Biochemistry Involving Carbon-Fluorine Bonds

Biochemistry Involving Carbon-Fluorine Bonds In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium...

Author: Robert Filler

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Biochemistry Involving Carbon-Fluorine Bonds

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


Biochemistry Involving Carbon-Fluorine Bonds R o b e r t F i l l e r , EDITOR

Illinois Institute of Technology

A symposium sponsored by the Divisions of Fluorine and Biological Chemistry at the 170th Meeting of the American Chemical Society, Chicago, Ill., Aug. 26, 1975.

28

ACS SYMPOSIUM SERIES

AMERICAN CHEMICAL WASHINGTON, D. C.

SOCIETY 1976


Library of Congress

Data

Biochemistry involving carbon-fluorine fonds. (ACS symposium series; 28, ISSN 0097-6156) Includes bibliographical references and index. 1. Organofluorine compounds—Physiological effect— Congresses. I. Filler, Robert, 1923. II. American Chemical Society. Division of Fluorine. III. American Chemical Society. Division of Biological Chemistry. IV. Series: American Chemical Society. ACS symposium series; 28. QP981.F55B56 ISBN 0-8412-0335-0

Copyright ©

612'.0157 ACSMC8 28 1-215

76-13037 (1976)

1976

American Chemical Society A l l Rights Reserved.

N o part of this book may

be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E UNITED STATES O F AMERICA


ACS Symposium Series R o b e r t F. G o u l d ,

Series

Editor


FOREWORD The ACS SYMPOSIU

a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.


PREFACE Τη recent years there has been increasing interest and research activity in the biochemistry and medicinal applications of compounds con taining the carbon-fluorine bond. The medicinal chemistry of fluorinated organic compounds was last discussed at a symposium held at the national ACS meeting in Washington, D.C. in September 1971. Simultaneously a Ciba Foundation symposium on the biochemistry and biological activities of carbon—fluorine compound and incisive discussions a symposium, promi nent contributors to the field participated, were published in 1972 by Elsevier. A survey of fluorinated compounds of medicinal interest was presented in the December 1974 issue of Chemical Technology. The biochemical aspects of C - F chemistry have developed rapidly since the pioneer studies in the early 1940s by Sir Rudolph Peters, who elucidated the mechanism of the toxic action offluoroacetateby invoking the concept of 'lethal synthesis." Special mention should also be made of the elegant studies since the late 1950s by Charles Heidelberger and his colleagues on the tumor-inhibitory effects of nucleotides of fluorinted pyrimidines. Important advances in the biochemistry of organofluorine compounds continue unabated and a symposium on this subject, cosponsored by the Divisions of Fluorine and Biological Chemistry, was held at the Chicago ACS meeting in August 1975. This volume includes all ten presentations and discussions at that symposium. The subjects are of broad interest, ranging from fluorocarboxylic acids as enzymatic and metabolic probes to the use of perfluorocarbons as "artificial blood." We hope that these reports will further stimulate those already active in the field and create a sense of excitement and enlightenment to the curious. To the newcomer we say "Welcome—don't be afraid of fluorine compounds—they're lots of fun." Finally, I wish to express my appreciation to all of the contributors for their patience and unstinting cooperation in making this book possible. Illinois Institute of Technology Chicago, Ill. January 1976

ROBERT FILLER

ix In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1 Fluorocarboxylic Acids as Enzymatic and Metabolic Probes E R N E S T KUN University of California, San Francisco, Department of Pharmacology, and the Cardiovascular Research Institute, San Francisco, Calif. 94143

The development of site specific reagents necessarily depends on the knowledge of specific biochemical reactions, which should include the chemical structure of catalysts, substrates, modifiers, and products of enzymatic processes. Very little is known about catalytic sites of most enzymes, therefore comparison of substrates with analogues may be one of the experimental approaches to study this problem. It follows also that extensive information related to analytical enzymology (i.e., catalytic properties of isolated enzymes, enzymatic composition of cells) has to preceed the meaningful application of specific probes in complex systems. This endeavor is conceptually a synthetic one and intends to elucidate biological functions. It is only in relatively rare cases that inhibitors of cellular systems act in a manner predicted from in vitro enzymology. For example, a linear multienzymatic process can be indeed regulated by a rate limiting enzymatic component, susceptible to a specific inhibitor. In this case the biological significance of the linear multienzyme system can be studied with success. If acute inhibition does not cause rapid irreversible changes in cellular economy, sustained inhibition may trigger a variety of compensatory cellular processes, encompassing both intermediary and macromolecular metabolism. Experimental pathology and toxicology may benefit from these studies, since pathophysiology of environmental toxic effects is likely to be traced to specific initiating reactions of inhibitors with specific cellular sites. This experimental subject is at present only in its beginning stages. In contrast to linear systems-distributive, branching,and cyclic multienzymatic processes (1) respond in a far more complex manner to perturbations by a specific inhibitor. Alterations of steady state concentrations of metabolites, and subcellular changes of topographic distribution of intermediates of metabolic pathways will occur. Most of these consequences are unpredictable from idealized metabolic charts (2). Experimental results obtained with relatively uncomplicated fluorocarboxylic acids indicate that major metabolic pathways are apparently not 1 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2

BIOCHEMISTRY

INVOLVING C A R B O N - F L U O R I N E

BONDS

regulated by properties of enzymatic components alone but more importantly by a v a i l a b i l i t y of substrates. The role of primary regulatory enzymes in c e l l u l a r systems, problems of molar ratios of substrate to enzymes in biological systems have been reviewed (3) but the majority of biochemical texts have not incorporated these advances at present time. The choice of F substitution of H or OH groups in well known carboxylic acid substrates to obtain specific inhibitors was based on straight forward chemical considerations (4). The present paper is restricted to experimental work related to the mode of action of fluorocarboxy l i c acids, developed in our laboratory. >

F1uoro-dicarboxylic acids A series of chemical and enzymological experiments (5,6,7, 8,9,10,11,12,13,14,15,16,17,18,19) with mono and difluoro oxalacetates, fluoroglutamates mal ate, fluorolactate and mal ate dehydrogenases, transaminases, glutamate and lactate dehydrogenases suggested a reasonably uniform pattern of interaction between fluorodicarboxylic acid substrates with respective enzymatic s i t e s . It is of interest that introduction of one or two F atoms did not change the conformation of the parent molecule to the extent that i t could not be recognized by enzymatic s i t e s . In generalênzyme-fluoro-substrate Michaelis-Menten complexes are readily formed, but in some instances the rates of conversion to products, i . e . , the process of enzymatic catalysis i t s e l f is d r a s t i c a l l y reduced by F-substitution. The molecular reasons for this inhibition by Fsubstitution are as yet unexplored, and constitute a significant problem of mechanistically oriented enzymology, worthy of more detailed investigations. As would be expected F-dicarboxylic acids behave as r e l a t i v e l y uncomplicated l i n e a r l y competitive inhibitors with respect to the non-fluorinated substrate molecule. Kinetic analyses of the effect of fluoro-oxalacetate in bisubstrate systems of mal ate dehydrogenases (19) and lactate dehydrogenase (14) support this conclusion. A summary of experimental results is shown in Table I. Whereas monofluoro oxalacetate is a slowly reacting substrate of MDH, difluoro subs t i t u t i o n converts this substrate homolog to an equally good substrate of MDH to oxalacetate i t s e l f with respect to Vmax,except difluoro-oxalacetate has a Km of 4.0 mM (about 4.10^ higher than Km of oxalacetate). Further exploration of this significant effect of difluoro substitution may shed light on the as yet unknown molecular mechanism of catalysis of MDH. Since the p r i mary purpose of our investigations was to obtain biological probes, the s t a b i l i t y of F-dicarboxylic acids were also i n v e s t i gated in biological systems. Despite the promising in v i t r o properties of e-monofluoro oxalacetatic acid as an i n ï ï ï b i t o r of MDH, i n s t a b i l i t y prevented i t s application in complex systems. As would be expected.monofluoro oxalacetate is susceptible to



330 μΜ 710 μΜ

1800 μΜ 640 μΜ

3 F-glutaric

a-F-glutamate(NADP )

a-F-glutamate(NAD )

Liver GDH

Liver GDH

Liver GDH

Liver malic enzyme (decarboxylating)

Kidney MDH

5

6

7

8

9

L(+) 3-fluorolactate

--

300 μΜ

13 μΜ

300 μΜ

330 μΜ

15.0

13.0

transaminative defluorination

45. μΜ

LEGEND TO TABLE I: MDH = malate dehydrogenase; GOT = glutamate oxalacetate amino transferase; GDH = glutamate dehydrogenase; LDH = lactate dehydrogenase; Ma = molecular a c t i v i t y ; (S) = with physiological substrate; (F-S) = with fluoro-analogue

10 Muscle LDH

(-)-erythrofluoromalate

33'-difluoromalate

+

+

3 F-oxalacetic

Liver GOT(mito)

4

33* -F2-oxalacetic

1.0

Liver GOT(mito)

4.0 mM

3

33'-F2-oxalacetic

Kidney MDH(mito)

η

2

1

SUBSTRATE AND INHIBITORY PROPERTIES OF F-CARBOXYLIC ACIDS Ma F-carboxylic acid 1^ Κ* MâXF-S) Kidney MDH(mito) 101 3-F-oxalacetic 0.5 μΜ 0.5 μΜ

Enzyme

No.

4

BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS

COOH

COOH I ÇH + Ε

I

FCH I c=o I COOH

2

HCNH I COOH ?

COOH

I HC II

C-NH COOH Figure 1.

2

COOH I CH

COOH I FCH I

2

C=0 I COOH

COOH ^ HCH I C=NH I COOH

HCNH

2

COOH

H0 2

COOH I CH I + NH, c=o I COOH 2

Transaminative degradation of monofluoro-oxahcetic acid


1.

KUN

Fluorocarboxylic

5

Acids as Probes

bivalent metal ion catalyzed decarboxylation to fluoro pyruvate. The kinetics of this reaction has been studied in detail (20). Interaction of monofluoro oxalacetate with glutamate-oxalacetate aminotransferase yields rapid elimination of F~ and Ν Η Λ from a system containing both aspartate and the fluoro-acid (b), repre senting a rare case of elimination reactions characteristic of pyridoxal-phosphate c a t a l y s i s , as well known from the work of Snell and his school. This i s shown in Figure 1. From a com bined use of fluoro-glutarate, a r e l a t i v e l y specific i n h i b i t o r of GDH (11) and of difluoro-oxalacetate, an i n h i b i t o r of GOT (10) the regulation of both transaminative and oxidative pathways of mitochondrial glutamate metabolism was further elucidated (12). Enzymatic reduction of monofluoro-oxalacetate by MDH on a prepar ative scale yielded (-)erythrofluoromalic acid (21) which i s an excellent i n h i b i t o r of malate dehydrogenases in v i t r o . In isolated hepatocytes this i n h i b i t o r acts only on cytosolic MDH isoenzyme because i t doe mitochondrial membrane (cf. 21), therefore i t i s useful as a c e l l u l a r probe of this MDH isoenzyme. Extensive t r i a l s with mitochondria and isolated hepatocytes - as models for the study of complex systems - indicated that only difluoro-oxalacetic and (-)erythrofluoromalic acidsproved useful. Difluoro-oxalacetate, by i n h i b i t i n g GOT,proved to be stable and highly s p e c i f i c in i t s action. Mono fluoro-malate, besides i n h i b i t i n g cytosolic MDH is also an effective activator of the c i t r a t e carrier system of the inner mitochondrial membrane, as shown in Figure 2 (cf. 21). When mitochondria are incubated with c i t r a t e and the exit +

f

.

Cit

I

i 2

F-Mal

ι 4

ι 6

Min.

ι 8

1 10

Molecular Pharmacology

Figure 2.

Spectrophotometric assay of citrate entry into mitochondria


6

BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS

of i s o c i t r a t e is monitored by an externally added NADP + Mg + isocitrate dehydrogenase i s o c i t r a t e detector system in the dual wave length spectrophotometer, rapid i s o c i t r a t e efflux is observed under a wide variety of experimental conditions. This process is greatly stimulated by (-)erythrofluoromalate (see Figure 2) as indicated by the large increase of the rate of extramitochondrial NADPH formation. Fluoromalate appears to act similar to malate (cf. 22) except i t s effect is not complicated by penetration and subsequent metabolism in the matrix - as i t is the case with malate. Consequently (-)erythrofluoromalate is a more specific and useful activator of the c i t r a t e - i s o c i t r a t e translocating membrane system than oxidizable dicarboxylic acids (compare with 22). It is a puzzle why c i t r a t e - i s o c i t r a t e exchange in energized mitochondria proceeds with the observed high efficiency (about 80% of added c i t r a t e appears as i s o c i t r a t e in the extramitochondrial compartment). We présumeras discussed more extensively l a t e r through the inner mitochondrial membrane is a more complex process than a carrier mediated carboxylic acid exchange (22) and may reflect an energy coupled membrane process of as yet unknown physiological significance. In v i t r o chemical and enzymatic studies thus far provided fluoro malate and difluoro-oxalacetate as candidates useful as probes in c e l l u l a r systems. The discovery of a valuable technique, that of isolation of hepatocytes by Berry and Friend (23), gave significant impetus to further studies. Gluconeogenesis from lactate was c h a r a c t e r i s t i c a l l y inhibited by difluoro-oxalacetate and to a lesser extent by (-)erythrofluoromalate,whereas glucose formation from pyruvate was more sensitive to fluoromalate than to difluoro-oxalacetate (24) as shown in Table II and Figure 3. Combination of both fluoro-dicarboxylic acids +

Tabl e II

Effects of fluoro-dicarboxylic acids on rates of gluconeogenesis in intact isolated liver cells Cells (125—225 mg wet weight), obtained from the livers of animals faeted 18 h, were incubated for 40 min at 37 °C. The initial substrate concentration was 10 mM and inhibitor concentration 2.5 m M . The values given are means ± S . E . with the number of observations in parenthesis. Rates of glucose formation have been calculated after subtraction of the average rate observed in the absence of added substrate. °· ± - ° ° n m o l x g ^ X m i n " (32 observations) T l l i 8

w

a

s

Substrate added

0 8

0

3

. ..... . . . Inhibitor added

1

_. . . Glucose formed μπιοί x g" x miu 1

L-Lactate L-Lactate L-Lactate L-Lactate Pyruvate Pyruvate Pyruvate Pyruvate

European Journal of Biochemistry

Fructose Fructose Fructose

0.45 Fluoromalate 0.34 Difluorooxaloacetate 0.24 Fluoromalate, Difluorooxaloacetate 0.11 — 0.51 Fluoromalate 0.21 Difluorooxaloacetate 0.38 Fluoromalate, 0.22 Difluorooxaloacetate 2.82 Fluoromalate 2.81 Difluorooxaloacetate 2.Si

Apparent i n h l b l t l o n -1

·/·

± 0.02 (20) ± 0.06 (4) ± 0.04 (4)

24 56

± 0 . 0 5 (3) ± 0.02 (20) ± 0.04 (5) ± 0.08 (4) ± 0 . 0 7 (4)

76 — 59 25 57

± 0.11 (14) (2) -(2)

— —


2+

1.

KUN

Fluorocarboxylic

0

Acids

1

as Probes

7

J I • . I 2 3 4 5 Inhibitor concentration (mM) European Journal of Biochemistry

Figure 3. on rate of cells from pyruvate (·) or foctate (O). Each point is the average of two experiments. Conditions were the same as given in the legend of Table 1. , with difluorooxaloacetate; , with fluoromalate.

i n h i b i t gluconeogenesis from both precursors by 76-80% without s i g n i f i c a n t l y altering c e l l u l a r processes other than those i n v o l ved in the transfer of reducing equivalents through the inner mitochondrial membrane (25, 26). In preliminary studies we found no noticable toxic effects of either fluoro carboxylic acids when injected intravenously (20 mg/100 g body weight) into mice. Marked changes in glutamate and aspartate concentrations in the l i v e r indicated that both fluoro carboxylic acids actually penetrated l i v e r parenchyma c e l l s . It would appear reasonable to undertake more extensive in vivo studies with both fluoro carboxy l i c aci-ds as a model for a possible experimental chemotherapeut i c approach to metabolic disorders l i k e diabetes, characterized by abnormally large gluconeogenesis. Studies with fluorocitrate a.)Structure of the inhibitory isomer. In contrast to fluorodicarboxylic acids of apparently low or undetectable acute toxicity,the most important fluorotricarboxylic acid: monofluoroc i t r i c acid, because of i t s remarkable t o x i c i t y , plays a h i s t o r ical significance in the f i e l d of biochemical lesions, an area established by S i r Rudolph Peters (cf. 27). In a series of classical experiments Peters and his school established that the site of action of fluorocitrate is localized at an i n i t i a l step of c i t r a t e metabolism (28). The enzymatic s i t e of action of fluorocitrate was proposed to be mitochondrial aconitase (27).


8


This mechanism appears to be incompatible with the results of Guarrierra-Bobyleva and Buffa (29), who found that after in vivo administration of toxic doses of fluorocitrate,only the metabolism of c i t r a t e was inhibited in mitochondria whereas c i s - a c o n i tate - which is also a substrate of aconitase - is oxidized nor mally. Because of the apparent uncertainties surrounding both the chemistry of the toxic isomer of f l u o r o c i t r i c acid and i t s subcellular mode of action this problem was reinvestigated. As i l l u s t r a t e d in Figure4 (cf. 30) the four possible isomers of monofluorocitric acid are formed either from fluoroacetyl-CoA and oxalacetic acid or from monofluoro-oxalacetic acid and acetyl CoA. It was shown (31,32) that the toxic isomer i s formed only by enzymatic condensation of fluoro acetyl CoA with oxalacetate whereas a l l other isomers had no s i g n i f i c a n t inhibitory effect on aconitase. Because of the r e l a t i v e l y weak inhibitory effects of fluorocitrate (Κ · * 50-80 μΜ) the d i s t i n c t i o n between "inhibi tory" and "non-inhibitory would wish for. Besides i n i t i a l velocity kinetic analyses, we, as well as others,observed a time dependent, anomalous i n h i b i t i o n of aconitase a c t i v i t y , but only in the i s o c i t r a t e d hydrogenase coupled test system, containing either Mg or Mn?+ (see l a t e r for more details ). Elucidation of the chemical structure of the toxic fluorocitrate isomer was successful despite the d i f f i c u l t i e s encountered in the enzymology of aconitase. Synthesis and r e s o l ution of isomers was accomplished in 1969 (33) and i t was shown that the electrophoretically separated erythro isomers (Figure 5 and Figure 6) contained the toxic species, which was further re solved and i d e n t i f i e d as (-)erythrofluorocitric a c i d , correctly defined as: 1R: 2Rl-fluoro-2-hydroxy-l,2,3-propane tricarboxylic acid. Crystallographic analysis of rubidium ammonium hydrogen fluorocitrate dihydrate (34) lent further support to our deduction, based o r i g i n a l l y on NMR and pk analyses of electrophoretically resolved diastereoisomers (31,32). Since the c i t r a t e condensing enzyme plays a key role in the biosynthesis of (-)erythrofluorocitric acid from fluoroacetic acid some kinetic characteristics of this enzyme were also determined with F-acetvl CoA as substrate. Results are shown in Table I I I , η

2+

TABLE

3

Summary of Kinetic Properties of Citrate Synthase from Pig Heart" Con stant K

Acetyl-CoA Fluoroacetyl-CoA F l uo roace ty 1-Co A Acetyl-CoA Fluoroacetyl-CoA

m

K K

m x

Kinetic constant

Substrate or inhibitor 25 μλί

23 μλί 2.2 μλί 2.77 (/xmolcs D P N H / m g / m i n ) 0.0084.5 (pinoles D P N H / m g / m i n )

v ' mil D P N , malate and malate dehydrogenase served as asourceof oxalacetate, and the reaction was followed by appearance of D P N H . β

'The Citric Acid Cycle"


KUN

Fluorocarboxylic

Acids

as Probes

F of mulot (•)-Mixture

Corbons derived from oxoloocetote or< fluoro-oxaloacetote

_ \

Λ

Λ

f COOH | H-C-H 1

|HOOC-C-OH

Carbons derived Γ from ocetote or < fluoroocetote L

H-C-F 2 1 C

0

0

H

(•)- Mixture

~\ COOH1 | H-C-F 2 1

HOOC-C-OH

H-C-H 1

1

C

COO" Newman projections " O O C ^ L OH olong C - 2 . C - 3 axis T A J (ionized forms) F-V^

C

O

coo" H O ^ L COO" JXj

1

F-C-H 2

HOOC-i-OH F-C-H

H

COOH 1

COOH 1 H-C-H

HOOC-fj-OH

2

H-Ç-H

COOH1

COOH

coo"

COO"

F ^ C >

Configurational P r e f i x * fi/S

System

(2S.3R)-

Racemates E «tensions of carbohydrote-omino acid rules ( C O O H group Ignored)

(2R.3S)-

(2R.3R)-

(2RS.3SR)-

L

0$Lg-

« 9" L

Racemates

erythro-L $

t

$

erythro-0 -

t

threo-Oj-

$

erythro-D L $

U Og0 L .LgC^-

^s^gLg"

Racemates

(2S.3Sh

(2RS.3RS)-

threo-L,-

fhreo-O L -

$

e

f

Provisional Assignments Weaker acids (Component A) H

Stronger octds (Component 8 )

I

From fluoroocetyl-CoA

From fluoro-/ oxaloacetate

«-

Not formed enzymaticolly è

enanttomers

"The Citric Acid Cycle"

Figure 4.

The isomers of monofluorocitric acid *

* T h e t e r m i n o l o g y o f L. D* a n d erythro D„ originates f r o m Η . B . V i c k c r y [ A Suggested N e w N o m e n c l a t u r e f o r the Isomers o f Isocitric A c i d , " / . Biol. Chan. 237, 1739 (1962)1. F o r c l a r i f i c a t i o n o f RS nomenclature, the reader is referred t o : C a h n , R . S., Ingold, C , a n d P r e l o g , V . : Angew. Client. Intern. Ed. Engl. 3 S 5 ( 5 ) , 511 ( 1 9 6 6 ) , a n d C a h n , R . S., 7. Chem. Educ. 1 1 6 ( 4 1 ) , 508 ( 1 9 6 4 ) . M


BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E

0

0

0

n

C

ο

Y

ο οο ο 0000 ο

ο ο 0 0 0 00

U χ

CL

I

Ο

·— —

—·

ο

ν

LJL

2 3 4 5 6

Journal of Biological Chemistry

Figure 5. Electrophoresis of fluorocitric acid from enzymatic and chemical syntheses. CS: chemically synthesized fluorocitric acid; P: picric acid marker; fluorocitric acids I and II (see text) (32).

6£

5Ό

I 1 l I

1

CNF I I I t

40

3.0

CHF C H 1

2

I

I I I I i 1 I » 1

CH 1 1 I I t I 1 1 1

2.0

ΙΌ

2

1

W i 1

1

1 I I 1

I

C H , I 1 t 1

I

0 ppm ( δ )

I I 1 I

1

I 1 1 I

t

1 I I I

Journal of Biological Chemistry

Figure 6.

Nuclear magnetic resonance spectrum of triethyl fluorocitrate (32)


1

1 1

BONDS

1.

KUN

Fluorocarboxylic

Acids

11

as Probes

(cf. 30). Whereas Km for acetyl CoA and i t s F-analog is the same, Vmax in the presence of F-acetyl CoA is about l/300th of V measured in the presence of the physiological substrate. Fluoroacetyl CoA acts also as an inhibitor of the condensation of acetyl CoA with oxalacetate. mx

b.)Mode of action of (-)erythrofluorocitrate. A molecular mechanism of fluorocitrate t o x i c i t y has to account for the i r r e versible cessation of c e l l function in certain s p e c i f i c c e l l s of the nervous system, known to be the probable anatomical organ s i t e of this poison (28). Whereas the question of organ s p e c i f i c i t y in the manifestation of pharmacological responses belongs to the domain of physiology, universality of metabolic enzymes in most organs s t i l l provides acceptable in v i t r o models for the study of biochemical toxicology. Consequently i f i n h i b i t i o n of aconitase is the mode of action of f l u o r o c i t r a t e , irreversible inhibition of this enzyme should b tions of the i n h i b i t o r becaus most probably present in vivo (compare with slow rates of fluoroc i t r a t e biosynthesis cf. 30). Even i f these requirements are met there is some uncertainty why aconitase i n h i b i t i o n should prove f a t a l , when inhibitors of other c i t r i c acid cycle enzymes causes r e l a t i v e l y small t o x i c i t y : eg. malonate is not a powerful poison (cf. 1), or as stated e a r l i e r , inhibition of malate dehydrogenase in vivo e l i c i t s no detectable toxic effects. This question is of particular significance, since the existence of cytoplasmic and mitochondrial isoenzymes of aconitase (35) make i t uncertain why the operation of the tricarboxylate cycle could not proceed undisturbed i f only one, i . e . , the mitochondrial isoenzyme^were inhibited (compare with 29). Because of the h i s t o r i c a l l y developed notion (36) identifying fluorocitrate t o x i c i t y with aconitase i n h i b i t i o n , the present evidence related to this question w i l l be examined in d e t a i l . Notorious problems of enzyme i n s t a b i l i t y at advanced stages of purification are well known to those concerned with the enzymology of aconitase. In a l l but one instance millimolar concentrations of ascorbate, cysteine, and F e are required to demonstrate f u l l aconitase a c t i v i t y in v i t r o . More recent studies concerned with the mechanism of aconitase action were performed almost entirely in this highly a r t i f i c i a l in v i t r o environment (37,38). This enzyme had a molecular weight of 68,000. Gawron et a l . (39,40) while unable to reproduce the i s o lation procedure of Villafranca and Mildvan (37,38) obtained an enzyme (mol. weight = 66,000) which had higher s p e c i f i c a c t i v i t y than reported by e a r l i e r workers. It appears therefore doubtful that previous in v i t r o studies (37,38) are entirely valid even within the framework of cuvette enzymology. Non-competitive i n hibition of heart aconitase (37) by transaconitate with c i t r a t e or i s o c i t r a t e as substrates (41) could not be reproduced (42)» therefore the explanatory mechanism of Villafranca (41) postulating two isomeric forms of aconitase appears to be without firm 2+


12


experimental foundation. One of the - until recently - unrecog nized complications of in v i t r o enzymology of aconitase l i e s in the frequently used NADP^ dependent i s o c i t r a t e dehydrogenase assay system i t s e l f . As demonstrated in 1974 (43)^various pre parations of aconitase are susceptible to time dependent i n a c t i vation by bivalent cations e g . , Mg or Mn^ , which are necessary constituents of the coupled assay system. Unawareness of this complication can result in double reciprocal plots of reaction rates, which can simulate competitive or non-competitive or mixed types of inhibitions by fluorocitrate with highly variable apparent Κ·,· values calculated from primary plots. We have also been misled by these types of experimental observations and pos tulated e a r l i e r a time dependent inactivation of aconitase by f l u o r o c i t r a t e , presumably by fluoro-aconitate or i t s defluorinated product (cf. 30). The very low Ki values for fluorocitrate reported by Brand et a l . (44), assaying aconitase a c t i v i t i e s of crude mitochondrial extract are apparently due to the inactivation of aconitase by Mg2 , which i s superimposed to the competitive inhibitory effect of fluorocitrate as reported more recently (43). In view of the a r t i f i c i a l i t y of the enzymology of aconitase, we pursued this problem by isolating an electrophoretically homogeneous enzyme which i s , for a f i n i t e period of time, f u l l y active without any a r t i f i c i a l activator whatsoever (43). Purification is shown in Table IV. The molecular weight of this cytoplasmic isoenzyme i s 2+

+

Table IV Purification of cytoplasmic aconitase from pig liwr Purification slq>

Total protein

Total acon itase activ ity at 25°

Specific activitv at 25°'

μηιοΐα/ηηη min/mg protein Step 1 (liver extract) Step 2 Step :> Step I

12,300 5(i0 180 8

1,045 730 500 85

0.134 1.32 2.70 10.0


111,000. considerably higher than purified preparations requir ing Fe^+ cysteine for activation. A much less purified mito chondrial isoenzyme has also been isolated under conditions which maintained maximal a c t i v i t y without added activators. From Figure 7 i t i s evident that fluorocitrate acted as a l i n e a r l y competitive reversible i n h i b i t o r . From suitable compu ter models substrate and inhibitory constants were calculated as summarized in Table V. It i s apparent that (-)erythrofluoro%


1.

KUN

Fluorocarboxylic

Acids

13

as Probes

Ε Ε 40θ|

//·> —

u

Jj

COO

I

2ΌΟΟ

I

I

OOO

L_

2000

l/S(M) Molecular Pharmacology

Figure 7. Competitive inhibition of cy toplasmic (A) and mitochondrial (B) aconitase of pig liver by (—)-erythrofluorocitrate. Rates of cis-aconitate for mation 240 nm at 25°. plasmic aconitase, and in B, 32 μ% of mitochondrial aconitase, were used per test system (5-cm light path; 3-ml vol ume) at varied concentrations of citrate (abscissa). Curve 1, 500 μΜ. fluorocitrate; 2,100 μΜ fluorocitrate; 3, no fluorocitrate.

Table V Siihslrutt: constants fitr citrate ami fl-ixoritralc and inhihilor constants f100 >100 >100 >100 >100 >100 >100

e

cone.

(pg/ml)

a

Ribavirin

C

_

-10 10 30 7 30

to reduce v i r u s - i n d u c e d c y t o p a t h o g e n i c i t y by 50%.

E s s e n t i a l l y s i m i l a r r e s u l t s were obtained f o r p - f l u o r o phenylalanine. An e s t a b l i s h e d a n t i v i r a l agent, Ι-β-Dribofuranosyl-1,2,4-triazole-3-carboxamide. Abbreviations are i d e n t i f i e d i n r e f . 32. At concentrations of 100 ug/ml, morphological a l t e r a t i o n of the host c e l l s was apparent only a f t e r 3 days. e


30


p-Glu-His-Pro-NH

2

(TRF)

p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH

2

(LHRF)

of the imidazole r i n g f o l l o w i n g i n t r o d u c t i o n of f l u o r i n e , the e x i s t e n c e of any a c t i v i t y i s s u r p r i s i n g , and these r e s u l t s suggest that r e c o g n i t i o n of the peptide by i t s receptor may depend more on o v e r a l l conformation than on imidazole b a s i c i t y . A - F l u o r o h i s t i d i n e . Despite the minimal s t e r i c consequences of f l u o r i n e s u b s t i t u t i o n and the s i m i l a r i t y i n r i n g b a s i c i t i e s of the isomers, 2 - f l u o r o - and A - f l u o r o h i s t i d i n e are r e a d i l y d i f f e r e n t i a t e d by the h i s t i d i n e t-RNA systems. To date, the evidence i n d i c a t e s that A - f l u o r o h i s t i d i n e does not s u b s t i t u t e f o r h i s t i d i n e i n p r o t e i n b i o s y n t h e s i s , nor does t h i s analogue show s i g n i f i c a n t b a c t e r i o s t a t i c or a n t i v i r a l a c t i v i t (Table d II) s t u d i e s suggest that th (17), f l u o r i n e at C-A being , perhaps, u l a r hydrogen bond. Yet, the A - f l u o r o isomer i s a s u b s t r a t e f o r b a c t e r i a l h i s t i d i n e decarboxylase (but not f o r the mammalian enzyme), while both isomers are modest substrates f o r h i s t i d i n e ammonia-lyase (Table I I I ) . The a b i l i t y of A - f l u o r o h i s t i d i n e to f u n c t i o n both as a weak s u b s t r a t e and a strong competitive i n h i b i t o r f o r the l a t t e r enzyme prompted a r e i n v e s t i g a t i o n of i t s mechTable I I I . E f f e c t s of F l u o r o h i s t i d i n e s on H i s t i d i n e Ammonia-Lyase (pH 9, 25°) Κ Compound

or (mM)

m

L-Histidine

2.7

A-F-L-Histidine

1.25

2-F-L-Histidine

170

K. 1

V (uM/mg/min)

30 0.85 1-2

anism of a c t i o n (2A). A n a l y s i s of k i n e t i c data, r e v e r s i b i l i t y , i s o t o p e i n c o r p o r a t i o n , and isotope e f f e c t s demonstrates that the r a t e - l i m i t i n g step f o r t h i s enzyme i s not the breakdown of an intermediate aminoenzyme (as p r e v i o u s l y supposed), but the almost concerted l o s s of a 3-hydrogen atom and the c o v a l e n t l y bound amino group. While the isomers l o s e ammonia at comparable r a t e s , Af l u o r o h i s t i d i n e i s bound much more e f f e c t i v e l y . Urocanase, the enzyme f o l l o w i n g the ammonia-lyase i n the c a t a b o l i c sequence f o r h i s t i d i n e (Figure 5), promotes the h y d r a t i o n rearrangement of urocanic a c i d (25). While n e i t h e r f l u o r o u r o c a n i c a c i d i s s i g n i f i c a n t as a s u b s t r a t e (Table IV), the 2 - f l u o r o isomer


2.

