Advances in Applied Microbiology, Volume 44

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ADVANCES IN

Applied Microbiology VOLUME 44

This Page Intentionally Left Blank

ADVANCES IN

Applied Microbiology Edited by SAUL L. NEIDLEMAN Oakland, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 44

Academic Press San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1997 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at thebottom of the first page of a chapter in this book indicates the Publisher's consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923) for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1997 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2164197 $25.00

Academic Press a division of Harcourt Brace 6.Company 15 East 26'h Street, 15* floor, New York, New York 10010, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Serial Number: 0065-2164 International Standard Book Number: 0-12-002644-9

PRINTED IN THE UNITED STATES OF AMERICA 97 98 99 00 0 1 02 BB 9 8 7 6 5

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CONTENTS

Biologically Active Fungal Metabolites

CEDIUCPEARCE 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. A Brief History of Fungal Products in Medicine. ......................

The Potential of Fungally Derived Chemical Diversity. . . . . . . . . . . . . . . . . . Approaches to Growth and Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammalian Enzyme Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cholesterol Biosynthesis and Lipid Metabolism Inhibitors . . . . . . . . . . . . . . Receptor Binding Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiinfective Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antitumor and Cytotoxic Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Miscellaneous Pharmaceutical Activity. ............................. XI. Agriculturally Active Compounds . . . . . . . . . . . XII. Summary and Conclusions. . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. IV. V. VI. VII. VIII. IX.

1 2 3 3 5 13

20 25 47 51 57 66 68

Old and New Synthetic Capacities of Baker’s Yeast

P. D’ARRIGO, G. PEDROCCHI-FANTONI, AND s. SERVI 11. Reducing Capa 111. The Formation

IV. Oxidations: Ge V. Hydrolytic Act VII. The Biogeneration of Aroma Compounds

IX. Conclusions. . . . ................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 119

Investigation of the Carbon- and Sulfur-Oxidizing Capabilities of Microorganisms by Active-Site Modeling

HERBERT L. HOLLAND I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Models for Microbial Hydroxylations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Models for Sulfoxidation Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. SummaryandPrognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

125 133 150 158 159

vi

CONTENTS

Microbial Synthesis of o-Ribose: Metabolic Deregulation and Fermentation Process

P. DE WULF AND E. J.

VANDAMME

I. Introduction. . . . . . ................ 11. Natural Occurrence d Its Derivatives . . 111. Physicochemical Characteristics of D-Ribose. . . . . . . . IV. Detection and Identification of D-Ribose . . . . . . . . . . V. Applications of D-Ribose . . . . . . . . . . . . . . . . . . . . . . VI. Nonmicrobial Production of D-Ribose . . . . . . . . . . VII. Microbial Production of D-Ribose . . . . . . . . . . . . . . . . . . VIII. Pleiotropic Properties of D-Ribose-Producing Tran Bacillus Mutant spp.. . . . . . . . . . . . . . . . . . . . . . . . IX. o-Ribose Production by Fermentation with Bacillus X. Kinetics of D-Ribose Production by Bacillus spp.. . . XI. Conclusions and Future Perspectives . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . ...

...........

168 168 171 172 172 176 180 188 191 203 204 205

Production and Application of Tannin Acyl Hydrolase: State of the Art P. K. LEKHAAND B. K. LONSANE 11. Historical Highlights. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Tannin-Hydrolyzing Enzymes . . . . . . . . . IV. Source ofTannase . . . . . . . . . . . . . . . . . . .

VIII. Location of Tannase

.......................

X. Properties of Tannase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................. XIV. Applications of Tannase

216 216 218 222 226 228 237 239 239 241 246 246 249 250 255 255

Ethanol Production from Agricultural Biomass Substrates

c.

RODNEYJ. BOTHASTAND BADAL SAHA I. Introduction. . . . ............................... ..... 11. Lignocellulosic Biomass . . . . . . . . . . . . . . . . . . . . . . . ................. 111. Pretreatment . . . . . . . . . . . . . . . . . . . . . .................

V. Fermentation. . . ............... VI. Technological Co p . . . . . . . . . . .. VII. Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .. ....

261 263 265 267 2 74 279 282 282

CONTENTS

vii

Thermal Processing of Foods, A Retrospective, Part I: Uncertainties In Thermal Processing and Statistical Analysis

1. 11. 111. IV. V. VI.

M. N. RAMEsH, S. G. PRAPULLA, M. A. KLJMAR, AND M. MAHADEVAIAH ..................... ..................... .................... tion . . . . . . . . . . . . . . . . . .............. usions . . . . . . . . . .............. rences . . . . . . . . . .........

.. .. ..

..

.. ..

*.

288 289 305 308 310 311 312

Thermal Processing of Foods, A Retrospective, Part I I: On-Line Methods for Ensuring Commercial Sterility

M. N. M E S H , M. A. KUMAR,S. G. PRAPULLA, AND M. MAHADEVAIAH I. 11. 111. IV. V.

Introduction.. . . . . . . . FO Integrators. . . . . . . . ........ On-Line Monitoring Sy s . . .............................. Semiautomatic Retort Control Systems for Optimum Sterilization. . Computer-Aided Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

VIII. Conclusions

.. ..

316 317 321 324 326 327 341 342 343

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

347

....

355

.. ..

.. ..

.. ..

CONTENTS OF PREVIOUS VOLUMES. ....................


Biologically Active Fungal Metabolites CEDRICPEARCE MYCOsearch, a subsidiary of Oncogene Science Inc. Durham, North Carolina 27707

I. Introduction 11. A Brief History of Fungal Products in Medicine 111. The Potential of Fungally Derived Chemical Diversity Iv. Approaches to Growth and Nutrition V. Mammalian Enzyme Inhibitors VI. Cholesterol Biosynthesis and Lipid Metabolism Inhibitors VII. Receptor Binding Antagonists VIII. Antiinfective Agents IX. Antitumor and Cytotoxic Activity X. Miscellaneous Pharmaceutical Activity XI. Agriculturally Active Compounds XII. Summary and Conclusions References

I. Introduction

The role of fungal metabolites in medicine and the unwanted biological effects of fungal metabolites have been observed and studied for many years. That being so, the intent of this chapter is to review more recent literature reporting significant biologically active compounds from fungi, drawing from those published up to December 1996. In many ways this is an exciting time for the discovery of bioactive metabolites from fungi, with new cultures being isolated from a wider variety of habitats and substrates and with the application of a greater understanding of fungal metabolism becoming more apparent by employing more sensitive and novel bioassays as screens, and by applying the recent advances in chemistry that have made the isolation and characterization of these compounds much more rapid. Analysis of the literature shows that proportionally more bioactive fungal products than actinomycete metabolites are being reported now than a decade ago. There are a number of excellent books and reviews of earlier work; for example, see Turner (1971), Turner and Aldridge (1983), Cole and Cox (1981),and Gloer (1995a, 1996). It is not the intention of this review to be encyclopedic nor to evaluate the relevance of any bioactivity to its translation into a mechanism of action for a new drug. Although 1 ADVANCES IN APPLIEO MICROBIOLOGY, VOLUME 44 Copyright 0 1997 by Academic Press, Inc. All rights of reproduction in any form reserved. 0065-2164/97$25.00

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mycotoxins are strictly bioactive, they have not been included unless the ones described in the literature have demonstrated some more useful medicinal or agricultural attribute.

II. A Brief History of Fungal Products in Medicine

In the past 50 years, there have been a number of highly successful drugs based on fungal metabolites, and these have enjoyed a huge global market. Penicillin, for example, a potent antibiotic against sensitive Gram-positive bacteria was first observed many years ago being produced by a Penicillium notatum by Fleming (1946). Subsequent research showed that penicillins are also produced by Penicillium chrysogenum and a variety of other organisms (Lechevalier, 1975). Penicillin G, the original metabolite isolated, is still used clinically, and the utility of this and the very important semisynthetic derivatives as crucial antibacterial weapons cannot be overestimated, even in the face of antibiotic resistance. Studies on the biosynthesis and other biochemical, physiological, and genetic aspects of the production of these antibiotics is ongoing in many academic laboratories, and results from this work will undoubtedly provide a better understanding of how and why these organisms produce such compounds. The work of Baldwin and colleagues on p-lactam antibiotics is a high point in this area (Roach et al., 1995, and the references therein). The immunosuppressant cyclosporine was first discovered as an antifungal agent produced by a Tolypocladium inflatums (Beauvaria nivea) and Cylindrocarpon lucidum (Dreyfus et al., 1976). It was subsequently discovered to have excellent immunosuppressive activity and is used for treatment following organ transplant (Goodman Gilman et al., 1985). Cyclosporine A has been reported from a variety of different common organisms, including many strains of Tolypocladium inflatum and ?: geodes, as well as Acremonium, Beauvaria, Fusarium, Paecillomyces, and Verticillium species (Sanglier et al., 1990). Finally, among of the most successful drugs derived from a fungal product are the cholesterol biosynthesis inhibitors related to Lovastatin, which was initially reported by Merck from an Aspergillus terreus (Vagelos, 1991; see Section VI). There were reports of related compounds from Penicillium brevicompactum and Penicillium citrinum from groups at Beecham’s (Brown et al., 1976) and Sankyo (Endo et al., 1976). These compounds inhibit the rate-limiting step in cholesterol biosynthesis, HMG-CoA reductase, and they are used clinically to reduce cholesterol levels.

