determination by GLC-MS, 74 metabolites, GLC-MS, 81 Ampicillin, 346, 349, 429 polarography, 286 Atmospheric pressure ionization, 36 Atomic mass of isotopes, 29 ATP-ase activity, assay, 309, 324 Barbiturates, determination by GLC-MS, 74, 75 metabolism, GLC-MS, 72 BB-K8, 382-387 activity, 383, 384 structure, 382 Beckmann glucose analyzer, 270 Beckmann oxygen analyzer, 256 479
Benzodiazepines, polarography, 283-285 Benzylpenicillin, 346, 429 Binding proteins, purification, 123 Bio-Gel P, 137, 139 Biogenic amines, purification, 139 GLC-MS, 79, 82 Biosynthesis, NMR in, 162 Bis-chloromethyl ether, GLC-MS, 88 BL-P1462, 346 BL-P1654, 346 BrdiEka catalytic waves, 264-268 Bronopol, 342 t-Butyldimethylsilyl derivatives for GLCMS, 13 Caffeine, determination by GLC-MS, 75 metabolism, GLC-MS, 70 Calcium, in walls of Pseudornonas, 339 release, metabolic effects, 305 Cambendazole, metabolism, GLC-MS, 72 Carbamazepine, 70 determination by GLC-MS, 74 metabolism, GLC-MS, 70 Carbenicillin, 344, 350, 429 combination with other antibiotics, 349 esters, 344-346 resistance, 360 Carbohydrates and GLC-MS, 57 Catecholamines, determination by GLCMS, 80 metabolites, separation, 141 Cefamandole, 430 Cefazolin, 432 Cefoxitin, 438 Cellulose in affinity chromatography, 1 1 1, 126 Cell wall synthesis, inhibition, 452 Cephacetrile, 432 Cephachlomezin, 432
480
INDEX
Cephalexin, 359-370, 432 Cephalocillin, 404 Cephaloglycin, 432 Cephaloram, 432 Cephaloridine, 359-370, 432 Cephalothin, 359-370, 432 derivatives, 431 Cephalosporinase, 43 1 Cephalosporins, 396 absolute configuration, 426 anhydride, 418 C, 431, 432, 436 A’-derivatives, 406 clinically used, 429 enlarged ring, 449 enzymes, resistance to, 440 mode of action, 451 Ps. aeruginosa and, 358-370 2-substituted, 398 4-substituted carboxyl, 415 6-substituted, 425 7-substituted, 429 spiro, 407 structure-activity relationships, 463 sulphones, 398 sulphonium ylides, 398 sulphoxides, 398, 401 tricyclic, 407 Cephamycin, 431, 436, 438, 441 resistance to enzymes, 440 Cephanone, 432 Cephapirin, 432 Ceruloplasmin in ESR, 219 Chemical ionization, 33,34,40,70,76,84,86 Chemical vapour analysis, 84 Chloramphenicol, polarography, 285 resistance, 384 structure, 384 Chlordiazepoxide metabolites, GLC-MS, 69 polarography, 282, 284 Chlorpromazine metabolites, GLC-MS, 69 Chlortetracycline, 356 polarography, 286 Chyrnotrypsin, purification, 119, 134 ESR, 234 Clinical use of GLC-MS, 52 Cloxacillin, 346, 365, 430 Cobalt proteins, ESR, 214
Codeine, determination by GLC-MS, 74 Column chromatography, 105 gel, 135, 140 high pressure, 144 hydrophobic, 132 Concanavalin A, 169 Conformation of proteins from NMR, 182 Conformation study with polarography, 269 Constitutive plactamase, 367 Coupling constants, NMR of peptides, 176 Coupling reactions in affinity chromatography, 109 Crypticity studies in resistance, 353 Cuproproteins, ESR, 209-21 1 Cyclic AMP, 299-301 action of hormones, 300 assay, 316-319 discovery, 294 Cyclic GMP, 297, 301 assay, 313-315, 319 3‘,5’-Cyclic nucleotide phosphodiesterase, 297, 298 assay, 320 Cyclic nucleotides, assays, 313 body fluids, 311 broken cell preparations, 306-3 10 intact cells, 310 Cytochrome c, NMR, 174 P-450, ESR, 203, 228 polarography, 269 Cytosine, polarography, 260 DAP I, 45 Dehydrogenases, purification, 120 Derivatives for GLC-MS, 11 Deoxyribonucleic acids, polarography, 261 Diazepam, determination by GLC-MS, 74, 75 metabolites in GLC-MS, 69, 70 polarography, 282 Dicloxacillin, 430 Differential pulse polarography, 286 Dinucleoside monophosphates, 139 Dipenicillins, 418 Dipeptidylaminopeptidase I, 45 Diphenylhydantoin, determination by GLCMS, 74, 75 metabolites, GLC-MS, 69 Disaccharides and GLC-MS, 60