KIRK AND COHEN

Ring-F luorinated

31

Imidazoles

Table IV. E f f e c t s of F l u o r o u r o c a n i c A c i d s on Urocanase (pH 7.4, 25°)

7

10 K Compound

Urocanic a c i d

m

or (M)

K

±

V (units/ml)

400

1.2

-

4-F-Urocanic

acid

-

2-F-Urocanic

acid

1

0.008

measurable a c t i v i t y as i n h i b i t o r or substrate.

i s now a very potent i n h i b i t o enzyme capable of d i r e c t removal of f l u o r i n e from the imidazole r i n g . The enzymes which degrade the imidazole r i n g of h i s t i d i n e , e v e n t u a l l y to glutamic a c i d , can a l s o operate on the f l u o r o h i s t i d i n e s , a l b e i t very slowly. Thus f a r , we have found no evidence f o r metabolites of these analogues i n v i v o . As already mentioned, the f l u o r o h i s t i d i n e s can be i n c o r p o r a t e d i n t o a p o l y p e p t i d e sequence v i a t o t a l s y n t h e s i s . Thus, the peptide fragment, r i b o n u c l e a s e - S - ( l - 1 5 ) , has been s y n t h e s i z e d with 4- f l u o r o - L - h i s t i d i n e r e p l a c i n g h i s t i d i n e - 1 2 (27). T h i s f l u o r i n e c o n t a i n i n g peptide (4-F-His-RNase-(l-15)) a s s o c i a t e s w i t h RNase-S(21-124) as w e l l as, and even more s t r o n g l y than, RNase-S-(l-20). A v a r i e t y of c r i t e r i a suggest that the f l u o r o h i s t i d i n e - c o n t a i n i n g aggregate has a three-dimensional s t r u c t u r e very s i m i l a r to that of the complex, RNase-S'. The noncovalent recombination of RNase5- (l-15) or -(1-20) w i t h RNase-S-(21-124) r e s t o r e s p r a c t i c a l l y a l l the enzymatic a c t i v i t y of the o r i g i n a l enzyme, RNase-A; the a n a l ogous complex with 4-F-His-RNase-(l-15) i s t o t a l l y devoid of a c t i v i t y , although i t i s probably s t i l l capable of b i n d i n g s u b s t r a t e . The r e s u l t s provide strong support f o r the proposal that His-12 f u n c t i o n s as a general a c i d - g e n e r a l base c a t a l y s t i n the enzymatic process — a r e d u c t i o n of 5 u n i t s i n the pK of the r i n g being more than ample to remove t h i s c a t a l y t i c c a p a b i l i t y . Fluorohistamines. Two types of r e c e p t o r , termed HI and H2, have been i d e n t i f i e d as mediators of histamine's b i o l o g i c a l a c t i o n s . C h a r a c t e r i z a t i o n of these r e c e p t o r s , and of histamine's b i o l o g i c a l r o l e s , have been hampered by l a c k of agents which s e l e c t i v e l y b i n d one type. Recently, i t has been found that v a r i o u s s u b s t i t u t i o n s at C-2 or C-4 of the histamine r i n g s e l e c t i v e l y decrease b i n d i n g f o r H2 or HI r e c e p t o r s , r e s p e c t i v e l y . Thus, 2phenylhistamine r e t a i n s some 20% of histamine's potency at HI r e c e p t o r s , but i s l e s s than 0.1% as e f f e c t i v e at H2 r e c e p t o r s . In a p r e l i m i n a r y study, 2-fluorohistamine was found (28) to have


32

BIOCHEMISTRY

INVOLVING C A R B O N - F L U O R I N E BONDS

/CH -CHC00H

CH=CHCOOH

2

NH

Lyase

2

-NH3 Urocanase +H2Q CL

£H CH C00H

H00CCHCH2CH C00 2

H

NH CH= NH Figure 5

-do HO-CHl

H0-CH2

ON

OH

5-FICAR 5-Fluoro-L β-D-ribofuranosylimidazole4-carboxamide

ribavirin 1 -β-D-ribofuranosyîl 2,4-triazoh-3carboxamide y

5-AICAR 5-amino-l -β-Ό-rihofuranosuUmidazole4-carboxamide

Figure 6


2.

ΚΓΑΚ AND COHEN

Ring-Fluonnated

Imidazoles

33

equal or greater a f f i n i t y f o r HI r e c e p t o r s ( i n guinea p i g ileum) than the p r e v i o u s l y most e f f e c t i v e HI a g o n i s t , 2-aminoethylthiaz o l e . By analogy, 4-fluorohistamine might act as a s e l e c t i v e agon i s t f o r H2 r e c e p t o r s , a p o s s i b i l i t y now under i n v e s t i g a t i o n . 2-Fluorohistamine was a l s o found to be an e f f e c t i v e s t i m u l a t o r of c y c l i c AMP formation i n b r a i n s l i c e s , which c o n t a i n both HI and H2 receptors (29). Fluoroimidazole Ribosides. R e l a t i v e l y simple imidazole d e r i v a t i v e s occupy key r o l e s as intermediates i n the b i o s y n t h e s i s of the purine n u c l e o s i d e s . A major approach to the design of a n t i v i r a l agents i s based on analogues of Imidazole species which might i n t e r f e r e with the b i o s y n t h e s i s of n u c l e o s i d e s or of n u c l e i c a c i d s (Figure 6 ) . Thus, r i b a v i r i n , a t r i a z o l e analogue of 5-AICAR, commands s e r i o u s a t t e n t i o n as a broad-spectrum a n t i v i r a l agent (30) . We have synthesized 5-FICAR a f l u o r o analogue of 5-AICAR (31) ; t h i s compound show s i m i l a r to that of r i b a v i r i to b l o c k the b i o s y n t h e s i s of both DNA and RNA. R i b a v i r i n has been shown to act by i n h i b i t i n g the enzyme, IMP dehydrogenase (33), and we assume, t e n t a t i v e l y , that 5-FICAR f u n c t i o n s at the same p o i n t i n the b i o s y n t h e t i c pathway. Future

Plans

The r e s u l t s of these and other s t u d i e s i n f l u o r o i m i d a z o l e chemistry and biochemistry have r a i s e d i n t e r e s t i n g questions f o r the f u t u r e . Some aspects of the p h y s i c a l and chemical p r o p e r t i e s of f l u o r o i m i d a z o l e s do not conform to expectations based on data f o r other imidazole systems and suggest, » that some sub s t i t u t e d imidazoles a r e , at b e s t , b o r d e r l i n e aromatic systems. Further understanding may be provided by a study of d i f l u o r o imidazoles: 4 , 5 - d i f l u o r o i m i d a z o l e has been synthesized and i s , indeed, found to have anomalous p r o p e r t i e s ; d e s p i t e a r a t h e r ex t e n s i v e e f f o r t , however, the 2 , 4 - d i f l u o r o isomer has not y e t been prepared. On the b i o l o g i c a l s i d e , we may ask, , why the isomeric f l u o r o h i s t i d i n e s show such marked d i f f e r e n c e s i n b i n d i n g and r e sponse to h i s t i d i n e - s p e c i f i c enzymes; whether the replacement of h i s t i d i n e by f l u o r o h i s t i d i n e i n an enzyme sequence i n v a r i a b l y leads to l o s s of a c t i v i t y ; whether the 2 - f l u o r o i m i d a z o l e s can be made s u f f i c i e n t l y r e a c t i v e to f u n c t i o n as covalent a f f i n i t y l a b e l s f o r receptor s i t e s . H o p e f u l l y , these and other questions w i l l have been answered before the next F l u o r i n e Symposium.


34


Literature Cited (1) Goldman, Peter, Science (1969), 164, 1123. (2) Ciba Foundation, "Carbon-Fluorine Compounds," Elsevier, Amsterdam, 1972. (3) Reference 2, p. 211. (4) Belsham, M. G., Muir, A. R., Kinns, Michael, Phillips, Lawrence, and Twanmoh, Li-Ming, J. Chem. Soc., Perkin Trans. 2 (1974), 119. (5) Pavlath, A. E. and Leffler, A. J., "Aromatic Fluorine Com pounds," Reinhold, New York, 1962. (6) Hudlicky, Miklos, "Organic Fluorine Chemistry," Plenum Press, New York, 1970. (7) Kirk, K. L. and Cohen, L. Α., unpublished observations. (8) Kirk, K. L. and Cohen, L. Α., Symposium on Fluorine in Medic inal Chemistry, 162nd National Meetin f th America Chem ical Society, Washington (9) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1971), 93, 3060. (10) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1973), 95, 4619. (11) Kirk, K. L. and Cohen, L. Α., J. Org. Chem. (1973), 38, 3647. (12) Nagai, Wakatu, Kirk, K. L., and Cohen, L. Α., J_. Org. Chem. (1973),38,1971. (13) Kirk, K. L., Nagai, Wakatu, and Cohen, L. Α., J. Amer. Chem. Soc. (1973),95,8389. (14) Lousberg, R. J. J. Ch. and Weiss, Ulrich, Experientia (1974), 30, 1440. (15) Kirk, K. L., manuscript in preparation. (16) Lowenbach, W. A. and King, M. M., unpublished data. (17) Yeh, H. J. C., Kirk, K. L., Cohen, L. Α., and Cohen, J. S., J. Chem. Soc., Perkin Trans. 2 (1975), 928. (18) Wong, J. L. and Keck, J. H., Jr., J. Org. Chem. (1974), 39 2398, and earlier references cited therein. (19) Kirk, K. L., McNeal, Elizabeth, Cohen, L. Α., and Creveling, C. R., manuscript in preparation. (20) Klein, D. C., Kirk, K. L., Weller, J. L., and Parfitt, A. G., Mol. Pharmacol., in press. (21) Furano, Α. V., Kirk, K. L., and Cohen, L. Α., unpublished data. (22) De Clercq, Erik, Kirk, K. L., and Cohen, L. Α., unpublished data. (23) Monahan, Μ. Α., Vale, Wylie, Kirk, K. L., and Cohen, L. Α., unpublished data. (24) Klee, C. Β., Kirk, K. L., Cohen, L. Α., and McPhie, Peter, J. Biol. Chem. (1975), 250, 5033. (25) Kaeppeli, Franz, and Retey, Janos, Eur. J. Biochem. (1971), 23, 198, and earlier references cited therein. (26) Klee, C. B., Kirk, K. L., and Cohen, L. Α., unpublished data.


2.

KIRK AND COHEN

Ring-Fluorinated

Imidazoles

35

(27) Dunn, Β. Μ., DiBello, Carlo, Kirk, K. L., Cohen, L. Α., and Chaiken, I. M., J. Biol. Chem. (1974), 249, 6295. (28) Dismukes, R. Κ., unpublished data. (29) Dismukes, R. K., Rogers, Michael, and Daly, J. W., Neurochemistry, in press. (30) Khare, G. P., Sidwell, R. W., Witkowski, J. T., Simon, L. N., and Robins, R. K., Antimicrob. Ag. Chemother. (1973), 3, 517. (31) Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Tetrahedron Letters, in press. (32) De Clercq, Erik, Luczak, Miroslav, Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Life Sciences (1975), 17, 187. (33) Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. Κ., and Simon, L. Ν., Proc. Nat. Acad. Sci. USA (1973), 70, 1174. Discussion Q. A.

Has there been a check of monitoring on whether C-F cleavage occurs? I assume you r e f e r to s t a b i l i t y i n b i o l o g i c a l systems. The normal pathway f o r h i s t i d i n e degradation i n v o l v e s conversion to urocanic a c i d , and, u l t i m a t e l y , to glutamic a c i d . As I had i n d i c a t e d , 2 - f l u o r o h i s t i d i n e i s a very poor s u b s t r a t e f o r the f i r s t two enzymes i n t h i s pathway; a t the t h i r d s t e p , f l u o r i d e i o n would be r e l e a s e d , together with innocuous degrada t i o n products. 4 - F l u o r o h i s t i d i n e can be transformed slowly to 4 - f l u o r o u r o c a n i c a c i d , which i s probably a dead end. Except f o r t h i s data, we have found no i n d i c a t i o n s f o r enzymatic removal of f l u o r i n e .

Q.

Have you t e s t e d the b i o l o g i c a l a c t i v i t y of f l u o r o a l k y l i m i d a -

A.

We have not made any C F ^ - s u b s t i t u t e d i m i d a z o l e s .

Q. A.

As f a r as you know, are any of them known? Yes. A s e r i e s of 4 - t r i f l u o r o m e t h y l i m i d a z o l e s have been reported by Baldwin and h i s colleagues a t Merck Sharp & Dohme [ c f . J . Med. Chem. (1975), l g , 895] and 2 - t r i f l u o r o m e t h y l imidazoles by Lombardino and Wiseman [ J . Med. Chem. (1974), 17, 1182.]

Q.

What do you think would be the long-term s t a b i l i t y of the C-F bond i n 2-fluoroimidazoles? Simple compounds, such as 2 - f l u o r o i m i d a z o l e and 2 - f l u o r o - 4 methylimidazole can be kept i n d e f i n i t e l y i n the s o l i d s t a t e at -80°, but t r i m e r i z e i n a month or two a t 0 ° . In c o n t r a s t , 2 - f l u o r o h i s t i d i n e appears to be i n d e f i n i t e l y s t a b l e a t room temperature. D i l u t e s o l u t i o n s of 2 - f l u o r o h i s t i d i n e i n phos phate b u f f e r (pH 7) have been kept at 0° f o r months without evidence of d e t e r i o r a t i o n .

z o l e s - r e p l a c i n g F by

A.

CF3?


36

Q. A.

Q. A.

Q.

A.

BIOCHEMISTRY INVOLVING

C A R B O N - F L U O R I N E BONDS

Can you say anything about the a n t i - a r t h r i t i c a c t i v i t y of the fluorohistidines ? T e s t i n g has not yet been done. We are aware of the p o s s i b i l i t i e s i n t h i s d i r e c t i o n and hope to get such a study going soon. I'd likÇgto ask whether j g u ever considered the use of r a d i o active F as a probe? F has a h a l f - l i f j g O f 110 minutes. Yes, we have. Studies on the s y n t h e s i s of F imidazoles were i n i t i a t e d some time ago at the Atomic Energy Commission i n Bucharest. You're acquaintecjgwith the work of A l Wolf and others a t Brookhaven with F fluor©phenylalanine and other b i o l o g i c a l l y i n t e r e s t i n g molecules? ^g Yes, I am. A l Wol p o i n t of p a r t i c u l a the b l o o d - b r a i n b a r r i e r very q u i c k l y , and so there i s c o n s i d e r a b l e i n t e r e s t i n t h i s compound f o r b r a i n s c i n t i g r a p h y .


3 2-Fluoro-L-Histidine: A Histidine Analog Which Inhibits Enzyme Induction DAVID C. KLEIN Section on Physiological Controls, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014 KENNETH L. KIRK Section on Biochemical Mechanisms of Arthritis, Metabolism, and Institutes of Health, Bethesda

f Chemistry Nationa Institut

Amino acid analogs have a long and interesting history (1,2) and have been extremely valuable to biochemists, pharmacologists, and clinicians as analytical tools and therapeutic agents. Some of the best established and most useful of these compounds are para-fluorophenylalanine, para-chlorophenylalanine, and alpha-methyldopa. The total number of such compounds which have been synthesized with the hopes of producing potent and specific tools probably is in the thousands. Potential Actions of Amino Acid Analogs The stratagem usually employed with an amino acid analog is to have i t enter cells and substitute for the parent compound (1,2); the structural modification characterizing the analog precludes the normal function or metabolic fate of the parent compound. Substitution of the analog for the parent compound could involve substitution in a metabolic pathway resulting in an altered metabolite. Additionally, a metabolic pathway could be inhibited by an amino acid analog by several mechanisms, including competitive or noncompetitive inhibition of a specific enzyme or false feedback. Lastly, substitution for the parent compound in the synthesis of proteins could result in altered proteins. Such replacement of an amino acid analog for the parent compound in the primary structure of a protein could alter a protein through a number of modes of action, including alteration of the secondary, tertiary, or quarternary structure, alteration of the active site of an enzyme, and alteration of the regulatory site of an enzyme. Role of Histidine in Enzymes Histidine is one of the most important amino acids involved in the catalytic action of enzymes (3,40. It functions via the imidazole group, which can act as a general acid, general base, 37 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

38


or n u c l e o p h i l e . This c a t a l y t i c r o l e represents the g e n e r a l l y recognized f u n c t i o n of h i s t i d i n e i n enzymes (5). Some of the enzymes i n which h i s t i d i n e i s a t the a c t i v e s i t e i n c l u d e RNAse, glucose-6-phosphatase, and chymotrypsin. Recently, evidence has appeared i n d i c a t i n g h i s t i d i n e may have a f u r t h e r r o l e i n p r o t e i n s (6). I t i s now g e n e r a l l y accepted that the a c t i v i t i e s of many enzymes are regulated v i a covalent chemical m o d i f i c a t i o n of enzyme p r o t e i n (5_ ]_>Q) » i n c l u d i n g phos phorylation. S p e c i f i c p r o t e i n kinases c a t a l y z e phosphorylations, and s p e c i f i c phosphatases c a t a l y z e dephosphorylation. As a r e s u l t , the a c t i v i t i e s of enzymes can be turned on and turned o f f q u i c k l y . The phosphorylation of p r o t e i n s occurs v i a covalent phosphorylation of s p e c i f i c amino a c i d s , with the r e s u l t i n g formation of both a c i d - s t a b l e and a c i d - l a b i l e phosphate bonds. The a c i d - s t a b l e bonds appear to be formed with the hydroxy groups of s e r i n e and threonine, whereas the a c i d - l a b i l e bonds appear to be formed with the imidazol The importance o obvious; i t i s a l s o p o s s i b l e that the imidazole group may be important i n the r e g u l a t i o n of enzyme a c t i o n . The a v a i l a b i l i t y of a h i s t i d i n e analog i d e n t i c a l i n s i z e and shape to h i s t i d i n e but l a c k i n g the f u n c t i o n a l c h a r a c t e r i s t i c s imparted by the imidazole group would be of general i n t e r e s t . 9

2-Fluoro-L-Histidine:

Stratagem

2 - F l u o r o - L - h i s t i d i n e was synthesized p r i m a r i l y t o provide an analog of h i s t i d i n e which would have the same s i z e and shape as h i s t i d i n e , but which would be devoid of the normal a c t i o n imparted by the imidazole group (9). (For a more d e t a i l e d d i s c u s s i o n see L. Cohen, t h i s Symposium Series.) Fluorine i s about the same s i z e as the hydrogen i t r e p l a c e s i n h i s t i d i n e

NH

2

ι

CHsCHCOOH

2-FLUORO-L-HISTIDINE (SYNTHESIS: Kirk ETAL., J. Am. Chem. Soc.,55,8389 (1973))


3.

KLEIN AND KIRK

39

2-Fluoro-L-Histidine

and thus w i l l not introduce major s t e r i c m o d i f i c a t i o n s of h i s t i dine. F l u o r i n e - s u b s t i t u t i o n , because of the strong e l e c t r o n withdrawing e f f e c t of f l u o r i n e , w i l l s u b s t a n t i a l l y a l t e r the pK of the imidazole group. The r e s u l t i n g compound w i l l be essent i a l l y i n a c t i v e , as compared to the parent compound, as a c a t a l y s t i n enzymes. T h i s e f f e c t of f l u o r i n e s u b s t i t u t i o n i s demonstrated i n ^ Table I. In t h i s experiment, the degree of t r a n s f e r of a [ C ] a c e t y l group from [ C ] a c e t y l coenzyme A to the aromaticâmine tryptamine was determined by measuring the amount of N-[ C]acetyltryptamine formed. Imidazole c a t a l y z e d t h i s t r a n s f e r , whereas 2 - f l u o r o i m i d a z o l e d i d not. Likewise, on a t h e o r e t i c a l b a s i s , f l u o r i n e s u b s t i t u t i o n would preclude the phosphorylation of imidazole. Thus, i f 2 - f l u o r o - L - h i s t i d i n e were incorporated by c e l l s i n t o p r o t e i n s i n p l a c e of h i s t i d i n e these p r o t e i n s could not be phosphorylated at a h i s t i d i n residue i f th histidin were p a r t i c i p a t i n g i n th 2-fluoro-L-histidine-containin Pineal N-Acetyltransferase:

Regulation

We have had a long standing i n t e r e s t i n an enzyme i n the p i n e a l gland, N - a c e t y l t r a n s f e r a s e (10). T h i s enzyme c a t a l y z e s the formation of N-acetylated d e r i v a t i v e s of a number of aromatic amines (10,11,12), such as N-acetyltryptamine and N - a c e t y l s e r o t o nin (N-acetyl-5-hydroxytryptamine). I t i s p h y s i o l o g i c a l l y important because the d a i l y 50-100 f o l d change i n the a c t i v i t y of t h i s enzyme r e g u l a t e s l a r g e changes i n the c o n c e n t r a t i o n of serot o n i n i n the p i n e a l gland and l a r g e changes i n the production of N - a c e t y l s e r o t o n i n and melatonin (5-methoxy-N-acetyltryptamine), the p u t a t i v e hormone of the p i n e a l gland. As i s true of the a c e t y l t r a n s f e r r e a c t i o n presented above, i t i s thought that N - a c e t y l t r a n s f e r a s e molecules i n general act through the imidazole group of h i s t i d i n e by a t t a c k on the t h i o a c e t y l group of a c e t y l CoA with the r e s u l t i n g formation of the unstable i m i d a z o l e - a c e t y l bond and the subsequent t r a n s f e r of the a c e t y l group to an acceptor amine (3). A f u r t h e r r o l e of imidaz o l e i n N - a c e t y l t r a n s f e r a s e molecules might be to accept phosphate groups, although d i r e c t evidence f o r t h i s does not e x i s t . The i n crease i n enzyme a c t i v i t y which occurs on a d a i l y b a s i s appears to depend not only upon p r o t e i n s y n t h e s i s but a l s o , i n yet an undefined way, upon the a c t i o n of adenosine 3 ,5 -monophosphate ( c y c l i c AMP). I t i s not c l e a r , however, whether c y c l i c AMP a c t i vates new enzyme molecules, as they are being synthesized, stimul a t e s the formation of new enzyme molecules, or has both a c t i o n s . We f e e l there i s some evidence to suggest that enzyme a c t i v i t y may be regulated by an a c t i v a t i o n - i n a c t i v a t i o n mechanism. T h i s i s p r i m a r i l y because N - a c e t y l t r a n s f e r a s e a c t i v i t y can be r a p i d l y destroyed, probably via enzyme i n a c t i v a t i o n (13,14). I f an a c t i v a t i o n - i n a c t i v a t i o n mechanism p l a y s a r o l e i n the r e g u l a t i o n f

f


40

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

Table I Comparison of the c a t a l y t i c a c t i v i t y of imidazole and 2 - f l u o r o imidazole i n an a c e t y l t r a n s f e r r e a c t i o n . 14 N-[ C]Acetyltryptamine formed at 37°C (nanomoles/60 min incubation)

Reaction a d d i t i o n s ( f i n a l concentrations) 14

[ C ] A c e t y l - C o A (0.5 tryptamine (10 mM)

Λ

mM), 0.14

14

[ C ] A c e t y l - C o A (0.5 mM) , tryptamine (10 mM), imidazole (100 mM)

Λ

13.4

14

[ C ] A c e t y l - C o A (0.5 mM) tryptamine (10 mM), 2 - f l u o r o i m i d a z o l e (10

X H

Tubes were prepared by adding 25 y l volumes of 2 mM [ C ] acetyl-CoA (S.A. = 1 yCi/ymole), 40 mM tryptamine, 400 mM imid a z o l e , and 400 mM 2 - f l u o r i m i d a z o l e (9), as i n d i c a t e d above. A l l chemicals were d i s s o l v e d i n .1 M sodium phosphate b u f f e r , pH 6.9. B u f f e r was added to the r e a c t i o n tubes to b r i n g the f i n a l volume to 100 y l . Data i s given as the mean of 3 determinations which were w i t h i n 10% of the mean. -, *The i d e n t i f i c a t i o n of the N-[ C]-acetyltryptamine e x t r a c t e d i n t o chloroform was confirmed by two dimensional TLC [chloroform, methanol, g l a c i a l a c e t i c a c i d (90:10:1) followed by e t h y l ace t a t e ] ; 88.7% of the r a d i o a c t i v i t y a p p l i e d to a pre-coated s i l i c a g e l p l a t e , F-254 (Brinkman Instruments Co.) was i s o g r a p h i c with s y n t h e t i c N-acetyltryptamine. Chlorof2£m e x t r a c t i o n of a tryptamine-free r e a c t i o n c o n t a i n ing 0.5 mM [ C]acetyl-CoA and 100 mM imidazole y i e l d e d a small amount^gf r a d i o a c t i v i t y equivalent to l e s s than 0.02 nanomoles of N-[ C]acetyltryptamine. Chloroform e x t r a c t i o n of a tryptamineTfree r e a c t i o n c o n t a i n ing 100 mM 2 - f l u o r o i m i d a z o l e and 0.5 mM [ C]acetyl-CoA y i e l d e d no r a d i o a c t i v i t y .


3.

KLEIN AND KIRK

2-Fluoro-L-Histicline

41

of N - a c e t y l t r a n s f e r a s e , i t may i n v o l v e c y c l i c AMP. As pointed out above, i t i s known that the a c t i v i t i e s of other enzymes are regulated by c y c l i c AMP (7). Furthermore, the a c t i v i t i e s of some of these enzymes a r e c l o s e l y c o r r e l a t e d with t h e i r phosphorylation state. In view of our s p e c u l a t i o n that h i s t i d i n e phosphorylation may be i n v o l v e d i n the r e g u l a t i o n of the a c t i v i t y of N - a c e t y l t r a n s f e r a s e and that h i s t i d i n e might be at the a c t i v e s i t e of Na c e t y l t r a n s f e r a s e , the study of the e f f e c t s of analogs of h i s t i dine on the a c t i v i t y of N - a c e t y l t r a n s f e r a s e became of p a r t i c u l a r interest. The E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Induction of P i n e a l N - A c e t y l t r a n s f e r a s e A c t i v i t y in vivo. A v a l u a b l e t e s t of the p r a c t i c a l use of any amino a c i d anal o g i s to determine i f i t w i l l a c t i n an i n t a c t normal animal without producing sever i n i t i a l experimental e f f o r t a c t i v i t y of N - a c e t y l t r a n s f e r a s e i n the p i n e a l gland can be stimul a t e d by catecholamines a c t i n g d i r e c t l y on the c e l l s c o n t a i n i n g t h i s enzyme (11). Predict-ably, the a c t i v i t y of t h i s enzyme can a l s o be stimulated i n the i n t a c t animal by the i n j e c t i o n of these and s i m i l a r compounds, i n c l u d i n g i s o p r o t e r e n o l (15). We found that s t i m u l a t i o n of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l was blocked by i n j e c t i o n s of 2 - f l u o r o - L - h i s t i d i n e (16). We d i d not, however, detect any acute adverse s i d e e f f e c t s of t h i s compound, nor d i d we f i n d that the i n j e c t i o n of a s i m i l a r dose of h i s t i d i n e could b l o c k the i n d u c t i o n of the enzyme. Although t h i s experiment showed that 2 - f l u o r o - L - h i s t i d i n e could act in v i v o , i t d i d not i n d i c a t e whether i t was a c t i n g d i r e c t l y on the p i n e a l gland, nor d i d i t provide any other i n f o r m a t i o n regarding the mode of a c t i o n of t h i s compound. In Vitro Studies on the Induction of P i n e a l N - A c e t y l t r a n s f e r a s e Activity. E a r l y i n our s t u d i e s on p i n e a l N - a c e t y l t r a n s f e r a s e we d i s covered that we could t r e a t p i n e a l glands i n organ c u l t u r e w i t h adrenergic agents, i n c l u d i n g a d r e n a l i n and i s o p r o t e r e n o l , and i n t h i s way i n c r e a s e the a c t i v i t y of N - a c e t y l t r a n s f e r a s e (11). T h i s provided an e x c e l l e n t model system f o r f u r t h e r s t u d i e s with 2fluoro-L-histidine. E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Adrenergic Induction of N - A c e t y l t r a n s f e r a s e A c t i v i t y . When p i n e a l glands are t r e a t e d with 2 - f l u o r o - L - h i s t i d i n e the adrenergic s t i m u l a t i o n of N - a c e t y l t r a n s f erase a c t i v i t y i s s u b s t a n t i a l l y reduced (Table I I I ) . T h i s b l o c k i n g e f f e c t of 2 - f l u o r o - L - h i s t i d i n e was seen w i t h glands which were removed from animals and t r e a t e d immediately with i s o p r o t e r e n o l and 2 - f l u o r o - L - h i s t i d i n e , or with glands which had


42

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

Table I I In vivo i n h i b i t i o n by 2-F-HIS of the isoproterenol-induced i n crease i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y .

Exp

Group

Treatment

1

A

Isoproterenol

Β

Isoproterenol, 2-F-HIS

2

C

Control

A

Isoprotereno

D

Isoproterenol, HIS

N-Acetyltransferase A c t i v i t y (nmole/gland/hour) 12.6

+1.42

3.0

+

.58

0.2

+

.011

8.33 + 1.05

Animals (100-gram male Sprague-Dawley Rats) were deprived of food overnight. Groups Β and D were i n j e c t e d subcutaneously i n the lower back r e g i o n with a s o l u t i o n of h i s t i d i n e (HIS) or a suspension o f 2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) (250 mg/kg; 25mg/0.25 ml s a l i n e ) a t 9:00 a.m. Groups A, B, and D were i n j e c t e d sub cutaneously i n the nape of the neck with i s o p r o t e r e n o l (20 mg/kg, 2.0 mg/0.1 ml s a l i n e ) at 10:00 a.m. Groups Β and D r e c e i v e d d u p l i c a t e 2-F-HIS or HIS i n j e c t i o n s at 10:30 a.m. A l l animals were k i l l e d a t noon and t h e i r p i n e a l glands were removed r a p i d l y . Values are based on 4 glands; data are presented as the mean + S.E. (From K l e i n e t a l . , 16).


3.

KLEIN AND KIRK

43

2~Fluoro-L-Histidine

been c h r o n i c a l l y denervated (16). Chronic denervation removes a l l n e u r a l elements from the gland; the observation that 2 - f l u o r o L - h i s t i d i n e acted i n denervated glands i n d i c a t e d that i t was a c t i n g on p i n e a l c e l l s d i r e c t l y . E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the C y c l i c AMP Induction of N - A c e t y l t r a n s f e r a s e . Catecholamines s t i m u l a t e the a c t i v i t y of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y by f i r s t s t i m u l a t i n g the production of c y c l i c AMP (10). One p o s s i b l e mechanism of a c t i o n of 2 - f l u o r o - L - h i s t i d i n e was i n h i b i t i o n of the a c t i o n of c y c l i c AMP. We evaluated t h i s by determining whether 2 - f l u o r o - L - h i s t i dine could block the s t i m u l a t i o n of N - a c e t g l t r g n s f e r a s e a c t i v i t y as caused by a d e r i v a t i v e of c y c l i c AMP, Ν ,2' d i b u t y r y l adeno s i n e 3 ,5 -monophosphate ( d i b u t y r y l c y c l i c AMP). The s t i m u l a t i o n of N - a c e t y l t r a n s f e r a s e a c t i v i t y caused by t h i s compound was block ed by 2 - f l u o r o - L - h i s t i d i n e (Table I I I ) T h i s i n d i c a t e d that 2f l u o r o - L - h i s t i d i n e was probabl sequence of events whic and f o l l o w s both the i n t e r a c t i o n of catecholamines w i t h a receptor and the production of c y c l i c AMP. f

f

S p e c i f i c i t y of 2 - F l u o r o - L - H i s t i d i n e

I n h i b i t i o n of Enzyme

Induction

Our f i n d i n g that the i n d u c t i o n of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y was blocked by 2 - f l u o r o - L - h i s t i d i n e r a i s e d the question of whether the i n h i b i t o r y e f f e c t s of t h i s compound were s p e c i f i c to t h i s enzyme, or whether the i n d u c t i o n of other enzymes could be i n h i b i t e d . To examine t h i s important question we studied the s t e r o i d i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e i n f e t a l l i v e r , the benz[a]anthracene i n d u c t i o n of a r y l hydrocarbon hydroxylase a c t i v i t y i n l i v e r c e l l s , and the spontaneous i n c r e a s e of o r n i t h i n e decarboxylase a c t i v i t y i n both mouse mammary t i s s u e and the p i n e a l gland. In a l l cases the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e were examined i n in vitro systems (16). S t e r o i d Induction of L i v e r Tyrosine Amino Transferase. The a c t i v i t y of t y r o s i n e amino t r a n s f e r a s e i n l i v e r can be stimulated by treatment with a low concentration of the s t e r o i d dexamethasone (17). We determined that the i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e a c t i v i t y i n f e t a l l i v e r expiants by s t e r o i d treatment was blocked by 2 - f l u o r o - L - h i s t i d i n e (Table IV, 16). Benz[a]anthracene Induction of L i v e r A r y l Hydrocarbon Hydroxylase A c t i v i t y . The a c t i v i t y of l i v e r a r y l hydrocarbon hydroxylase, a membrane bound enzyme, can be induced by low con c e n t r a t i o n s of a number of compounds (18), i n c l u d i n g benz[a]anthracene. Using a c e l l c u l t u r e system, i t was found that the i n d u c t i o n of the a c t i v i t y of t h i s enzyme was s u b s t a n t i a l l y blocked by 2 - f l u o r o - L - h i s t i d i n e (Table V, 16).