BIOLOGICALLY ACTIVE FUNGAL METABOLITES

3

Fungal natural products still provide important lead structures for drug development, a recent example being mycophenolic acid, mofetil. Mycophenolic acid was initially discovered from a Penicillium brevicompacturn in 1896 and subsequently found from a number of other penicillia. The structure was reported in 1952. Various bioactivities associated with this compound were discovered, including its immunosuppressive action (Wu, 1994). The mofetil derivative is a pro-drug that is broken down in the body to liberate mycophenolic acid, which inhibits the biosynthesis of certain precursors of nucleic acids and thereby inhibits proliferation of the cells involved in the immune response. Syntex/Roche has developed mycophenolic acid mofetil as an immunosuppressive drug, and this has been recently approved for use following kidney transplantation (Anonymous, 1995). This is a good example of the need to test the bioactivity of as many known natural products as possible in any new drug discovery screen, since a previously known compound can have the activity required and be the lead sought. Ill. The Potential of Fungally Derived Chemical Diversity

Hawksworth (1991) estimated that there are one and a half million fungi in existence, although at that time only about 69,000 had been described in the literature. Assuming that the actual rate of description of organisms by fungal taxonomists is approximately linear, that number may now be between 75,000 and 80,000. This is still small compared to the one and a half million thought to exist, and, apparently, the majority of fungi inhabiting the world have not been described. By implication, most of these fungi have not been screened in drug discovery programs. The potential chemical diversity of this vast untapped resource is surely one of the great driving forces behind today’s search for novel metabolites for use as drugs or leads to those drugs. IV. Approaches to Growth and Nutrition

Fungi require carbon, nitrogen, phosphorus, sulfur, minerals, vitamins, and other growth factors. They are sensitive to temperature and oxygen/carbon dioxide in their environment; pH is also a critical factor (Jennings, 1995). Nutrition to support growth is generally not difficult to satisfy, especially since many of cultures are isolated by growing out from substrates. Many of the nutritional requirements can simply be satisfied by using plant material. The challenge faced is to provide the organisms with conditions that allow expression of secondary metabo-

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lism and accumulation of unusual metabolites (Martin et al., 1982; Bushell, 1989; Tanaka, 1992; Demain, 1992). There is probably no such thing as the best culture medium that will allow any fungus to express its secondary metabolic capability, and it is impossible to predict what a freshly isolated fungus will require for metabolite accumulation. In large screening programs, in order to manage the risk that the cultures as a group are being given a fair chance to express secondary metabolism, a number of media are generally employed. Initial evaluation of media is usually made on three levels: (1) suitability for fungal growth and metabolite accumulation; (2) compatibility with the screen being used; and (3) effect on isolation chemistry. Using HPLC in conjunction with a high-hit-rate bioassay, such as Bacillus subtilis, the suitability of a medium can be determined; this should probably be considered as a short-term fix that has to be confirmed by constantly monitoring the positive rate in the assays being employed for the drug discovery program. Selection of media is complex since the possible variations are so large. Simple media such as potato dextrose work very well as broth and agar, and this has been validated many times, with novel bioactive compounds being produced. Variations using potato dextrose media have been employed, including starting with fresh potato, although the most efficient is to simply employ commercial media, possibly supplementing with other nutrients. A more complex but commonly employed approach is to add a rapidly utilized sugar, for example, dextrose, and a more slowly utilized carbohydrate, for example, mannitol, together with a nitrogen source and minerals, in a medium. Fungi will frequently grow rapidly using the glucose and then enter a slower-growing phase favoring secondary metabolism. Many good ideas can be obtained from the literature (Jennings, 1995). Ammonia levels seem to control biosynthesis of many compounds. When fermentations were carried out using ammonia-trapping agents, up to tenfold increases in titers were observed (Tanaka, 1992). One approach to controlling this is to use either a complex slowly utilized nitrogen source such as soy meal or to use an ammonium salt of an organic acid, with the latter acting as the carbon source. If each part of the salt is utilized at the same rate, no accumulation of ammonium results and no dramatic change in pH is observed. Media containing an inorganic nitrogen source such as ammonium nitrate could be used providing the cultures are incubated long enough so that a stationary phase is entered well before the fermentation is processed. There is also a large amount of literature showing that phosphate levels control secondary metabolism (Tanaka, 1992) and that metabolite


5

accumulation is triggered once this has been depleted. Examples include the production of cephalosporins, a number of aminoglycosides, and chloramphenicol. This led to the development of media containing low phosphate and to media containing phosphate trapping agents. Use of these agents resulted in fivefold increases in antibiotic production in some cases. However, some groups deliberately used high-phosphate media and proceeded to discover novel compounds whose biosynthesis was not controlled by this mechanism. The implication is that by using such an approach they increased their chances of finding novel compounds; certainly, many new compounds were found. Some of these approaches have been used to grow actinomyces only, but would be worth investigating for fungal secondary metabolism. A variety of solid substrates can be usefully employed. These included nutrient grains such as rice or corn, or nonnutrient vermiculite containing a nutrient liquid medium. This type of environment has a number of advantages, most importantly that this reflects the natural state for a substrate encountered in the field and provides a suitable environment for partial or complete expression of the life cycle along with the associated chemical signaling agents. Further variations can be introduced by changing the incubation time and temperature; with a lean medium the incubation time can usually be shortened since the organisms become nutritionally stressed earlier. Temperatures either lower or higher than the traditional ZZOC are known to have a profound effect, but again there is no way of predetermining this. There are many other combinations of carbon source, nitrogen source, phosphate, sulfur, minerals, etc., which can be tried, and should be. Resins can be added to the media that bind certain metabolites and thus increase their production, or alter metabolism in a way that leads to novel compounds accumulating. This often results in dramatic changes in titers and types of end-products. V. Mammalian Enzyme Inhibitors

There have been many enzyme inhibitors isolated from microorganisms and fungi, and the mechanism of action for many bioactive compounds can be interpreted in terms of the inhibition of some key reaction. The search for an anticancer fungal metabolite is constant and is rejuvenated periodically by the identification of new targets. Protein kinases are a group of enzymes that phosphorylate proteins as a means of regulating their conformation and activity. Two such families of enzymes that play a vital role in cellular physiology are the protein kinase C group (PKC)and the protein tyrosine kinases (PTKs).The PKCs

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(2)Emodin

(1 ) Balanol

0

H STRUCTURES 1, 2.

phosphorylate serine or threonine, while the PTKs are responsible for the phosphorylation of certain tyrosine residues. Both of these enzymes have been identified as playing crucial roles in cell proliferation and differentiation (Nishizuka, 1988; Chen et al., 1987). The PTK enzymes appear to be deregulated during tumor transformation to malignancy, as have PKCs in certain circumstances. PKC is the receptor for phorbol esters that promote tumor formation. The role of these enzymes in the development of cancer makes them an ideal target for screening natural product extracts. One of the more potent protein kinase C inhibitors ever to be reported is balanol (I), which was discovered from a fungus, Verticillium balanoides isolated by Barry Katz of MYCOsearch from a rhizomorph of a pine. Fermentations of this culture initially produced in MYCOsearch laboratories showed activity against PKC, the active compound (balanol) being isolated and characterized by the Sphinx Pharmaceuticals natural products group (Kulanthaivel et. al., 1993). Balanol was shown to inhibit PKC isozymes in the nanomolar range, having equal or better potency than the actinomyces product staurosporin, as well as showing some activity against PKA (Kulanthaivel, 1995). Three hundred analogues were subsequently prepared for structure activity studies. Unfortunately, the selectivity observed in the reported experiments was not sufficient to warrant pursuing this compound. A number of fungally derived anthraquinones related to emodin have been reported to inhibit protein tyrosine kinase. The anthraquinone emodin (2) was reported by Jayasuriya et al. (1992) from the roots of a Chinese medicinal plant, Polygonium caspiatum, and related

7

BIOLOGICALLY ACTIVE FUNGAL METABOLITES Paecilcquinones

OH

0

OH

HOO *H

OH HO

0

OH

(5)c

(9) Versiconol OH I

roH

0 STRUCTURES 3-9

bioactive compounds have been reported from fungi (Fredenhagen etal., 1995a,b). Some of the more potent of these were the paeciloquinones @-9), which were isolated from fermentations of Paecilornyces carneus, a culture isolated from a soil sample collected in a jungle region in Bolivia (Petersen et al., 1995). The production of paeciloquinones was closely dependent on the conditions of the fermentations. It was reported that shake-flask fermentations using a complex medium containing saccharose, mannitol, meat extract,