INDEX Disulphides in polarography, 264 Dopamine, determination by GLC-MS, 80 Dropping mercury electrode, 248, 249, 259 EDTA, 352, 353 use in bacterial resistance, 347 use with other antibacterial agents, 347. 348 Electrocyclization, 275 Electron capture detection, 79, 80 Electron impact ionization, 33, 40, 70 ENDOR, 200, 219-221, 227 Environmental toxicology, GLC-MS, 86 Enzacryl, 142 Enzymatic resistance in bacteria, 358 Enzyme deficiency disorders, 63 defects, 61 Enzymes, affinity chromatography, 107 cephalosporins and, 456 ESR and, 223 NMR and, 163 penicillin and, 456 polarography, 233, 270 purification, 119 Epicillin, 430 ESR spectra, 167, 170, 192 Ethanol, determination by GLC-MS, 74 Fats and GLC-MS, 53 Fatty acid derivatives in GLC-MS, 22 Ferredoxin in ESR, 197, 206 Field ionization, 33 desorption, 37 Flucloxacillin, 430 Formulations of drugs, analysis, 279 Free radicals in biology, 193, 217, 218 NMR, 175 Functionalization in polarography, 278 Galactosidase, purification, 121 Gamma-globulin, purification, 134 Gel chromatography, 136, 140 Gentamicin, antibacterial action, 344, 349, 377-383 structure, 378 GLC-MS, 1 analysis of complex mixtures, 70 applications, 37 clinical uses, 51 combination, 7 computers, 18
481
deficiency disorders, 63 derivatives suitable for, 11, 13, 70, 76, 80 environmental uses, 86 forensic uses, 84 high resolution MS, 16 instrumentation, 2, 8, 15, 27 metabolic studies, 51, 64, 69 pharmacological applications, 68 principles, 2 quantitative drug assay, 74 toxicological applications, 68, 84 Glucuronides detected by GLC-MS, 70 Glycogen phosphorylase, 304-306 Glycogen synthase, 306 assay, 322 Guanethidine, determination by GLC-MS, 74 Guanine, polarography, 261 Guanylate cyclase, 297, 298 assay, 308, 310 g-Values, 195 Haemin enzymes, polarography, 269 Haemoglobin, spin labelling, 233 Haemoproteins, 204-206 Halogens, polarographic determination, 258 Heavy isotope tracers, GLC-MS, 72 Hetacillin, 430 High pressure chromatography, 144 instrumentation, 149 supports, 148 Histones, NMR, 182 Hydrazide-agarose, 110 Hydrogen bonding in chromatography, 138 Hydrophobic chromatography, 132 Hydroxynalidic acid, determination, 147 Imipramine, determination by GLC-MS, 74 Immunochemistry, ESR in, 240 Indolealkylamines, determination by GLCMS, 81 Indoramin, determination by GLC-MS, 74 Inorganic compounds, determination by polarography, 252 Ion monitoring in GLC-MS, 25 Ionization, atmospheric pressure, 36 chemical, 33, 34, 40, 70, 77, 84, 86 electron impact, 33, 40, 70 field, 33
482
INDEX