1

Experiment

1.6

18.8

-HIS, 2-F-HIS, (0-11 hours); I s o p r o t e r e n o l (3-11 hours) -HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours) -HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)

None

SCGX

SCGX

2.5

10.4

-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)

+ 0.52 *

+ 3.00

+ 0.09 *

+ 2.11

3.2 + 0.56 *

16.4 + 2.74

None

-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)

None

4.9 + 0.31 *

-HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)

HIS, 2-F-HIS (0-11 hours); I s o p r o t e r e n o l (3-11 hours)

None

10.5 + 1.5.

0.2 + 0.03

N-Acetyltransferase A c t i v i t y (nmole/gland/hour)

None

HIS (0-11 hours); I s o p r o t e r e n o l (3-11 h r s )

None

(0-11 hours)

HIS

Treatment i n Organ C u l t u r e

None

Surgical Pretreatment

I n h i b i t i o n by 2-F-HIS of the drug-Induced increase i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y i n organ culture

Table I I I


-HIS (0-11 hours); d i b u t y r y l c y c l i c AMP (3-11 h r s ) -HIS, +2-F-HIS (0-11 hours); d i b u t y r y l c y c l i c AMP (3-11 hrs)

None

Treatment i n Organ C u l t u r e

None

Surgical Pretreatment

2.6 + 0.64*

22.7 + 3.43


Superior c e r v i c a l ganglionectomy (SCGX) was performed 14 days p r i o r t o organ c u l t u r e . P i n e a l glands were removed between 10:00 and 12:00 a.m. and placed i n t o c u l t u r e . The c o n c e n t r a t i o n o f 2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) was 3 mM; and, when present, the c o n c e n t r a t i o n of h i s t i d i n e (HIS) was 0.1 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 M HC1 t o a f i n a l c o n c e n t r a t i o n of 10 yM. D i b u t y r y l c y c l i c AMP was added i n 5 y l of c u l t u r e medium t o a f i n a l c o n c e n t r a t i o n of 1 mM. Each value i s based on 4 glands. Data a r e given as the mean + S.E. * S t a t i s t i c a l l y l e s s than glands t r e a t e d with e i t h e r i s o p r o t e r e n o l or d i b u t y r y l c y c l i c AMP Ρ < .01. S t a t i s t i c a l a n a l y s i s was performed using Student's " t " t e s t . (From K l e i n et a i . , 16).

3

Experiment

46

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

Table IV The e f f e c t of 2-F-HIS on the dexamethasone i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e a c t i v i t y i n f e t a l r a t l i v e r expiants i n c u l t u r e Treatment i n c u l t u r e (hours) (0-3) (3-11)

Tyrosine amino t r a n s f e r a s e a c t i v i t y (nmole/mg protein/min)

Control

Control

Control

Dexamethasone

2-F-HIS

2-F-HIS

2-F-HIS

2-F-HIS + dexamethasone

6.5 54.2 6.6

10.4

A f t e r a 24-hour i n c u b a t i o n period with 1 mM h i s t i d i n e , the t i s s u e was t r a n s f e r r e d to f r e s h medium c o n t a i n i n g 0.1 mM h i s t i d i n e f o r the i n d i c a t e d treatments. The concentration of 2 - f l u o r o - L h i s t i d i n e (2-F-HIS) was 3 mM. Dexamethasone treatment was i n i t i a t e d by adding 5 y l of c u l t u r e medium c o n t a i n i n g dexamethasone r e s u l t i n g i n a f i n a l medium concentration of 5 yM. Each value i s the average of the means of d u p l i c a t e determinations performed on d u p l i c a t e c u l t u r e s . The d u p l i c a t e means d i d not d i f f e r by more than 3 nanomoles/mg protein/minute. (From K l e i n e t a l . , 16).

Spontaneous Increase of O r n i t h i n e Decarboxylase A c t i v i t y . O r n i t h i n e decarboxylase i s involved i n the s y n t h e s i s of p o l y amines, which appear to play a r o l e i n the process o f c e l l d i v i s i o n and gene r e g u l a t i o n (19). The a c t i v i t y of t h i s enzyme i s g e n e r a l l y high under two circumstances: f o l l o w i n g traumatic treatment o f t i s s u e , such as during regeneration of l i v e r or immediately a f t e r t i s s u e i s placed i n t o c u l t u r e , and during normal growth of t i s s u e . The a c t i v i t y of o r n i t h i n e decarboxylase increases i n both mouse mammary t i s s u e expiants (20) and i n p i n e a l glands (unpublished r e s u l t s , D. C. K l e i n and T. Oka) soon a f t e r these t i s s u e s are placed i n t o c u l t u r e . We examined the e f f e c t of 2f l u o r o - L - h i s t i d i n e on the spontaneous increase of o r n i t h i n e decarboxylase a c t i v i t y i n these systems. I t was found that 2f l u o r o - L - h i s t i d i n e d i d not block the spontaneous increase of t h i s enzyme i n the mouse mammary t i s s u e (Table VI) but d i d block i t i n the p i n e a l gland (Table V I I ) . The l a c k of an e f f e c t of 2 - f l u o r o - L - h i s t i d i n e on the increase of o r n i t h i n e decarboxylase i n the mouse mammary t i s s u e may be due


3.

KLEIN AND

KTRK

47


Table V The e f f e c t of 2-F-HIS on the benz[a]anthracene i n d u c t i o n of a r y l hydrocarbon hydroxylase i n l i v e r hepatoma (Hepa-1) c e l l s A r y l hydrocarbon hydroxylase hydroxylase a c t i v i t y (pmoles/ ιmg protein/min)

Treatment i n C u l t u r e (hours) (0-3)

(3-11)

Control

Control

Control

Benz[a]anthracene

2-F-HIS

2-F-HIS

2-F-HIS

2-F-HIS + benz[a]anthracen

19 159 6.2

C e l l s were obtained from a stock maintained i n c u l t u r e and t r a n s f e r r e d to 15 X 60 mm Falcon dishes c o n t a i n i n g 3 ml of Waymouth MAB medium with 10% f e t a l c a l f serum f o r 72 hours u n t i l 75-80% confluency was achieved. The medium was then replaced with 2 ml of BGJ c o n t a i n i n g 0.1 mM h i s t i d i n e f o r the treatments d e t a i l e d above. The c o n c e n t r a t i o n of 2 - f l u o r o - L - h i s t i d i n e (2-FHIS) was 3 mM. Benz[a]anthracene was added i n a concentrated s o l u t i o n r e s u l t i n g i n a f i n a l c o n c e n t r a t i o n of 13 μΜ. Data are presented as the means of d u p l i c a t e determinations (18) performed on d u p l i c a t e c u l t u r e s . The i n d i v i d u a l values d i d more than 5%. (From K l e i n et al., 16).

American Chemical Society Library 1155

16th

St.

N.

W.

Washington, D. C. 20036 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BIOCHEMISTRY

48


BONDS

Table VI E f f e c t o f 2-F-HIS on the spontaneous increase of o r n i t h i n e decarboxylase a c t i v i t y i n midpregnant mouse mammary expiants.

Treatment Not incubated

Ornithine ^ c a r b o x y l a s e a c t i v i t y (picomoles of C 0^ produced/mg t i s s u e / h r ) 5.0

Incubated (0-3 hours) Control

41.2

+ HIS (3 mM)

33.6

+ 2-F-HIS (3 mM)

33.

Midpregnancy mouse (CH~/Hen) mammary expiants were used (20) ( 2 - f l u o r o - L - h i s t i d i n e , 2-F-HIS; h i s t i d i n e , HIS). The c u l t u r e medium used was M 199 ([HIS] - 0.175 mM). Data a r e presented as the mean enzyme a c t i v i t y i n two c u l t u r e s . Enzyme a c t i v i t y i n each c u l t u r e i s based on d u p l i c a t e determinations, which a r e w i t h i n 1% of the mean. (From K l e i n et a i . , 16).


In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 4.37 + 0.84

2-F-HIS (0-11 hrs) + I s o p r o t e r e n o l (3-11 hrs)

P i n e a l organ c u l t u r e s were prepared j s d e s c r i b e d i n Table I I . Each datum i s presented as the mean (+ S.E.) of 4 to 6 determinations. For the determination of o r n i t h i n e decarboxylase a c t i v i t y , i n d i v i d u a l glands were sonicated i n 100 y l of the assay b u f f e r (20) Immediately a f t e r being removed from c u l t u r e . The homogenate was stored at -20C f o r 24 hours, thawed, and t r a n s f e r r e d t o a r e a c t i o n v i a l f o r enzyme assay. The f i n a l volume of the r e a c t i o n was 125 y l and c o n t a i n e d , p y r i d o x a l phos phate (40 yM), d i t h i o t h r e i t o l (5 mM) , EDTA (4 mM) , T r i s - H C l (50 mM), and DL-[1-C ] o r n i t h i n e (20 yM, s p e c i f i c a c t i v i t y = 43 yCi/ymole). * S i g n i f l e a n t l y lower than incubated glands not t r e a t e d with 2F-HIS, ρ > 0.01. (From K l e i n e t a l . , 16).

13.24 + 4.17

C o n t r o l (0-3 hrs) + I s o p r o t e r e n o l (3-11 hrs)

34.52 + 9.5*

6.38 + 0.86

2-F-HIS (0-11 hrs)

.02

67.45+6.47

0.15 +

Ornithine^decarboxylase a c t i v i t y (picomoles of C 0^ produced/gland/hr)

C o n t r o l (0-11 hrs)

Incubated

Not incubated

Treatment

N-Acetyltransferase activity (nmols/gland/hr)

The e f f e c t of 2-F-HIS on the spontaneous i n c r e a s e of o r n i t h i n e decarboxylase a c t i v i t y i n p i n e a l glands.

Table VII


50

to the higher c o n c e n t r a t i o n of h i s t i d i n e i n the c u l t u r e medium. T h i s would block the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e , as discussed below. A l t e r n a t i v e l y , mammary t i s s u e may contain more h i s t i d i n e or destroy 2 - f l u o r o - L - h i s t i d i n e more q u i c k l y . General Metabolic

E f f e c t s of


The observation that 2 - f l u o r o - L - h i s t i d i n e blocked the induct i o n of s e v e r a l enzymes r a i s e d the p o s s i b i l i t y that t h i s compound, although not apparently t o x i c to the i n t a c t animal, was i n f a c t h i g h l y t o x i c to t i s s u e i n general. To i n v e s t i g a t e t h i s p o s s i b i l i t y we examined the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e on s e v e r a l metabolic parameters (16). We found that a f t e r 24 hours of treatment w i t h 2 - f l u o r o - L h i s t i d i n e , RNA s y n t h e s i s was not decreased (Table V I I I ) . T h i s was remarkable i n view of th dependency of i t upon enzym s y n t h e s i s a small i n h i b i t i o due to 2 - f l u o r o - L - h i s t i d i n e was apparent immediately. T h i s i n h i b i t o r y e f f e c t d i d not, however, i n c r e a s e during the next 24 hours of treatment. E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on Hydroxyindole-O-Methyltransferase A c t i v i t y . Another t o p i c of i n t e r e s t was the e f f e c t of 2 - f l u o r o - L - h i s t i d i n e on the steady-state l e v e l s of a t h i r d enzyme i n the p i n e a l gland, hydroxyindole-O-methyl-transferase (10). I t i s g e n e r a l l y thought that the steady s t a t e l e v e l of an enzyme depends upon both the ongoing production and d e s t r u c t i o n of enzyme molecules. Thus, even though a l a r g e increase i n hydroxyindole-O-methyltransferase a c t i v i t y could not be s t u d i e d , as i n the case of the enzymes examined above, i t seemed probable that the steady-state l e v e l s of t h i s enzyme d i d i n f a c t r e f l e c t enzyme production at a r a t e equal to that of enzyme degradation. The e f f e c t of a 24-hour treatment w i t h 2 - f l u o r o - L - h i s t i d i n e on the a c t i v i t y of t h i s enzyme, which converts N - a c e t y l s e r o t o n i n to melatonin, was examined. 2 - F l u o r o - L - h i s t i d i n e d i d not a l t e r the a c t i v i t y of t h i s enzyme (Table IX). E f f e c t of 2 - F l u o r o - L - H i s t i d i n e on the I n c o r p o r a t i o n of Histidine into Protein. The l a c k of a n o n s p e c i f i c e f f e c t of 2f l u o r o - h i s t i d i n e on s e v e r a l of the metabolic parameters examined l e d us to suspect that t h i s amino a c i d analog may be a c t i n g on a s p e c i f i c mechanism, which was i n v o l v e d i n the i n d u c t i o n of a l l the enzymes we examined. Whereas i t d i d not seem that p r o t e i n s y n t h e s i s was h a l t e d by 2 - f l u o r o - L - h i s t i d i n e , i t d i d seem p o s s i b l e that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by v i r t u e of being i n c o r porated i n t o p r o t e i n i n p l a c e of h i s t i d i n e . We approached t h i s hypothesis by f i r s t determining i f the i n h i b i t i o n of enzyme i n d u c t i o n by 2 - f l u o r o - L - h i s t i d i n e was accompanied by an i n h i b i t i o n of the i n c o r p o r a t i o n of h i s t i d i n e .


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51


Table V I I I The e f f e c t of long term 2-F-HIS treatment i n organ c u l t u r e on the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o macromolecules i n p i n e a l glands incubated w i t h [ C]LEU and [ H ] u r i d i n e f o r a three hour period. R a d i o a c t i v e precursor i n TCA , insoluble material [ C]LEU [ H]Uridine (nanomoles/gland) (picomoles/gland) 14

Treatment i n Organ Culture

J

C o n t r o l (0-3 hours)

0.34

+

0.041

1.04

+

.098

2-F-HIS (0-3 hours)

0.27

+

0.031

0.87

+

.167

0.28

+

0.018*

1.30

+

.122

C o n t r o l (0-24 hours) 2-F-HIS (0-27 hours)

P i n e a l glands were removed between 10:00 and 12:00 A.M. and placed i n t o organ c u l t u r e . The c u l t u r e medium contained no HIS. The concentration-of 2-F-HIS was 3 mM. [ H ] U r i d i n e (3μΜ, S.A.= 21yCi/mole) and [ C]LEU (0.38 mM,S.A.= 8.62 yCi/ymole) were present f o r only the f i n a l three hours of the i n c u b a t i o n p e r i o d . Values, which are based on 4 glands, are computed from the S.A. of the r a d i o a c t i v e p r e c u r s o r s and the r a d i o a c t i v i t y pgr gland p r e c i p i t a t e . Data are presented as the means + S.E. Statisti c a l l y l e s s than the value f o r c o n t r o l glands incubated f o r 27 hours (p < 0.01) but not s i g n i f i c a n t l y l e s s than [ C]LEU i n c o r p o r a t i o n i n t o glands treated f o r 3 hours with 2-F-HIS. (From K l e i n et a l . , 16).


52


Table

IX

Lack of an e f f e c t of long-term treatment with 2-F-HIS on a c t i v i t y of hydroxyindole-O-methyltransferase.

Treatment i n Organ Culture Not

incubated

the

Hydroxyindole-O-methyltransferase activity(picomoles/gland/hour) 69.2

+

11.1

C o n t r o l (0-24

hours)

109.2

+

19.1

2-F-HIS (0-24

hours)

132.8

+

8.4

C o n t r o l (0-48

hours)

88.8

+

6.0

2-F-HIS (0-48

hours

P i n e a l glands were removed between 10:00 and 12:00 A.M. and placed i n t o organ c u l t u r e i n medium c o n t a i n i n g 0.1 mM HIS. Each value i s based on 4 glands and i s presented as the mean (+ S.E.). The concentration of 2-F-HIS was 3 mM (from K l e i n et a l . , 16).

14 IJ^was found that i n the presence of 7 μΜ [ C ] h i s t i d i n e that [ C ] h i s t i d i n e could be incorporated i n t o p r o t e i n . More i n t e r e s t i n g , however, was the f i n d i n g that i n the presence of 2 - f l u o r o L - h i s t i d i n e t h i s i n c o r p o r a t i o n was blocked (Table X). The e f f e c t s of^glevated e x t r a c e l l u l a r concentrations of h i s t i d i n e on both [ C ] h i s t i d i n e i n c o r p o r a t i o n and i n d u c t i o n of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l were examined i n the presence of 2 - f l u o r o - L - h i s t i d i n e (Figure 1). I t was found that the i n h i b i t o r y e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e could be overcome by higher concentrations of h i s t i d i n e . This observation pointed to the explanation that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by d i r e c t l y competing with h i s t i d i n e as a substrate i n p r o t e i n synthesis. 3 I n c o r p o r a t i o n of [ Η]2-Fluoro-L-Histidine i n t o P r o t e i n A d i r e c t method of determining whether 2 - f l u o r o - L - h i s t i d i n e was a c t u a l l y being incorporated i n t o p r o t e i n was to incubate glands with r a d i o l a b e l e d 2 - f l u o r o - L - h i s t i d i n e and recover the l a b e l l e d amino a c i d from p r o t e i n . We have synthesized [ H]2f l u o r o - L - h i e t i d i n e and have found that i t i s incorporated i n t o p r o t e i n and that t h i s i n c o r p o r a t i o n can be blocked by c y c l o h e x i mide (an i n h i b i t o r of p r o t e i n synthesis) and by high concentra t i o n s of h i s t i d i n e . In a d d i t i o n we have been able to enzymatic a l l y d i g e s t l a b e l l e d p r o t e i n and recover a r a d i o a c t i v e compound which has the same chromatographic and e l e c t r o p h o r e t i c c h a r a c t e r -


3.

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AND

KIRK

53


i s t i c s as does a u t h e n t i c ^ 2 - f l u o r o - L - h i s t i d i n e . This r a d i o l a b e l l e d compound apparently i s [ H ] 2 - f l u o r o - L - h i s t i d i n e (21). T h i s set of observations provide necessary support f o r the hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s incorporated i n t o newly synthesized p r o t e i n . A l t e r n a t i v e Mechanisms of A c t i o n of


Although the above evidence i s necessary to accept the hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s incorporated i n t o p r o t e i n , t h i s f i n d i n g might be c o i n c i d e n t a l and not causative. We have thus examined other p o s s i b l e modes o f a c t i o n of 2 - f l u o r o - L - h i s t i d i n e . F i r s t l y , i t seemed p o s s i b l e that 2 - f l u o r o - L - h i s t i d i n e was a c t i v e only a f t e r decarboxylation to 2-fluorohistamine. This, however, was proven improbabl becaus 2-fluorohistamin itself was without e f f e c t on th f e r a s e a c t i v i t y (16). Secondly f l u o r o - L - h i s t i d i n e to assays of N - a c e t y l t r a n s f e r a s e d i d not i n h i b i t enzyme a c t i v i t y , e l i m i n a t i n g the p o s s i b i l i t y that 2-fluoro-Lh i s t i d i n e was a competitive or noncompetitive i n h i b i t o r of enzyme a c t i v i t y (16). T h i r d l y , we thought that 2 - f l u o r o - L - h i s t i d i n e might have induced the production o f an i n h i b i t o r of N - a c e t y l t r a n s f e r a s e a c t i v i t y . We examined t h i s p o s s i b i l i t y by adding


Figure 1. The effect of histidine (HIS) on N-acetyltransf erase activity and [ C]HIS incorporation in pineal glands treated with isoproterenol and 2-fluoro-L-histidine (2-FHIS). Data was collected from 4 experiments; a different concentration of HIS was used in each. Percent inhibition is calculated from the following: 100 [isoproterenol value—(isoproterenol + 2-F-HIS value)] isoproterenol value. In each experiment a set of 4 glands was treated with 10 Μ isoproterenol and a second set of 4 glands was treated with 10 Μ isoproterenol and 3 mM 2-F-HIS (16). 14

μ

μ


In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 1.17

1.65

1.43

2-F-HIS (0-11 hours)

Control (0-3); Isoproterenol (3-11 hours)

2-F-HIS (0-11 hours); Isoproterenol (3-11 hours) + .075

+ .076

+ .158

+ .071

~ [ H]LEU

-,

+ .019

C]HIS

+ .022

.126 + .005'

.41

.117 + .018*

.30

[

3.68

14.3

0.18

0.34

+

+

+

+

.548

.034

.034

.068


P i n e a l glands were removed between 10:00 and 12:00 and were placed into,organ c u l t u r e . The c u l t u r e medium contained 0.38 mM [ H]LEU (S.A. = 51.2 yCi/umole) and 1 mM [ C]HIS (S.A. = 24 yCi/ymole). The c o n c e n t r a t i o n of 2-F-HIS was 3 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 mM HCl to a f i n a l c o n c e n t r a t i o n of 10 yM. At the end of the experiment glands were sonicated i n 100 y l of 2 mM p e n i c i l l a m i n e i n 0.01 M sodium phosphate b u f f e r , pH 6.9, at 4°C. T h i s treatment preserves enzyme a c t i v i t y d u r i n g handling. A 50 y l sample of each s o n i c a t e was used f o r enzyme assay; a 25 y l sample was used f o r p r e c i p i t a i o n of TCA i n s o l u b l e m a t e r i a l . Values, which are based on 4-5 glands, are computed from the S. A. of the r a d i o a c t i v g amino a c i d and the r a d i o a c t i v i t y per gland p r e c i p i t a t e . ^ Data are presented as the mean + S.E. S i g n i f i c a n t l y l e s s than c o n t r o l value (P < .01). S i g n i f i c a n t l y l e s s than group t r e a t e d with i s o p r o t e r e n o l alone (P < .01) (From K l e i n et a l . , 16).

1.25

Control (0-11 hours)

Treatment i n Organ Culture

R a d i o a c t i v i t y i n TCA-insoluble m a t e r i a l (nmoles of r a d i o a c t i v e amino acid/gland)

3 E f f e c t of 2-F-HIS on r a d i o a c t i v i t y incorporated i n t o p r o t e i n of glands incubated with [ H]LEU and r\:]HIS.

Table Χ

3.

KLEIN

A N D KIRK


55

homogenates of glands t r e a t e d with i s o p r o t e r e n o l and 2 - f l u o r o - L h i s t i d i n e to homogenates of glands t r e a t e d only with i s o p r o t e r e n o l (16). The r e s u l t i n g enzyme a c t i v i t y was the sum of the a c t i v i t i e s o f each homogenate, i n d i c a t i n g that an i n h i b i t o r probably was not present i n the i s o p r o t e r e n o l + 2 - f l u o r o - L - h i s t i d i n e homo genate. F i n a l l y , we added 2 - f l u o r o - L - h i s t i d i n e to glands which had already been treated with i s o p r o t e r e n o l f o r 3 hours. I f 2f l u o r o - L - h i s t i d i n e were i n a c t i v a t i n g newly formed enzyme mole c u l e s , i t would have caused a r a p i d i n a c t i v a t i o n of enzyme a c t i v ity. T h i s , however, was not observed (16). Conclusions The r e s u l t s o f our s t u d i e s i n d i c a t e that 2 - f l u o r o - L - h i s t i d i n e c e r t a i n l y i s not an a c u t e l y t o x i c compound but that i t can i n h i b i t the i n d u c t i o n of s e v e r a l without b l o c k i n RNA synthe s i s , without i n h i b i t i n out a l t e r i n g the a c t i v i t One explanation of these r e s u l t s i s that 2 - f l u o r o - L - h i s t i d i n e i s incorporated i n t o enzyme p r o t e i n , and that i n c o r p o r a t i o n r e s u l t s i n the s u b s t i t u t i o n of 2 - f l u o r o - L - h i s t i d i n e f o r h i s t i d i n e i n the primary s t r u c t u r e o f p r o t e i n s . The s u b s t i t u t i o n could block the i n d u c t i o n of enzyme a c t i v i t y by causing changes i n the s t r u c t u r e of the enzyme, by producing n o n - f u n c t i o n a l a c t i v e s i t e s i n the enzymes, or by b l o c k i n g phosphorylation o f h i s t i d i n e at a r e g u l a t o r y s i t e on the enzyme. We have not, however, shown whether i n c o r p o r a t i o n of 2f l u o r o - L - h i s t i d i n e i n t o complete enzyme p r o t e i n a c t u a l l y occurs. T h i s i s one of our f u t u r e goals. We may f i n d , a l t e r n a t i v e l y , that 2 - f l u o r o - L - h i s t i d i n e blocks the production o f complete molecules of any p r o t e i n i n which i t i s incorporated. Net i n c o r p o r t a t i o n of r a d i o l a b e l l e d l e u c i n e might appear n e a r l y normal because an increased number of p r o t e i n fragments were synthesized. I t a l s o has not been shown whether the phosphoryl a t i o n of enzyme p r o t e i n i s blocked by the s u b s t i t u t i o n of h i s t i dine with 2 - f l u o r o - L - h i s t i d i n e . T h i s and other f a s c i n a t i n g problems regarding the a c t i o n of 2 - f l u o r o - L - h i s t i d i n e are present l y being studied. The s o l u t i o n s of t h i s problem w i l l not only provide us with information regarding the a c t i o n of 2 - f l u o r o - L h i s t i d i n e , but w i l l a l s o provide b a s i c information about b i o l o g i c a l processes i n general.

LITERATURE CITED 1. Shive, W. and Skinner, C. G. "Metabolic Inhibitors, A Comprehensive Treastise" pp. 1-60, Academic Press, New York (1963). 2. Fowden, L., Lewis, D., and Tristram, Η., Adv. Enz. (1968) 29, 89-163. 3. Jencks, W. P. "Catalysis in Chemistry and Enzymology,"


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pp 163-168, McGraw Hill, New York (1969). 4. Kirsch, J., Ann. Rev. Biochem. (1973) 42, 205-234. 5. Lehninger, "Biochemistry" pp 217-248, Worth Publishers, Inc., New York (1975). 6. Chen, C. C., Smith, D. L., Bruegger, B. B., Halpern, R. Μ., and Smith, R. Α., Biochemistry (1974) 13, 3785-3790. 7. Segal, H. L., Science (1973) 180, 25-32. 8. Langan, Τ. Α., Proc. Nat. Acad. Sci. USA (1968) 64, 12671271. 9. Kirk, K. L., Nagai, W. and Cohen, L. Α., J. Am. Chem. Soc., (1973) 95, 8389-8392. 10. Klein, D. C. "The Neurosciences: Third Study Program" pp. 509-519, MIT Press, Cambridge, Mass. (1974). 11. Klein, D. C. and Weller, J. L., J. Pharmacol. Exp. Ther. 186, 516-527. 12. Deguchi, T., J. Neuroche 13. Klein, D. C. and Weller 14. Binkley, S., Klein, D. C. and Weller, J. L., J. Neurochem. (1976) (in press). 15. Axelrod, J., Science (1974) 184, 1341-1348. 16. Klein, D. C., Kirk, K. L., Weller, J. L., Oka, T., Parfitt, Α., and Owens, I. S., Molec. Pharmacol. (1976) (in press). 17. Kenny, F. T., "Mammalian Protein Metabolism" pp 131-145, Academic Press, New York (1970). 18. Gielen, J. E., and Nebert, D. W. J. Biol. Chem. (1972) 247, 7591-7801. 19. Russel, D. Η., "Polyamines in Normal and Neoplastic Growth" Raven Press, New York (1973). 20. Oka, T. and Perry, J., J. Biol. Chem. (1976) (in press). 21. Klein, D. C., Kirk, K. L. and Weller, J. L. (manuscript in preparation).


4 Thymidylate Synthetase: Interaction with 5-Fluoro and 5-Trifluoromethyl-2'-Deoxyuridylic A c i d DANIEL V. SANTI, ALFONSO L. POGOLOTTI, THOMAS L. JAMES, YUSUKE WATAYA, KATHRYN M. IVANETICH, and STELLA S. M. LAM Department of Biochemistry and Biophysics, and Department of Pharmaceutical Chemistry, University of California, San Francisco, Calif. 94143

Thymidylate synthetas of 2'-deoxyuridylate (dUMP) to thymidylate (TMP) with the concomitant conversion of 5, 10-methylene tetrahydrofolate (CH FAH ) to 7, 8-dihydrofolate (FAH ) as depicted in F i gure 1 (for a recent review, see reference 1). In this process the hydrogen at C-6 of FAH is directly transferred to the methyl group of TMP (2). One of our objectives over the past few years has been to establish the mechanism of catalysis of thymidylate synthetase. Extensive investigations of chemical counterparts (3-6) have in dicated that the reaction is initiated by attack of a nucleophile at the 6-position of dUMP and that many, if not all, reactions along the pathway are facilitated by analogous nucleophilic catalysis. The proposed mechanism of this enzyme, as derived from investigations of chemical models is illustrated in Figure 2. It is proposed that the reaction is initiated by attack of a nucleophilic group of the enzyme to the 6-position of dUMP. In this manner, the 5-position of dUMP could be made sufficient ly nucleophilic (viz I, Figure 2) to react with CH FAH or an equivalent reactive species of formaldehyde. Thus, the initial condensation product between dUMP and CH F A H is now generally accepted (1, 7) to be one which is covalently bound to the enzyme and saturated across the 5, 6-double bond of dUMP (II). Proton abstraction from II would give the intermediate 2

4

2

4

2

2

4

4

enolate III. As with the chemical models, III should readily undergo a β-elimination to produce the highly reactive exocyclic methylene intermediate IV and FAH , bound to the enzyme in close proximity. Intermolecular hydride transfer from FAH to IV would yield dTMP, FAH , and the native enzyme. It should be emphasized that all of the aforementioned reactions and inter mediates have direct chemical counterparts, and are in complete accord with all available biochemical data. With the availability of a stable enzyme from an amethop terin resistant strain of Lactobacillus casei (8, 9) and facile methods for its purification (9-11), we undertook studies which 4

4

2

57 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Figure 2. Suggested sequence for the thymidylate synthetase reaction. All pyrimidine structures have a l-(5-phospho-2'-deoxyribosyl) substituent and R = CH NHC H COGlu. t

6

k


BONDS

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might p r o v i d e d i r e c t s u p p o r t f o r p r o p o s a l s w h i c h w e r e b a s e d on the a f o r e m e n t i o n e d n o n e n z y m i c m o d e l s . A l t h o u g h a n u m b e r of d i r e c t i o n s h a v e b e e n p u r s u e d t o w a r d s t h i s o b j e c t i v e , the f o l l o w i n g s u m m a r i z e s o u r i n v e s t i g a t i o n s of the i n t e r a c t i o n of t h y m i d y l a t e synthetase w i t h 5 - f l u o r o - 2 ' - d e o x y u r i d y l a t e ( F d U M P ) and 5 - t r i f l u o r o m e t h y l - 2 - d e o x y u r i d y l a t e ( C R d U M P ) . Studies of these two f l u o r i n a t e d n u c l e o t i d e s have p r o v i d e d c o n v i n c i n g e v i d e n c e for the m e c h a n i s m of this e n z y m e ; i n a d d i t i o n , they h a v e p r o v i d e d i n s i g h t into how these d r u g s act, and how the r e a c t i v i t y of f l u o r i n a t e d m o l e c u l e s m i g h t be u t i l i z e d i n the d e s i g n of e n z y m e i n h i b i t o r s . We e m p h a s i z e that a n u m b e r of other l a b o r a t o r i e s have been engaged i n s i m i l a r i n v e s t i g a t i o n s , and r e g r e t that s p a c e does not p e r m i t c o m p l e t e c i t a t i o n of a l l the e x c e l l e n t s t u d i e s p e r f o r m e d i n this a r e a . f

5 - F l u o r o - 2 ' -deoxyuridylat s o m e t i m e ( 12, 13) tha of t h y m i d y l a t e s y n t h e t a s e , but the n a t u r e of i n h i b i t i o n has b e e n the topic of c o n s i d e r a b l e c o n t r o v e r s y (_1_4). S i n c e the 6 - p o s i t i o n of 1 - s u b s t i t u t e d 5 - f l u o r o u r a c i l s i s quite s u s c e p t i b l e t o w a r d n u c l e o p h i l i c attack ( 15- 17), we s u s p e c t e d that F d U M P m i g h t e x e r t its i n h i b i t o r y effect by r e a c t i o n w i t h the p r o p o s e d n u c l e o p h i l i c c a t a l y s t of t h y m i d y l a t e s y n t h e t a s e . Studies f r o m this (18, 19) and o t h e r (20, 21 ) l a b o r a t o r i e s h a v e s i n c e d e m o n s t r a t e d this to be the c a s e . A s i m p l i f i e d d e p i c t i o n of the i n t e r a c t i o n s of F d U M P and C H ^ F A H ^ is g i v e n below i n F i g u r e 3.