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and ammonium nitrate and grown at 30°C yielded 50 mglliter of paeciloquinone A, but using similar conditions in a bioreactor the yield was very much reduced. Investigations showed this was probably due to insufficient oxygenation, and by carrying out the bioreactor experiment at a higher temperature, 33"C, comparable concentrations of the desired product were observed. Of the seven components produced by the fungus, the exact nature of products from a given fermentation could be changed by manipulating the medium. Thus, on the medium described above, paeciloquinones A, C, and D, and versiconol were produced, whereas on a medium containing sucrose, mannitol, peanut oil, meat extract, and ammonium nitrate inoculated and incubated in the same manner produced paeciloquinones B, E, and F. One explanation is that the peanut oil contains a six carbon-containing precursor for the side chain, but a more complex argument is needed to explain the origins of the four carbon fragment seen in components A, C, and D. This type of observation is commonly made with fermentations. However, without extensive biosynthetic and biochemical investigation, the cause is really speculative. These metabolites were inhibitors of v-abl, c-src, and EGF-R PTK, with IC50values in the micromolar range. Paeciloquinones A, C, and D were most potent against EGF-R with IC50values between 6.7 and 11 pM; A, C and F were most potent against v-abl with IC50values between 0.56 and 3.6 r-LM; A and C were most potent against c-src with IC50 values of 2 and 9 pM, respectively. None of the paeciloquinones displayed antimicrobial activity. During the invasive and metastatic phase of cancer, the tumor cells must penetrate the basement membranes of the tissue under attack. This degradative step is caused in part by protease and glycosidase. One of these enzymes is heparinase, which degrades the heparan sulfate of basement membranes. Heparinase has been identified in B16 melanoma, and its activity is associated with the metastatic capacity of malignancy. Trachyspic acid (lo) a, metabolite from Talaromyces trachyspermus that had been isolated from a Japanese soil sample, grown in a complex medium containing glycerol, potato, yeast extract, and malt extract, inhibits heparinase with an IC50of 36 pM (Shiozawa et al., 1995). Trachyspic acid showed no inhibitory activity towards bovine liver P-glucuronidase, thus demonstrating a degree of selectivity, although the activity in an invasive assay was not reported. Trachyspic acid is similar to three other reported polycarboxylic acids containing long alkyl side chains: (+)- and (-)-decylcitric (Gatenbeck and Mahlen, 1968; Brandage et al., 1976) and spiculisporic (Clutterbuck et al., 1931) acids, isolated fiom Penicillium spiculisporum, later reclassified as T


9

(10) Trachyspic acid

0

(11) Pachyrnic acid

II

(12) Dehydroturnulosicacid

II

STRUCTURES 10-12.

Trachysperrnus. The bioactivity of these previously reported compounds was not given, but, since they predate the discovery of the significance of the heparinase, it would be interesting to investigate their bioactivity in the assays employed here. Phospholipase A2 is responsible for producing fiee fatty acids and lysophospholipids. PLA2-I is present in secretions hom the pancreas, whereas PLA2-I1is involved in the inflammatory process. Inhibitors of PLA,-II might be useful antiinflammatory agents. PLA2 inhibitors have been isolated from a few fungi, including the Chinese fungus Poria cocos Wolf, a polyporaceae that is well known in traditional medicine for its diuretic, sedative, and tonic effects. The active compounds, pachymic acid (11)and dehydrotumulosic acid (12)were isolated and characterized (Cuellar et al., 1996) and were shown to inhibit snake venom PLA2 with ICs0 values of 2.9 and 0.84 pM, respectively.

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CEDlUC PEARCE R1

I

OH

H3C'

.CH3

(16) MR 304A

STRUCTURES 13-16.

Acetylcholinesterase (AChE) is the enzyme responsible for the breakdown and subsequent inactivation of the neurotransmitter acetylcholine. From a screening program looking for microbial metabolites that inhibit AChE, Omura et al. (1995) found a Penicillium sp. FO-4259 isolated from a Japanese soil sample that produced such activity when grown in a medium containing saccharose, glucose, corn steep liquor, meat extract, agar (not enough to produce a solid medium) potassium was phosphate, and calcium carbonate. The compound, arisugacin (s), characterized and shown to be similar to two tremorgenic compounds, territrems B (14)and C (G),previously reported from Aspergillus terreus (Ling et al., 1979, 1984). Arisugacin had an of 1 nM against human erythrocyte AChE, approximately 200 times more potent than tacrine, an inhibitor that has potential for use in Alzheimer's disease (Summers et al., 1986). Arisugacin was 1500 less potent than tacrine against butyryl-cholinesterase and is thus far more selective than the latter. The territrems were also shown to be effective AChE inhibitors. Not all novel fungal metabolites are large complex structures with multiple stereochemical centers, as is shown by the compound known This compound was discovered from a program seekas MR304A (16). ing melanin biosynthesis inhibitors (Lee et al., 1995). The culture producing MR304A is Trichoderma harzianum isolated from a Korean soil sample. Bioactivity was determined using three assays: against mush-


11

(17 ) Melanoxadin

STRUCTURE 17.

room tyrosinase, melanin production in Streptomyces bikiniensis, and melanin formation in €316 melanoma cells. MR304A inhibited all three. It was shown to be a noncompetitive inhibitor of mushroom tyrosinase with an IC5,, of 7.5 pg/ml and at 1pg/ml inhibited melanogenesis in B16 melanoma without showing cytotoxicity. Other isonitrile fungal metabolites have been reported from Trichoderma spp. (Boyd et al., 1991; Brewer et al., 1979; Fujiwara et al., 1982); one of these, trichoviridin, was also shown to be a melanin biosynthesis inhibitor (Hashimoto et al., 1994). Using the larval haemolymph of the silkworm Bombyx mori as the basis for a melanin biosynthesis inhibitor, Hashimoto et al. (1995) When the haemolymphs are exposed, to discovered melanoxadin (17). air they change color from yellow to black, and this is due to the formation of melanin. These workers found a Trichoderma sp. that was isolated from a Japanese soil sample that produced the oxazole melanoxadin when grown in a complex medium. The IC5,, value in the haemolymph assay was 22.3 pg/ml compared to 13.1 pg/ml for trichoviridin. The IC5,, against mushroom tyrosinase was 98 pg/ml. The inhibition of aldose reductase is a new approach to the treatment of diabetes mellitus (Sarges, 1989). A number of microbial products have been isolated that inhibit this enzyme. The tricyclic salfredins 18-24 have been isolated from Crucibilum sp. RF-3817 by Matsumoto et al. (1995). The culture was isolated from a piece of rotten wood, and the active compounds produced in a complex medium containing starch, sucrose, and yeast extract. Seven related metabolites were produced. The most potent of these in a rat lens aldose reductase assay were salfredins A4 and C2, both of which contain a glycine moiety. When the producing organism was grown in a medium containing glycine, the yield of the A4 component was increased 25-fold and the C2 component 15-fold. By using a medium containing corn starch, glucose, corn steep liquor, peanut oil, dried yeast, and calcium carbon-

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Salfredins

(18) A 3

R=-CH(COOH)CH2CH&OOH

(19) A 4

R=-CHSOOH

(20)A 7 R=-CH(CH&OOH

(21)Ci R=-H

(22)C2 R=-CH2COOH (23) C3 R=-CH(CH&OOH

STRUCTURES 18-24.

BIOLOGICALLY ACTIVE FUNGAL METABOLITES (25)Compactin

13

(26)Lovastatin

STRUCTURES

25, 26.

ate, glycine supplementation at a rate of 1%gave the best yields: 2579 yg/ml A2 and 798 yg/ml C2. VI. Cholesterol Biosynthesis and Lipid Metabolism Inhibitors

Some of the most potent and clinically useful cholesterol metabolism effectors have been derived from fungal products, most notably the hydroxymethyl coenzyme A reductase (HMG-CoAreductase) inhibitors related to compactin (25) and produced by Penicillium brevicompactum (Brown et a]., 1976), and Lovastatin (26) from Aspergillus terreus (Alberts et al., 1980), which had also previously been reported from Monascus ruber (Endo, 1979). An interesting account of the discovery of Lovastatin is given in a review by Vagelos (1991). Subsequent to their initial discovery, these compounds have been shown to be common fungal metabolites but are uncommon from the otherwise prolific actinomycetes, and it is possible that in fungi that contain sterols as an integral part of their cell membranes the steroid biosynthesis inhibitors play some role in the control of the production of ergosterol and related metabolites. From the initial discovery of the HMG-CoA reductase inhibitors, there has been considerable attention paid to the fungi as a source of compounds for use in the control of other facets of mammalian steroid biochemistry. (The squalene synthase inhibitors squalestatins and zaragozic acids are reviewed in Section VIII). Acyl-CoA:cholesterol acyltransferase (ACAT) esterifies cholesterol and thereby has a central role in regulating intracellular free cholesterol levels in humans. In atherosclerosis, foam cells are formed that contain large quantities of cholesteryl esters, the presence of which is directly related to ACAT activity. It is thought that inhibitors of this enzyme will be potential antiatherosclerotic drugs (Sliskovic and White, 1991).

14

CEDlUC PEARCE

H

(30)Terpencble D

STRUCTURES 27-30.