Ionisation (continued) field desorption, 37 Iron-sulphur proteins, ESR and, 206-209. 224, 225 Iron-proteins, ESR, 204-209 Irradiated molecules, ESR, 221 Isotope tracers, GLC-MS, 72 Kanamycin, 349, 370, 371 Keto acids in GLC-MS, 68 P-Lactamase, 350, 354, 359 Lanthanide shift reagents, 172 Lecithins, ESR, 236 NMR, 182, 184 Lectins, insolubilized, 127 Librium, polarography, 282 Lidocaine, determination by GLC-MS, 74 Lilacillin, 345, 346 Line-width spectrum, 198 Lipids and GLC-MS, 53 Lividomycin, 372-380 structure, 379 Lorazepam, polarography, 273 Lumiflavin, ESR, 220 Lysozyme and gram-negative bacteria, 352 NMR, 172 purification, 137, 143 Mafenide, 357 Magnesium, requirement for adenylate cyclase, 295 requirement for ATP, 302 Manganese proteins in ESR, 216 Marine lobster, leg nerve, 238 Mass spectrometry, 2 Mechanisms of resistance to antibiotics, 352 exclusion, 352-357 Membranes, ESR, 236, 237 NMR, 182 spin labelling, 236, 237 Metabolic disorders, 61 Metaclopramide, mass spectrum, 35 Metalloproteins, ESR, 200-216 Metals, polarographic determination, 252 Methadone, GLC-MS, 85 Methaemoglobin, ESR, 202 Methaqualone, GLC-MS, 85 Methaemoglobin, polarography, 269 Methicillin, 346, 350, 429 Methoxime-TMS derivative, 47
Methsuxinimide metabolites, GLC-MS, 69 Metmyoglobin, polarography, 269 Mogadon, polarography, 282 Molybdenum proteins, ESR, 212 Morphine, determination by GLC-MS, 74, 75 Murein in cell walls, 336, 337, 338, 340 Mutation studies in bacterial resistance, 357 Myokinase, 3 I5 Nafcillin, 349, 430 Nalidixic acid, 147, 348 Neomycin, 372-376 structure, 376 Nitrazepam, polarography, 282, 284 Nitrosamines in food, GLC-MS, 88 Nitroxides, spin labelling, 231,233,234,241 NMR spectroscopy, 159 13C, 164, 174, 175, 178, 179, 183 I9F, 163 14N, 178 "P, 172, 178, 183 tissue studies, 185 Noradrenaline, determination by GLC-MS, 80 metabolites, GLC-MS, 80 Nortryptyline, 82 determination by GLC-MS, 74 metabolites, GLC-MS, 69, 72 Nuclear-hyperfine splitting, 1% Nuclear Overhauser effect, 181 Nucleic acids, electrochemical properties, 259 Nucleosides, NMR, 166 5-Nucleotidase, 325 Nucleotides, conformation, 172 insolubilized, 126 Oestrogen identification by GLC-MS, 48 Organic acids in GLC-MS, 65, 66 compounds, polarography, 275 Oxacillin, 430 Oxazepam, 271 polarography, 282, 283 Oxygen, poiarographic determination, 254 Paramagnetic ions, 193 probes, NMR, 167 Parathyroid hormone, GLC-MS, 40 Parebers, 342 Parkinsonian patients, urine analysis, 82
INDEX Penicillic acid, biosynthesis, 163 Penicillins, 396 absolute configuration, 426 3-carbonitrile, 4 18 G, 285, 286, 429 G dimer, 444 in clinical use, 429 mode of action, 451 N, 429 polarography, 285, 286 Ps. aeruginosa and, 358-370 resistance, 354, 361 ring expansion, 399 spiro derivatives, 440 structure-activity relationships, 463 2-substituted, 398 3-substituted carboxyl, 415 5-substituted, 425 6-substituted, 429 sulphones, 398 sulphoxides, 398 thioanhydride, 419 V, 429 Pentafluoropropionyl derivatives in GLCMS, 26 Pentobarbitone, determination by GLC-MS, 75 Peptides, GLC-MS, 40 Peroxidases, mechanism of action, 218 Pesticide residues, GLC-MS, 87 Pethidine, determination by GLC-MS, 75 Phenbenicillin, 429 Phenethicillin, 429 Phenobarbitone, determination by GLCMS, 75 metabolites, GLC-MS, 69 Phenols, antibacterial activity, 342 Phenoxymethylpenicillin, 429 Phentermine, determination by GLC-MS, 74 Phenytoin metabolism, GLC-MS, 70 Phosphofructokinase, NMR, 176 Phospholipids, GLC-MS, 56 Phosphorylase, activation by catecholamines, 302 assay, 321 fluorotyrosine, NMR, 163 kinase, 303, 304, 321 types a and b, 305
483