E-FdUMP

Ε

E-FdUMP-CH FAH '4 2

-

ι—ι

1

Ê.FdUMP.CH FAH

Figure 3


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U s i n g the i s o t o p e t r a p p i n g m e t h o d (2_2), we have found that the two l i g a n d s , F d U M P and C H ^ F A H ^ , i n t e r a c t w i t h the e n z y m e i n a r a n d o m f a s h i o n , a n d that f o r m a t i o n of the i n i t i a l t e r n a r y c o m p l e x ( E ^ F d U M P ^ C H ^ F A H ^ ) i s at l e a s t p a r t i a l l y r a t e d e t e r mining. F r o m e q u i l i b r i u m b i n d i n g techniques we h a v e a s c e r tained that the d i s s o c i a t i o n constantes of both b i n a r y c o m p l e x e s (K^ a n d K ^ ) a r e a p p r o x i m a t e l y 10" M . T h e f i r s t t e r n a r y c o m plex which is f o r m e d with F d U M P , C H ^ F A H . and enzyme is d e p i c t e d as E - F d U M P ' C H ^ F A H ^ a n d does not i n v o l v e c o v a l e n t bonds. Interestingly, analogous r e v e r s i b l e t e r n a r y complexes m a y be f o r m e d u s i n g a n a l o g s of the c o f a c t o r . T h r o u g h s t u d i e s of the i n t e r a c t i o n of F d U M P and a n a l o g s of C H ^ F A H ^ we h a v e a s c e r t a i n e d that t h e r e i s a s t r i k i n g s y n e r g i s m i n b i n d i n g of l i g a n d s to this p r o t e i n . T h a t i s , the a f f i n i t y of e i t h e r l i g a n d f o r the cognate b i n a r y c o m p l e x i s c a . two o r d e r s of m a g n i t u d e g r e a t e r than the a f f i n i t y f o r the f r e e e n z y m e With many a n a l o g s of C H ^ F A H ^ , thi that i t m a y be p h y s i c a l l T h e s e t e r n a r y c o m p l e x e s show i n t e r e s t i n g u l t r a v i o l e t d i f f e r e n c e s p e c t r a w h i c h m a y be u s e d f o r t h e i r q u a n t i t a t i o n a n d characterization. Shown i n F i g u r e 4 a r e d i f f e r e n c e s p e c t r a o b tained w i t h C H 2 F A H ^ a n d 5, 8 - d e a z a f o l i c a c i d ; s i m i l a r d i f f e r e n c e s p e c t r a h a v e a l s o b e e n o b t a i n e d w i t h a n u m b e r of other a n a l o g s of f o l i c a c i d . C h a r a c t e r i s t i c of these d i f f e r e n c e s p e c t r a i s a peak at c a . 330 n m a n d , u s u a l l y , a t r o u g h at c a . 290 n m . A l t h o u g h the e x a c t r e a s o n f o r the s p e c t r a l changes w h i c h o c c u r upon f o r m a t i o n of the r e v e r s i b l e t e r n a r y c o m p l e x e s i s y e t u n k n o w n , we s u g g e s t that they r e s u l t f r o m p e r t u r b a t i o n s of the p a m i n o b e n z o y l g l u t a m a t e m o i e t y of the c o f a c t o r a n a l o g s w h i c h r e sult f r o m t h e i r e n v i r o n m e n t w i t h i n the t e r n a r y c o m p l e x . T h i s p e r t u r b a t i o n i s b e l i e v e d to be a m a n i f e s t a t i o n of a c o n f o r m a t i o n a l change w h i c h i s r e l a t e d to the a f o r e m e n t i o n e d s y n e r g i s m i n b i n d i n g of the two l i g a n d s . T h e d i f f e r e n c e s p e c t r u m of the E » F d U M P C H F A H c o m p l e x ( F i g u r e 4) i s s i m i l a r to those o b s e r v e d f o r the c o f a c t o r a n a l o g s , s u g g e s t i n g that s i m i l a r c h a n ges i n e n v i r o n m e n t o c c u r w i t h the n a t u r a l c o f a c t o r , C H ^ F A H . . T h e r e i s one s t r i k i n g d i f f e r e n c e i n that t h e r e i s a l o s s of d i f f e r e n t i a l a b s o r b a n c e at 269 n m i n the c o m p l e x f o r m e d w i t h C H ^ F A H ^ (18, 1 9 ) w h i c h w e h a v e not o b s e r v e d i n c o m p l e x e s f o r m e d w i t h c o f a c t o r a n a l o g s ; the r e a s o n f o r this w i l l b e c o m e a p p a r e n t i n the e n s u i n g d i s c u s s i o n . e

2

4

The complex f o r m e d with thymidylate synthetase, F d U M P , and the n a t u r a l c o f a c t o r C H J A H . , h a s b e e n e x t e n s i v e l y i n v e s t i g a t e d . T h i s c o m p l e x i s e x t r e m e l y tight a n d m a y r e a d i l y be i s o l a t e d b y a v a r i e t y of t e c h n i q u e s (18, 1 9 , 21, 23, 24). Using r a d i o a c t i v e F d U M P and C H , F A H ^ , the e n z y m e h a s b e e n t i t r a t e d a n d shown to p o s s e s s two F d U M P a n d two c o f a c t o r b i n d i n g s i t e s p e r m o l e ( 1 9 ) . T h e s t o i c h i o m e t r y of b i n d i n g h a s b e e n v e r i f i e d i n a n u m b e r of l a b o r a t o r i e s b y a v a r i e t y of m e thods ( 11, 25, 26). T h i s i s i n a c c o r d w i t h the e a r l i e r f i n d i n g


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that t h y m i d y l a t e s y n t h e t a s e f r o m L . c a s e i has two a p p a r e n t l y i d e n t i c a l subunits of M W 35, 000 e a c h (9, 27). Studies of the r a t e of a s s o c i a t i o n of F d U M P w i t h the E - C H - , F A H . c o m p l e x (k ) and its d i s s o c i a t i o n (k ) have 2 4 ^ on off ' v

7

x

E-CHÂH^+f îl]FdUMP

Ε-Cr^FArJ·

r r

[hi]FdUMP

off a l l o w e d us t o ^ c ^ l c u l a t e the d i s s o c i a t i o n c o n s t a n t of the c o m p l e x to be c a . 10 M . T h i s p r o v i d e s a n e x p l a n a t i o n f o r the d i s c r e p a n c i e s i n R e v a l u e s r e p o r t e d f o r F d U M P in the l i t e r a t u r e ; n a m e l y , p r e v i o u s e x p e r i m e n t s w e r e u s i n g c o n c e n t r a t i o n s of e n z y m e h i g h e r than the and w e r e i n c o n c e n t r a t i o n r a n g e s w h e r e F d U M P was b e h a v i n g as a s t o i c h i o m e t r i c i n h i b i t o r . The kinetically determined i s a p p r o x i m a t e l y 10 - f o l d l o w e r than that f o r the b i n a r y c o m p l e x ; i n e f f e c t the p r e s e n c e of the c o f a c t o r i n c r e a s e s the 10 k c a l / m o l i n b i n d i n g t u r e of the i n t e r a c t i o n of F d U M P and t h y m i d y l a t e s y n t h e t a s e i n v o l v e s changes w h i c h o c c u r w i t h i n the bound t e r n a r y c o m p l e x . S e v e r a l l i n e s of e v i d e n c e d e m o n s t r a t e r a t h e r c o n c l u s i v e l y that a c o v a l e n t bond i s f o r m e d b e t w e e n F d U M P and t h y m i d y l a t e s y n t h e t a s e w i t h i n the c o m p l e x , (a) T h e E - ^ l F d U M P ^ C r L F A H ^ c o m p l e x m a y be t r e a t e d w i t h a n u m b e r of p r o t e i n d é n a t u r a n t s ( u r e a , g u a n i d i n e h y d r o c h l o r i d e , e t c . ) without a p p a r e n t l o s s of p r o t e i n - b o u n d r a d i o a c t i v i t y . W i t h few e x c e p t i o n s , s u c h t r e a t m e n t i s s u f f i c i e n t to d i s r u p t n o n c o v a l e n t i n t e r a c tions b e t w e e n low m o l e c u l a r w e i g h t l i g a n d s and t h e i r p r o t e i n receptors, (b) U p o n f o r m a t i o n of the c o m p l e x , t h e r e i s a d e c r e a s e of a b s o r b a n c e at 269 n m w h i c h c o r r e s p o n d s to s t o i c h i o m e t r i c l o s s of the p y r i m i d i n e c h r o m o p h o r e of F d U M P . This res u l t s t r o n g l y s u g g e s t s that the 5, 6 - d o u b l e bond of the p y r i m i dine is s a t u r a t e d i n the bound c o m p l e x , (c) T h e r a t e of d i s s o c i a t i o n of [6- H ] F d U M P f r o m the c o m p l e x shows a s e c o n d a r y t r i t i u m i s o t o p e effect ( k ^ / k ^ ) of 1.23. T h i s would c o r r e s p o n d to k £ j / k j 3 1. 15 and c l e a r l y α errions traite s that the 6 - c a r b o n of the h e t e r o c y c l e u n d e r g o e s sp to sp r e h y b r i d i z a t i o n d u r i n g the p r o c e s s as r e q u i r e d i f the 5, 6 - d o u b l e bond of F d U M P i s s a t u r a t e d i n the c o m p l e x , (d) P r o t e o l y t i c d i g e s t i o n of the c o m p l e x y i e l d s a p e p t i d e w h i c h i s c o v a l e n t l y bound to both F d U M P and C H ^ F A H ^ . T h e u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a of this p e p t i d e a r e c h a r a c t e r i s t i c of 5 - a l k y l t e t r a h y d r o f o l a t e s a n d , as w i t h the n a t i v e c o m p l e x , t h e r e i s no e v i d e n c e of u l t r a v i o l e t a b s o r p t i o n of the F d U M P c h r o m o p h o r e . k

=

F r o m t h e s e l i n e s of e v i d e n c e , together w i t h i n f o r m a t i o n g a t h e r e d f r o m m o d e l c h e m i c a l c o u n t e r p a r t s , the s t r u c t u r e of the e n z y m e ' F d U M P ' C H ^ F A H ^ c o m p l e x i s c u r r e n t l y b e l i e v e d to be as d e p i c t e d i n F i g u r e 5. H e r e , a n u c l e o p h i l e of the e n z y m e has a d d e d to the 6 - p o s i t i o n of F d U M P , and the 5 - p o s i t i o n of the p y r i m i d i n e i s c o u p l e d to the 5 - p o s i t i o n of F A H . v i a the m e t h y l e n e


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Figure 4. Dashed line: Ultraviolet difference spectra of FdUMP, CH FAH and thymidylate synthetase vs. CHgFAHi and thymidylate synthetase. Solid line: FdUMP, 5,8-deazofohte and thymidylate synthetase vs. enzyme and 5,8-deazafolate. t

Figure 5.

h

Structure of the FdUMP · CH · F A t f j thymidylate synthetase ternary complex where X represents a nucleophile of one of the enzyme amino acids t


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g r o u p of the c o f a c t o r . A s i m i l a r structure was proposed f r o m e v i d e n c e o b t a i n e d i n d e p e n d e n t l y i n another l a b o r a t o r y (20). R e f e r r i n g to F i g u r e 5, it i s noted that the a s s i g n e d s t r u c ture f o r the E F d U M P * C H > F A r J c o m p l e x i s a n a l o g o u s to one of the p r o p o s e d s t e a d y state i n t e r m e d i a t e s of the n o r m a l e n z y m i c r e a c t i o n ( v i z II, F i g u r e 2). T h e y d i f f e r i n that II p o s s e s s e s a p r o t o n at the 5 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a b s t r a c ted i n a s u b s e q u e n t s t e p , w h e r e a s the E ' F d U M P - C H ^ F A H ^ c o m p l e x ( F i g u r e 5) p o s s e s s e s a s t a b l e f l u o r i n e at the c o r r e s p o n d i n g position. T h u s , i t a p p e a r s that F d U M P behaves as a ' q u a s i substrate f o r this r e a c t i o n . T h a t i s , it e n t e r s into the c a t a l y t i c r e a c t i o n as d e p i c t e d f o r the s u b s t r a t e d U M P i n F i g u r e 2 up to the p o i n t w h e r e a n i n t e r m e d i a t e i s f o r m e d w h i c h c a n p r o c e e d no f u r t h e r ; i n effect, a c o m p l e x i s t r a p p e d w h i c h r e s e m b l e s a steady state i n t e r m e d i a t e ( v i z II, F i g u r e 2) of the n o r m a l catalytic reaction. e

1

11

We have r e c e n t l y obtaine a n FdUMP* C r L F A H . * peptid t i o n of the F O U M P M C H ^ A H ^ • t h y m i c j v l a t e s y n t h e t a s e c o m p l e x . A s shown i n F i g u r e 6, the 94 M H z F s p e c t r u m c o n s i s t s o f a doublet of t r i p l e t s l o c a t e d 87. 2 p p m u p f i e l d of the e x t e r n a l r e f e r e n c e / t r i f l u o r o a c e t i c a c i d . O u r c u r r e n t i n t e r p r e t a t i o n of this s p e c t r u m i s as f o l l o w s : T h e d o u b l e t i s c a u s e d b y s p l i t t i n g of the F r e s o n a n c e b y the p r o t o n at the 6 - p o s i t i o n of the u r a c i l r i n g (H . ) w i t h a c o u p l i n g constant ^ of 32. 5 H z . E a c h c o m p o n e n t of m e d o u b l e t i s s p l i t f u r t h e r into a t r i p l e t ( i n t e n s i t y r a t i o (1:2:1) c a u s e d b y c o u p l i n g of the f l u o r i n e w i t h the a d j a c e n t m e t h y l e n e p r o t o n s ( H ^ ) of the c o f a c t o r w i t h the m a g n i t u d e of the c o u p l i n g constant b e i n g Λ 9 . 2 H z . T h e i n n e r m o s t l i n e s of the t r i p l e t s o v e r l a p s o the F r e s o n a n c e a p p e a r s to be a quintet w i t h i n t e n s i t y r a t i o 1:2:2:2:1. It h a s b e e n w e l l e s t a b l i s h e d that the t r i g o n a l g e o m e t r y of the c a r b o n y l a t o m s i n u r a c i l d e r i v a t i v e s s a t u r a t e d a c r o s s the 5, 6 - d o u b l e bond r e s u l t s i n a h a l f - c h a i r c o n f o r m a t i o n w i t h s u b stituents o n c a r b o n a t o m s 5 a n d 6 s t a g g e r e d (28-30) as c o m m o n l y fowjid i n c y c l o h e x a n e . The F spectrum, in conjunction with p r e v i o u s l y reported u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a l data (3J.), of the F d U M P » C H ^ F A H . * peptide y i e l d s d e f i n i t i v e e v i d e n c e f o r i t s s t r u c t u r e . T h e d o u b l e t of t r i p l e t s i m p l i e s that the f l u o r i n e - b o n d e d c a r b o n i s f l a n k e d b y C H a n d C H g r o u p s ( i . e. C H C F C P ^ ) . T h e C H , of c o u r s e , o c c u r s at the 6 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a t t a c h e d to the n u c l e o p h i l e of the e n z y m e . T h e m o s t l o g i c a l a s s i g n m e n t f o r the C H - , i s the b r i d g i n g g r o u p between the n u c l e o t i d e and c o f a c t o r a s d e p i c t e d f o r the n a t i v e c o m p l e x i n F i g u r e 5. B a s e d o n the s t a b i l i t y of the F d U M P ' C r ^ F A H . » peptide i n the a b s e n c e o f a n t i o x i d a n t s , we h a v e s u r m i s e d that the C F ^ g r o u p b r i d g e s the n u c l e o t i d e to the 5 - a n d n o t to the 1 0 - n i t r o g e n o f the cofactor. It i s p o s s i b l e to a s s i g n the s t e r e o c h e m i s t r y of the 2


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64

19

Figure 6.

F

INVOLVING

CARBON-FLUORINE

BONDS

NMR

94 MHz

Fluorine-19 nmr spectrum

of the

FdUMP

· CH FAH t

k

· peptide

substituents w h i c h have a d d e d a c r o s s the 5, 6 - d o u b l e bond of F d U M P to g i v e the t e r n a r y c o m p l e x by c o m p a r i n g the o b s e r v e d c o u p l i n g constants w i t h those f r o m e x t e n s i v e l y s t u d i e d m o d e l s . A s shown i n F i g u r e 7, the 5 - f l u o r o and 6 - h y d r o g e n of the F d U M P * C H ^ F A H ^ * peptide a r e p r o p o s e d to be i n a t r a n s p s e u d o axial conformation. T h e e n z y m e n u c l e o p h i l e and c o f a c t o r a r e t h e r e f o r e trans_ p s e u d ο e q u a t o r i a l . T h e a d d i t i o n of a n u c l e o p h i l e of t h y m i d y l a t e s y n t h e t a s e to the 6 - p o s i t i o n of d U M P is a p r i m a r y event i n the e n z y m e - c a t a l y z e d r e a c t i o n ( F i g u r e 8). T h e r e s u l t a n t c a r b a n i o n (1) r e a c t s w i t h C H , F A H ^ to p r o d u c e a n i n t e r m e d i a t e w i t h a s t r u c t u r e analogous to tnat of the t e r n a r y c o m p l e x f o r m e d w i t h F d U M P and the c o f a c t o r (2, 3). A b s t r a c t i o n of the 5 - h y d r o g e n f o l l o w e d by a s e r i e s of steps i n v o l v i n g r e d u c t i o n of the one c a r b o n u n i t a n d e l i m i n a t i o n of the n u c l e o p h i l e r e s u l t s i n the o b s e r v e d p r o ducts of the r e a c t i o n . T h e s e steps h a v e b e e n p r e v i o u s l y d e p i c ted i n d e t a i l i n F i g u r e 2. W i t h the l o g i c a l a s s u m p t i o n that the n o r m a l e n z y m e - c a t a l y z e d r e a c t i o n o c c u r s i n a m a n n e r s i m i l a r to the f o r m a t i o n of the t h y m i d y l a t e s y n t h e t a s e « F d U M P · C r | F A H ^ c o m p l e x , s e v e r a l d e t a i l s c o n c e r n i n g the m e c h a n i s m of the n o r m a l e n z y m i c r e a c t i o n m a y be i n f e r r e d . F i r s t , the o v e r a l l s t e r e o c h e m i c a l pathway of the e n z y m e c a t a l y z e d r e a c t i o n m a y be d e d u c e d . T h e a d d i t i o n of the n u c l e o p h i l e and c o f a c t o r a c r o s s the 5, 6 - d o u b l e bond m u s t o c c u r i n a t r a n s f a s h i o n . C o n s e q u e n t l y , the s u b s e q u e n t e l i m i n a t i o n of the 5 - h y d r o g e n and the e n z y m i c n u c l e o p h i l e m u s t o c c u r as a c i s elimination .


SANTi E T A L .

Thymidyhte

Synthetase

R

Figure 7. Stereochemical projection of the FdUMP · CH FAH · peptide as determined hy its F nmr spectrum; R = 5-phospho-2'deoxyribosyl t

h

19

Figure 8


66

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

S e c o n d , the s t e r e o c h e m i s t r y c o n c e r n i n g the t r a n s i e n t i n t e r m e d i a t e s shown i n F i g u r e 8 m a y be i n f e r r e d . T h e m e c h a n i s tic d e t a i l s d i s c u s s e d below f o l l o w f r o m the p r i n c i p l e that a g r o u p r e a c t i n g w i t h the π - s y s t e m of the u r a c i l h e t e r o c y c l e ap p r o a c h e s a p p r o x i m a t e l y p e r p e n d i c u l a r to the plane of the r i n g ; by m i c r o s c o p i c r e v e r s i b i l i t y , a s i m i l a r o r i e n t a t i o n i s r e q u i r e d w h e n a g r o u p d e p a r t s to r e f o r m the π - s y s t e m . T h u s , the i n i t i a l attack of the n u c l e o p h i l e of the e n z y m e at the e l e c t r o p h i l i c 6 - c a r b o n of d U M P s h o u l d be p e r p e n d i c u l a r to the plane of the heterocycle. T h e r e s u l t a n t c a r b a n i o n (1) w i l l be d e l ^ c a l i z e d throughout the c a r b o n y l g r o u p s and w i l l be h i g h i n sp c h a r a c ter. T h e a p p r o a c h of C H ^ F A H ^ to the 5 - p o s i t i o n w o u l d ^ p e r p e n d i c u l a r to the plane of the r i n g a n d , b a s e d on the F data p r e s e n t e d a b o v e , t r a n s to the n u c l e o p h i l e attached to the 6 - p o s i t i o n . A s a r e s u l t , as shown i n s t r u c t u r e 2, the c o f a c t o r w o u l d e x i s t i n a p s e u d o a x i a l p o s i t i o n , and the 5 - h y d r o g e n w o u l d be p s e u d o e q u a t o r i a l . F o the p r o t o n f r o m the 5 - p o s i t i o s i t i o n (3) p r i o r to its a b s t r a c t i o n to f o r m the c a r b a n i o n (4). This mechanistic interpretation necessitates a previously u n r e c o g n i z e d c o n f o r m a t i o n a l c h a n g e , w h i c h o c c u r s a f t e r a d d i t i o n of the c o f a c t o r but b e f o r e a b s t r a c t i o n of the 5 - p r o t o n ; and r e s u l t s i n r i n g i n v e r s i o n about the 5 - a n d 6 - p o s i t i o n s of the n u c l e o t i d e 19

i n t e r m e d i a t e s {i.e. 2-*\3). T h e r e s u l t s of F n m r studies d e s c r i b e d h e r e a r e p r e l i m i n a r y ; a d e t a i l e d n m r study of the i n t e r a c t i o n of F d U M P w i t h t h y m i d y l a t e synthetase w i l l be p u b lished elsewhere. 5 - T r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d y l a t e (C ξ d U M P ) . CEdUMP i s a potent i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e . R e y e s aria H e i d e l b e r g e r have r e p o r t e d that u p o n p r e i n c u b a t i o n C E ^ d U M P c a u s e s i r r e v e r s i b l e i n h i b i t i o n of t h y m i d y l a t e synthetase f r o m E h r l i c h A c s i t e s c e l l s (32). B a s e d on the o b s e r v a t i o n that t r i f l u o r o m e t h y l u r a c i l ( C F ~ U ) a c y l a t e s a m i n e s i n aqueous media to give u r a c i l - 5 - c a r b o x a m i d e s (33), it was s u g g e s t e d that the i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase m i g h t r e s u l t f r o m a s i m i l a r a c y l a t i o n of a n a m i n o g r o u p at o r n e a r the a c t i v e site of the e n z y m e as shown i n F i g u r e 9 (32). A q u e s t i o n that a r o s e i s why the tr i f l u o r o m e t h y l g r o u p at the 5 - p o s i t i o n of u r a c i l d e r i v a t i v e s s h o u l d be at a l l s u s c e p t i b l e to these r e a c t i o n s . T h e c a r b o n - f l u o r i n e bond i s quite s t r o n g (34) and a n outstanding c h a r a c t e r i s t i c of m o s t t r i f l u o r o m e t h y l groups is their unusual r e s i s t a n c e toward c h e m i c a l d e g r a d a t i o n . A s r e l e v a n t e x a m p l e s , it is noted that b e n z o t r i f l u o r i d e s , d e r i v a t i v e s of 6 - t r i f l u o r o m e t h y l u r a c i l s (35), d e r i v a t i v e s of 2t r i f l u o r o m e t h y l - 4 - o x o p y r i m i d i n e s (36) and 5 - t r i f l u o r o m e thy Ι ό - a z a u r a c i l (37) a r e quite stable t o w a r d h y d r o l y t i c r e a c t i o n s . In c o n t r a s t , U is r a p i d l y c o n v e r t e d into 5 - c a r b o x y u r a c i l ( C U ) (33 ) i n b a s i c m e d i a a n d , although s o m e w h a t s l o w e r , n u c l e o s i d e s of C F ^ U a r e c o n v e r t e d into the c o r r e s p o n d i n g


4.

SANTi E T A L .

Thymidylate

Synthetase

Figure 9

n u c l e o s i d e s of C U (38-40) and C F g d U R p r o v i d e s C f r o m p y r i m i d i n e s (41). A n u m b e r of other c o m p o u n d s have been r e p o r t e d to h a v e r e a c t i v e C - F g r o u p s (see r e f e r e n c e 42). T h e r e a c t i v i t y of C - F bonds i n m o s t c a s e s has b e e n a t t r i b u t e d to h y p e r c o n j u g a t i v e e f f e c t s (43, 44), h y d r o g e n bonding effects (44, 45), and d i r e c t d i s p l a c e m e n t (S.^2) r e a c t i o n s (46). M o d e l r e a c t i o n s i n v o l v i n g the h y d r o l y s i s o f c o m p o u n d s p o s s e s s i n g C - F bonds w e r e e x a m i n e d in this l a b o t a t o r y i n an a t t e m p t to u n d e r s t a n d the u n d e r l y i n g f e a t a r e s w h i c h r e s u l t e d i n the r e a c t i v i t y of s o m e of these m o l e c u l e s (42). F r o m s u c h s t u d i e s we p r o p o s e d that C - F bond l a b i l i z a t i o n u s u a l l y i n v o l v e s one of s e v e r a l g e n e r a l m e c h a n i s m s , (a) A s d e p i c t e d i n F i g u r e 10a, p r o t o n r e m o v a l i s at an a t o m a to the c a r b o n b e a r i n g the f l u o r i n e a t o m w i t h the r e s u l t a n t negative c h a r g e , e i t h e r i n a s t e p w i s e o r c o n c e r t e d m a n n e r , a i d i n g i n the f o r m a t i o n of an i n t e r m e d i a t e (fluoro) a l k e n e . D e p e n d i n g on the s t a b i l i t y of the a l k e n e , it m a y o r m a y not react with solvent, (b) T h e p r o t o n m a y be s i t u a t e d on a n a t o m s u c h that the n e g a t i v e c h a r g e r e s u l t i n g f r o m the i o n i z a t i o n of the p r o t o n can e x e r t its i n f l u e n c e t h r o u g h an extended π - s y s t e m ( F i g u r e 10b). (c) W h e n the c o m p o u n d i s an a l l y l i c f l u o r i d e i n c a pable of u n d e r g o i n g either of the m e c h a n i s m s d e s c r i b e d a b o v e , it m a y u n d e r g o n u c l e o p h i l i c ( M i c h a e l - t y p e ) attack at the β - c a r bon w i t h a s s i s t a n c e by the d e v e l o p i n g c a r b a n i o n to give an i n t e r m e d i a t e s i m i l a r to those p r e v i o u s l y d e s c r i b e d ( F i g u r e 10c). In any of the a b o v e , t r i f l u o r o m e t h y l g r o u p s give c a r b o x y l i c a c i d s o r d e r i v a t i v e s , d i f l u o r o m e t h y l g r o u p s give a l d e h y d e s or k e t o n e s , and f l u o r o m e t h y l g r o u p s give a l c o h o l s or alkenes. M o s t of the C - F bond c l e a v a g e s thus f a r r e p o r t e d c a n be e x p l a i n e d then i n t e r m s of the a f o r e m e n t i o n e d m e c h a n i s m s ; the a b i l i t y of the c o m p o u n d s to f o r m o l e f i n i c i n t e r m e d i a t e s of the type d e s c r i b e d a p p e a r s n e c e s s a r y f o r s u c h r e a c t i o n s to o c c u r . T h e m e c h a n i s m ( s ) b y w h i c h the o l e f i n i c i n t e r m e d i a t e s a r e t r a n s f o r m e d to p r o d u c t s i s b e l i e v e d to i n v o l v e a l t e r n a t e a d d i t i o n of


68


H

I I — X-C—F

5=t -X^-C-^F —X=C

—product

(a)

H

ι

I

Α Γ \ Ι Α - X - * ( C = C ) i-C-L-F — •

— X-(C=C) - C - F

— X=C—(C=C) —C=C

> - » product

n l

ι

(b)

—c=Q-c—F -*• —c—cic-ί —» N u :

>

ι

/

—C—C=C —»—^-product

I

(c)

\

Nu

Figure 10

—C—CF. I J

ι

— C = C

/ \

H

II

F

HO

I II

—C—COH -*• — C — C — F

II _ ! =

yl /

\ c

O

H

,

v

H

\

I

^ - U o

Figure 11

n u c l e o p h i l e (or solvent) to the i n t e r m e d i a t e , and e l i m i n a t i o n of f l u o r i d e i o n . A p o s s i b l e m e c h a n i s m f o r h y d r o l y s i s of the C F ^ g r o u p is d e p i c t e d i n F i g u r e 11 a n d , as shown, m a y i n v o l v e the i n t e r m e d i a c y of a c y l f l u o r i d e s and k e t e n e s i n the t r a n s f o r m a tion of a t r i f l u o r o m e t h y l g r o u p to a c a r b o x y l a t e f u n c t i o n , a l though these i n t e r m e d i a t e s h a v e , as yet, not been d e t e c t e d .


4.

SANTi E T A L .

Thymidylate

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69

T h e above p r o v i d e d i n s i g h t into the p o s s i b l e m e c h a n i s m s by w h i c h C F ^ d U M P m i g h t a c t as an a c y l a t i n g agent. T o o b tain d i r e c t s u p p o r t i n g e v i d e n c e , the m e c h a n i s m s of h y d r o l y t i c r e a c t i o n s of 5 - t r i f l u o r o m e t h y l u r a c i l a n d its N - a l k y l a t e d d e r i v a t i v e s w e r e e x a m i n e d i n d e t a i l (47). T h e r e s u l t s of these studies a r e s u m m a r i z e d b e l o w , and d e p i c t e d i n F i g u r e s 12-14. A l l r e a c t i o n s a p p e a r to p r o c e e d by f o r m a t i o n of a h i g h l y reactive intermediate having an exocyclic difluoromethylene g r o u p at the 5 - p o s i t i o n w h i c h s u b s e q u e n t l y r e a c t s w i t h w a t e r o r h y d r o x i d e i o n i n a s e r i e s of r a p i d steps to give c o r r e s p o n ding 5 - c a r b o x y u r a c i l s . F o r those d e r i v a t i v e s w h i c h p o s s e s s an i o n i z a b l e p r o t o n at the 1 - p o s i t i o n , the p r e d o m i n a n t m e c h a n i s m i n v o l v e s i o n i z a t i o n to the conjugate b a s e and a s s i s t a n c e by the 1 - a n i o n i n the e x p u l s i o n of f l u o r i d e i o n ( F i g u r e 12). W h e n i o n i z a t i o n at the 1 - p o s i t i o n i s p r e c l u d e d by the p r e s e n c e of a n a l k y l substituent ( F i g u r e 13), a c y l a t i o n r e a c t i o n s p r o c e e d by r a t e d e t e r m i n i n g attac the n e u t r a l o r n e g a t i v e l v i d e the r e a c t i v e i n t e r m e d i a t e . In o r d e r to o b t a i n s u i t a b l e i n t r a m o l e c u l a r m o d e l s , and to v e r i f y the p r i m a r y s i t e of r e a c t i o n of 1 - s u b s t i t u t e d d e r i v a t i v e s , a s e r i e s of 1-(a>-aminoalkyl)trif l u o r o m e t h y l u r a c i l s w e r e p r e p a r e d and t h e i r h y d r o l y s e s e x a m i n e d ( F i g u r e 14). N e i g h b o r i n g g r o u p p a r t i c i p a t i o n was a p p a r e n t w h e r e attack of the a m i n o g r o u p on the 6 - p o s i t i o n of the h e t e r o c y c l e r e s u l t s i n the f o r m a t i o n of f i v e - , s i x - , and s e v e n - m e m b e r e d r i n g s ; i n the c a s e of 1 - ( 3 - a m i n o p r o p y l ) - 5 - t r i f l u o r o m e t h y l u r a c i l , apparent f i r s t - o r d e r constants w e r e m o r e than 10 t i m e s g r e a t e r than s i m p l e 1 - a l k y l d e r i v a t i v e s not possessing a neighboring nucleophile. W i t h r e g a r d to the e n z y m i c r e a c t i o n , the s a l i e n t f e a t u r e of these studies i s that the t r i f l u o r o m e t h y l g r o u p of C F ^ d U M P d e r i v a t i v e s w o u l d o n l y b e h a v e as an a c y l a t i n g agent when a s e c o n d a r y d r i v i n g f o r c e i s f u r n i s h e d by r e a c t i o n s w h i c h o c c u r at other p a r t s of the h e t e r o c y c l e . T h a t i s , it i s n e c e s s a r y that a n u c l e o p h i l e i s a d d e d to the 6 - p o s i t i o n of the h e t e r o c y c l e ; i n this m a n n e r , the n o r m a l l y i n e r t t r i f l u o r o m e t h y l g r o u p w o u l d be c o n v e r t e d into a h i g h l y r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e w h i c h m i g h t then a c y l a t e a n u c l e o p h i l i c g r o u p of the e n z y m e . In a c c o r d w i t h p r o p o s a l s f o r the i n v o l v e m e n t of n u c l e o p h i l i c c a t a l y s i s i n the e n z y m i c r e a c t i o n (viz I, F i g u r e 2), these studies l e d us to p r o p o s e a r e l a t e d m i n i m a l m e c h a n i s m f o r the r e p o r t e d i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase b y CFjdUMP. In the pathway d e p i c t e d i n F i g u r e 15, it was s u g g e s t e d that j u x t a p o s e d w i t h i n the a c t i v e s i t e , a n u c l e o p h i l i c g r o u p of the e n z y m e (:Z) adds to the 6 - p o s i t i o n of C F ~ d U M P , p r o m o t i n g the e x p u l s i o n of f l u o r i d e i o n and the f o r m a t i o n of a r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e s i m i l a r to those e n c o u n t e r e d i n o u r m o d e l s t u d i e s . The reactive interm e d i a t e w o u l d then be t r a p p e d by a n u c l e o p h i l i c g r o u p of


70

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Figure 13


BONDS

4.

SANTI E T A L .