A number of groups have reported inhibitors of ACAT from fungi, including compounds from a new genus. (For this review, as a general rule, only those most recently published are included, although some groups have published prolifically on this topic in the past few years.) Huang et al. (1995a,b) discovered in a new fungi, Albophoma yarnanashiensis, a coelomycete isolated from a soil sample collected in Yamanashi, Japan, which produced a series of metabolites, the terpendoles 27-30 when grown in a medium containing starch, glycerol, soybean meal, yeast, potassium phosphate, calcium carbonate, magne-


15

sium sulfate, and potassium chloride. Four of these products were novel, and two, paspaline (Fehr and Acklin, 1966) and emindole (Nozawa et al., 1987), had been reported previously from other fungi. In an ACAT assay using rat liver microsomes, terpendoles C and D were the most potent inhibitors, showing ICs0 values of 2.1 and 3.2 pM, respectively. In addition, in a bioassay designed to test the effect on both cholesteryl ester formation and cell viability in macrophage J774, terpendole D showed an IC50 of 0.048 pA4 and a CD50 (a measure of cytotoxicity) of >24.8 pA4. Terpendole C and D showed no acute toxicity in ddY mice at 100 mg/kg. It was postulated that the sterol-like side chain of the terpendoles might compete with the natural substrate of ACAT, cholesterol. When a different medium containing maltose, Ebios, yeast, potassium phosphate, magnesium sulfate, and potassium bromide was fermented with A. Yamanashiensis, eight new terpendoles were produced (Tomoda et al., 1995d); all other parameters appeared to be the same as the earlier fermentation, when some or all of the new products were produced, but at a level that made isolation and characterization impractical. None of the new metabolites approached the potency of terendole D, the closest being approximately one-tenth as active, and none of the terpendoles showed any antibacterial or antifungal activity. A second group of ACAT inhibitors, the pyripropenes 3 1 4 2 (Omura et al., 1993b; Tomoda et al., 1994; Kim et al., 1994; Tomoda et al., 1995a) and the GERI-BP001 series (4345)(Jeong et al., 1995), with a similar motif to the terpendoles, that is, the presence of a steroid-like moiety, were both reported from Aspergillusfumigatus. The most potent inhibitor in the whole pyripropene series, substance A, had an IC50 value of 0.16 pA4. While attempting to produce enough material for further biological work, the group exposed the fungus to N-methy1-N’nitro-N-nitrosoguanidine and isolated mutants, one of which was shown to produce 10 times the amount of pyripyropene than the parent did when grown in a medium containing starch, glycerol, soybean meal, yeast, potassium chloride, potassium phosphate, magnesium sulfate, calcium carbonate, and nicotinic acid (Tomoda et al., 1995a). Biosynthetic studies demonstrated that the nicotinic acid added to the fermentation medium was incorporated directly into the metabolites. This mutant strain also produced eight new compounds in the series. Pyripropene L was the most potent of these in the ACAT inhibition assay, with an value of 0.27 pM.None of these compounds showed antibacterial activity. Similar compounds were discovered in an independent investigation in Korea (Jeong et al., 1994, 1995). In this case, the producing organism, isolated from a soil sample collected from

16

CEDRIC PEARCE pyripropene

(43)GERI-BPWI M

R1&. R d H 3

(44) GERI-EP001 A

R1=OH, Rz=CH3

(45) GERl-BPOOl B Rt=H, R*=CH&H3 STRUCTURES

43-45.


17

Mountain Dukyou, Korea, was cultured in a complex medium containing starch and soytone wherein it produced ACAT inhibitory activity, which leveled off at 6 days. The purified components, GERI-BP001 M, A and B, which are closely related to the pyripropenes, had IC50values against rat liver microsome enzyme of 42, 94, and 40 pM, respectively, with pyripyropene A, a known inhibitor included as a control, having an IC,, of 43 nM. These compounds showed no antibacterial or antiviral activity. The azaphilone group of ACAT-inhibiting metabolites, the isochromophilones 46-51 were discovered by the Kitasato Institute group (Arai et al., 1995). These compounds were produced by Penicillium rnulticolor FO-3216 isolated from a Japanese soil sample collected in Tokyo. Fermentation in a medium including starch, glycerol, soybean meal, yeast, potassium chloride, calcium carbonate, magnesium sulfate, and potassium phosphate for 3 days at 27°C in a fermenter led to the production of isochromophilones 3-5, with six being produced in the same medium but in shaken Erlenmeyer flasks. Isochromophilones 4 and 6 were the most potent of the group against ACAT, with IC50values of 50 pM. 4 also showed inhibitory activity against cholesteryl ester transfer protein, with an IC50 of 98 pM. 3, 5, and 6 had some weak antimicrobial activity as well as inhibiting the growth of B16 melanoma cells with IC50 values of 33, 36, and 30 pM, respectively. An alternative approach to decreasing circulating cholesterol levels by reducing its biosynthesis is to inhibit the absorption of dietary cholesterol in the intestine. Intestinal absorption involves the enzyme cholesterol esterase, which esterifies cholesterol prior to the generation of chylomicrons, the form in which lipids are transported around the body (Goodman Gilman et al., 1985). It has been shown that cholesterol absorption is markedly decreased in rats when they are depleted of cholesterol esterase (Gallo et al., 1984). Sakai and colleagues (1995) isolated a Stachybotrys sp. that produced a series of triprenylphenol cholesterol esterase inhibitors, two of which, K-76 (52)and stachybotryhad previously been reported from other strains of Stachydial (s), botrys complementi (Kaise et al., 1979) and Stachybotrys cylindrospora (Ayer and Miao, 1993), respectively. Ten of the remaining metabolites (54-63) were novel triprenylphenols that were produced when the Stachybotrys originally isolated fiom a soil sample collected in Japan was grown in a medium containing glucose, soybean meal, polypeptone, meat extract, yeast extract, potassium phosphate, and magnesium sulfate. The activity of the purified metabolites was determined using porcine pancreatic enzyme catalyzing the formation of cholesterol oleate. Of the 10 compounds isolated, stachybotrydial was the most potent,

lsochromophllones

OAc (49) IV

0 STRUCTURES 46-51.

(51) VI

19

BIOLOGICALLY ACTIVE FUNGAL METABOLITES Triprenylphenols

0

HO'"' HJC

"CH3 (52) 1 R>=OH, R&HO

(676)

(55) 3 Ri=OH. Rp=H, X=NH

(53)2 Rl=H, Rz=CHO (Stachybotrydial)

(56) 4 R,=OH. Rp=OCH,, X=NH

(54) 9 Ri=H, R&3i3

(57)5 Rl=OH. Rz=OCHs, X=NH (58) 7 Rl=OH, Rz=H, X=N(CHp)flH (59) 8

Rl=OH, Rz=H, X=N(CH,),COOCH,

(60)10 R+I,

Rz=H, X=(CHz)&DOCH3

(61) 12 Rl=H, Rz=H, X=O

(62)11

STRUCTURES 52-63

having an IC,, of 60 pM, with two other metabolites having IC,, values of 200 (K-76) and 270 (IX) yM. Stachybotrydial inhibited cholesterol esterase in a time-dependent manner and was irreversible. Stachybotrydial also inhibited the dietary absorption of cholesterol in rats and significantly reduced the serum levels of cholesterol in mice fed a diet containing 1%cholesterol. Since these compounds had no effect on ACAT, another enzyme postulated to control cholesterol absorption, their regulatory effect is presumed to work through control of the esterase. Other facets of lipid metabolism that have been targets for fungal metabolites include fat biosynthesis. The enzyme diacylglycerol acyltransferase catalyzes acylation from acyl-CoA to diacylglycerol, thus producing triacylglycerol. This is the last step in fat formation. Reports

20

CEDlUC PEARCE

of inhibitors of this pathway are rare. In their screening program to find metabolites that would inhibit this enzyme, Tomoda and co-workers (Tomoda et al., 1995b,c) found a Humicola sp. isolated from a Japanese soil sample in which a series of compounds, amidepsines A-D (64-67), was produced when grown in a liquid medium containing sucrose, glucose, corn steep liquor, meat extract, potassium phosphate, magnesium sulfate, ferrous sulfate, manganese chloride, zinc sulfate, copper sulfate, cobalt chloride, calcium carbonate, and agar. Amidepsines A-C consist of a tridepside and an amino acid, and as such are a new type of fungal metabolite, although tridepsides have been isolated from a variety of fungi and lichens and shown to have a number of bioactivities. Amidepsine D was previously reported from the lichen Parmelia damaziana (Elix et al., 1981). These compounds inhibited the target enzyme with IC5,, values of 10.2, 17.5, 19.2, and 51.6 pM (A-D, respectively). The effects of these compounds in Raji cells confirmed that they inhibit production of triacylglycerol formation with IC5,,values of 15.5, 3.35, 17.2, and 2.82 W(A-D, respectively). Compound B showed more selectivity since it had little inhibitory effect on the production of either phosphatidylcholine or phosphatidylethanolamine in Raji cells. While these compounds showed no cytotoxicity at the concentrations used in the assays, they were mildly antibacterial against Bacillus subtilis. In a second strain of Humicola sp. FO-5969 grown in the same medium, this same research group (Tomoda et al., 1996) discovered a methylated derivative (68) of amidepsine A, which was coproduced with B. This strain did not yield the other three amidepsines. The new metabolite, amidepsine E, is a weak inhibitor of the target enzyme, with an value of 124 pM. VII. Receptor Binding Antagonists

A number of fungal products have been shown to have neuropharmacological properties. Extreme examples include the well-known lysergic acid diethylamide (LSD) from Claviceps purpurea and psilocybin from, among others, Psylilocybe mexicana. Not surprisingly, other neuropharmacologically active compounds with various bioactivities have been reported. In a program screening fungal extracts for compounds that effect binding to the GABA-benzodiazepam receptor, Ainsworth et al. (1995) discovered xenovulene hom a strain of Acremonium strictum. This culture had been isolated from a foam sample collected from a tropical stream. When the fungus was cultured in a medium containing glucose, yeast extract, MES, Tween 80, Antifoam A,

BIOLOGICALLY ACTIVE FUNGAL METABOLITES OH

21

0

(64) Amidepsine A

(65) Amidepsine 6

-H

(66)Amidepsine C

-H

I

-H

0 (67)Amidepsine D

- CH3

(68)Amidepsine E

- CH3

- OH

-H

STRUCTURES 64-68

and carboxymethylcellulose, the humulene-related compound xenovulene (69)was produced. Humulene is a recognized plant product and is present in, for example, hops, but it has also been isolated from Coriolus consors (Nokoe et a]., 1976), and derivatives from other fungi (Turner and Aldridge, 1983), including the bioactive compounds pycnidione and upenifeldin (Harris et a]., 1993; Mayerl et al., 1993, 1994). Xenovulene inhibited the binding of flunitrazapam to the GABA-benzodiazepam receptor from ox brain with an ICs0 value of 40 nhl; the IC,, for the sodium salt was 10 nM.