use in cyclic AMP assay, 313 Phosphorylase phosphatase, assay, 322,324 Photosynthesis and ESR, 226 Piribedil, determination by GLC-MS, 74 Platinum electrode, 255 Polarography, 248 analytical uses, 271 automatic analysis, 272 molecular conformation, 269 organic compounds, 275 organic synthesis, 273 study of reactions with, 269, 271 tablets analysis, 280 Polarometric titration, 278 Pollution, GLC-MS in, 87 Polyacrylamide in chromatography, 112, 136 Polyacryloylmorpholine in chromatography, 142 Polymixins, 343 antibacterial activity, 343 resistance, 355 Polyribonucleotides, polarography, 261 Polysaccharides matrices, 109 coupling, 109, 110 Propicillin, 429 Propoxyphene metabolites, GLC-MS, 72 Propranolol metabolism, GLC-MS, 69, 72 Prostaglandin, derivatives for GLC-MS, 11, 13, 14, 73, 75 separation, 140 Protein kinase, 301-303 assay, 320 Proteins, NMR, 163, 175, 181 polarography, 266-269 Proteolysis in polarography, 270 Pseudomonas aeruginosa, 334 antibiotics and, 356-384 cell envelope, 335 characteristics, 335 drugs against, 340-351 mechanism of resistance, 352 structure, 336 Pyridoxamine, electrolytical preparation, 274 Pyruvate kinase, 315 Quaternary ammonium compounds, antibacterial activity, 342
484
INDEX
Quinacillin, 430 Quinonoid vitamins, ESR, 219 Receptor site, purification, 123 Redox enzymes, 201 R factor-mediated p-lactamase, 365 Resonant frequency, 194, 195 Ribonuclease, NMR, 164, 165, 180 purification, 137 Ribonucleic acids, polarography, 262 Ribose, polarography, 259 Rubredoxins, ESR, 206, 207 Salbutamol, determination by GLC-MS, 74 Salsolinol, detection by GLC-MS, 82 pentafluoropropionyl derivative in GLC-MS, 26 Selected ion monitoring, 25, 27, 32, 75, 79, 81, 86, 88 Sephadex, 108, 124,131,136, 138,140,143 Sepharose, 115, 118, 124-126, 128, 129, 132, 133, 141 Sepheron, 142 Serenid D, polarography, 282 Serotonin, 83 Sodium fluoride, stimulation of adenylate cyclase, 296 Spacer gels in chromatography, 112 Spectroscopy, ESR, 192-241 NMR, 159-190, 192 Sphingophospholipids and GLC-MS, 56 Spin-label techniques, 193, 230-233 Spin-orbit coupling, 195, 198 Spins, number detected, 199 Stable isotopes, 29, 30 drug metabolism, 71 Steroids, derivatives for GLC-MS, 17, 47 fragmentation, 46 separation, 140 Streptomycin, 377 derivatives, 377 polarography, 285, 286 Ps.aeruginosa and, 358 structure. 377
Stripping analysis, 249 Succinate dehydrogenase, ESR, 226 Sugars and GLC-MS, 57 anomers, 58 Sulbencillin, 429 Sulphamylan, 357 Sulphite oxidase, ESR, 212 Sulphoamino penicillins, 345 Sulphonamides, Ps. aeruginosa and, 357 Sulphydryl compounds, polarography, 262-269 Synergism against Ps. aeruginosa, 349 Terbutaline, determination by GLC-MS, 74 Tetracyclines, polarography, 285, 286 resistance of Ps. aeruginosa, 356 Tetrahydrocannabinol, determination by GLC-MS, 74 Tetrahydroquinolines, GLC-MS, 81 Thiazepines, antibacterial activity, 449 Thienopyrimidine metabolism, GLC-MS, 72 Thiothixene, determination by GLC-MS, 74 Thymine, polarography, 261 Ticarcillin, 345, 346 Tobramycin, 372, 379-381 antibacterial activity, 380-383 structure, 381 Tocopherols, separation, 139 Transferrin, ESR, 208, 209 Tranxillum, polarography, 282 Triacylglycerol lipase, 306 assay, 323 2,4,6-Trichlorotriazine,1 10 Trimethoprim, 349 Trimethylsilyl derivatives for GLC-MS, 12, 17, 38, 44, 70, 71, 76 Trypsin, purification, 119 Uracil, polarography, 261 Valium, polarography, 282 Vanadium proteins, ESR, 216 Vitamin BIZ,ESR, 214 Vitamin Q, ESR, 220