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Synthetase

Figure 15


71

72


the e n z y m e to g i v e , a f t e r a n u m b e r of s t e p s , the a c y l a t e d enzyme. Subsequent to c o m p l e t i o n of these m o d e l s t u d i e s , the i n t e r a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase was r e i n v e s tigated i n another l a b o r a t o r y u s i n g the e n z y m e f r o m L . c a s e i (21). T h e s e w o r k e r s o b s e r v e d that C F ^ U M P , C H . F A F L t h y m i d y l a t e synthetase f o r m e d a tight t e r n a r y c o m p l e x w n i c h was i s o l a t a b l e by d i s c g e l e l e c t r o p h o r e s i s u n d e r n o n - d e n a t u r i n g c o n d i t i o n s . H o w e v e r , u n l i k e the F d U M P * C H ^ F A H ^ e n z y m e c o m p l e x , no change i n the d i f f e r e n c e s p e c t r a w a s o b s e r v e d w h e n C F g d U M P was u s e d . F u r t h e r m o r e , g e l e l e c t r o p h o r e s i s i n the p r e s e n c e of a p r o t e i n d é n a t u r a n t r e s u l t e d i n a p p a r e n t d e s t r u c t i o n of the c o m p l e x . A f t e r d e n a t u r a t i o n of the c o m p l e x , the n u c l e o t i d e p r o d u c t was o b s e r v e d to m i g r a t e i d e n t i c a l l y w i t h authentic C F ^ d U M P on D E A E - c e l l u l o s e p a p e r . F r o m these r e s u l t s i t was c o n c l u d e d that C - F bonds of the n u c l e o t i d n u c l e o p h i l e of the e n z y m C F dUMP. R e c e n t r e s u l t s o b t a i n e d i n this l a b o r a t o r y a r e not i n a c c o r d w i t h t h e s e f i n d i n g s . A l t h o u g h the m e c h a n i s m of r e a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase has not b e e n a s c e r t a i n e d at this t i m e , i t a p p e a r s c e r t a i n that C - F bond c l e a v a g e i s c a t a l y z e d by the e n z y m e , p r o b a b l y v i a n u c l e o p h i l i c c a t a l y s i s . O u r p r e l i m i n a r y r e s u l t s w h i c h l e a d to this c o n c l u s i o n are s u m m a r i z e d below. C o n t r a r y to the p r e v i o u s r e p o r t (2 1), we o b s e r v e that s u b t r a c t i o n of the u l t r a v i o l e t s p e c t r u m of e n z y m e a n d C F L F A F L f r o m that of the e n z y m e , C H ^ F A H ^ a n d C F ^ d U M P p r o d u c e s a d i f f e r e n c e s p e c t r a ( F i g u r e 16a) w h i c h i s v e r y s i m i l a r to that o b s e r v e d w i t h the F d U M Ρ · C H ^ F A H ^ · e n z y m e t e r n a r y c o m p l e x ( F i g u r e 4). A s w i t h the t h y m i a y l a t e s y n t h e t a s e ^ F d U M P C H ^ F A H ^ c o m p l e x , t h e r e i s the c h a r a c t e r i s t i c i n c r e a s e of a b s o r b a n c e at 330 n m and a d e c r e a s e at 261 n m ; the l a t t e r is i n a c c o r d w i t h s a t u r a t i o n of the 5, 6 - d o u b l e bond of the n u c l e o t i d e . U p o n a d d i t i o n of s o d i u m d o d e c y l s u l f a t e , the o n l y d i f f e r e n t i a l a b s o r b a n c e i s that c h a r a c t e r i s t i c of a n u c l e o t i d e w i t h λ » 267 n m ; although this i s u p f i e l d f r o m the m a x i m u m of C F ^ d U M P , we have not y e t a s c e r t a i n e d w h e t h e r a n a l t e r a t i o n of s t r u c t u r e i s i n v o l v e d o r whether the s h i f t i s a r t i f a c t u a l . In the a b s e n c e of C H ^ F A H the i n c u b a t i o n of t h y m i d y l a t e s y n t h e t a s e and C F ^ d U M F ^ ( r a t i o 1:6) f o r 20 m i n u t e s at 2 2 ° r e s u l t s i n 89% i n a c t i v a t i o n of the e n z y m e . T h i s i s i n a c c o r d w i t h p r e v i o u s r e p o r t s on the effect of this n u c l e o t i d e on the e n z y m e f r o m E h r l i c h a s c i t e s c e l l s (32). The difference spec t r a of C F g d U M P and e n z y m e vs e n z y m e i s shown i n F i g u r e 16b. T h e m a x i m u m of C F ^ d U M P at 261 n m d e c r e a s e s , and a t r a n sient b r o a d peak a p p e a r s w h i c h has a b s o r b t i o n u p to c a . 340 n m . A f t e r 1 h o u r , the f i n a l s p e c t r u m e x h i b i t s a m a x i m a at 276 n m , r e s e m b l i n g 5 - a c y l d e r i v a t i v e s of d U M P . Paper a


n

d

4.

SANTI E T A L .

Thymidylate

73

Synthetase

ι 260

ι

ι 300

ι

ι 340

W A V E L E N G T H Figure 16. Ultraviolet difference spectra, (a) Dashed line: CF dUM? CH FAH and thymidylate synthetase vs. CH FAH and thymidylate synthetase. Solid line: after treatment with sodium dodecyl sulfate, (b) Dashed line: CF dUMP and thymidylate syn thetase vs. thymidylate synthetase after 20 seconds. Solid line: after 1 hr. Broken line: ultraviolet spectrum of CF dUMP. s

t

y

k

s

s


t

k

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BONDS

c h r o m a t o g r a p h y of this r e a c t i o n m i x t u r e shows a s i n g l e spot w h i c h m o v e s s l i g h t l y s l o w e r than the s t a r t i n g m a t e r i a l (C^dUMP). A l t h o u g h this p r o d u c t h a s not yet b e e n i d e n t i f i e d , i t i s not 5 - c a r b o x y - d U M P . W h e n C R d U M P was t r e a t e d w i t h a l i m i t i n g a m o u n t of t h y m i d y l a t e synthetase (50:1) f o r 23 h o u r s at 2 2 ° , we w e r e a b l e to d e t e c t that at l e a s t 0.3 e q u i v a l e n t s of F ~ w e r e r e l e a s e d , d e m o n s t r a t i n g that C - F bonds of the n u c l e o tide w e r e i n d e e d l a b i l i z e d . A l t h o u g h these r e s u l t s a r e too p r e l i m i n a r y to p e r m i t d e f i n i tive i n t e r p r e t a t i o n , c e r t a i n c o n c l u s i o n s m a y be r e a c h e d a n d s p e c u l a t i o n s m a y be f o r w a r d e d . It i s c l e a r that the i n t e r a c t i o n of C I ^ d U M P and t h y m i d y l a t e synthetase i n the a b s e n c e of c o f a c tor m a y r e s u l t i n c l e a v a g e of C - F bonds of the n u c l e o t i d e as w e l l as i n a c t i v a t i o n of the e n z y m e . F r o m the a f o r e m e n t i o n e d m o d e l s t u d i e s , it i s m o s t r e a s o n a b l e to p r o p o s e that a c t i v a t i o n of the C - F bond r e q u i r e s a d d i t i o n of a n u c l e o p h i l e of the e n z y m e tu the 6 - p o s i t i o n of th i n a c t i v a t i o n i s not k n o w n than the r e a c t i o n l e a d i n g to C - F bond c l e a v a g e . It i s a l s o a p p a r e n t that the p r e s e n c e of C H ^ F A H ^ m o d u l a t e s this r e a c t i o n i n s o m e y e t unknown m a n n e r ; tne e n z y m e * C F ^ d U M P C H 2 F A H ^ c o m p l e x h a s u l t r a v i o l e t s p e c t r a l q u a l i t i e s quite s i m i l a r to those of the c o m p l e x f o r m e d w i t h F d U M P , i n d i c a t i n g that the 5, 6 - d o u b l e bond of d U M P i s s a t u r a t e d i n the ternary complex. Further experiments are i n progress which a i m to e l u c i d a t e the m e c h a n i s m of i n t e r a c t i o n of d U M P with thymidylate synthetase. #

Literature Cited 1. Friedkin, Μ., (1973), Advan. Enzymol. 38, 235. 2. Pastore, E. J., and Friedkin, M. (1962), J. Biol. Chem. 237, 3802. 3. Santi, D. V., and Brewer, C. F. (1973), Biochemistry 12, 2416. 4. Pogolotti, A. L., and Santi, D. V. (1974), Biochemistry 13, 456. 5. Santi, D. V., and Brewer, C. F. (1968), J. Amer. Chem. Soc. 90, 6236. 6. Kalman, T.I. (1971), Biochemistry 10, 2567. 7. Benkovic, S. J., and Bullard, W. P. (1973), Prog. Bioorg. Chem. 2, 134. 8. Crusberg, T.C., Leary, R., and Kisliuk, R. L. (1970), J. Biol. Chem. 245, 5292. 9. Dunlap, R. Β., Harding, N. G. L., and Huennekens, F. M. (1971), Biochemistry 10, 88. 10. Leary, R. P., and Kisliuk, R. L. (1971), Prep. Biochem. 1, 47. 11. Galivan, J. H., Maley, G. F., and Maley, F. (1975), Bio chemistry 14, 3338.


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75

12. Cohen, S. S., Flaks, J. G., Barner, H. D., Loeb, M. R., and Lichenstein, J. (1958), Proc. Nat. Acad. Sci. U. S. 44, 1004. 13. Heidelberger, C., Kaldor, G., Mukherjee, K. L., and Danenberg, P. B. (1960), Cancer Res. 20, 903. 14. Blakley, R. L. (1969). The Biochemistry of Folic Acid and Related Pteridines, New York, Ν. Υ., American Elsevier. 15. Fox, J. J., Miller, N.C., and Cushley, R.J. (1966), Tetrahedron Lett., 4927 16. Otter, B.A., Falco, Ε. A., and Fox, J.J. (1969), J. Org. Chem. 34, 1390. 17. Reist, E. J., Benitez, Α., and Goodman, L. (1964), J. Org. Chem. 29, 554. 18. Santi, D. V., and McHenry, C.S. (1972), Proc. Nat. Acad. Sci. U.S. 69, 1855. 19. Santi, D. V., McHenry, C.S., and Sommer, H. (1974), Biochemistry 13 20. Langenbach, R. J. C. (1972), Biochem. Biophys. Res. Commun. 48, 1565. 21. Danenberg, P. V., Langenbach, R. J., and Heidelberger, C. (1974), Biochemistry 13, 926. 22. Rose, I. Α., O'Connell, E. L., Litwin, S., and Bar Tana, J. (1974), J. Biol. Chem. 249, 5163. 23. Santi, D. V., McHenry, C.S., and Perriard, E.R. (1974), Biochemistry 13, 467. 24. Aull, J. L., Lyon, J. A., and Dunlap, R. B. (1973), Bio chem. Jour. 19, 210. 25. Sharma, R. Κ., and Kisliuk, R. L. (1973), Fed. Proc., Fed. Amer. Soc. Exp. Biol. 31, 591. 26. Aull, J. L., Lyon, J.A., and Dunlap, R. B. (1974), Arch. Biochem. Biophys. 165, 805. 27. Loeble, R.B., and Dunlap, R. B. (1972), Biochem. Bio phys. Res. Commun. 49, 1671. 28. Rouillier, P., Delmau, J., and Nofre, C. (1966), Bull. Soc. Chim. (France), 3515. 29. Furburg, S., and Janson, L. H. (1968), J. Amer. Chem. Soc. 90, 470. 30. Katritzky, A. R., Nesbit, M.R., Kurtev, B. J., Lyapova, Μ., and Pojarlieff, I. G. (1969), Tetrahedron 25, 3807. 31. Wahba, A. J., and Friedkin, M. (1961), J. Biol. Chem. 236, PC 11. 32. Reyes, P., and Heidelberger, C. (1965), Mol. Pharma col. 1, 14. 33. Heidelberger, C., Parsons, D. G., and Remy, D. C. (1964), J. Med. Chem. 7, 1. 34. Pauling, L. (1960), The Nature of the Chemical Bond. Ithaca, Ν. Υ., Cornell University Press, 85. 35. Giner-Sorolla, Α., and Bendich, A. (1958), J. Amer. Chem. Soc. 80, 5744. 36. Barone, J.A. ( 1963), J. Med. Chem. 6, 39.


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37. Dipple, Α., and Heidelberger, C. (1966), J. Med. Chem. 9, 715. 38. Shen, T. Y., Ruyle, W. V., and Lewis, Η. M. (1965), J. Org. Chem. 30, 835. 39. Khwaja, Τ. A., and Heidelberger, C. (1969), J. Med. Chem. 12, 543. 40. Ryan, K. J., Acton, Ε. Μ., and Goodman, L. (1966), J. Org. Chem. 31, 1181. 41. Heidelberger, C., Boohar, J., and Kampschroer, D. (1965), Cancer Res. 25, 377. 42. Sakai, Τ. Τ., and Santi, D. V. (1973), J. Med, Chem. 1079. 43. Roberts, J. D., Webb, R. L., and McElhill, E. A. (1950), J. Amer. Chem. Soc. 72, 408. 44. Filler, R., and Novar, H. (1960), Chem. Ind. (London), 1273. 45. Jones, R. (1947) 46. Nestler, H. J., an 57, 1117. 47. Santi, D. V., and Sakai, T. T. (1971), Biochemistry 10, 3598.


5 The Effect of Aliphatic Fluorine on Amine Drugs RAYW.FULLERandBRYANB.MOLLOY The Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Ind. 46206

Fluorine is th (Table I), and its presence in a molecule can greatly affect the ionization of acids and bases (2). The withdrawal of electrons toward fluorine on a carbon atom adjacent to a carboxyl carbon or an amino-bearing carbon increases the carboxyl group's ability to re lease a proton and decreases the amine's ability to accept a proton. The carboxylic acid becomes a stronger acid, and the amine becomes a weaker base. For example, the pK of ethylamine is >10 compared to 5.7 forβ,β,β-trifluoroethylamine(3). We have studied some sympathomimetic amine drugs having reduced basicity due to fluorine substituted on the β carbon. The influence of ionization on the ac tivity of sympathomimetic amines has previously been considered (4), but with the exception of compounds having substituents directly on the nitrogen, no such amines having pK values below physiological pH appear to have been available (5). An advantage of the β,βdifluoro compounds is that the substitution is in a position of the molecule that is not a major site of metabolic attack nor a site known to be involved in binding to physiological receptors, and the small size of the fluorine atoms (Table I) results in minimal steric alteration in the molecule. Thus the changes in the pharmacological properties of the amines as a result of β,β-difluoro substitution probably are due to the change in ionization. In solution an amine exists in the following equilibrium, a

a

-H+ RNH3+

^

V

RNH2

+H+


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the p o s i t i o n o f t h e e q u i l i b r i u m depending on t h e p K o f t h e amine and t h e pH o f t h e s o l u t i o n . When t h e pH i s e q u a l t o t h e p K , 50% o f t h e drug m o l e c u l e s a r e p r o t o n a t e d ( c a t i o n i c ) and 50% a r e n o n p r o t o n a t e d ( n e u t r a l ) . When t h e pH i s one u n i t below t h e p K , j u s t o v e r 90% o f t h e m o l e c u l e s e x i s t i n t h e c a t i o n i c form, and when the pH i s two u n i t s below t h e p K more t h a n 99% o f t h e m o l e c u l e s a r e charged. I n t h i s paper we compare some p r i m a r y amines w i t h p K v a l u e s w e l l above p h y s i o l o g i c a l pH t o t h e i r 3 , 3 - d i f l u o r o d e r i v a t i v e s h a v i n g p K v a l u e s below p h y s i o l o g i c a l pH. The p a r e n t amines a r e n e a r l y c o m p l e t e l y c a t i o n i c whereas t h e 3 , 3 - d i f l u o r o derivat i v e s e x i s t p r e d o m i n a n t l y as n e u t r a l m o l e c u l e s a t p h y s i o l o g i c a l pH. Presumably as a r e s u l t o f t h i s charge d i f f e r e n c e , t h e p h a r m a c o l o g i c p r o p e r t i e s o f t h e d r u g s — b o t h t h e b i o l o g i c a l f a t e and t h e b i o l o g i c a l actions of the d r u g s — a r a

a

a

a

a

a

TABLE Fluorine

Atom

in

Van

Relation

der radius,

to

Waals

I Other

Halogen

Atoms

1

Electronegativity value

0

A

F

1.

35

4. 0

Cl

1.

80

3. 0

Br

1.95

I

2.

H

1.2

1

Data

from

2.8 15

2.5 2. 1

(1).

Amphetamine The e f f e c t on i o n i z a t i o n o f amphetamine produced by t h e p r e s e n c e o f one o r two f l u o r i n e atoms on t h e 3 carbon i s shown i n T a b l e I I . A s i n g l e f l u o r i n e atom reduces t h e p K v a l u e by more than one pH u n i t . The p K o f t h e monofluoro d e r i v a t i v e o f amphetamine i s s t i l l above p h y s i o l o g i c a l pH, however, so t h a t most o f a

a


5.

FULLER

Aliphatic

AND MOLLOY

79

Fluorine

the monofluoro compound i s i o n i z e d a t p h y s i o l o g i c a l pH. More t h a n 99% o f t h e m o l e c u l e s o f amphetamine e x i s t as c a t i o n s a t p h y s i o l o g i c a l pH. In c o n t r a s t , t h e d i f l u o r o compound has a p K below p h y s i o l o g i c a l pH and thus would e x i s t m a i n l y as a n e u t r a l m o l e c u l e r a t h e r t h a n as a c a t i o n i n t h e body. a

TABLE Effect

of ^ Fluorine Ioni zation

οf

II Substution on Amphetamine Distribution

Compound

at RNH

2

\ \=y

the

of Ion ic pH 7. 4

Forms

RNH

z+

y-CH CHNH

1

2

2

CH

3

(/

\=/

X \=y

y-CHCHNH

1 1

F

CH

8.

35

10

90

73

27

2

y-CF CHNH 2

2

1

6.97

CH

3

The i n t e r a c t i o n o f amphetamine w i t h b i o l o g i c a l macromolecules ought t o be p r o f o u n d l y a f f e c t e d by t h e f l u o r i n e s u b s t i t u t i o n and a t t e n d a n t i o n i c changes, s i n c e t h e t r a n s p o r t and enzymic systems t h a t determine d i s t r i b u t i o n and m e t a b o l i s m o f t h e drug s h o u l d a c t d i f f e r e n t l y on a n e u t r a l u n p r o t o n a t e d amine compared to a c a t i o n . A number o f s t u d i e s on t h e i n t e r a c t i o n o f amphetamine and i t s f l u o r i n a t e d d e r i v a t i v e s w i t h enzymes and o t h e r p r o t e i n s i n v i t r o and o f t h e pharma c o l o g i c c h a r a c t e r i s t i c s o f t h e s e drugs i n v i v o have borne o u t t h e e x p e c t e d a l t e r a t i o n s i n p r o p e r t i e s o f amphetamine r e s u l t i n g from f l u o r i n e s u b s t i t u t i o n . In V i t r o I n t e r a c t i o n s . β,β-Difluoro substitution i n c r e a s e s t h e a c t i v i t y o f amphetamine as an i n v i t r o s u b s t r a t e f o r l u n g N - m e t h y l t r a n s f e r a s e (6) o r l i v e r microsomal deaminase (Ί). On t h e o t h e r H a n d , β , β -


80

BIOCHEMISTRY


BONDS

d i f l u o r o s u b s t i t u t i o n d e c r e a s e s t h e a c t i v i t y o f amphet amine as an i n h i b i t o r o f m i t o c h o n d r i a l monoamine o x i dase (Table I I I ) . L i k e w i s e , β , β - d i f l u o r o s u b s t i t u t i o n d i m i n i s h e s t h e b i n d i n g o f amphetamine t o b o v i n e serum albumin (Table I I I ) . Thus r e d u c i n g t h e p K o f amphet amine by β , β - d i f l u o r o s u b s t i t u t i o n can e i t h e r enhance or i n h i b i t i t s i n t e r a c t i o n s with v a r i o u s b i o l o g i c a l macromolecules i n v i t r o . a

TABLE Effect

of &jβ-Difluoro Amphetamine : and Binding

of

III

Substitution on Monoamine Oxidase to Bovine Serum

Two

Properties Inhibition Albumin

Drug

Amphetamine ft,

β-Difluoroamphetamine

3.

23

82

2.

56

45

MAO inhibition was measured with rat liver mito chondrial enzyme and ^C'-tryptamine as substrate. The pl50 value is the negative log of the molar concentra tion of inhibitor producing 50% inhibition. Binding to 4% bovine serum albumin in pH 7.4 sodium phosphate buf fer was measured with 10 micromolar drug concentration; after f i l t r a t i o n through Aminco Centriflo u l t r a f i l t r a tion cones drug levels were assayed colorimetrically with methyl orange.

In V i v o P r o p e r t i e s . The e f f e c t o f β - f l u o r i n e sub s t i t u t i o n on t h e d i s t r i b u t i o n o f amphetamine among v a r i o u s body t i s s u e s a f t e r d r u g a d m i n i s t r a t i o n t o ex p e r i m e n t a l a n i m a l s was s t r i k i n g . F i g u r e 1 shows t h e t i s s u e d i s t r i b u t i o n o f amphetamine, β-fluoroamphetamine, and β , β - d i f l u o r o a m p h e t a m i n e i n r a t s one hour a f t e r t h e drugs were i n j e c t e d i n t r a p e r i t o n e a l l y a t e q u i m o l a r doses. H i g h e s t t i s s u e l e v e l s o f amphetamine were i n l u n g , and l o w e s t l e v e l s were i n t h e f a t , a l l t i s s u e s h a v i n g h i g h e r l e v e l s than b l o o d . The r e l a t i v e d i s t r i b u t i o n o f t h e monofluoro d e r i v a t i v e was s i m i l a r , l e v e l s i n a l l t i s s u e s e x c e p t l u n g b e i n g about t h e same as t h o s e o f amphetamine. T h i s s i m i l a r i t y i s e x p e c t e d s i n c e t h e monofluoro compound, l i k e amphetamine, i s m o s t l y p r o t o n a t e d a t p h y s i o l o g i c a l pH. I n marked con t r a s t was t h e d i s t r i b u t i o n o f t h e d i f l u o r o d e r i v a t i v e .


5.

FULLER

Aliphatic

A N D MOLLOY

81

Fluorine

H i g h e s t l e v e l s o f t h i s d r u g were i n f a t , t h e t i s s u e t h a t c o n t a i n e d l o w e s t l e v e l s o f t h e o t h e r two amines. L e v e l s o f t h e d i f l u o r o compound i n o t h e r t i s s u e s were c o n s e q u e n t l y reduced; f o r example, t h e l e v e l s i n b r a i n were o n l y about o n e - f o u r t h t h o s e o f amphetamine. This d i s t r i b u t i o n a t one hour i s s i m i l a r t o t h a t seen a t o t h e r times ( 7 ) , i . e . t h e r e i s l i t t l e r e d i s t r i b u t i o n o f drug and t K e r a t e o f d i s a p p e a r a n c e o f t h e drugs from a l l t i s s u e s i s about t h e same. S i n c e t h e predominant p h a r m a c o l o g i c e f f e c t s o f amphetamine r e s u l t from i t s a c t i o n on t h e b r a i n , we compared r e g i o n a l d i s t r i b u t i o n o f amphetamine and 3 , 3 d i f l u o r o a m p h e t a m i n e i n b r a i n (Table I V ) . The drugs were g i v e n a t doses chosen t o produce comparable whole brain levels. Some d i f f e r e n c e s i n d i s t r i b u t i o n among the v a r i o u s anatomi region noted but thes not as g r e a t as t h v a r i o u s o r g a n s . Th amphetamine i n b r a i n was n o t s i g n i f i c a n t l y a l t e r e d by d i f l u o r o s u b s t i t u t i o n (1) . TABLE Regional Distribution 3, β-Dif'luoroamphetamine

Brain (% of

Total

Region Brain

IV of

Amphetamine in Rat

Drug

Level,

hemispheres (62-67%)

Cerebellum Midbrain Brain

(14-17%) (8-10%)

stem

Hypothalamus

nanomoles/g

Weight) Amphetamine

Cerebral

and Brain

(6-10%) (3-4%)

3> $-Difluoro amphetamine

46

± 4

57

± 2

18

± 6

33

±

54

± 4

92

± 6

65

± 20

71

±

86

±

84

± 6

11

Drugs were injected i . p . (amphetamine at 10 3,3-difluoroamphetamine at 40 mg/kg) 1 hour rats were killed. Mean values and standard 5 rats per group are shown.

mg/kg, before errors


3

7

the for

82

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

J u s t as t h e t i s s u e d i s t r i b u t i o n o f amphetamine i s changed by β, β - d i f l u o r o s u b s t i t u t i o n , so a l s o i s t h e metabolism o f t h e drug changed. A l t h o u g h t h e h a l f l i v e s o f amphetamine and β , β - d i f l u o r o a m p h e t a m i n e a r e about t h e same i n r a t s , t h e pathways o f m e t a b o l i s m f o r the two drugs a r e d i f f e r e n t (Table V ) . Amphetamine i s m e t a b o l i z e d i n t h e r a t p r e d o m i n a n t l y by h y d r o x y l a t i o n on t h e p a r a p o s i t i o n o f t h e a r o m a t i c r i n g , whereas β , β - d i f l u o r o a m p h e t a m i n e appears n o t t o be m e t a b o l i z e d by p a r a - h y d r o x y l a t i o n a t a l l (7). Instead the d i f l u o r o compound i s m e t a b o l i z e d r a p i d l y by o x i d a t i v e d e a m i n a t i o n (7). T i s s u e l e v e l s o f amphetamine and i t s h a l f - l i f e a r e i n c r e a s e d by agents l i k e d e s m e t h y l i m i p r a mine t h a t i n h i b i t a r o m a t i c h y d r o x y l a t i o n , whereas t i s sue l e v e l s o f β , β - d i f l u o r o a m p h e t a m i n e a r e i n c r e a s e d by t y p i c a l i n h i b i t o r s o f microsomal enzymes l i k e SKF 525A and DPEA {7)· Inductio by c h r o n i c phénobarbita the r a t e o f removal o f β , β - d i f l u o r o a m p h e t a m i n e from t i s s u e s a f t e r i t i s i n j e c t e d i n t o r a t s b u t has no e f f e c t on t h e r a t e o f removal o f amphetamine (0). β , β - D i f l u o r o s u b s t i t u t i o n increases the a c t i v i t y of amphetamine as a s u b s t r a t e f o r microsomal deaminases, an e f f e c t t h a t can be shown i n v i t r o . Apparently the d i f l u o r o s u b s t i t u t i o n abolisKes the a c t i v i t y of amphetamine as a s u b s t r a t e f o r t h e a r o m a t i c hydroxyl a t i n g system, though t h i s system i s d i f f i c u l t t o study i n v i t r o . The f a i l u r e o f t h e d i f l u o r o compound t o be H y d r o x y l a t e d i l l v i v o may be due t o one o r b o t h of the f o l l o w i n g reasons: (a) i t i s more r e a d i l y deaminated t h a n i s amphetamine, (b) i t may be l e s s r e a d i l y h y d r o x y l a t e d t h a n amphetamine. TABLE Effect of &,$-Difluoro Amphetamine Metabolism

Drug

Half-life in Brain

V Substitution in the

Rat

Major

Metabolic

Amphetamine

1.δ

hrs

Ring

β, $-Difluoro amphetamine

1.5

hrs

Oxidative

on

Route

hydroxylation deamination


5.

FULLER

AND

MOLLOY

Aliphatic

Fluorine

83

3,3-Difluoroamphetamine has been shown t o i n c r e a s e locomotor a c t i v i t y i n mice t o the same e x t e n t as amphetamine (9), though a p p r o x i m a t e l y f o u r t i m e s h i g h e r doses o f the 3 i f l u o r o compound have t o be g i v e n . L i k e w i s e about f o u r times h i g h e r doses o f t h e d i f l u o r o compound a r e r e q u i r e d t o produce e q u i v a l e n t drug l e v e l s i n b r a i n compared t o amphetamine, so t h e i n t r i n s i c a b i l i t y o f t h e d i f l u o r o compound t o cause CNS s t i m u l a t i o n seems about e q u a l t o t h a t o f amphetamine. In mice, t h e h a l f - l i f e o f t h e d i f l u o r o compound (0.3 hours) i s much s h o r t e r than i s t h a t o f amphetamine (0.9 h o u r s ) . A p p a r e n t l y t h i s d i f f e r e n c e i s r e l a t e d t o the f a c t t h a t mice have t h e enzymic machinery t o metabo l i z e amphetamine by o x i d a t i v e d e a m i n a t i o n (10), and s i n c e t h e d i f l u o r o d e r i v a t i v e i s a much b e t t e r s u b s t r a t e f o r d e a m i n a t i o n i t i s m e t a b o l i z e d much more r e a d i l y i n mice t h a of amphetamine and 3 , 3 s i m i l a r r e s u l t s i n mice and i n r a t s . One use t h a t has been made o f 3 , 3 - d i f l u o r o a m p h e t amine i s as a t o o l t o e l u c i d a t e i n t e r r e l a t i o n s h i p s among the v a r i o u s a c t i o n s o f amphetamine. Gessa, C l a y and B r o d i e (11) had s u g g e s t e d t h a t the h y p e r t h e r m i a f o l l o w i n g ampEetamine i n j e c t i o n i n t o r a t s was a d i r e c t consequence o f t h e e l e v a t i o n o f plasma f r e e f a t t y acids. We found t h a t 3 , 3 - d i f l u o r o a m p h e t a m i n e injected at an e q u i m o l a r dose e l e v a t e d f r e e f a t t y a c i d s t o t h e same e x t e n t as amphetamine but produced no h y p e r t h e r m i a (12), showing t h a t t h e s e two e f f e c t s o f amphetamine were c o m p l e t e l y d i s s o c i a b l e by v i r t u e o f d i f l u o r o substitution. Presumably the h y p e r t h e r m i c response t o amphetamine i s due t o an a c t i o n i n b r a i n , hence 3*3d i f l u o r o a m p h e t a m i n e produces h y p e r t h e r m i a o n l y when i t i s i n j e c t e d a t higher doses. Another d i f f e r e n c e between amphetamine and 3,3d i f l u o r o a m p h e t a m i n e i s t h e i n a b i l i t y o f t h e l a t t e r drug to cause d e p l e t i o n o f n o r e p i n e p h r i n e l e v e l s i n b r a i n and h e a r t (Table V I ) . H i g h doses o f amphetamine have l o n g been known t o d e p l e t e n o r e p i n e p h r i n e i n t h e s e t i s s u e s (13). We gave t h e d i f l u o r o compound a t f o u r times the~cTose o f amphetamine t o produce e q u i v a l e n t drug l e v e l s i n b r a i n and h e a r t but s t i l l found no reduction of norepinephrine l e v e l s . The p r e c i s e mechanism(s) by which amphetamine lowers n o r e p i n e p h r i n e l e v e l s i s s t i l l u n c e r t a i n , but h y d r o x y l a t e d m e t a b o l i t e s may p l a y a r o l e i n t h i s e f f e c t . The f a i l u r e o f 3/3d i f l u o r o a m p h e t a m i n e t o lower n o r e p i n e p h r i n e c o u l d t h e n be e x p l a i n e d because i t i s not m e t a b o l i z e d by hydroxylation.


84

BIOCHEMISTRY

TABLE Inability

of

VI

β,ë-Difluoroamphetamine

Amphetamine-Like

Treatment


Depletion

of

to Tissue

Norepinephrine Heart

Group

Cause Norepinephrine

Levels,

vg/g Brain

Control

0.89±.05

dl-Amphetamine 0.1 mmole/kg

0.58±.01

(P-Difluoro-pChloroamphetamine

6.8

RNH3+

1

99

80

20

e q u i v a l e n t t o those t o n i n and 5 - h y d r o x y i n d o l e a c e t i were d e p l e t e d t o about t h e same e x t e n t as by p - c h l o r o amphetamine a t 6 hours (Table I X ) . However, t h e r e was TABLE Brain Levels

Drug

Serotonin and after Injection or its β,β-Di

IX

5-Hydroxyindoleaoetic Acid of p-Chloroamphetamine fluoro Derivative

Treatment

Serotonin

5HIAA % of

Control

p-Chloroamphetamine (0.1 mmole/kg) 6 24

hrs

46

± 3 *

54

+

2*

hrs

43

± 3 *

50

t

2*

3*

β, ë-Difluorop-chloroamphetamine (0.4 mmole/kg) 6

hrs

44

±

1*

56

±

24

hrs

97

t

6

95

t

^Significant

drug

effect,

P-difluoro-phydroxy amphetamine but have not been able to synthesize it. There may be a problem of stabil ity with compounds having both a hydroxy I and -CF2R group on the ring. Q. Is fluoride ion a metabolite of these difluoro compounds? y

A.

We do not know whether any of the fluorine is removed metabolically.

Q. About the possibility of changing the basicity of the nitrogen in the difluoro compounds—have you looked at the effect of the 3,3-difluoro substituent on transport across biological membranes? A.

Mostly in indirect ways. I think the large change in tissue distribution produced by the 3,3difluoro substitution i s a manifestation of altered transport across various biological membranes in the complex system of the whole animal. Dr. David Wong has looked at the ability of 3*3difluor ο-p-chlorο amphetamine to inhibit the trans port of monoamines across synaptosomal membranes in vitro; the difluoro compound is less effective as an inhibitor than is p-chloroamphetamine itself.


98

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

Q.

Do you know what t h e p r o d u c t s a r e from o x i d a t i v e d e a m i n a t i o n o f t h e β , β - d i f l u o r o compounds?

A.