22

CEDRIC PEARCE

Neuropeptide Y is a peptide regulator with a wide variety of biological effects that is distributed in catecholamine-containing neurones. It has been associated with the intake of food and water as well as regulation of pituitary function, vasoconstriction, and anxiolysis/sedation (Grundemar and Hakanson, 1994). Three classes of neuropeptide Y receptor have been described (Gehlert, 1994). Kodukula and co-workers (1995) isolated a tetracyclic compound, BMS-192548 (701, from Aspergillus niger that inhibits the binding of neuropeptide Y to NPYl and NPY2 receptors, with IC50values of 24 and 27 yM, respectively. BMS-192548 (Shu et al., 1995) is related to a number of known fungal metabolites and is a regio- and possibly a stereoisomer of the known compound TAN-1612 (71),a metabolite isolated from Penicilliurn claviforme (Ishimaru et al., 1994). TAN-1612 was shown to be a substance P inhibitor. These compounds are closely related to the actinomyces product tetracycline, although BMS-192548 showed no activity against a range of Gram-positive and -negative organisms at 100 yg/ml. Endothelins are a group of peptide vasoconstrictors of potent and long lasting effect. Three endothelin isopeptides are secreted by endothelial cells. Endothelins are thought to have a role in a variety of cardiovascular problems, including hypertension (Saito et al., 1989), congestive heart failure (Watanabe et al., 1990), and various related diseases. A number of streptomyces products are known to be antagonists of endothelin (Ihara et al., 1991; Miyata et al., 1992). In a screening program looking for endothelin antagonists from fungi, Nakamura et QI. (1995) discovered a series of novel metabolites, the stachybocins (72-74),from cultures of Stachybotrys sp. M6222. This culture was isolated from a Japanese soil sample, and, when grown in glucose, peptone, corn steep liquor, potassium phosphate, magnesium sulfate, and celite, produced the stachybocins. These compounds consist of two spirobezofurans, each attached to a decalin derivative with both units connected by a lysine (Ogawa et al., 1995b).The three metabolites inhibited the binding of radiolabeled endothelin-1 to human endothelin receptors type A with IC50values of 13, 1 2 , and 15 yM, respectively, for isomers A, B, and C, and with values for inhibiting binding to the type B receptor of 7.9, 9.5, and 9.4 yM, respectively. When these compounds were tested for relaxation activity in rabbit aortae treated with endothelin-1, contraction was relaxed 86, 70, and 68% for metabolites A, B, and C, respectively, at 30 yM, thus demonstrating that these compounds are endothelin antagonists. These compounds showed no antimicrobial effects. A second group of fungal metabolites, the azaphilones (Pairet et al., 1995), have been reported as metabolites from Penicillium


23

(69) Xenovulene A

0

OH

0

OH

CH30

0

OH (70) EMS-192518

OH

OH

CH3O

0

0

0

OH (71) TAN-1612

0

0

(72)A Rj=RpH (73) B Rj=OH, R2=H (74) C Rl=H. R2=OH

STRUCTURES 69-74.

sclerotiorurn, these also being antagonists of endothelin-A and endothelin-B. Two of these azaphilones (75 and 76) are novel sclerotiorin analogues and are similar to the isochromophilones, inhibitors of acyl-CoA:cholesterol acyltransferase, as discussed above. In binding studies with human ET, and ETb receptors using radiolabeled

24

CEDlUC PEARCE

Azaphih

(75)l R=CI

(76)2 R=H

(77) RES-12141 R*=OH, RFH, R,=OCH, (78) RES-1214-2

Rl=OH. R d I . R & C H 3

(79) Dihydroepiepofonin

I

OH

STRUCTURES 75-79.

endothelin-1, the IC,, values ranged between 56 and >250 pA4. In a third study, Ogawa and co-workers (1995) discovered two endothelin-1 antagonists from a Pestalotiopis sp. that had been isolated from a Japanese and -2 (781,which soil sample. These two compounds, RES-1214-1 (77) are simple aromatic bicyclic metabolites, showed IC5, values of 1.5 and 20 pA4 using ET, receptors and endothelin-1. In a screening program aimed at the discovery of interleukin-1 receptor binding antagonists, Kuo and colleagues (1995) found a PenicilIium patulum that produced bioactivity, and bioactivity-guided isolation led to the known compounds gentisyl alcohol and epiepoformin. Subsequent fermentations including HP-20 resin led to production of a novel compound, dihydroepiepoformin (79).The inclusion of resins in fermentations to improve titers and to enhance production of otherwise


25

very minor components is a previously reported approach used in a number of programs (e.g., Marshall et al., 1990). Mechanistically this approach probably works by removing feedback inhibition from metabolic pathways and by sequestering intermediates. The compounds described had IC50 values of 0.7 mM. VIII. Antiinfective Agents

There is a chronic unmet need for new antifungal agents to be used clinically along with amphotericin and the azoles. There have been a large number of reports of fungally produced antifungal agents; however, one of the biggest issues is to find agents that are selective against fungi without effecting the host. The structurally similar zaragozic acids and squalestatins, both inhibitors of squalene synthase and discovered by the groups at Merck and Glaxo independently, are some of the most potent and promising antifungal agents discovered. The squalestatins were discovered from fermentations of a Phoma sp. (Dawson et al., 1992; Sidebottom et al., 1992; Baxter et al., 1992) by investigators who had engaged in screening fungi for the production of inhibitors of squalene synthase. The zaragozic acids were initially discovered as metabolites from four fungi, Leptodontidium elatius, two strains of Sporormiella intermedia, and a sterile mycelia (Bergstrom et al., 1993).The L. elatius was isolated from a wood substrates collected in North Carolina, the S. intermedia were both isolated from dung collected in Arizona, and the Mycelia sterilia was isolated from a Spanish water sample. An excellent review of the discovery and structural, biosynthetic, and mechanistic studies on this group of compounds have been recently published by Bergstrom et al. (1995), and these exciting metabolites will not be discussed in depth herein. Zaragozic acids are a group of compounds with a similar basic structure with a considerable variety of minor modifications within these groups, and structures of A through F are given here (80-84). Zaragozic F is the first metabolite of this series to have a nonaromatic alkyl side chain attached to position 1 (Dufresne et d., 1996). The zaragozic acids are potent inhibitors of squalene synthase, with zaragozic acid A giving Kj values against rat liver microsoma1 enzyme of 78 pM (Bergstrom et al., 1993) to 1.6 nM (Hasumi et a]., 1993), and an IC5,, value of 12.5 nM (Baxter et al., 1992). At least 20 separate fungal isolates have been shown to produce members of the zaragozic acid/squalestatin group (Bills et al., 1994). An enlightening example of the importance of careful media preparation was given by

STRUCTURES 80-83.


27

Connors et al. (1995),who demonstrated that, when a batch of medium was sterilized at a low heat input (R, = 33.4 min), the products from a subsequent fermentation differed from a batch prepared using a higher heat input (R, = 50.5 min), indicating significant thermal decomposition of some ingredient(s1. In the latter case, the yield of 4’-desacetoxy zaragozic acid C (85)was approximately 130 yg/ml compared to 60 yglml from normally sterilized media, although biosynthesis of the coproduced 4’-O-desacetyl zaragozic acid C (86)and zaragozic acid C remained the same. The different sterilization techniques also led to detectable changes in growth and metabolism of the fungus. The production medium contained lactose, glycerol, primatone, yeast extract, sodium citrate, and magnesium sulfate. The polyketide origins of zaragozic acid have been determined, and it was determined that the acetoxy group is derived from acetate (Byrne et a!., 1993). The 4‘-desacetoxy zaragozic acid C is probably a biosynthetic precursor to the other two compounds, and under the conditions established in this heat-altered medium, the production of this intermediate is in some way greater than the ability of the fungus to carry out the two-step conversion to zaragozic acid C. The related squalestatins (squalestatin 1= zaragozic acid A = 80)were first reported in 1992 (Dawson et al., 1992; Sidebottom et al., 1992; Baxter et al., 1992); these compounds are also very potent squalene synthase inhibitors. In addition to the naturally occurring compounds, to expand their analogue series the Glaxo group carried out microbial biotransformations on the squalestatins using actinomycetes and fungi and were able to prepare five novel compounds, all of which retained bioactivity (Middleton et al., 1995). Three thousand and five hundred cultures were screened to find these biotransforming microorganisms. Four of the fungi tested were able to deacetylate squalestatin (87),20% of the fungi examined were able to carry out the desacylation reaction (88),and a fusarium produced the methyl ester 89.The modified squalestatins were potent inhibitors of mammalian and fungal squalene synthase, with IC5,, values ranging from 3 to 30 nM. The echinocandins are also a very promising and potent group of antifungal agents, these lipopeptides being inhibitors of fungal glucan biosynthesis. Recent reviews have been published and should be consulted for in-depth information (Current et al., 1995; Debono, 1994, 1995; Tkacz, 1992). Briefly, echinocandin B is a product of Aspergillus nidulans and is a potent inhibitor of P-1,3-glucan biosynthesis, which results in it being also a potent inhibitor of Candida and Pneumocystis carinii (Buss and Waigh, 1995; Weinberg, 1996).