Dr. John Parti has isolated the oxime> the and the alcohol as in vitro metabolites of difluoroamphetamine. He has found that the of difluorophenylacetone i s formed as a in vivo in rats and is excreted in urine in and conjugated form.


ketone, βoxime metabolite free

6 Metabolic and Transport Studies with Deoxyfluoro-monosaccharides N.F.TAYLOR,A.ROMASCHIN,andD.SMITH Department of Chemistry, University of Windsor, Ontario N9B 3P4 Canada

The rational fluorocarbohydrates and related compounds as probes for the study of enzyme specifity, carbohydrate metabolism and transport in biological systems has been elaborated in a previous symposium (1). Such compounds have now been used to study carbohydrate metabolism in yeast cells (2), Ps. fluorescens (3) and E. coli (4); enzyme specificity of glycerol kinase (5), yeast hexotinase (6), phosphoglucomutase and UDPG pyrophosphorylase (7) and glycerol-3-phosphate dehydrogenase (8); carbohydrate transport in hamster intestine (9) and the human erythrocyte (10), (11). The wide range of synthetic fluorinated carbohydrates and related compounds now available has been extensively reviewed (12). As will be evident from our recent studies, however, many detailed biochemical studies will be limited until the introduction of C and/or into fluorocarbohydrates has been accomplished. l 4

The Transport of D-Glucose Across the Human Erythrocyte Membrane. The question of the exact nature of the carrier protein(s) and translocation mechanism for the transport of D-glucose across the erythrocyte membrane is still debated (13). However, the saturation kinetics obtained for D-glucose and glucose analogues are in accordance with a facilitated transport mechanism which allows binding of the sugar molecule to one or more sites of a receptor protein in the membrane. A comprehensive study of the specificity of this binding has been undertaken by a number of workers (14), (15), (16), (17), In particular the comparative inhibition and comparative transport studies of D-glucose with a number of monodeoxy-D-glucoses and monodeoxymonofluoro99 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

100

BIOCHEMISTRY


BONDS

D - g l u c o s e s p r o v i d e k i n e t i c parameters which p e r m i t assignment o f hydrogen bonds between s p e c i f i c oxygen atoms i n D-glucose and r e c e p t o r s i t e s i n t h e c a r r i e r protein. Thus u s i n g the o p t i c a l method o f Sen and Widdas (1_5) and t h e s i m p l i f i e d r a t e e q u a t i o n (18) f o r the e x i t o f g l u c o s e from p r e - l o a d e d e r y t h r o c y t e s , we have shown (11_) t h a t r e p l a c i n g the oxygen f u n c t i o n a t C 3 o f D-glucose by f l u o r i n e t o g i v e 3 - d e o x y - 3 - f l u o r o D-glucose does n o t s i g n i f i c a n t l y change t h e h a l f - s a t u r a t i o n c o n s t a n t (K ) f o r t h e c a r r i e r p r o t e i n (Table 1). In c o n t r a s t 3-deoxy-D-glucose has l o s t t h i s a b i l i t y t o hydrogen bond a t C 3 and c o n s e q u e n t l y has a lower a f f i n i t y f o r the c a r r i e r p r o t e i n ( h i g h e r Κ v a l u e ) . In a d d i t i o n the K v a l u e f o r 5 - t h i o - D - g l u c o s e ( T a y l o r , N.F. & Gagneja, G.L. u n p u b l i s h e d r e s u l t ) s u g g e s t s t h a t t h e r i n g oxygen a t C 5 o f D-glucose i s a l s o i n v o l v e d w i t h hydrogen bonding (Tabl x

χ

x

T a b l e 1.

K and V v a l u e s o f D-glucose and d e r i v a t i v e s a t 37° x

m a x

V Sugar

Κ (mM) χ

D-Glucose 3-Deoxy-3-fluoroD-glucose 3-Deoxy-D-glucose 5-Thio-D-glucose

max (mmol. L i t r e

3.9 2.3

640 600

15.3 15.0

340 500

^ min

^)

K i n e t i c parameters were d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d (11). These r e s u l t s a r e i n agreement w i t h B a r n e t t e t a l . (10} who showed by i n h i b i t i o n s t u d i e s t h a t u n l i k e 3-deoxyD - g l u c o s e , 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e b i n d s t o the c a r r i e r p r o t e i n as w e l l as D - g l u c o s e . Furthermore, t h e importance o f the β - o r i e n t a t i o n o f -OH a t in D-glucose was s u g g e s t e d by the h i g h K i v a l u e s f o r 1-deoxy-D-glucose and α - D - g l u c o s y l f l u o r i d e and t h e low Ki value f o r 3-D-glucosylfluoride. These t r a n s p o r t and i n h i b i t i o n s t u d i e s p r o v i d e e v i d e n c e f o r t h e p r o p o s a l by Kahlenburg and Dolansky (19) t h a t t h e oxygen f u n c t i o n s l o c a t e d a t C i , C 3 and t h e r i n g oxygen a t C 5 o f the C l - c o n f o r m a t i o n o f 3-D-glucopyranose ( F i g u r e 1 ) , a r e c o n s i d e r e d t o be n e c e s s a r y f o r e f f e c t i v e hydrogen bonding o f D-glucose t o t h e t r a n s p o r t protein. T h i s model a g r e e s w i t h the d e t e c t i o n o f three d i f f e r e n t r e c e p t o r groups (- N H 2 , - SH and i m i d a z o l e ) on t h e p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t (20) and i s a l s o c o n s i s t e n t w i t h a r e c e n t l y proposed model


6.

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E T

A L .

Deoxyfluow-monosaccharides

101

(21) f o r t h e mode o f a c t i o n o f c y t o c h a l a s i n Β (22) i n h i b i t i o n o f g l u c o s e t r a n s p o r t i n t h e human e r y t h r o cyte. Thus a D r i e d i n g m o l e c u l a r model ( F i g u r e 2) o f c y t o c h a l a s i n Β (I) r e v e a l s an almost i d e n t i c a l s p a t i a l d i s t r i b u t i o n o f t h e f o u r oxygen atoms l o c a t e d a t C I , C19, C18 and C4 t o t h o s e l o c a t e d a t C5, C l , C2 and C3 o f t h e C l - c o n f o r m a t i o n o f 3-D-glycopyranose ( F i g u r e 1 ) . At l e a s t t h r e e o f t h e s e s i t e s a t R, R l and R3 ( F i g u r e 2) a r e i m p l i c a t e d i n hydrogen bonding t o t h e c a r r i e r p r o t e i n f o r D-glucose and p a r t i a l l y e x p l a i n why c y t o c h a l a s i n Β i s a c o m p e t i t i v e i n h i b i t o r o f D-glucose t r a n s p o r t i n t h e human e r y t h r o c y t e w i t h K i , 1.2 χ 10"' M (21) . A f u r t h e r point o f i n t e r e s t r e s i d e s i n the f a c t t h a t when whole r e d b l o o d c e l l s a r e i n c u b a t e d w i t h 3d e o x y - 3 - f l u o r o - D - g l u c o s e f o r 24 h r s a t 37°C a s m a l l but s i g n i f i c a n t r e l e a s ( H a l t o n , D. & T a y l o r 200mM 3 - d e o x y - 3 - f l u o r o g l u c o s e C-F c l e a v a g e r e a c h e s a maximum (^ 1%) a f t e r 24 hours i n c u b a t i o n ( F i g u r e 3 ) . U n l i k e t h e c o n t r o l s such c e l l s c o m p l e t e l y l o s e t h e i r a b i l i t y to transport glucose. The s i g m o i d a l c u r v e i n d i c a t e s m u l t i p l e k i n e t i c s f o r t h e mechanism o f f l u o r i d e r e l e a s e and one p o s s i b l e e x p l a n a t i o n f o r t h e œ r e s u l t s would be t h e concommittant c o - v a l e n t attachment o f t h e g l u c o s e r e s i d u e t o one o f t h e r e c e p t o r s i t e s o f the c a r r i e r p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t . Such a mechanism i s shown ( F i g u r e 4) i n which 3-deoxy3-fluoroglucose (II) hydrogen bonds t o t h e r e c e p t o r p r o t e i n , e l i m i n a t e s HF t o produce t h e e p o x i d e ( I I I ) which i s a t t a c k e d by t h e n u c l e o p h i l i c p r o t e i n t o p r o duce ( I V ) . We have r e c e n t l y s y n t h e s i s e d 3H-C-(3)-3d e o x y - 3 - f l u o r o - D - g l u c o s e (Lopes, D. & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) i n o r d e r t o e s t a b l i s h whether g l u c o s y l a t i o n o f a membrane p r o t e i n has i n f a c t o c c u r r ed. Such a r e a c t i o n may p e r m i t i s o l a t i o n o f t h e c a r r i e r p r o t e i n f o r D-glucose. M i c r o b i a l Metabolism o f Deoxyfluoro-D-glucoses Our p r e v i o u s s t u d i e s (3) demonstrated t h a t 3-deaxyfluoro-D-glucose (II) i s m e t a b o l i s e d by whole r e s t i n g c e l l s o f P £ . f l u o r e s c e n s , w i t h r e t e n t i o n o f t h e C-F bond, t o produce 3 - d e o x y - 3 - f l u o r o - D - g l u c o n i c a c i d ( V ) . C e l l - f r e e e x t r a c t s o f t h i s organism o x i d i s e d 3FG f u r t h e r t o 3 - d e o x y - 3 - f l u o r o - 2 - k e t o - D - g l u c o n i c a c i d (VI). I t has a l s o been shown t h a t t h e same enzymes t h a t o x i d i s e D-glucose ( g l u c o s e o x i d a s e and g l u c o n a t e dehydrogenase) o x i d i s e (II) and t h a t (II) and (V) a r e c o m p e t i t i v e i n h i b i t o r s o f g l u c o n o k i n a s e (23) . In o r d e r t o study f u r t h e r t h e s p e c i f i c i t y o f t h e s e enzymes


102

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INVOLVING

CARBON

Figure 1. Stereospecific binding sites of β-D-glucopyranose in the Cl-conformation to a transport protein . . . Hydrogen bonds. R, Rl and R3 represent receptor sites on the transport protein.


6.

T A Y L O R

E T

A L .

103

Deoxyfluoro-monosaccharides

Ί9Ν

Figure 2.

R1

Model of cytochalasin B

gen bonds. X = -CH ?h. R, RI, R2 and R3 represent receptor sites on the transport protein. t

R2

R3

C

Figure 3. Fluoride ion released after incubation of human erythrocytes at 37° with different concentrations of 3-deoxy-3fluoro-D-glucose: A 50 mM, · 100 mM, Ο ^50 mM, • 200 mM. Fluoride ion determinations were by the fluoride elec trode (26) and the preparation of the erythrocytes as previ ously described (11). The initial control fluoride ion concent ration was 1.0 X 10' M. 4


104


towards o t h e r d e o x y f l u o r o - D - g l u c o s e s we have examined the b i o c h e m i c a l e f f e c t s o f t h e i s o m e r i c 4-deoxy-4fluoro-D-glucose (VII) {24) on whole r e s t i n g c e l l s and c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s ( H i l l , L. & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) .

CH OH 2

COOH

COOH

-H H—I—OH

H-f-OH

CH OH 2

(ID

(V)

(VI)

(VII)

Warburg r e s p i r o m e t r y i n d i c a t e d t h a t u n l i k e (II) (VII) i s n o t o x i d i s e d by whole c e l l s o f P£. f l u o r e s c e n s b u t t h e r e i s an immediate r e l e a s e o f f l u o r i d e a n i o n f F i g u r e 5). U s i n g 2.5mM o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e t h e r a t e o f C-F c l e a v a g e i s l i n e a r o v e r t h e f i r s t f o u r hours and a f t e r 24 hours 94% o f t h e c o - v a l e n t f l u o r i n e i s r e l e a s e d as f l u o r i d e a n i o n (no F " was d e t e c t e d i n the absence o f c e l l s ) . The p o s s i b l e d e f l u o r i n a t e d products o f t h i s r e a c t i o n are D-glucose, D-galactose, 4-deoxy-D-glucose and 3,4-anhydro-D-galactose. Only the l a t t e r two compounds a r e l i k e l y s i n c e D - g a l a c t o s e and D - g l u c o s e , even i n t h e p r e s e n c e o f f l u o r i d e a n i o n , are o x i d i s e d by t h e organism. T . l . c . a n a l y s i s o f t h e c e l l s u p e r a t a n t s and i n t r a c e l l u l a r c o n t e n t s , however, has f a i l e d t o r e v e a l any new c a r b o h y d r a t e components. When c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s a r e c h a l l e n g e d w i t h 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) we f i n d t h a t no s i g n i f i c a n t de-flùorination o c c u r s . Thus i n the c o n c e n t r a t i o n range 2 - 2 0 ymoles t h e i n i t i a l r a t e and e x t e n t o f o x i d a t i o n o f (VII) by c e l l - f r e e e x t r a c t s is shown (Table 2 ) . The o x i d a t i o n o f (VII) i s comp l e t e w i t h i n 2 hours and 2 g atoms o f oxygen/mole o f s u b s t r a t e a r e consumed.


6.

TAYLOR ET A L .

H" (III)

(ID

105


(IV)

Figure 4. Possible mechanism of fluoride release from 3deoxy-3-fluoro-D-glucose. · protein X = Ν or S.

60

Hours Figure 5. Release of fluoride anion from 2.5 mM 4-deoxy-4-fluoro-Dglucose by whole cells of Ps. fluorescens. • Fluoride anion. A Oxygen uptake. Six Warburg flasks were used. Each flask contained 5 fimoles 4-deoxy-4-fluoro-D-glucose, 20 mg dry wt cells and 0.67M phosphate buffer to 2.0 ml in main well. Flask contents were incubated at 30°C with shaking. At time intervals the contents of each flask were centri fugea at 4 040 g for 20 min and fluoride ion determinations made with a fluoride electrode (26) on the supernatants. Warburg conditions for oxygen uptake: 30°C, reaction vol 2.0 ml. Fhsk contained 1.0 ml 0.67M phosphate buffer and 0.5 ml 5 ^moles of 4-deoxy-4-fluoro-D-glucose in main well and 0.2 ml 20% KO H aq. in center well. Reaction was initiated by tipping 0.5-ml cell suspension (24 mg dry wt) in 0.67M phosphate buffer from side arm. Endogenous respiration subtracted (1434 μΐ in 480 min).


106

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

T a b l e 2.

O x i d a t i o n o f 4-deoxy-4-fluoro-D-glucose by c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s . Rate o f Moles 0 / Amount o f oxidation mole o f Net 4-fluoroglucose y l oxygen substrate ymoles O 2 / ymoles oxidised hr/mg p r o t e i n consumed 2

2.0 5.0 10.0 20.0 5.0

(glucose)

0.07 0.18 0.26 0.34 0.38

1.12 0.99 1.00 0.96 1.03

50 110 225 440 115

Warburg c o n d i t i o n s : 30°C, gas phase, a i r . R e a c t i o n volume, 2.0 m l . Each f l a s k c o n t a i n e d 37 mg c e l l - f r e e e x t r a c t p r o t e i n , 1 ymole NAD, 0.67 M phosphate b u f f e r , pH, 7.0 made up t o 1.5 l i th mai compartment S i d e arm c o n t a i n e d 0. w e l l 0.2 ml 20% KOHag pape was i n i t i a t e d by t y p i n g c o n t e n t s from t h e s i d e arm. Endogenous r e s p i r a t i o n (317 y l oxygen i n 150 min) subtracted. A f t e r o x i d a t i o n o f (VII) was complete, s i l i c a g e l t . l . c . a n a l y s i s (EtOAC:ACOH:H 0 3:3:1) o f t h e c e l l - f r e e e x t r a c t r e v e a l e d t h e absence o f ( V I I ) ( R , 0.6) and t h e presence o f a new component (RF 0.45). By analogy w i t h t h e e s t a b l i s h e d m e t a b o l i c pathway o f g l u c o s e (27) and 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e {23) i n t h i s organism, t h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e two s t e p o x i d a t i o n o f (VII) by g l u c o s e o x i d a s e and g l u c o n a t e dehydrogenase t o 4 - d e o x y - 4 - f l u o r o - D - g l u c o n i c a c i d (VIII) and 4-deoxy-4-fluoro-2-keto-D-gluconic a c i d (IX) r e s p e c tively. 2

F

ÇOOH -OH

HCH-H (VII)

H-

H CH °H 2

(VIII)

COOH H H-

h o

H &H2OH

(IX)


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ET

AL.

107


I s o l a t i o n and c h a r a c t e r i s a t i o n o f (IX) i s c u r r e n t l y being i n v e s t i g a t e d . The e x t e n s i v e d e f l u o r i n a t i o n o f (VII) by whole c e l l s o f Ps. f l u o r e s c e n s and the r e t e n t i o n o f the C-F bond on t r e a t m e n t o f fVTI) w i t h c e l l f r e e e x t r a c t s suggest t h a t C-F c l e a v a g e o c c u r s a t the cell-wall/membrane l e v e l o f the organism. The mode o f uptake o f D-glucose by Ps. f l u o r e s c e n s i s not known. However, i n a c l o s e l y r e l a t e d s p e c i e s Ps. a e r u g i n o s a , i t has been shown (28) t h a t a l t h o u g h the phosphoenolp y r u v a t e p h o s p h o t r a n s f e r a s e system (29) i s not i n v o l v e d , the t r a n s p o r t o f D-glucose i s energy dependant and, t h e r e f o r e , l i k e l y t o have a c a r r i e r p r o t e i n system. A s i m i l a r g l u c o s e t r a n s p o r t system may be p r e s e n t i n Ps. f l u o r e s c e n s and t h e f a i l u r e o f t h e whole c e l l t o o x i d i s e 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) may be due t o a s t e r e o s p e c i f i c r e a c t i o n of (VII)with a c a r r i e r p r o t e i n system i n whic i s r e l e a s e d as f l u o r i d p e r m i t the sugar r e s i d u e (at C 4 ) t o become a t t a c h e d t o p r o t e i n i n the membrane and account f o r the f a c t t h a t we a r e unable t o d e t e c t any m e t a b o l i t e e i t h e r o u t s i d e o r i n s i d e the c e l l d u r i n g the i n c u b a t i o n p e r i o d . Some s u p p o r t f o r t h i s p o s s i b i l i t y i s p r o v i d e d by (a) the f a c t t h a t when whole c e l l s are p r e - i n c u b a t e d w i t h 2.5mM 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e f o r 12 hours and c h a l l e n g e d w i t h 2 - 8mM g l u c o s e s i g n i f i c a n t i n h i b i t i o n o f the r a t e o f r e s p i r a t i o n o c c u r s ( F i g u r e 6 ) . T h i s would be c o n s i s t e n t w i t h a b l o c k e d g l u c o s e t r a n s p o r t s i t e ( s ) a l t h o u g h as t h e g l u c o s e c o n c e n t r a t i o n i s i n c r e a s e d t o 20 ymoles some r e c o v e r y o f r e s p i r a t i o n i s apparent. The p o s s i b i l i t y t h a t a s m a l l u n d e t e c t a b l e amount o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e o r a n o n - o x i d i s able f l u o r i n a t e d metabolite i s i n h i b i t i n g glucose metabolism and/or t r a n s p o r t i s o f c o u r s e not e x c l u d e d by t h e s e r e s u l t s . (b) Ps. f l u o r e s c e n s i s unable t o grow on a m i n e r a l s a l t s medium w i t h 4 - d e o x y - 4 - f l u o r o g l u c o s e (VII) as a s o l e carbon source a l t h o u g h f l u o r i n e i o n i s r e l e a s e d i n t o the medium. S e v e r a l examples a r e known where the r e l e a s e o f f l u o r i d e from a C-F compound by a b a c t e r i u m a l l o w s the f r e e n o n - f l u o r i n a t e d fragment t o s e r v e as a source o f carbon and energy f o r growth. Thus a Pseudomonad has been i s o l a t e d which grows on f l u o r o a c e t a t e as a r e s u l t o f C-F c l e a v a g e (30). S i m i l a r l y , a Pseudomonad has been i s o l a t e d which w i l l grow on m o n o f l u o r o c i t r a t e a f t e r f l u o r i d e r e l e a s e (31). The i n a b i l i t y o f 4 - d e o x y - 4 - f l u o r o g l u c o s e ( V I I ) , t o a c t as a carbon source f o r Ps. f l u o r e s c e n s , d e s p i t e C-F c l e a v a g e , may be due t h e r e f o r e , t o the attachment o f the sugar r e s i d u e t o some c e l l u l a r component. It

i s expected

t h a t the s y n t h e s i s o f

^-^C-(l)-4-


108


Figure 6. Oxidation of D-glucose by rest ing whole celh of Ps.fluorescensafter pre incubation with 2.5 mM 4-deoxy-4-fluoroD-glucose. Pre-incubation conditions: 30°C, reaction volume 2.0 ml, time 12 hr. Each Warburg flask contained 1.0 ml 0.67M phos phate buffer, 0.5 ml 5 μmoles of 4-deoxy-4fluoro-D-glucose or 5 ^moles D-glucose in main well and 0.5-ml cell suspension (28 mg dry wt) in 0.67M phosphate buffer in side arm. Pre-incubation was initiated by tipping contents from side arm. Oxidation of added D-glucose, Warburg conditions: 30°C, reaction volume 2.5 ml, gas phase, air. After preincubation period, 0.5 ml of μmole quantities of D-glucose were added from the side arm, 0.3 ml 20% KOHag added to the center well. Endogenous res piration subtracted (879 μΐ in 240 min). I Average oxidation of 5, 10, μτηοΙβ8 glucose after pre-incubation 5 μmole glucose. • 10 μmole, Δ 15 μmole, A 20 μmole glu cose added after pre-incubation of celh with 2.5 mM 4-deoxy-4-fluoro-D-glucose.

Temp.

^

Figure 7. (a) Temperature programmed gas chromatogram of locust haemolymph and (b) Locust fat body utilizing OV-17 (7.5%) phase at 170°C followed by a 4°C/min heating rate. Peaks: A = a-D-glucose, Β = β-D-glucose, C = trehalose.


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f l u o r o - D - g l u c o s e , based on a K i l i a n i e x t e n s i o n o f 3- d e o x y - 3 - f l u o r o - D - a r a b i n o s e {22) with Na CN, w i l l a l l o w us t o a s c e r t a i n the a c t u a l m e t a b o l i c f a t e o f 4- d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) and i t s d e f l u o r i n a t e d product. In o r d e r t o examine the b a c t e r i a l s p e c i f i c i t y o f t h i s C-F c l e a v a g e we have r e c e n t l y examined the b i o c h e m i c a l e f f e c t s o f (VII) on E. c o l i (ATCC 11775) and shown t h a t no s i g n i f i c a n t C-F c l e a v a g e by whole c e l l s or c e l l - f r e e e x t r a c t s occurs. A s m a l l but s i g n i f i c a n t uptake o f (VII) i s o b s e r v e d (0.06 mg/mg d r y weight o f b a c t e r i a ) . U s i n g a n o t h e r s t r a i n o f E. c o l i (ATCC 1494 8) we have a l s o demonstrated ( L o u i e , L i - Y u & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) t h a t (VII) p r e v e n t s u t i l i z a t i o n o f l a c t o s e i n t h i s o r g a n i s m by u n c o m p e t i t i v e i n h i b i t i o n o f the i n d u c t i o n o f β galactosidase. Our r e s u l t s are s i m i l a r t o t h o s e r e p o r t e d f o r the e f f e c t on E. c o l i ( 4 ) . 14

T o x i c i t y of 3-deoxy-4-fluoro-D-glucose i n Locusta înigratoria and S c h i s t o c e r c a g r e g a r i a A l t h o u g h 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e (II) d i s p l a y s s e v e r a l p h y s i o l o g i c a l and b i o c h e m i c a l e f f e c t s i n r a t s (33), i t i s not t o x i c . Coupled w i t h the f a c t t h a t r a t s e x c r e t e l a r g e q u a n t i t i e s o f unchanged (II) v i a u r i n e and f a e c e s and a l s o the r e l a t i v e l y l a r g e q u a n t i t i e s o f (II) (5g/Kg body weight) n e c e s s a r y t o evoke a b i o c h e m i c a l response i t was c o n s i d e r e d t o be o f some i n t e r e s t t o s t u d y an a n i m a l o r g a n i s m which r e q u i r e d a s m a l l e r dosage o f (II) and r e t a i n e d water t o a g r e a t e r extent. Two c l o s e l y r e l a t e d A f r i c a n l o c u s t s p e c i e s , S c h i s t o c e r c a g r e g a r i a and L o c u s t a m i g r a t o r i a were chosen f o r t h i s p u r p o s e . In b o t h 10-14 day a d u l t i n s e c t s (II) was t o x i c (LDCQ 5mg/g l o c u s t t i s s u e ) when i n j e c t e d i n t o the haemocoel. T h i s compound was a l s o found t o be t o x i c when o r a l l y i n g e s t e d . T o x i c i t y was e v i d e n c e d by p r o g r e s s i v e l o s s o f m o t o r a c t i v i t y w i t h d e a t h o c c u r r i n g between 30 hours t o 4 d a y s . These symptoms s u g g e s t e d t h a t a slow m e t a b o l i c p o i s o n i n g was occurring. U s i n g the gas c h r o m a t o g r a p h i c p r o c e d u r e o u t l i n e d by F o r d and Candy (34) f o r o b t a i n i n g a c a r b o h y d r a t e scan, l e v e l s of v a r i o u s steady s t a t e n e u t r a l carboh y d r a t e m e t a b o l i t e s were assayed from v a r i o u s l o c u s t t i s s u e s (Figure 7). I t was found t h a t l e v e l s o f (II) d i s a p p e a r e d r a p i d l y from hemolymph, f a t body and f l i g h t muscle w i t h i n two hours o f i n j e c t i o n . A n a l y s i s of e x c r e t a i n d i c a t e d t h a t a s i g n i f i c a n t p o r t i o n o f the i n j e c t e d dose was l o s t . Gas c h r o m a t o g r a p h i c r e s u l t s


BIOCHEMISTRY


Figure 8. (a) Temperature programmed gas chromatogram of Locust haemolymph after 3deoxy-3-fiuoro-D-glucose injections. M = me tabolite, A = a-D-glucose, Β = β-D-glucose, C = trehalose, (b) Locust fat body after similar treatment.

T i me

Figure 9. (a) Gas chromatogram (isothermal conditions) of locust hemolymph after 3-deoxy-dfluoro-D-glucose and glucose administration using OV-17 stationary support. M = 3-deoxy3-fluoro-glucitol G = glucitol, A = a-D-glucose Β = β-D-glucose. (b) Using E301 stationary support. y

y


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AL.

i n d i c a t e d t h a t a new m e t a b o l i t e appeared i n the n e u t r a l carbohydrate f r a c t i o n f o l l o w i n g a d m i n i s t r a t i o n of ( I I ) . T h i s c a r b o h y d r a t e was shown t o be the a l d i t o l o f ( I I ) , namely, 3 - d e o x y - 3 - f l u o r o - D - g l u c i t o l (X) by gas chroma t o g r a p h i c and TLC comparison o f the s y n t h e t i c a l l y made a l d i t o l (Lopes, D. and T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) . t o the one o b t a i n e d from l o c u s t t i s s u e e x t r a c t s ( F i g u r e s 8 and 9 ) . Furthermore, a f t e r i n s e c t s were p o i s o n e d w i t h a 10.8 mg dose o f (II) and 12 hours l a t e r hemolymph g l u c o s e l e v e l s a r t i f i c a l l y r a i s e d by a 10.8 mg dose o f g l u c o s e the presence o f 0.4 - 0.5 mg/100mg t i s s u e o f (X) and 0.3 - 0.6 mg/100mg t i s s u e D - g l u c i t o l was d e t e c t e d . T h i s i s the f i r s t example o f s o r b i t o l metabolism t o be d e t e c t e d i n e i t h e r o f t h e s e species. Under normal c o n d i t i o n s s o r b i t o l was not d e t e c t a b l e i n the n e u t r a l carbohydrate f r a c t i o n . This phenomenon may hav (a) the r a t e o f norma g r e a t e r than i t s r a t e o f f o r m a t i o n , (b) T h i s was not an important m e t a b o l i c pathway i n the l o c u s t and was o n l y d e t e c t a b l e due t o i n h i b i t i o n . S o r b i t o l m e t a b o l i s m has been r e p o r t e d i n the s i l k worm (35) and mosquito (36). An e x t e n s i v e r e v i e w o f p o l y o l s and t h e i r metabolism has been o f f e r e d by T o u s t e r and Shaw (37) . Our r e s u l t s suggested t h a t the pathway from g l u c o s e t o f r u c t o s e v i a s o r b i t o l was a c t i v e i n t h e s e l o c u s t s p e c i e s and t h a t (II) b l o c k e d the subsequent c o n v e r s i o n o f s o r b i t o l t o f r u c t o s e t h r o u g h the i n h i b i t o r y a c t i o n o f (X) on t h e enzyme s o r b i t o l dehydrogenase ( F i g u r e 10). A precursor product r e l a t i o n s h i p i s e v i d e n t between (II) and (X) i n b o t h hemolymph and f a t body ( F i g u r e 11). Initially i t was thought t h a t the enzyme s o r b i t o l dehydrogenase was found i n t h e hemolymph o f the l o c u s t . T h i s i s the case i n silkworm (35). No a c t i v i t y , however, was a s s a y a b l e i n l o c u s t hemolymph u s i n g 0.1M s o r b i t o l and lOmM n i c o t i n a m i d e adenine d i n u c e l t i d e (NAD ) as substrates. Subsequent study r e v e a l e d t h a t the enzyme a c t i v i t y was c o n f i n e d t o the f a t body. U s i n g about 10 f o l d p u r i f i e d enzyme p r e l i m i n a r y r e s u l t s have suggested t h a t t h i s enzyme i s not v e r y a c t i v e i n l o custs having a K f o r s o r b i t o l i n the o r d e r o f 0.05M. P r e l i m i n a r y r e s u l t s suggest t h a t (X) i s a c o m p e t i t i v e i n h i b i t o r o f the enzyme w i t h a KiÔ.lM. These r e s u l t s a l s o suggest t h a t the pathway o f s o r b i t o l m e t a b o l i s m from g l u c o s e t o f r u c t o s e i s not v e r y important t o the t o t a l m e t a b o l i s m o f the l o c u s t . Therefore, i t i s d i f f i c u l t t o r a t i o n a l i z e how the i n h i b i t i o n o f such a minor pathway can m a n i f e s t t o x i c a c t i o n . It i s q u i t e p o s s i b l e t h a t some h i t h e r t o u n d e t e c t e d +

M


112


Dehydrogenase

Figure 10.

Sorbitol conversion pathway.

Time

in m i n u t e s

Figure 11. Levels of 3-deoxy-3-fluoro-D-glucose (II) and 3-deoxy-3-fluoro-D-glucitol (X) in locust hemolymph and fat body after a 3.6-mg injection of (II). (II) in hemolymph (X) in hemolymph (II) in fat body.


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metabolites are responsible for t o x i c i t y . C h e f u r k a (38), (39.) has suggested a means o f e v a l u a t i n g glucose metabolism i n i n s e c t s using 1 - C and 6 - C l a b e l l e d D-glucose by examining t h e r e l a t i v e r a t e s o f ^ C02 e v o l u t i o n . T h i s t e c h n i q u e may be a u s e f u l t o o l f o r m o n i t o r i n g any o t h e r changes i n t h e pathways o f g l u c o s e m e t a b o l i s m induced by (II) o r i t s metabolites. Our s t u d i e s on t h e mode o f t o x i c i t y w i l l be f u r t h e r e d when and/or H - l a b e l l e d f l u o r o g l u c o s e s become a v a i l a b l e b u t t h e s e p r e l i m i n a r y r e s u l t s i n d i c a t e t h a t some f l u o r i n a t e d c a r b o h y d r a t e s a r e n o t o n l y t o x i c t o i n s e c t s b u t may a l s o a c t as probes f o r t h e d e t e c t i o n o f unsuspected m e t a b o l i c pathways. 14

14

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3

Acknowledgment T h i s work i s s u p p o r t e C o u n c i l o f Canada.

Abstract (a) The use of deoxyfluoro-D-glucose as probes for the study of glucose transport across the human erythrocyte membrane is discussed. These studies are also related to the mode of action of cytochalasin Β inhibition of D-glucose across this membrane, (b) Evidence is presented to show that 4-deoxy-4fluoro-D-glucose is not oxidised by whole resting cells of Ps. fluorescens (ATCC 12633) but an extensive release of fluoride anion occurs. With cell-free extracts of Ps. fluorescens however, 4-deoxy-4-fluoroD-glucose is oxidised to the extent of 2 g atoms of oxygen/mole of substrate. Possible reasons for this C-F cleavage are discussed. (c) 3-Deoxy-3-fluoro-Dglucose is toxic to Locusta migratoria (L.D.50 5mg/g and is metabolised by an NAD-linked sorbitol dehydro genase, which is located in the fat body of the insect, to 3-deoxy-3-fluoro-D-glucitol. The activity of the partially purified enzyme is low with Km, 0.05M for sorbitol. 3-Deoxy-3-fluoro-glucitol is a competitive inhibitor with K i , 0.1M.


114


Literature Cited. 1. Ciba Fdn. Symposium: "Carbon-Fluorine Compounds: Chemistry, Biochemistry and Biological Activities." pp 1-417. Associated Scientific Publishers, New York, 1972. 2. Woodward, B., Taylor, N.F. & Brunt, R.V., Biochem. Pharmacol. (1971), 20 1071-1077. 3. Taylor, N.F., White, F.H. & Eisenthal, R.E., Biochem. Pharmacol. (1972), 21 347-353. 4. Miles, R.J. & S.J. J. Gen. Microbiol. (1973) 76 305-318. 5. Eisenthal, R.E., Harrison, R., Lloyd, W.J. & Taylor, N.F., Biochem. J. (1972), 130 199-205. 6. Bessel, E.M. & Thomas, P., Biochem. J. (1973) 131 843-850. 7. Wright, J.A., Taylor N.F. Brunt R.V & Brownse R.W., Chem. Commun 8. Silverman, J.B., Barbiatz, P.S., Mahajan, K.P., Buschek, J. & Foudy, T.P., Biochemistry (1975) 14 2252-2258. 9. Barnett, J.E.G., Ralph, A. & Monday, K.A., Biochem. J. (1970) 118 843-850. 10. Barnett, J.E.G., Holman, J.D. & Monday, K.A., Biochem. J. (1973)131211-221. 11. Riley, G.J. & Taylor, N.F., Biochem. J. (1973) 135 773-777. 12. Kent, P.W., Ciba Fdn. Symposium: "Carbon-Fluorine Compounds". pp 169-208. 13. Lieb, W.R. & Stein, W.D., Biochim. Biophys. Acta (1972), 265 187-207. 14. LeFevre, P.G., Pharmacol. Rev. (1961) 13 39-70. 15. Sen, A.K. & Widdas, W.F., J. Physiol (London) (1962) 160 392-403. 16. Lacko, L. & Burger, M. Biochem. J. (1962) 83 622-625. 17. Baker, G.F. & Widdas, W.F., J. Physiol. (London) (1972) 271 10p. 18. Miller, D.M. in "Red Cell Structure and Function". (Jamieson, G.A. & Greinwalt, Τ.A. Eds.) pp 240-292, J.B. Lippincott & Co. Philadelphia & Toronto, 1969. 19. Kahlenberg, A. & Dolansky, D., Canad. J. Biochem. (1972) 50 638-643. 20. Bloch, R.J. Biol. Chem. (1974) 249 1814-1822. 21. Taylor, N.F. & Gagneja, G.L., Proc. Canad. Fed. Biol. Soc. (1975) 18 p 4. 22. Aldridge, D.C., Armstrong, J.J. & Speake, R.N., J. Chem. Soc. (1967) 1667-1676. 23. Taylor, N.F., Hill, L. & Eisenthal, R.E., Canad. J. Biochem. (1975) 53 57-64.


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24. Barford, A.D., Foster, A.B., Westwood, J.H., Hall, L., & Johnson, R.N., Carbohydr. Res. (1971), 19 49-61. 25. Umbreit, W.W., Burris, R.H. & Stauffer, J.F., "Manometric Techniques" pp 1-61. Burgess Publish ing Co., Minneapolis, Minn. 1964. 26. Woodward, B., Taylor, N.F., & Brunt, R.V., Analyt. Biochem. (1971) 36 303-309. 27. Wood, W.A. & Schwerdt, R.F., J. Biol. Chem. (1953) 201 501-511. 28. Midgley, M. & Dawes, E.A., Biochem. J. (1973) 132 141 29. Kundig, W., Ghosh, S. & Roseman, S., Proc. Nat. Acad. Sci. (1964) 52 1067-1074. 30. Goldman, P. J. Biol. Chem. (1965) 240 3434-3438. 31. Kirk, Κ., Goldman P. Biochem J (1970) 117 409-410. 32. Wright, J.A., an (1967) 3 333-339. 33. Riley, G.J., Ph.D. Thesis University of Bath U.K. (1973). 34. Ford, W.C.L., Candy, D.J., Biochem. J. (1972) 130, 1101. 35. Faulkner, P., Biochem. J. (1958) 68 375. 36. Handel, E. Van, Comp. Biochem. Physiol. (1969) 29 1023. 37. Touster, Ο., Shaw, D.R.D., Physiol. Rev., (1962) 42 181. 38. Chefurka, W., in "The Physiology of Insecta"., Volume 2, pages 641-656, Academic Press Inc., New York, 1965. 39. Chefurka, W., Ela, R., and Robinson, J.R., J. Insect Physiol. (1970) 16 2137-2156. 40. Hall, L.D., Johnson, R.N., Foster, A.B. and Westwood, J.H., Canad. J. Chem. (1971) 49 236-240. 41. Lin, S. and Spudich, J.A., J. Biol. Chem. (1974) 249 5778-5783.


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BONDS

Q.

Is the conformation o f glucose, 3-deoxy-3-fluoro g l u c o s e and 3-deoxy-glucose t h e same?

A.

Although there is no direct experimental evidence, t h e s e t h r e e hexoses p r o b a b l y have t h e same p r e dominate C l - c o n f o r m a t i o n i n aqueous s o l u t i o n w i t h a l l t h e s u b s t i t u e n t s equitorial. There is some e v i d e n c e , however, t h a t t h e i n t r o d u c t i o n o f f l u o r i n e can d i c t a t e which c o n f o r m a t i o n t h e sugar r i n g w i l l adopt. Thus Hall et al (40) have shown t h a t i n the case o f t h e a c e t y l a t e d D - x y l o s y l f l u o r i d e d e r i v a t i v e s t h e ß-anomer adopts t h e unexpected c o n f o r m a t i o n i n which all t h e s u b s t i t u e n t s a r e axially disposed.

Q.

Does g l u c o s e b i n d w i t h c y t o c h a l a s i n B?

A.

No. The b i n d i n and our own kinetic results (21) in which c y t o c h a l a s i n Β is shown t o be a c o m p e t i t i v e inhibitor ( K i , 10- M) o f g l u c o s e t r a n s p o r t in t h e human e r y t h r o c y t e suggest t h a t c y t o c h a l a s i n Β b i n d s t o a c a r r i e r p r o t e i n and n o t t o g l u c o s e . 7


7 Organic Fluorocompounds in Human Plasma: Prevalence and Characterization W. S. GUY Department of Basic Dental Sciences, University of Florida, Box J424, Gainsville, Fla. 32610 D. R. TAVES Department of Pharmacology and Toxicology, University of Rochester, Rochester, N.Y. 14642 W. S. BREY, JR. Department of Chemistry, Universit Taves discovered tha tained two distinct forms of fluoride (1-4). Only one of these was exchangeable with radioactive fluoride. The other, non -exchangeable form was detectable as fluoride only when sample preparation included ashing. This paper is concerned with three aspects of this newly discovered, non-exchangeable form: 1) its prevalence in human plasma, 2) how its presence in human plasma affects the validity of certain earlier conclusions about the metabolic handling of the exchangeable form of fluoride, and 3) its chemical nature. Preliminary work in this laboratory suggested that the non -exchangeable form was widespread in human plasma but did not exist in the plasma of other animals. Ashing increased the amount of fluoride an average of 1.6 ± 0.25 SDμM(range 0.4-3.0) in samples of plasma from 35 blood donors in Rochester, N.Y. (5). No such fluoride was detectable (above 0.3 μM) in blood serum from eleven different species of animal including horse, cow, guinea pig, chicken, rabbit, sheep, pig, turkey, mule and two types of monkey (6). Standard methods for analysis of exchangeable fluoride in serum have in the past included ashing as a step in sample preparation (7). Taves showed that the amount of fluoride in serum that would mix with radioactive fluoride was only about one-tenth the amount generally thought to be present based on analyses using these older methods (4). When plasma samples from individuals living in cities having between 0.15 and 2.5 ppm fluoride in their water supply were analysed by these older methods, no differences were found between the averages for the different cities. This led to the conclusion that "homeostasis of body fluid fluoride content results with intake of fluoride up to and including that obtained through the use of water with a fluoride content of 2.5 ppm" (8). If the non-exchangeable form of fluoride predominated in these samples, differences in the exchangeable fluoride concentration would probably not have been apparent, and it would be unnecessary to postulate such rigorous 117 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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homeostatic c o n t r o l mechanisms f o r f l u o r i d e . In t h i s study plasma samples were c o l l e c t e d from a t o t a l of 106 i n d i v i d u a l s l i v i n g i n f i v e d i f f e r e n t c i t i e s with between 0.1 and 5.6 ppm f l u o r i d e i n t h e i r p u b l i c water supply. These were analyzed f o r both forms of f l u o r i d e . I n t h i s way the r e l a t i o n ship between exchangeable f l u o r i d e c o n c e n t r a t i o n i n the plasma and the consumption of f l u o r i d e through d r i n k i n g water was r e evaluated, and the prevalence of the non-exchangeable form was further studied. With respect to the chemical nature of the non-exchangeable form of f l u o r i d e s e v e r a l l i n e s of evidence suggested that i t was some s o r t of organic fluorocompound of intermediate p o l a r i t y , t i g h t l y bound to plasma albumin i n the blood. I t migrated with albumin during e l e c t r o p h o r e s i s of serum a t pH nine (3) and was not u l t r a f i l t e r a b l e from serum ( 2 ) . Attempts at d i r e c t e x t r a c t i o n from plasma with s o l v e n t petroleum ether and e t h y Treatment of albumin s o l u t i o n (prepared by e l e c t r o p h o r e s i s of plasma) with c h a r c o a l a t pH three d i d remove the bound f l u o r i n e f r a c t i o n . And f i n a l l y , when plasma p r o t e i n s were p r e c i p i t a t e d with methanol a t low pH the f l u o r i n e f r a c t i o n o r i g i n a l l y bound to albumin appeared i n the methanol-water supernatant i n a form which s t i l l r e q u i r e d ashing to r e l e a s e f l u o r i n e as i n o r g a n i c f l u o r i d e (5) . Based on these c o n s i d e r a t i o n s the non-exchangeable form of f l u o r i d e i n human plasma i s r e f e r r e d to as "organic f l u o r i n e " throughout the r e s t of t h i s paper. In order to f u r t h e r c h a r a c t e r i z e the organic f l u o r i n e f r a c t i o n , i t was p u r i f i e d from 20 l i t e r s of pooled human plasma and c h a r a c t e r i z e d by f l u o r i n e nmr. M a t e r i a l s and Methods A n a l y t i c a l Methods. Values f o r organic f l u o r i n e were c a l c u l a t e d by taking the d i f f e r e n c e between the amount of i n o r g a n i c f l u o r i d e i n ashed and unashed p o r t i o n s of the same m a t e r i a l . The f o l l o w i n g procedure was used to prepare ashed samples: 1) samples (sample s i z e f o r plasma was 3 ml) were placed i n platinum c r u c i b l e s and mixed with 0.6 mmoles of low f l u o r i d e MgCl and 0.1 mmoles of NaOH, 2) these were d r i e d on a h o t p l a t e and then ashed (platinum l i d s i n place) f o r 2-4 h r a t 600° C i n a muffle furnace which had been modified so that the chamber received a flow of a i r from outside the b u i l d i n g (room a i r increased the blank and made i t more v a r i a b l e ) , and 3) ashed samples were d i s s o l v e d i n 2 ml of 2.5 Ν H S0^ and t r a n s f e r r e d t o polystyrene d i f f u s i o n dishes using 2 r i n s e s with 1.5 ml of water. The f o l l o w i n g procedure was used f o r s e p a r a t i o n of f l u o r i d e from both ashed and unashed samples: 1) samples (sample s i z e f o r unashed plasma was 2 ml) were placed i n d i f f u s i o n dishes (Organ C u l t u r e Dishes, Falcon P l a s t i c s , Oxnard, C a l i f . , absorbent 2

2


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119

Pfosma

removed, r i n s e d with water), a c i d i f i e d with 2 ml of 2.5 Ν H SO^, and a g i t a t e d with a g e n t l e s w i r l i n g a c t i o n on a l a b o r a t o r y shaker f o r 30 min to remove C0 ; 2) f o r each sample the trapping s o l u t i o n (0.5 ml, 0.01 Ν NaOH + p h e n o l t h a l e i n - p - n i t r o p h e n o l i n d i c a t o r ) was placed i n a small polystyrene cup i n the c e n t e r - w e l l of the d i f f u s i o n d i s h , 1 drop of 10% T r i t o n - X 100 was added to the sample to decrease surface t e n s i o n and make the d i f f u s i o n r a t e more uniform between samples c o n t a i n i n g plasma and those not, the l i d with a small hole made near i t s l a t e r a l margin was sealed i n t o p l a c e with petroleum j e l l y , 0.02 ml of 4% hexamethyldisiloxane (Dow Corning, F l u i d 200, 0.65 c s , Midland, Mich.) i n ethanol was i n j e c t e d through the hole i n the l i d i n t o the sample, and the hole was sealed Immediately with petroleum j e l l y and a s t r i p of p a r a f f i n f i l m ; and 3) samples were d i f f u s e d with g e n t l e s w i r l i n g f o r a t l e a s t 6 hr, d i f f u s i o n was terminated by breaking the s e a l and trapping s o l u t i o n checked a t t h i s p o i n t t and d r i e d i n a vacuum oven (60° C, 26 in-Hg vacuum, i n the presence of a NaOH d e s i c c a n t ) . 2

2

F l u o r i d e was determined by potentiometry with the f l u o r i d e e l e c t r o d e . The system used c o n s i s t e d of a f l u o r i d e e l e c t r o d e o r i e n t e d i n an i n v e r t e d p o s i t i o n (model 9409A, Orion Research Inc. Cambridge, Mass.), a calomel reference e l e c t r o d e ( f i b e r t y p e ) , a p l a s t i c vapor s h i e l d which j u s t f i t t e d over the bodies of both e l e c t r o d e s forming an enclosed sample chamber i n which watersaturated t i s s u e paper was placed above the sample to prevent e v a p o r i z a t i o n of the sample, and a high impedence voltmeter (model 401, O r i o n ) . Samples were prepared and read i n the f o l l o w i n g way: 10 μΐ of 1 M HAc was drawn i n t o a polyethylene m i c r o p i p e t t e (Beckman Micro Sampling K i t , Spinco Div., Beckman I n s t . Co., Palo A l t o , C a l i f . ) and deposited i n t o the cup c o n t a i n i n g the r e s i d u e from the trapping s o l u t i o n a f t e r d r y i n g ; the f l e x i b l e t i p of the m i c r o p i p e t t e was used to wash down the w a l l s of the cup; and the s o l u t i o n was then t r a n s f e r r e d to the surface of the f l u o r i d e e l e c t r o d e and the reference e l e c t r o d e brought i n t o p o s i t i o n . Surfaces of the two e l e c t r o d e s were b l o t t e d dry between samples. Samples were read i n order of i n c r e a s i n g expected concentra t i o n and sets of samples were read between bracketing c a l i b r a t i o n standards. These standards were used i n two d i f f e r e n t ways during a run. F i r s t , they were flooded onto the e l e c t r o d e surfaces to e q u i l i b r a t e them to concentrations expected f o r samples and to make them uniform. This procedure permitted the a n a l y s t to take reasonably s t a b l e readings f o r samples w i t h i n one minute. Secondly, they were used i n 10 y l volumes f o r readings used i n preparing the standard curve. Values f o r i d e n t i c a l samples ( u s u a l l y t r i p l i c a t e s ) were averaged and the average blank was subtracted from sample means. These were then d i v i d e d by the average f r a c t i o n a l recovery of


120


f l u o r i d e (usually 90 to 95%) i n standards treated the same way as the sample s e t . P l a s t i c w a r e (Falcon P l a s t i c s ) was used f o r a l l a n a l y t i c a l procedures to avoid contamination by f l u o r i d e from g l a s s . Liquid volume measurements were made with 1, 5 and 10 ml p o l y s t y r e n e p i p e t t e s and a polycarbonate volumetric f l a s k (100 ml). Reagents were p u r i f i e d to i n s u r e uniformly low blanks. Water was r e d i s t i l l e d and d e i o n i z e d . A c e t i c a c i d and ammonia were r e d i s t i l l e d . F l u o r i d e contamination i n MgCl2 ( a n a l y t i c a l grade) was reduced by preparing a 1 M s o l u t i o n c o n t a i n i n g HC1 to pH 1 and scrubbing with hexamethyldisiloxane vapor i n a column through which the s o l u t i o n was continuously r e c y c l e d . Following scrubbing the s o l u t i o n was b o i l e d to one t h i r d volume to remove any r e s i d u a l v o l a t i l e s i l i c o n e s and then made j u s t b a s i c with NHi+OH. F l u o r i d e contamination i n I^SO^ was reduced by repeated e x t r a c t i o n s of a 6.7 Ν then b o i l i n g to one t h i r Buffered c a l i b r a t i o n standards were made from the same NaOH and HAc stock s o l u t i o n s as f o r samples. The blanks f o r ashed samples ranged between 0.2 and 1.5 nmoles f l u o r i d e and were t y p i c a l l y about 0.5 nmoles. The blanks were smaller f o r unashed samples; these ranged between 0.05 and 0.2 nmoles f l u o r i d e and were t y p i c a l l y about 0.1 nmoles. Factors a f f e c t i n g recovery of f l u o r i d e during d i f f u s i o n were i n v e s t i g a t e d w i t h F~ t r a c e r . Recovery during d i f f u s i o n was 97% a f t e r 80 min from 5 ml containing 2 ml of plasma. Increasing the a c i d i t y of the sample up to 5 N, the volume of the sample up to 7.5 ml, the amount of c o l d F~ up to 1 pmole, the amount of f l u o r i d e complexors up to 1 ymole of ThiNC^)^ had no m a t e r i a l e f f e c t on the r a t e of f l u o r i d e d i f f u s i o n . The absence of both plasma and detergent i n the sample compartment markedly slowed the r a t e of d i f f u s i o n . Not shaking the sample a l s o slowed the r a t e of d i f f u s i o n . Increasing the a l k a l i n i t y of the trapping s o l u t i o n to 0.1 Ν increased the r a t e of d i f f u s i o n but the lower concentration, 0.01 N, was r e q u i r e d here to permit a lower i o n i c strength i n the sample reading s o l u t i o n . O v e r a l l recovery of added cold f l u o r i d e was measured. In samples containing n e i t h e r plasma nor detergent the recovery a f t e r 6 hr d i f f u s i o n averaged 93% and 95% f o r ashed and unashed samples, r e s p e c t i v e l y . In samples containing plasma the recovery was 95% a f t e r 3 hr d i f f u s i o n . The degree to which f l u o r i n e from organic fluorocompounds could be f i x e d as i n o r g a n i c f l u o r i d e by ashing v a r i e d from l e s s than 1% f o r v o l a t i l e compounds l i k e p - a m i n o b e n z o t r i f l u o r i d e , m-hydroxybenzotrifluoride, b e n z y l f l u o r i d e and benzotrifluoride to over 80% f o r l e s s v o l a t i l e compounds l i k e 5 - f l u o r o u r a c i l , f l u o r o a c e t a t e and p - f l u o r o p h e n y l a l a n i n e . Methods used here f o r s e p a r a t i o n of f l u o r i d e ( d i f f u s i o n at rm. temp.) (9) and i t s q u a n t i t a t i o n ( f l u o r i d e e l e c t r o d e ) (10) are considered to be q u i t e s p e c i f i c f o r f l u o r i d e . One p o t e n t i a l l y


7.

GUY E T A L .

Fluorocompounds

in Human

121

Plasma

important i n t e r f e r e n c e , however, was c o d i f f u s a b l e organic a c i d s which might p a r t i a l l y n e u t r a l i z e the trapping s o l u t i o n and thus lower the pH of the b u f f e r e d reading s o l u t i o n . Indeed, i t was found that samples c o n t a i n i n g r e l a t i v e l y l a r g e concentrations of a c e t i c acid (e.g., f r a c t i o n s 2, 3 and 4 from step 4 i n the p u r i f i c a t i o n system) completely n e u t r a l i z e d the trap w i t h i n a few hours. The s i g n i f i c a n c e of t h i s problem i n the a n a l y s i s of f l u o r i d e i n blood plasma was i n v e s t i g a t e d i n two ways. F i r s t , four samples of human plasma were allowed to d i f f u s e f o r three weeks, and no change i n the c o l o r of the phenolphthalein i n d i c a tor i n the trapping s o l u t i o n was observed. Secondly, samples c o n t a i n i n g the same plasma were d i f f u s e d f o r d i f f e r e n t periods up to 158 hr and the apparent f l u o r i d e was determined. No changes were observed between samples which c o r r e l a t e d with d i f f u s i o n time. The s e n s i t i v i t y o the blank r a t h e r than th R e p r o d u c i b i l i t y v a r i e d with the amount of f l u o r i d e being measured. The c o e f f i c i e n t of v a r i a t i o n averaged 55% i n the low range (samples c o n t a i n i n g 0.25 to 0.75 nmoles F ) and 6.6% i n the high range (10-12 nmoles F " ) . Blood Plasma. Human plasma was obtained from blood banks i n f i v e c i t i e s . According to p u b l i c records these c i t i e s had not changed the f l u o r i d e c o n c e n t r a t i o n of t h e i r p u b l i c water supply f o r a t l e a s t s i x years p r i o r to o b t a i n i n g the samples. Samples were r e c e i v e d i n i n d i v i d u a l polyethylene bags which were p a r t of the Fenwall ACD blood c o l l e c t i o n system. In blood c o l l e c t i o n using t h i s system 450 ml of blood i s drawn i n t o a bag containing 67.5 ml of anticoagulant a c i d c i t r a t e dextrose (ACD) s o l u t i o n . When the c e l l s are removed the ACD s o l u t i o n remains i n the plasma. Because of t h i s d i l u t i o n of plasma a c o r r e c t i o n f a c t o r of 1.3 was a p p l i e d to values obtained here f o r the c o n c e n t r a t i o n of f l u o r i d e . The p o t e n t i a l e r r o r i n t h i s f a c t o r was ± 0 . 1 because of v a r i a t i o n between standard l i m i t s f o r hematocrit and minimum volume of the blood donation. Bovine blood was obtained a t slaughter and mixed immediately with ACD s o l u t i o n i n 1 l i t e r polyethylene b o t t l e s . E l e c t r o p h o r e s i s . A continuous flow e l e c t r o p h o r e t i c separator (model FF-3, Brinkman I n s t . , Inc., Westbury, N.Y.) was employed. Sample flow r a t e was 2.3 ml/hr, b u f f e r flow r a t e was 72 ml/hr, v o l t a g e was 0.67 kv, and current was 140 mamp. Separa t i o n took 19 hr. P l a t e s e p a r a t i o n was 1 mm and operating tempera ture was between 2 and 4° C. The b u f f e r was 0.12% (NHi ) C0 , made by bubbling C 0 from dry i c e i n t o r e d i s t i l l e d ΝΗίψΟΗ u n t i l the pH reached 9.0. +

2


2

3

122

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

P u r i f i c a t i o n System. Steps i n the p u r i f i c a t i o n system are summarized i n t a b l e I. In the f i r s t step one l i t e r of plasma (pooled from 5-6 i n d i v i d u a l s ) was d i a l y s e d i n seamless c e l l u l o s e tubing (1 i n . diameter) against 20 l i t e r s of water at 4° C. The d i a l y s a t e was changed twice at 24 hr i n t e r v a l s . In the second step d i a l y s e d plasma was f r e e z e d r i e d . In the t h i r d step the d r i e d powder from e l e c t r o p h o r e s i s was extracted with methanol i n a soxhelet e x t r a c t i o n apparatus (model 6810 G, Ace Glass, Inc., Vineland, N.J.). C e l l u l o s e e x t r a c t i o n thimbles (model 6812 G, Ace Glass) were soaked overnight i n methanol. Operating c o n d i t i o n s were 25° to 30° C under a vacuum of 24 in-Hg. Coolant f o r the condenser was 80% ethanol; i n l e t temperature was -10° to -20° C and o u t l e t tempera ture was -10° to 0°. Two l i t e r s of methanol were r e f l u x e d through the apparatus f o r a period of 4 hr and approximately 400 ml were l o s t to evaporatio were placed i n the f l a s In the f o u r t h step the r e s i d u e from the methanol e x t r a c t was f r a c t i o n a t e d according to the method described by Siakotos and Rouser (11) f o r separating l i p i d and n o n - l i p i d components. The method i s based on l i q u i d - l i q u i d p a r t i t i o n i n a column containing a dextran g e l (Sephadex G-25, coarse, beaded, Pharmacia F i n e Chemicals, Inc., N.Y.). Four eluents are used: 1) 500 ml chloroform/methanol, 19/1, saturated with water, 2) 1000 ml of a mixture of 5 p a r t s of chloroform/methanol, 19/1, and 1 part of g l a c i a l a c e t i c a c i d , saturated with water, 3) 500 ml of a mixture of 5 p a r t s chloroform/methanol, 19/1, and 1 part g l a c i a l a c e t i c a c i d , saturated with water, and 4) 1000 ml of methanol/water, 1/1. T h e i r method was modified f o r use here by i n c r e a s i n g the column length to that a t t a i n e d by using a f u l l 100 grams of dextran beads. Sample s i z e corresponded to that from 2.5 l i t e r s of the o r i g i n a l plasma. In the f i f t h step the residues from eluents 2 and 3 from two runs of step four were combined, a p p l i e d to a s i l i c i c a c i d column, and e l u t e d by reverse flow with an exponential gradient of i n c r e a s i n g amounts of methanol i n chloroform. The column (model SR 25/45, 2.5 cm i . d . χ 45 cm, Pharmacia) was f i l l e d to a height of 30 cm with s i l i c i c a c i d ( U n i s i l , 100-200 mesh, Clarkson Chem. Co., Inc., W i l l i a m s p o r t , Pa., heat a c t i v a t e d at 110° C f o r 2 days) and was washed with a complete set of e l u t i o n s o l v e n t s before use. The gradient maker (model 5858, set 4, Ace Glass Co.) was f i l l e d with 1 l i t e r of methanol i n the upper chamber and 2 l i t e r s of chloroform i n the lower. The flow r a t e was adjusted by the height of the s o l v e n t r e s e r v o i r s to an average of 3 ml/min f o r the f i r s t l i t e r of eluent. The sample had to be t r a n s f e r r e d to the column by repeated washings with chloroform because of i t s low s o l u b i l i t y i n t h i s s o l v e n t . This u s u a l l y r e q u i r e d about 30 ml of chloroform t o t a l . Dead volume for the system as 90 ml. F r a c t i o n s of 15 ml volume were c o l l e c t e d i n c a r e f u l l y cleaned g l a s s tubes.


7.

Fluorocompounds

GUY E T A L .

in Human

123

Plasma

Table I PROCEDURE FOR PURIFICATION OF FLUOROCOMPOUNDS FROM BLOOD PLASMA Fraction Treated blood plasma

plasma p r o t e i n s & protein-bound substances i n water s o l u t i o n plasma p r o t e i n s & protein-bound substances

Treatment step 1: exhaustive d i a l y s i s against d i s t i l l e d water step 2:

lyophilization

Fraction Removed smaller, waters o l u b l e components

water

extraction—soxh e l e t , 25°C, 24 in-Hg vacuum

plasma l i p i d s

step 4: column chromatography— liquid-liquid p a r t i t i o n on Sephadex

polar l i p i d s

step 5: column chromatography— adsorption on s i l i c i c acid

l i p i d s of low p o l a r i t y and residual polar contaminants

unknown: severa1 yellow f r a c t i o n s

Figure 1. Relationship between the concentration of fluoride in human plasma and the concentration of fluoride in the drinking water



Figure 2. Relationship between the concentration of organic fluorine in human pfosma and the concentration of fluoride in the drinking water

TUBE NO. Figure 3. Separation of fluoride and organic fluorine in human plasma by electrophoresis. A sample (about 45 ml) of human phsma was electrophoresed in pH 9 buffer and fractions between the sampling port (near tube 72) and the positive pole (near tube 1) were analyzed for the fluoride content of both ashed and unashed aliquots. Relative concentrations of proteins were estimated by absorbance at 280 nm.


BONDS

7.

GUY ET AL.

Fluorocompounds

in Human

Plasma

125

Tubing and f i t t i n g s to the columns were p o l y t e t r a f l u o r o e t h y lene (supplied l a r g e l y by Chromatronix, Inc., Berkeley, C a l i f . ) . A l l solvents were r e d i s t i l l e d . Methanol and chloroform were ACS c e r t i f i e d ( F i s h e r S c i e n t i f i c Co.) and a c e t i c a c i d was a n a l y t i c a l reagent grade, U.S.P. ( M a l l i n c k r o d t Chemical Works, St. L o u i s ) . Solvents were removed from samples i n a f l a s h evaporator. NMR. The nmr spectrum was obtained on a V a r i a n XL-100 spectrometer with N i c o l e t Technology F o u r i e r Transform accessory. The sample was d i s s o l v e d i n an approximately 1/1 mixture of CH OH and CDCI3 and s p e c t r a were run i n a 5 mm tube. External r e f e r e n c i n g to CFCI3 was used f o r the chemical s h i f t s , and these are expressed with p o s i t i v e numbers to lower f i e l d ( i . e . , higher frequency). E x t e r n a l l o c k was used. T y p i c a l c o n d i t i o n s were a pulse length of 15 microseconds c y c l e s of 2.5 sec, and a processing. 3

Results Values f o r i n o r g a n i c f l u o r i d e (F~) and organic f l u o r i n e (R-F) i n 106 plasma samples from humans l i v i n g i n f i v e c i t i e s are shown i n t a b l e I I . These data show that the average f l u o r i d e c o n c e n t r a t i o n i n plasma i s d i r e c t l y r e l a t e d to the f l u o r i d e c o n c e n t r a t i o n i n the water supply, and that the average organic f l u o r i n e concentration i n plasma i s not. No r e l a t i o n s h i p between f l u o r i d e i n plasma and organic f l u o r i n e i n plasma was apparent by i n s p e c t i o n of values f o r i n d i v i d u a l samples. The d i s t r i b u t i o n s of the values w i t h i n c i t i e s are shown i n f i g u r e s 1 and 2. In both cases the d i s t r i b u t i o n s appear to be l o g normally d i s t r i b u t e d with only 3 or 4 i n d i v i d u a l s s u r p r i s i n g l y deviant. In the cases of the two i n d i v i d u a l s with l i t t l e or no apparent organic f l u o r i n e ( f i g u r e 2, Andrews group, l e f t margin), the i n o r g a n i c f l u o r i d e l e v e l s were both i n excess of 7 μΜ, making the d i f f e r e n c e measure ment f o r organic f l u o r i n e d i f f i c u l t . The o v e r a l l mean value f o r organic f l u o r i n e was 1.35 ± 0.85 SD μΜ. Plasma was electrophoresed i n an attempt to reproduce the f i n d i n g s of Taves (3) using plasma from another i n d i v i d u a l . Results shown i n f i g u r e 3 c l o s e l y match those found e a r l i e r i n that a predominant form of organic f l u o r i n e appeared to migrate with albumin at pH 9, and i n that organic and i n o r g a n i c forms were c l e a r l y separated. The recovery, mass balance and p u r i f i c a t i o n f a c t o r s f o r steps i n the p u r i f i c a t i o n system l i s t e d i n t a b l e I are recorded i n t a b l e I I I . These data show that about o n e - t h i r d of the o r i g i n a l amount of organic f l u o r i n e i n plasma i s recovered i n the major peak from s i l i c i c a c i d chromatography. Another t h i r d i s accounted for i n other f r a c t i o n s and the r e s t i s not accounted f o r , presumably because of adsorption to surfaces of containers i n


126

BIOCHEMISTRY

INVOLVING CARBON-FLUORINE

BONDS

Table I I CONCENTRATION OF FLUORIDE (F~) AND ORGANIC FLUORINE (R-F) IN BLOOD PLASMA SAMPLES FROM FIVE CITIES HAVING DIFFERENT FLUORIDE CONCENTRATIONS IN THEIR WATER SUPPLY

3

a

[F"] i n Plasma , uM C i t y ([F~] i n Water, ppm)

Mean± SD(n)

Albany, N.Y. (— •—^ **—. ^ » rH > Φ Φ rH s Xi CO CO φ Β 4-> CO rH Φ φ r>% cd CO 4-1 Ό Η U

•

00

ο

9* ο

00 m rH CM rH

m co* •—^ rH

•

ο

^· α ο CM vO

< vD 00

rH

rH rH

CO CO

00 m

formation of

0.5

as the major p r o d u c t .

1,3-dioxolane,

^4,

suggests

that

t r i f l u o r o - 1 , 3 - d i o x o l a n e , ^3, f o l l o w e d by

r e a c t i o n a s we carbonate,

will

see

4

ratio

from

ΊΛ

i s an

i n the

4-chloro-2,2,5-

a dehydrochlorination addition

important

fluorination

reaction.

finding

in

The

this

of 4-chloroethylene

3.

B e c a u s e we

observed

the monochlorinated totally

formation of

results

halogen

4,5-dichloroethylene carbonate,

The

the c a t a -

The c h l o r i n a t e d - u n s a t u r a t e d

the

a hydrogen f l u o r i d e

unsaturated-1,3-dioxolane,

on

4-chloro-2,2,5-trifluoro-l,3-

d i o x o l a n e , J^3,

reaction

dependent

i n the c a t a l y s t / S F

we

began o u r

d e r i v a t i v e 3 u s i n g an H F / S F

unexpected

result

2,2,5-trifluoro-1,3-dioxolane,

3

exchange i n the 4,

was 13,

4

fluorination

of

investigation

of

ratio

of

0.45.

the formation of 4-chloroas the o n l y i s o l a t e d

product.

13


10.