28 CEDRIC PEARCE


29

0

OH

STRUCTURES 87-89.

Other antifungal lipid-containing glucan biosynthesis inhibitors have been reported. The mechanistically related fusacandins 90 and 91 were isolated from Fusarium sambucinum (Jackson et al., 1995; Hochlowski et al., 1995). The producing fungi were isolated from a polypore fruit body collected in Illinois and was cultured in a production medium containing glucose, mannitol, glycine, dried lard water, soybean meal, sodium citrate, potassium phosphate, and cobalt chloride. The fusacandins, which are related to the papuolacandins, contain two galactose units, one glucose, and an aromatic moiety, with fusacandin A esterified with two long-chain fatty acids and fusacandin B with one. Fusacandin A had MIC values against a range of Candida and other fungi between

30

CEDNC PEARCE Fusacandins

STRUCTURES 90, 91.

3.12 and 6.25 pg/ml compared to papuolacandin B, which was generally twice as active as fusacandin B, which was much less active than the A component. These metabolites did not show any antimicrobial activity. Fusacandin A was also shown to be a potent inhibitor of glucan synthase, and the anti-Candida action was antagonized in the presence of the osmoprotectant sorbitol. When macromolecules were synthesized by Candid0 albicans in the presence of fusacandins, only glucan biosynthesis was significantly depressed, and it seems very probable that the fusacandins are inhibitors of (1,3)-P-glucansynthesis. Using a screen for identifying inducers of microbial differentiation in Phoma destructiva, Dornberger et al. (1995) discovered a strain of Apiocrea chrysosperma that produced the bioactive peptaibol metabolites chrysospermins 92-95 when incubated in a static medium contain-

W

CI

g

Chrysosperims and Trichorzins Chrysosperims and Trichorzins

93

AcPhe

94 - _ IAcPhe

Aib

Ser

Aib

I Aib I Ser I Aib

Aib Iva

Leu

Leu

Gln

Gly

I Gln I Gly

G r:

Aib

Aib

Ala

Aib

Aib

Pro

Iva

Aib

Aib

Gln

Trpol

Aib

Aib

Ala

Aib

Aib

Pro

Aib

Aib

hi Gln

Trwl Tpl

95

AcPhe

Aib

Ser

Aib

Iva

Leu

Gln

Gly

Aib

Aib

Ala

Aib

Aib

Pro

Iva

Aib

Aib

Gln

96

AcAib

Gly

Ala

Aib

A%

Gln

Aib

Val

fib

Gly

Leu

Aib

Pro

Leu

Aib

Aib

GIn

Leu01

STRUCTURES 92-97. STRUCTURES 92-97.

$D

F

32

CEDRIC PEAFCE

ing malt extract, glucose, yeast extract, and ammonium sulfate. The screening bioassay consisted of malt agar plates containing vegetative mycelia of r! destructiva. Samples were spotted on the surface of these plates, and cytodifferentiation was measured by the zone of pigmentation produced around the site of sample application. Chrysospermins were subsequently shown to be active against a variety of Gram-positive bacteria and yeast with MIC values from 5 pg/ml against Gram-positive organisms to 10-12.5 pg/ml against Sporobolomyces salmonicolor and Phoma destructiva. Nine somewhat related antifungal peptaibols, the trichorzins and 97,have been isolated from Trichoderma harzianum (Goulard et al., 1995; Hlimi et al., 1995). In this case, the metabolites contained 18 amino acid residues compared to 19 in the chrysospermins, so that belong to the recognized group of long-sequence peptaibols. Both groups contain similar residues at positions 4, 5, 9, 15, and 16. The trichorzins, which were isolated from two strains of T harzianum from soil collected in Uruguay and Malaysia, were produced in a defined medium containing glucose as the carbon and energy source and potassium nitrate as the nitrogen source, together with inorganic salts. Clearly, in a defined medium as the one described, the opportunity for directed biosynthesis of novel analogues exists, although these authors did not report on such experiments. The trichorzins were not active against Gram-negative bacteria, although they were active against Staphylococcus aureus. Since MIC values were not reported, an accurate determination of potency could not be acquired, although the zones of inhibition for the amount of material added did not seem great. Taking the approach into account, the results are not simple to interpret. These authors also performed experiments to determine the membrane effects of the various components isolated and by measuring the leakage of fluorescent dye from liposomes demonstrated a direct relationship between increased hydrophobicity and increased potency. Employing a physicochemical screening approach, Grafe et al. (1995) discovered the lipopeptide helioferins 98 and 99,which mediated the transfer of ions from an aqueous environment to an organic phase. The producing organism, Mycogone rosea, was isolated from the fruiting body of a Macrolepiota sp. and was grown as a static surface culture in a medium containing malt extract, glucose, yeast extract, and ammonium phosphate. Helioferin A and B are potent antibacterial and antifungal agents with an MIC value against C. albicans of 5 pg/ml. These compounds are toxic to chicken embryos at 0.5 mg/kg, cause hemolysis at > l o 0 pg/ml, and are cytotoxic against L I Z 1 0 leukemia and L929 mouse fibroblasts, with IC,, values of 0.01-0.04 pg/ml. The helioferins

I"


O = ? ( :I2

33

34

CEDRIC PEARCE

OH

OH

(105) (2-12371

R=H

(106) CJ-12372 R=OH

STRUCTURES 100-106.

are linear aminolipopeptides and are thought to work as protonophoric compounds disrupting ion flux and energy metabolism. A number of compounds containing bicyclic decalin and related structures with a variety of bioactivity, but commonly antifungal, have been reported recently. Thus, the diepoxins 100-103 (Schlingmann et


35

(107) Australifungin

OH (108) Australifunginol

STRUCTURES 107, 108.

al., 1993) were obtained from fermentations of a Mycelia sterilia initially isolated from a Panamanian tree sample at MYCOsearch. Of the four diepoxins reported, 101was inactive, but the remaining three had some antibacterial activity and inhibited C. albicans and Rhodotorula rubra. As well as producing the diepoxins, these fermentations contained the very active allenic polyacetylene, 3,4,5,6-tetrahydro-6-hydroxymycomycin (104)(Schlingmann et al., 1995). The MIC values for this compound were 2 4 pg/ml against Staphylococcus aureus and B. subtilis and 10 pg/ml against C. albicans. The allenic polyacetylene are very unstable compounds and would be difficult to develop further. Compounds structurally related to the diepoxins, CJ-12,371(105)and CJ-12,372 were isolated by Sakemi et al. (1995) as inhibitors of DNA gyrase. These compounds were also active against Gram-positive bacteria with MIC values ranging from 25 to 100 pg/ml. No other activity was reported. The diepoxins CJ-12,371/2 and Sch-50673/Sch-50676 (discussed in the Section IX) are all related to the preussomerins, which are antifungal agents isolated by Weber and Gloer (1991) from a coprophilous fungus. Biosynthetically, these compounds could be products from arrested or diverted melanin production and as such could be common metabolites from pigmented fungi. A more complex group containing the decalin-type motif, australiand australifunginol m), has been reported by Mandala fungin (107) and co-workers (1995) from fermentations of Sporomiella australis. This culture, isolated from moose dung, was initially cultured in a solid

(m),

36

CEDRIC PEARCE Fusarielins

HO

\

HO

\

HO

HO

H

+ '...,

...'

OH

H

0

(110) B

H

& 0...."

H

0

/

.. (112) D

(1ll)C

STRUCTURES 109-112.

medium containing cracked corn, ardamine, and inorganic salts, but, subsequent to the discovery of the activity, several liquid media were tested and one containing either mannitol or fructose, oat flour, fibco yeast, L-glutamic acid, and MES proved to support a four- to fivefold increase in metabolite. Australifungin was a very potent antifungal agent, giving MIC values of less than 1pg/ml against Candida pseudotropicalis, C. tropicalis, Cryptococcus neoformans, C. Albicans, and Aspergillus fumigatus. Australifunginol was a relatively weak antifungal agent.


37

Certain antimitotic drugs cause deformation of Pyricularia oryzae, and this was used as the basis for a bioassay to screen for compounds interfering with microtubule function (Kobayashi et al., 1995). This approach led to the discovery of a series of antifungal agents from a Fusarium sp. originally isolated from a soil sample. When this fungus was cultured in a stationary potato dextrose broth, fusarielin A-D (109-112) were produced. Biosynthetic studies using I3C-labeled precursors demonstrated that these compounds are derived via a decaketide that undergoes five methionine-derived methylation reactions. Significantly, these results show that, while fusarielin contains the decalin motif, albeit somewhat disguised, it is not derived from a decalin precursor since the labeling pattern reported would be different if it were. Fusarielin A showed some antifungal activity but was not highly potent and had a narrow spectrum. This compound also did not inhibit microtubule assembly, thus showing the deformation assay employed probably has a more complex mechanism than speculated. The respiration inhibitors strobilurins and oudemansins are an exciting group of methoxyacrylic acids produced by a number of higher fungi, which have received considerable attention because of their antifungal, insecticidal, antiviral, and antitumor activities. These compounds were first reported in 1978, and work in this area has been reviewed elsewhere (Anke, 1995; Clough, 1993). New bioactive members of this class are still being discovered. A submerged culture of the basidiomycetes Favolaschia pustulosa was shown to produce 9-methoxystrobilurin L (113)and 9-methoxystrobilurin E (114) along with (Wood et al., 1996). The fungus had been isolated oudemansin L from fruit body tissue collected in a tropical forest. These compounds had some antibacterial and antifungal activity. 9-methoxystrobilurin L was shown to be cytotoxic, with an ICs0 against the human B lymphoblastoid cell line of 1.8 nM. 9-methoxystrobilurin A and K have been reported as metabolites from Favolaschia species (Zapf et al., 1995a). This latter group also isolated a sterol, favolon (m), from the same fungus (Anke et al., 1995). Favolon was produced in a medium containing yeast extract, malt extract, and glucose, and was present in the mycelia. Favolon displayed potent antifungal activity when tested against ascomycetes, basidiomycetes, oomycetes, and zygomycetes. The sterol was not active against bacteria, neither was it active against L1210 cells. This research group also isolated a new strobilurin, hydroxystrobilurin A (117),which was reported from basidiomycetes Pterula spp. (Engler et al., 1995). A mycelial culture obtained from the fruiting body of a fungus collected from a German forest was grown in a medium containing yeast extract, malt extract, and glucose in an aerated fermen-

m)

38

CEDRIC PEARCE

OCH3

(115)

STRUCTURES 113-115.

ter. The antifungal activity of hydroxystrobilurin A was similar to that reported for other strobilurins and oudemansins, with a wide spectrum of filamentous fungi and yeasts being inhibited, although, significantly, the additional hydroxyl group in hydroxystrobilurin results in decreased potency. New compounds have been discovered by investigating mutants of known producing strains in certain cases. Such was the case when


39

( 1 16) Favolon

CH3

STRUCTURE 116.

Sakuda et al. (1995) investigated a mutant of the patulin-producing Penicillium urticae, which was shown to produce a new azaphilone epoxide, patulodin (117a). This compound was shown to be weakly antifungal, having an MIC value of 50 p g h l against Pyricularia oryzae. As with the discovery of antiinsectan compounds (discussed in Section XI), Gloer and co-workers (1995a, 1996) have used an elegant discovery approach directed by nature and have sought antifungal compounds from fungi living in environments wherein the fungal inhabitants might be reasonably expected to compete for nutrients with each other. This area of research has been reviewed (Gloer, 1995a,b, 1996) and is only briefly discussed here. That there is interspecies chemical inhibition has been observed in many fungal communities. Gloer and his associates have been examining fungal isolates from dung that is sequentially colonized by different organisms. By looking for interspecies antagonism, a very large number of antifungal activities have been observed. Of approximately 250 coprophilous fungi examined, more than 60% were shown to produce antifungal metabolites (for more details of this work, see Gloer, 1996). The preussomerins previously noted as being the first of a new class of fungal metabolite, were isolated from Preussia isomera (Weber et a]., 1990; Weber and Gloer, 1991). Several of the preussomerins showed antifungal activity against fungi with which the producing culture may have to compete in nature, although activity against C. albicans was not observed. More recently, the antifungal agents cercophorins A-C (118m)were obtained from the coprophilus fungus Cercophora areolata in an isolate hom porcupine dung, along with the known trichothecene roridin E (Whyte et al., 1996). This fungus was used to ferment potato

m,

40

CEDRIC PEARCE

(1 17) Hydroxystrobilurin

(117a)Patulodin

(1 17b) Preussomerin A

OH

OH

OH STRUCTLJRES 3 17,11 7A,11 7B.

dextrose broth in shaken flasks at 25-28°C. The cercophorins were tested against dung from early successional fungi Sordaria fimicola and Ascobolus furfuraceus, and shown to inhibit the growth. These compounds were also active against some Gram-positive bacteria, with


41

(3343 (1 18) CerwphorinA

CHg (119) Cercophorin6

c33-43

(120) CerwphorinC

cercophorin A being the most potent, and had limited activity in the NCI tumor cell line. This appears to be a very promising and productive area of research. Mycophilic fungi associated with the fruiting bodies of either ascomycetes or basidiomycetes have been the subject of investigation (Zapf et al., 1995b), and this has led to the discovery of some interesting antifungal compounds. Sphaerellopsis filum, a widespread rust fungus mycoparasite, had been considered a biocontrol agent, although the chemistry of the toxins produced and the precise mechanism for toxicity was unknown. This culture produced antimicrobial bioactivity, which was isolated and shown to be due to the production of two xanthocillins, darlucins A (121) and B (122)(Zapf et al., 1995b). The darlucins were active against yeast and filamentous fungi, with the most potent MIC values being 2.5-5 yg/ml. These compounds also displayed antibacterial activity, with MIC values between 2.5 and 20 yg/ml against both Gram-positive and -negative bacteria, and showed some cytotoxicity against the BHK21, HeLaS3, L1210, and HL60 cell lines.

42

CEDRIC PEARCE Darlucins

OH

(121) A

NC

OH

(122) B

HO (1 23)Sch-57404

STRUCTURES 121-123.

In a recent preliminary report, an antifungal agent, Sch-57404 (123) containing the uncommon sodaricin nucleus was isolated from an unidentified fungus by Coval et al. (1995). This compound is only the second fungal metabolite having the unusual tricyclic nucleus shown. Sch-57404 was shown to be active against C. albicans, with MIC values of 16 pg/ml. As was demonstrated with the cyclosporines, fungally derived antifungal agents sometimes also demonstrate immunosuppressive effects with startling significance. Thus, cyclosporine A was initially identified


43

as an antifungal, and its effects on the immune system were recognized during further pharmacological investigation. The known ISP-1 (124) was isolated from Zsaria sinclairii by Fujita et al. (1994). This had previously been identified by two groups (Kluepfel et al., 1972; Aragozzini et al., 1972) as an antifungal agent, but Fujita and co-workers discovered this compound in a screen for immunosuppressive agents, and ISP-1 was reported to be 10 to 100 times more potent than cyclosporine A in suppressing lymphocyte proliferation in vitro and in other in vivo bioassays. This same research group also discovered the mycestericins 125-128 from a Mycelia sterilia (Sasaki et al., 1994; Fujita et al., 1996). These compounds were not as potent as ISP-1 on mouse allogenic mixed lymphocyte reaction, with mycestericins D and E being perhaps half as active, but this series of metabolites did allow for some speculative SAR conclusions. Fungi have been the source of many important antibacterial agents, including the penicillins and cephalosporins, both of which are used heavily 50 to 60 years after their discovery. However, from the reports in the literature there are not many novel antibacterial agents being isolated from fungi. Some of the antibacterial activity reported has been incidental to other biological activities, but these data demonstrate a level of interest in the area. With the emergence of antibiotic resistance, however, it would not be unexpected if more attention was paid to this area by drug discovery groups, The producing fungi may be in collections waiting to be tested. For example, a strain of the penicillin-producing fungus Penicillium chrysogenum was recently shown to produce a quinone, sorrentanone (m), that was active against Gram-positive and -negative bacteria, with MIC values of 16 pg/ml and higher (Miller and Huang, 1995). This is an area that may be worth revisiting simply because the targets have changed. There are a number of major viral diseases for which there is little or no treatment. Common viral diseases that affect huge populations include influenzas and colds. Progress has been made in some areas (e.g., human immunodeficiency virus and herpes), although none of the current usual chemotherapeutics are from natural sources. Of the few antiviral microbial products reported in the literature, the majority have been isolated from bacteria rather than fungi (Takeshima, 1992), and the rate of new antiviral fermentation products appearing in the literature seems to be increasing, which may be a reflection of better approaches to screening. In the HIV area, two reported screening strategies have led to the discovery of new products. During the HIV replication cycle there is a complex series of interactions between the virus and the host, including

44

CEDRIC PEARCE

0

HO

(124) ISP-1, Myriocin, Thermozymocld ' in

-

(125) Mycestericin D R= CH=CH-(CH2)6-Co-(CH2)5-CH3

0

0

0 (129)sonentanone

STRUCTURES 124-129.

specific binding of viral RNA with viral and host proteins. The viral protein, REV (for regulation of virion expression), regulates transport of viral RNA into cytoplasm, and the interaction of REV with the REV response element is essential for REV action. Using an approach based on the assumption that inhibition of this interaction could yield antivi-


45

(130) Harziphilone

0

(131) Fleephilone STRUCTURES 130, 131.

ral compounds, a productive screening program was established at Bristol-Myers Squibb. Thus, from a Trichoderma hQrZiQnZZmthat had been isolated from a Floridian soillplant sample, two active comwere obtained when pounds, harziphilone (130)and fleephilone the fungus was cultured in a medium containing glycerol, glucose, polypeptone, yeast extract, sodium chloride, and calcium carbonate (Qian-Cutrone et ~ l .1996a). , Interestingly, these metabolites show only slight structural similarity, although both inhibit binding of REV protein to REX response element, with ICs0 values of 2.0 pM for harziphilone and 7.6 pMfor fleephilone. In a secondary evaluation used to determine the effectiveness against virus in infected cells, in this case CEM-SS cells infected with HIV-1, neither compound had an effect using concentrations up to 760 pM harziphilone and 450 pM fleephilone. In this regard, it is interesting to note that an equally potent plant product, niruriside, which gave an ICs0value of 3.3 pMin the REV/REV response element assay, also failed to protect chronically infected cells at 260 pA4 (Qian-Cutrone et d., 1996b). In a second approach, inhibitors of gplZ0 binding to CD4 receptors were sought. In this case, it is speculated that, during the entry phase of virus to cell, the viral protein gplZ0 binds to the cellular surface CD4