DENSON

For t h i s be

Fluorinated

ET AL.

compound t o form,

occurring.

halogen

There

Volatile

197

Anesthetics

a hydrogen s u b s t i t u t i o n

i s no r e p o r t e d e v i d e n c e

exchange o r a d e h y d r o c h l o r i n a t i o n h y d r o g e n

tion

reaction.

tion

a n d o c c u r s w h e t h e r HF o r T i F

precursor results

This result

carbonate

with either

i n no r e a c t i o n .

with SF

i s independent

of the s t a r t i n g

carbonate,

3.

Heating the

c a r b o n a t e , 3^,

f o r 24 h r r e s u l t s

Hydrogen

addi-

concentra-

4

a t 150°C f o r 24 h r

4-chloroethylene

i n t h e absence o f c a t a l y s t

4

4

must

a direct

fluoride

of cat/SF

i s the catalyst.

4

HF o r T i F

Heating

reaction

f o reither

i n recovery

s u b s t i t u t i o n by S F

4

has

b e e n r e p o r t e d by A p p l e q u i s t a n d S e a r l e ( 1 3 ) . Experiments either

conducted

a t lower

temperatures

resulted i n

no r e a c t i o n o r i n t h e f o r m a t i o n o f 2 , 2 , 5 - t r i f l u o r o - 1 , 3 -

d i o x o l a n e , ^15, a s t h e m a j o r

product.

Cl-,

= 0 + SF

A

Cat./125°C

-0' 15

Hydroquinone

A

similar

ethylene prepare

was o b t a i n e d

2,

t o determine

to biological

i n attempts

The o b j e c t i v e o f t h i s

2,2-difluoro-1,3-dioxolane

dioxolane moiety

result

carbonate,

r a t i o was a g a i n m a i n t a i n e d

of the difluoroformal

In these

b e l o w 0.5.

experiments,

t h e HF/SF

4

T h e f o r m a t i o n o f 2,2,4,5-

t e t r a f l u o r o - 1 , 3 - d i o x o l a n e , J^6, was i n d e p e n d e n t t u r e and c a t a l y s t

was t o

f o r c o m p a r i s o n w i t h 1,3-

the contribution

activity.

to fluorinate experiment

o f both

tempera-

concentration.

16

The

hydrogen

substitution

of 4 - c h l o r o e t h y l e n e

reaction

carbonate,

observed

f o r the fluorination

3, i s t h e p r e d o m i n a n t

this

case.

pare

2,2-difluoro-1,3-dioxolane.

reaction i n

We have been u n s u c c e s s f u l i n a l l o u r a t t e m p t s

investigation

t o determine

This reaction

the f e a s i b i l i t y

t o pre-

i s under

of preparing

further this

compound.


198

BIOCHEMISTRY INVOLVING

Structure-Activity The

•

Relationships

major s t r u c t u r e - a c t i v i t y

anesthetics

relationships

potency

o f h y d r o c a r b o n s and

i n the

order I >

•

Unsaturation

•

F l u o r i n e a d d i t i o n decreases point

and

Increased an

•

increases

volatile

halogen

potency

ethers

Br >

Cl >

increase

F.

potency.

f l a m m a b i l i t y and

of adjacent •

for

a r e as f o l l o w s :

Halogenation in

C A R B O N - F L U O R I N E BONDS

potency, increases

boiling stability

atoms.

i n a homologous s e r i e s

increase i n molecular

weight,

and

oil/gas

One

o r more h y d r o g e n atoms a r e

follows

boiling

point,

coefficient. necessary

for

CNS

depression. Since

molecules

oxygen are introduce 16.

not one

c o n t a i n i n g only carbon, u s u a l l y very

potent

chlorine into

These experiments are

fluorine,

hydrogen,

a n e s t h e t i c s , we

and

attempted

to

2,2,4,5-tetrafluoro-1,3-dioxolane, summarized

i n the

following

equations

s

16

Photochemical at

chlorination

room t e m p e r a t u r e

results

of

16

tetrafluoro-1,3-dioxolane, maximum y i e l d ther in

of t h i s

chlorination

the

with

one

significant

Photochemical 16,

with

chlorination excess

of

4-chloro-2,2,4,5products.

to date

results

The

i s 53%.

i n an

i n c r e a s e i n the

4-chloro-2,2,4,5-tetrafluoro-l,3-dioxolane,

oxolane,

of

five

obtained

r e a c t i o n mixture no

e q u i v a l e n t of c h l o r i n e

formation

along with

p r o d u c t , ^17,

of the

by-products,

with

i n the

Fur-

increase desired

17.

2,2,4,5-tetrafluoro-1,3-di-

chlorine results

i n the

4,4,5,5,-tetrachloro-2,2-difluoro-1,3-dioxolane,

formation JL8, a s

the


of major

Fluorinated

DENSON E T A L .

10.

product be

(>95%).

reported

This

Volatile

reaction

i n a future

199

Anesthetics

i s under

publication.

further

s t u d y and w i l l

We do n o t b e l i e v e

4,4,5,5-tetrachloro-2,2-difluoro-1,3-dioxolane, potent

anesthetic

cule.

Generally

since

t h e r e a r e no h y d r o g e n

one o r more h y d r o g e n s

that

18, w i l l

be a

atoms i n t h e m o l e -

are required

f o r CNS

depression. The that atom. the

structure-activity

relationships

a more p o t e n t member o f t h i s We have a t t e m p t e d

because

weak bond

i n metabolic environments.

determine

how w e l l

structure

reactivity

our c l a s s

reaction

suggest

bromine

potency without

i t i s a thermodynamically However i n a n e f f o r t t o

of d i e t h e r s adheres

r e l a t i o n s h i p s g i v e n above,

f l u o r i n a t e 4,5-dibromoethylene this

contain a

t o maximize a n e s t h e t i c

i n c o r p o r a t i o n o f bromine,

from

p r e s e n t e d above

c l a s s would

carbonate

11

t o the reported we a t t e m p t e d t o

The major

product

i

19.

11

As

19

i n the case o f the f l u o r i n a t i o n

of 4,5-dichloroethylene

carbonate,

3, we have o b s e r v e d h a l o g e n e x c h a n g e ,

conversion

i s independent

been a b l e t o i s o l a t e

of cat/SF

4

but here

concentration.

t h e u n s a t u r a t e d bromine

this

We h a v e n o t

c o n t a i n i n g 2,2-

d i f l u o r o - 1 , 3 - d i o x o l a n e , 20.

20

Experiments with T i F exchange i n t h e absence in

t h e absence

experiments ethylene 6

f

4

4

of catalyst

involving

a t 1 0 0 ° and 150°C

of SF .

Similarly,

resulted

i n no e x c h a n g e .

the f l u o r i n a t i o n

c a r b o n a t e , 11,

resulted

i n no

e x p e r i m e n t s w i t h SF, Additional

o f b o t h 4,5-dibromo-

and 4 - b r o m o - 5 - f l u o r o e t h y l e n e

carbonate,

are i n progress.


200


Physiological The search

potential

a

by

Burgison

flushed with

synthesized

onto the stance

to the

the

of

the

j a r , and Every

after

15

0.5

the

interval

The

the

j a r was

by Ough and

Stone

j a r was

sub-

calculated (15).

compound t h a t w o u l d The

of

syringe

amount o f a t e s t

not

closed quickly

facilitated

by

gentle

F i v e m i c e were q u i c k l y d r o p p e d

but

noted.

no

later

t o become a n e s t h e t i z e d Since

the

t h a n 5 min

occurrence

into

between t h e s e

two

time

(loss

of

i s considered

i n d u c t i o n t i m e f o r known p o t e n t

m i c e were k e p t on

a pan.

the

of

induction to

represent

anesthetic

l i m i t s was

i n the

The

being

removed

every

15

record The

agents,

u s e d as

tail

a

crite-

latent

In the

toxicity

first

performed to determine

each of

the

another dose-range

e a c h compound were e x p r e s s e d

as

The

by

an

point.

hour

each

5 0

and

three

second

stage,

lower

con-

selected

anesthetic

margin of

(AC

of the

in

was

a n i m a l s were

In the

anesthetic

median l e t h a l

range,

t o chose t h r e e

p o t e n c y and

w h i c h 50%

hours

conducted

Fifteen

medium e f f e c t i v e

an

If

after

administered

concentration

median a n e s t h e t i c c o n c e n t r a t i o n concentration

A

end

i n m i c e was

a n i m a l s were t e s t e d a t

anesthetic

by

minutes.

compounds.

conducted

t o determine the

anesthetized.

was

concentrations.

was

concentration

The

the

was

tested

a dose-range experiment

lethal

three

removed

f o r twenty-four

were s e l e c t e d .

Fifteen

estimated

five

as

test

substances

the

study

of the

centrations.

The

pinch

stage,

concentrations

concentration.

every

s u b j e c t s were k e p t

e v a l u a t i o n of the

stages.

at

a n a l g e s i a was

considered

j a r , the

The

then

(to righting)

showed a n a l g e s i c r e s p o n s e a h a l f

from the

any

was

minutes,

recovery

Postanesthetic

the

minutes.

appropriate

j a r f o r 10

time of

base of each a n i m a l ' s t a i l

still

aminal

be

from a

seconds

an

to

ejected the

glass jar

a m e a s u r e d amount

bottl

pressing

tested

s c r e w cap,

and

" a i r " i n the

s u b s t a n c e was

container.

s q u e a k on

two

Initially the

of each t e s t

f o r each animal.

pinching

to

minute,

re-

those

induction.

placed

noted

jar.

f o r each animal

min

optimal

r i o n of

of

r e f l e x ) was

the

and

the

saturate

similar

A wide-mouth,

methods r e p o r t e d

the

time r e q u i r e d righting

to

" a i r " were e v a l u a t e d .

evaporation

rotation

in this

procedures

i n a s y r i n g e was

concentrations

saturate

the

compound

bottom of

according

(14).

agents synthesized

o x y g e n f o r one

t h a t would

Several

and

anesthetic

were t e s t e d i n m i c e by

described was

Evaluation

safety

of

index. )

i s defined

animals are

concentration

(LC


as

an

expected 5 0

)

is

10.

DENSON

an

E T

Fluorinated

AL.

Volatile

201

Anesthetics

e s t i m a t e d c o n c e n t r a t i o n by w h i c h 5 0 % o f t h e a n i m a l s a r e

expected to

AC

5 0

to die.

.

safety.

The lower

available

the A C

5 0

,

the lower

of

LC

safer.

5 0

of

the c o n c e n t r a t i o n o f drug

However, a d r u g

i s not n e c e s s a r i l y

methoxyflurane

(AI) i s the r a t i o

the a n e s t h e t i c index t h e g r e a t e r margin

f o r metabolism.

centration

for

The a n e s t h e t i c i n d e x

The l a r g e r

acting

at lower

con-

As a l r e a d y d i s c u s s e d ,

i s nephrotoxic, yet i t s A C

i s about

5 0

half

that

halothane. Before

reviewing are

p r e s e n t i n g our p h y s i o l o g i c a l

some d a t a a v a i l a b l e

summarized

i n Table

1 (2, 8 ) .

nephrotoxicity

data are a v a i l a b l e

1,3-dioxolane,

i t i s interesting

larger

than halothane

fluorine,

hydrogen,

reported

by T e r r e l l

noteworthy

that

(8)

data,

i t i s worthwhile

f o r other anesthetics. While

These

data

no h e p a t o c e l l u l a r o r

for 2,2-(bis)trifluoromethylt o note

that

T h i s molecule

i t s AI i s s l i g h t l y

contains only

carbon

an (9)

halogenate

t h e r e was

very

little

analogs,

increase i n activity.

Table 1 ANESTHETIC POTENCY FOR FLUORINATED

ANESTHETICS

B.P. Anesthetic

(°C)

AC

50

Halothane

0.3

Fluroxene

43

1.2-8

Enflurane

57

2-4

100

0.5

2,2

(Bis)trifluoro-

AI

MAC

5 0

2.74

0.78

105

Methoxyflurane

LC

5 0

0.78

3.50

-

-

0.23 6.0 2.2

-

4.70

2.38

methyl-1,3-dioxolane

We

began o u r p h y s i o l o g i c a l

standard. is

slightly

Table ized

1).

gained rapid

we o b t a i n e d a n A I o f 4.75,

h i g h e r t h a n t h e A I o f 3.50

previously

by p t o s i s

was c h a r a c t e r i z e d

the r i g h t i n g

reflex,

accompanied

by " f l a t

r e c o v e r y t i m e s were o b s e r v e d ,

elimination.

(Rapid e l i m i n a t i o n

lipophilicity.)

At l e t h a l

e a r s were o b s e r v e d

tails."

they maintained

by

i n addition

i s character-

lacrimation.

Once t h e a n i m a l r e it.

indicating

In a d d i t i o n , rapid

halothane

i s often associated with

c o n c e n t r a t i o n s , dark

T a b l e 2 summarizes our f i n d i n g s

t o p t o s i s and

as a which

reported (see

At a n e s t h e t i c concentrations, halothane

during induction

Recovery

e v a l u a t i o n s using halothane

In these experiments

eyes

lower

and b l a n c h e d

lacrimation.

f o r halothane.


202

BIOCHEMISTRY

INVOLVING

CARBON-FLUORINE

BONDS

Table 2 EFFECTS OF HALOTHANE CF -CHBrCl, ON MICE (AI = 4.75) 3

Cone,

No of

Vol

Animals

%

Time, min 0.78

Deaths 0

5

0.83

0

1.00 1.06

15 15

-

7 12

1.03

0

4.00

15

8

15

7.72

3

5.00

15

13

15

8.13

9

15

8.83

12

15

14

anesthetic concentration 12, minimum h i n d

l e g movement and p t o s i s were

no l a c r i m a t i o n o r c o n v u l s i v e b e h a v i o r .

terized

by t h e a n i m a l s

had

a bouncy g a i t .

for

halothane,

running

Recovery

in circles.

times

elimination

suggests

M i c e were w o b b l y and

Ptosis

than

t h a t , e v e n a t low

a higher

4 , 5 - d i c h l o r o - 2 , 2 - d i f l u o r o - 1 , 3 - d i o x o l a n e , 12.

t r a t i o n s were c h a r a c t e r i z e d by i r r e g u l a r bradycardia.

charac

f o r 1£ were much l o n g e r

i s not n e a r l y as r a p i d

Slower e l i m i n a t i o n

observed,

R e c o v e r y was

a s shown i n T a b l e 3, s u g g e s t i n g

concentrations, halothane. for

Mean Recovery

15

dioxolane, but

Induced

0.94

5.50

At

Induced

before 30 sec before 5 min

as i t i s f o r

lipophilicity L e t h a l concen

respiration

a n d l a c r i m a t i o n were o b s e r v e d

and

apparent

i n a few

animals.

Table 3

EFFECTS OF

:c> |

\ F„ ON MICE

o

/

12

(ΑΙ = 7.88) Cone,

No of

Vol

Animals

%

Induced

Induced

before 30 sec before 5 min

Mean Recovery time, min

0

Deaths

0.25

15

-

0.30

15

2

0.63

0

0.35

15

-

13

10.60

0

2.00

15

-

15

60.08

0

2.50

15

-

15

60.47

6

3.00

15

1

15

120

0

14


10.

DENSON

ET

Anesthetic dioxolane, siveness, animals

Fluorinated

AL.

Volatile

concentrations of 4-chloro-2,2,5-trifluoro-1,3-

13, were c h a r a c t e r i z e d by h y p o a c t i v i t y , s l i g h t ptosis,

and o c c a s i o n a l l a c r i m a t i o n .

often ran i n c i r c l e s

Recovery times,

given

and some l o s t

i n Table

t h e AI d e t e r m i n e d

13^, was a p p r o x i m a t e l y

halothane,

recovery

mos)

times

those

reflexes.

reported

(see Table 3 ) .

t h e same a s t h e A I d e t e r m i n e d

were somewhat

the animals'

longer

f o r 13.

e y e s became e x t r e m e l y

and deep j e r k y r e s p i r a t o r y

breathing

righting

half 12

convul-

recovery the

f o r4-chloro-2,2,5-trifluoro-1,3-

dioxolane,

concentrations,

During

their

4, were a b o u t

for 4,5-dichloro-2,2-difluoro-1,3-dioxolane, Although

203

Anesthetics

response

was

At

dark

for

lethal

(exophthal-

f o l l o w e d by

shallow

and b r a d y c a r d i a .

Table

4

0
^0

X

Compound

Y

AC

Halothane

0.99

4.7

4.75

CI

Cl

0.33

2.6

7.88

13

CI

F

0.88

4.1

4.66

16

F

_

_

The

that

results

d i e d at

compound.

of the

a d m i n i s t r a t i o n of

the

LC

Further

pound

computing

three

high

the

high

concentrations

percentage of

concentration

p e r c e n t a g e s were p l o t t e d a g a i n s t 2.

From t h e

three

a n a l y s i s showed t h a t

indicating

i s considerably

derived

t h e AI

of

the

higher

levels

of

each

concentrations

as

margin of

than that

listed

than that

s a f e t y of

of

in

Table

4,5-dichloro-2,2-

i s s u b s t a n t i a l l y higher that

of

animals

concentration-response

o f e a c h compound was

5 0

difluoro-1,3-dioxolane halothane,

by

each of the

The

shown i n F i g u r e

curves, 6.

5 0

12

compounds were a n a l y z e d

as

AI

LC

5 0

the

of

new

com-

halothane.

Summary From t h e trends

o x o l a n e s do present. rine

summary o f o u r

data

in structure-activity not

As

contain

could

i s replaced

be

by

6,

we

can

see

some

A l t h o u g h our d i -

b r o m i n e , good a n e s t h e t i c a c t i v i t y

predicted,

fluorine.

chlorine i s replaced

cients will

be

determined

and

activity

increases

new

c l a s s of

potent

The

structure activity

the

The

d e c r e a s e as

Anesthetic

i n Table

relationships.

activity

lipophilicity

by

fluorine

reported as

the

in a

is

d e c r e a s e s as appears

(partition

chlo-

to coeffi-

later publication).

boiling

point

of

the

series

increases. A oped.

volatile

pounds a p p e a r t o f o l l o w t h o s e compounds i n t h i s

c l a s s are

anesthetics

has

r e l a t i o n s h i p s of t h i s reported

being

i n the

prepared

and

more d e t a i l e d p h y s i o l o g i c a l e x a m i n a t i o n s w i l l

been

devel-

c l a s s of

literature. evaluated, be

and

conducted.


com-

Other

10.

DENSON

Fluorinated

E T A L .

Volatile

207

Anesthetics

Acknowledgement s The search

authors

gratefully

by I n s t i t u t e s

Division,

acknowledge t h e support

of Health,

under Grant

General

Medical

of this re

Sciences

Number 5-R01-GM-20082-02.

Discussion Q:

How l o n g d i d y o u o b s e r v e

the animals

f o r evidence

of latent

toxicity? A:

In a l l cases

Q:

A r e t h e compounds s t a b l e t o base?

A:

Y e s ! You c a n c o n v e r t carbonate are

Q:

f o r at least

using

t h e d i f l u o r o f o r m a l group

concentrated

also stable t o mild

sulfuric

acid.

back t o t h e

T h e s e compounds

acid.

I s t h e r e any s p l i t t i n

A:

No, a t l e a s t

Q:

The p o s s i b i l i t y on

A:

24 h o u r s .

we hav exists

f o r other

isomers.

C a n y o u comment

that?

We c a n n o t

predict

or d e a l i n g with isomers

w h e t h e r we a r e s t u d y i n g one a c t i v e

unknown m i x t u r e s .

a r e present,

We c a n s a y t h a t

we a r e n o t o b s e r v i n g

an a c u t e

isomer

i f other lethal

effect.

Literature Cited 1. Cascorbi, H. F., "Anesthesia Toxicity," in 1974 Annual Refresher Course Lectures, American Society of Anesthesi ologists Annual Meeting, Washington, D. C., October 12-16, 1974, Lecture number 227 and references cited therein. 2. Larsen, Ε. R., "Fluorine Compounds in Anesthesiology," in Fluorine Chemistry Review, P. Tarrant, Vol. 3, pp. 1-44 (1969) and references cited therein. 3. Robbins, J. H., J. Pharmacol. Exptl. Therap. (1946), 86 197. 4. Brown, B. R., Jr., "Enzymes and Anesthesia," in 1974 Annual Refresher Course Lectures, American Society of Anesthesiolo gists Annual Meeting, Washington, D.C., October 12-16, 1974, Lecture Number 225 and references cited therein. 5. Van Dyke, R. A. and Chenoweth, Μ. Β., Anesthesiology (1965), 26, 348. 6. Tucker, W. K., Rackstein, A. D., and Munson, E. S. Brit. J. Anaesth., (1974) 46, 392. 7. Cohen, E., et al., Anesthesiology (1974), 41, 321. 8. Gilbert, Ε. E., (to Allied Chemical Co.), U.S. Pat. 3,314,850 (1967).


208


BONDS

9. Terrell, R. C., and Moore, G. L., (to Airco, Inc.), U.S. Pat. 3,749,791 (1973). 10. Virtue, R. W., Proc. Soc. Exptl. Biol. Med., (1950), 73, 259. 11. (a) Newman, M. S., and Addor, R. W., Amer. Chem. Soc. (1953) 75 1263. (b) Newman, M. S., and Addor, R. W., J. Amer. Chem. Soc., (1955), 77, 3789. 12. Olah, G., Nojima, Μ., and Kerekes, I., Synthesis, (1973) 779. 13. Applequist, D. E., and Searle, R., J. Org. Chem. (1964), 29, 987. 14. Burgison, R. Μ., "Animal Techniques for Evaluating Anesthetic Drug," in Animal and Clinical Pharmacologic Techniques in Drug Evaluation, J. H. Nodine and P. E. Siegler, eds., Yearbook Medical Publishers Inc, Chicago, Ill. (1964), pp. 369-372. 15. Ough, C. S., and Stone, , , (1961), , 16. Litchfield, J. T., Jr., and Wilcoxon, F. Α., J. Pharm. Exptl. Ther. (1949), 96, 99.


INDEX A AC 200 ACD 121 N-Acetylserotonin 39 iV-Acetyltransferase 39 adrenergenic induction of 41 cyclic A M P induction of 43 pineal 3 induction of 4 N-Acetyltryptamine 3 Acid(s) amino, analogs 37 electrophoresis of fluorocitric 10 erythrofluorocitric 8 fluoroacetic 8 fluorocarboxylic 1-19 fluoro-dicarboxylic 2 fluorourocanic, effects on urocanase 31 inhibitory properties of F-carboxylic 3 monofluorocitric 7 isomers of 9 nicotinic 90,91 perfluoro-octanoic 129 substrate properties of F-carboxylic 3 Aconitase, cytoplasmic 12 Actinomycin 28 Activities of fluorohistidines, antiviral 29 Activity, ornithine decarboxylase 46 Adenosine 3',5'-monophosphate 39 Adipose tissue 91 Adrenalin 41 Adrenergenic agents 41 Adrenergenic induction of N-acetyltransferase 41 AI 201 5-AICAR 32,33 Alditol Ill Aliphatic fluorine 77 Alphamethyldopa 37 Alveolographic studies with R F C 178 Amethopterin 57 Amine drugs 77 Amino acid analogs 37 p-Aminobenzoylglutamate 60 3-Aminomethyl-pyridine 90,91 A M P , cyclic 43 Amphetamine 78 in rat brain 81 A M P induction of N-acetyltransferase, cyclic 43 8 0

Analgesia Analogs, amino acid Anesthesia, surgical Anesthetic(s) concentration, median cyclic diether fluorinated volatile hazards of fluorinated

190 37 190 200 193 190-206 191

3,4-Anhydro-D-galactose 104 Antilipolytic drug 91 Antiviral activities of fluorohistidines 29 A T P synthesis 17

Β Benz[a]anthracene induction 43 Biochemistry of ring-fluorinated imidazoles 23-33 Biological disposition of R F C 181 Bonds, phosphate 38 Brain, amphetamine in rat 81 Brain, /?,/?-difluoroamphetamine in rat 81 Brominated fluorocarbons 171-186 Bronchography with R F C emulsions 180

C F-Carboxylic acids inhibitory properties of 3 substrate properties of 3 Catalysis of M D H 2 Catecholamines 41 CF dUMP 66 Chlorination, photochemical 198 p-Chloroamphetamine 88 Chloroform 191 Cholecystography 186 Chymotrypsin 38 Cis-trans perfluorodecalin emul sions 135-167 Citrate-isocitrate exchange 6 Complexes, Michaelis-Menten 2 Cyanosis 190 Cyclic A M P induction of N-acetyl transferase 43 Cyclic diether anesthetics 193 Cycloheximide 28 N-Cyclopropyl amines 92 Cytochalasin Β 101 Cytoplasmic aconitase 12 3


212

BIOCHEMISTRY INVOLVING CARBON—FLUORINE BONDS

D Deaminase, liver microsomal 79 Deamination, oxidative 82 Defluorination of fluorocitrate 14 4- Deoxy-D-glucose 104 Deoxyfluoro-D-glucoses 101 Deoxyfluoro-monosaccharides 99-113 Dephosphorylation 38 Dibutyryl adenosine 3',5'-monophosphate 43 Dibutyryl cyclic AMP 43 Dibutyryl adenosine 3',5'-monophosphate 43 Dibutyryl cyclic AMP 43 Diether anesthetics, cyclic 193 Difluoro-oxalacetate 2 0,/?-Difluoro substitution 79-80 β,/^Difluoroamphetanune 83,86 in rat brain 81 Drug, antilipolytic 9 Drugs, amine 7 dUMP 57

Fluoro-dicarboxylic acids 2 Fluorohistidines, antiviral activities of 29 Fluorohistidines, effects on histidine ammonia-lyase 30 2-Fluorohistidine 26 4-Fluorohistidine 30 2-Fluoro-L-histidine 37 Fluoroimidazole(s) 26 ribosides 33 4-Fluoroimidazoles, synthesis of 24 Fluorourocanic acids, effects on urocanase 31 Fluroxene 191,192

G D-.Galactose Gastroenerography GI tract, RFC in the

104 173 175

Gluconeogenesi Glucose-6-phosphatase Gregaria, Schistocerca

38 109

£ E. coli, effect offluorocompounds on 29 Efflux, inhibition of isocitrate 16 Efflux, rates of isocitrate 15 Electrophoresis offluorocitricacid 10 Emulsions bronchography with RFC 180 cis-trans perfluorodecalin 135-167 of RFC 180 Enflurane 191,192 Enzymatic probes 1-19 Enzyme induction 37, 43 Enzymes, histidine in 37 Erythrocyte membrane, human 99 Erythrofluorocitrate 11 Ervthrofluorocitric acid 8 Etners, synthesis of fluorinated 193 Ethylamine 77 Ethylene carbonate 194 Exchange, citrate-isocitrate 6 F FdUMP 59 5- FICAR 32,33 Fluorinated volatile anesthetics 190-206 Fluorine, aliphatic 77 Fluoroacetic acid 8 Fluoro compounds, effect on E. coli 29 Fluorocarbons, brominated 171-186 Fluorocarbons of high vapor pressure 176 Fluorocarboxylic acids 1-19 Fluorocitrate 7 defluorination of 14 Fluorocitric acid, electrophoresis of 10 5-Fluoro-2'-deoxyuridylate 59

H Halothane 191 effects on mice 202 Hazards offluorinatedanesthetics .... 191 Hepatocytes 143 Hepatography 185 Histidine 28 ammonia-lyase, effects of fluoro histidines on 30 in enzymes 37 incorporation into protein 50 Human erythrocyte membrane 99 Human plasma 117-133 HydTOxylindole-O-methyltransferase.

Hyperlipoproteinemia Hyperthermia Hypotensive effect

50

90 83 137

I Imidazole(s) 26 biochemistry ofring-fluorinated..23-33 pharmacology of ring-fluorinated 23-33 Incorporation of ( H) 2-fluoro-Lhistidine into protein 52 Incorporation of histidine into protein 50 Induction of N-acetyltransf erase, adrenergic .. 41 of Ν-acetyltransferase, cyclic AMP 43 benz [ a ] anthracene 43 enzyme 37,43 of pineal N-acetyltransferase 41 steroid 43 Inhibition of isocitrate efflux 16 Inhibition of monoamine oxidase 94 3


213

INDEX

Inhibitory properties of F-carboxylic acids 3 Intraabdominal surgery 190 Isocitrate efflux, inhibition of 16 Isocitrate efflux, rates of 15 Isomers of monofluorocitric acid 9 Isoproterenol 41,55

L Lactobacillus casei LC Lethal concentration, median Lipolysis Lipophilicity Liver microsomal deaminase Locusta migratoria Lymphography 5 0

57,72 200 200 90 201 79 109 181

M M D H , catalysis of 2 Median anesthetic concentration 200 Median lethal concentration 200 Melatonin 39 Membrane, human erythrocyte 99 Metabolic probes 1-19 Metabolism, sorbital Ill Methoxyflurane 191,192,201 N-Methyltransferase 79 Mice, halothane effects on 202 Michaelis-Menten complexes 2 Microsomal deaminase, liver 79 Migratoria, Locusta 109 Mitochondrial monoamine oxidase .... 80 Monoamine oxidase, inhibition of 94 Monochloroethylene carbonate 194 Monodeoxy-D-glucoses 99 Monodeoxymonofluoro-D-glucoses 99-100 Monofluorocitric acid 7 isomers of 9 Monofluoro-malate 5 Mosquito 111 Multienzymatic processes 1 Muscle relaxation 190

Ν Narcosis Nicotinic acid Norepinephrine

190 90, 91 83,92 Ο

Organic fluoro compounds Ornithine decarboxylase activity Oxidase, inhibition of monoamine Oxidative deamination

117-133 46 94 82

Ρ Pancreatography Para-chlorophenylalanine Para-fluorophenylalanine Perfluorochemicals Perfluorodecaline emulsions, cis-trans Perfluorohexylbromide Perfluoro-octanoic acid Peritoneal contamination PFC PFD Phagocytosis of R F C Pharmacology of ring-fluorinated imidazoles Phenethylamine C-Phenethylamine Phosphate bonds Phospholipid 14

186 37 37 135 135,147 135-167 185 129 175 135 135 176 23-33 84 86 38 147

Pineal N-acetyltransferase 39 induction of . 41 Pineal gland 39 Polyols HI Potency for fluorinated anesthetics 201 Pressure, fluorocarbons of high vapor 176 Probes, enzymatic 1-19 Probes, metabolic 1-19 Processes, multienzymatic 1 Protein, incorporation of ( H ) 2 fluoro-L-histidine into 52 Protein, incorporation of histidine into 50 Ps. aeruginosa 107 Ps. fluorescens 107 Pseudomonad 107 Ptosis 203 Pyrimidines 67 3

R Radiopaque fluorocarbon 173 Rat brain, amphetamine in 81 Rat brain, /?,^difluoroamphetamine in 81 Rates of isocitrate efflux 15 Relaxation, muscle 190 RFC 173 alveolographic studies with 178 biological disposition of 181 emulsions 180 bronchography with 180 in the GI tract 175 phagocytosis of 176 submersion in 176 Ribavirin 32 Ring-fluorinated imidazoles biochemistry of 23^33 pharmacology of 23-33 RNAse 38


214

BIOCHEMISTRY

S Schistocerca gregaria 109 Serine 38 Silk worm Ill Sorbitol dehydrogenase Ill Sorbitol metabolism Ill Splenography 185 Steroid induction 43 Submersion in R F C 176 Substrate properties of F-carboxylic acids 3 Surgery, intraabdominal 190 Swallowing reflex 190 Synthesis ATP 17 of fluorinated ethers 193 of 4-fluoroimidazoles 24 Synthetase, thymidylate 57-74


BONDS

Thymidylate synthetase 57-74 Tissue, adipose 91 TMP 57 Toxicity of 3-deoxy-4-fluoro-D-glucose 109 Triethyl fluorocitrate, N M R of 10 β,β,β-Trifluoroethylamine 77 5-Trifluoromethyl-2'-deoxyuridylate .. 66 Tryptamine 39

U Urocanase, effects of fluorourocanic acids on Urography

31 186

V

Τ Tachypnea Threonine

203 38

Ventriculomyelography Vinylidene carbonate


185 195

Biochemistry Involving Carbon-Fluorine Bonds

Involving Parents

Differential systems involving impulses

Silken Bonds

Mating Bonds

Primal Bonds

Primal Bonds

Chemical Bonds

Primal Bonds

Silken Bonds

Biochemistry

Biochemistry

Primal Bonds

Soul Bonds

Beloved Bonds

Soul Bonds

Differential systems involving impulses

Hydrogen Bonds

Soul Bonds

Silken Bonds

Primal Bonds

Silken Bonds

Beloved Bonds

Primal Bonds

Biochemistry

Biochemistry

Biochemistry

Biochemistry

Biochemistry (Biochemistry (Berg))

Biochemistry

Biochemistry

Biochemistry Involving Carbon-Fluorine Bonds

Involving Parents

Differential systems involving impulses

Silken Bonds

Mating Bonds

Primal Bonds

Primal Bonds

Chemical Bonds

Primal Bonds

Silken Bonds

Biochemistry

Biochemistry

Primal Bonds

Soul Bonds

Beloved Bonds

Soul Bonds

Differential systems involving impulses

Hydrogen Bonds

Soul Bonds

Silken Bonds

Primal Bonds

Silken Bonds

Beloved Bonds

Primal Bonds

Biochemistry

Biochemistry

Biochemistry

Biochemistry

Biochemistry (Biochemistry (Berg))

Biochemistry

Biochemistry

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