(m),

46

CEDRIC PEARCE (132) lsochromophilone I

(133) lsochromophilone II

(135)Trypostatin B

( 134) TrypastatinA

STRUCTURES 132-135.

molecule, and compounds that inhibit this process should be antiviral (Johnson and Hoth, 1993). Omura et al. (1993a) and Matsuzaki et al. (1995a,b) reported the first hngally derived inhibitor of this process that also showed significant effects on viral replication. Fermentation of Penicillium multicolor isolated hom a Japanese soil sample produced together with the known inacisochromophilones I (132)and I1 tive metabolites sclerotiorin, ochrephilone, and rubrorotiorin. Isochro-

(m),


47

mophilones I and 11 inhibited gp120-CD4 binding with ICs0 values of 6.6 and 3.9 pA4, respectively. Isochromophilone I1 significantly inhibited HIV replication, as determined by measuring viral core protein p24 production in human lymphocytes. None of the isolated compounds had any anti-Gram-positive or -negative bacterial or antifungal activity. IX. Antitumor and Cytotoxic Activity

It is well known that natural products have antitumor activity and that some of the most useful drugs available are, either directly or via derivatization, from these sources (e.g., Vinca alkaloids). The podophyllotoxins etoposide and teniposide; taxol from the Pacific yew; actinomycin D, bleomycin, mithramycin, mitomycin, daunorubicin, and doxorubicin all from Streptomyces species (Goodman Gilman et a]., 1985). A variety of novel fungal products have been reported that add to the spectrum of potential approaches to the chemotherapy of cancer. Tryprostatins 134 and 135 are inhibitors of the mammalian cell cycle produced by a marine fungus (Cui et al., 1995). The producing culture, Aspergillus fumigatus, was isolated from a sediment sample collected at a depth of 760 m at the mouth of the Oi river in Japan. Fermentation of a medium containing glucose, starch, soybean meal, potassium phosphate, and magnesium sulfate produced the two active components that inhibited the cell cycle progression of the mouse tsFT210 cell line in the G2/M phase at concentrations of 50 pg/ml (A) and 12.5 pg/ml (B). In a screen to discover compounds that would inhibit metastasis, two novel compounds of preussomerin type were isolated from Nattrassia mangiferae (Chu et al., 1995a). This culture, also from the MYCOsearch collection, was isolated from dead leaves collected from an arid region of Guatemala. Fermentation of a medium containing neopeptone, cerelose, and calcium carbonate led to the production of Sch-50673 (136) and 50676 (137).These compounds are related to a number of recently discovered bioactive fungal metabolites, including the antimicrobials CJ-12371/12372 and the diepoxins discussed earlier, and were also isolated from fungi from the MYCOsearch collection. Sch-50673 and 50676 were active in a chamber invasion assay using HT1080 human fibroblast cells, with ICs0 values of 6.2 and 2.8 pM,respectively. Less than 10% toxicity was reported at 25 pM. Further evaluation of these compounds was not reported. Many tumors are either not susceptible to cytotoxic agents or develop resistance once exposed to the agent. A large amount of research has been carried out to overcome this latter problem. Nozawa et al. (1995)

48

CEDlUC PEARCE

(137) sch 50676

(136) sch 50673

+

CH3

(138) FD-211

CH3

(139)SCh 52900 R=CH(OH)CHa (140) SCh 52901 R=CH*CHs (141) Verticillin R Z H 3

STRUCTURES 136-141.

discovered a strain of Myceliophthora lutea isolated from a Chinese soil sample that produced a lactone compound, FD-211 which was active against adriamycin-resistant human promyelocytic leukemia (HL-60) cells. Thus, while the IC5,, values against a number a cell lines were about 8 to 10 times less potent than adriamycin, against the adriamycin-resistant cells FD-211 was 20 times more potent than adriamycin. FD-2 11inhibited incorporation of radiolabeled precursors into DNA, RNA, and protein in HeLa cells. It would be very instructive to learn more about the mechanism of this compound.

(m),


49

During the transition from quiescent to growing state, one of the earliest biochemical events is for the c-fos proto-oncogene to be induced, and this is triggered by a variety of mitogens. significantly, it has been shown that some oncogene products also stimulate production of c-fos,and in certain tumors c-fos is constitutively elevated. For this reason it has been postulated that inhibition of c-fos induction could be an important target for drug discovery. In the program at ScheringPlough, a Gliocladium sp. was shown to produce compounds Sch52900 (139)and Sch-52901 (140)that displayed such activity (Chu et al., 1995b). The culture, Gliocladium sp. SCF-1168, was isolated in the MYCOsearch program from fresh leaf litter collected in a Puerto Rican rain forest. When a medium consisting of neopeptone, cerelose, and calcium carbonate was fermented, the Gliocladium produced three bioactive compounds, two of which were novel, and a third, verticillin that had been reported previously from a Verticillium sp. (Katagiri et al., 1970). Using a validatedfos/lac Z reporter murine system, the two new metabolites were shown to be inhibitory, with ICS0values of 1.5 and 18 pM for 52900 and 52901, respectively, while verticillin had an ICS0of 0.5 pM. Activation of the Ras gene is associated with many human cancers. Biochemical studies have shown that Ras functions following localization within the membrane and binds with GTP prior to transforming cells. Protein farnesyltransferase is involved in farnesylation of Ras proteins, and inhibitors of this enzyme are predicted to change membrane localization and activation of Ras proteins, and will possibly have utility as anticancer agents (Gibbs, 1991). A number of fungal metabolites have been isolated that show this activity, including the wellknown gliotoxins (Van Der Pyl et al., 1992), the andrastins (Shiomi et al., 1996), kuraosins (Uchida et al., 1996a,b) and fusidienol (Singh et al., 1994a,b), for example. The kuraosins 142 and 143 (Uchida et al., 1996a,b)were produced by a Paecilomyces strain that had been isolated from a Japanese soil sample. When the fungus was grown in a medium containing starch, glycerol, soybean meal, fermipan, potassium chloride, calcium carbonate, magnesium sulfate, and potassium phosphate, karaosins A and B were produced. Structurally these compounds are relatively simple and were subsequently synthesized by the discovery group (Uchida et al., 1996b). The ICS0values against protein farnesyltransferase were 59 and 58.7 pM for the A and B isomers, respectively. In a second study, the French group at Rhone-Poulenc Rorer (Van Der Pyl et al., 1995) isolated a structurally more complex metabolite from Chrysosporium lobatum, also found in a soil sample. This compound, RPRl13228 (M), was produced in a liquid medium

(m),

CEDlUC PEARCE

50

HO

(1 42)Kurasoin A OH

(143)Kurasuin B

(144) RPR113228

(145) Fusidienol

&

HO

STRUCTURES 142-145.

containing malt extract and agar. Inhibition of farnesyl protein transferase using either lamin B terminal sequence peptide or recombinant p21H-ras protein was examined, and IC,, values of 0.83 and 2.1 pMwere obtained. In an investigation by Merck scientists, the tricyclic compound fusidienol (145) was discovered from a Fusidium griseum originally isolated at MYCOsearch (Singh et al., 1994a,b). E griseum produced fusidienol when cultured on a millet-based medium, and purified material was active against bovine and human enzyme, with IC5, values of 0 . 3 and 2.7 pM, respectively.


51

(146) Taxol (147) 2a-hydroxydimeninol

HO.,,

STRUCTURES 146,147.

Supply is one of the inherent problems encountered while working towards bringing natural products to the clinic, and this is frequently the case with more exotic sources as well as with plant material. This is somewhat less of an issue with microbial fermentations since these are renewable, but they can never be ignored as a potential snag. This was highlighted during the development of the plant antitumor product taxol (146). With the discovery of an endophytic fungus, Taxomyces andreanae, associated with the Pacific yew Taxus brevifolia, which would produce taxol in culture (Stierle et al., 1993),certain expectations were raised about potential relatively facile production methods. This work has stimulated more investigations on the fungi associated with ?: brevifolia. In a report from Pulici et al. (1996),a Pestalotiopsis sp. isolated from Pacific yew leaves collected in Bozeman, Montana, was shown to produce the sesquiterpene 2-a-hydroxydimeninol (W), a compound related to a number of plant and fungal drimanes. This and related observations are very interesting in light of the speculation concerning the transfer of biosynthetic information between plants and fungi, although definitive proof that this occurs is lacking. In this report, 2-a-hydroxydimeninol was not shown to have any particular activity, but related compounds have been shown to be antitumor and antifeedants, and it would be interesting to test for these activities. X. Miscellaneous Pharmaceutical Activity

Platelet activating factor, l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF),is a phospholipid involved in a number of physiological

52

CEDlUC PEARCE

(148) Phomactin E

(149) Phomactin F

(150)PhomactinG

(151) Phomactin B

STRUCTURES 148-151.

events, both normal and disease-associated. These responses include degranulation of a variety of blood cells, smooth muscle contraction and bronchoconstriction, vascular permeability and hypotension, and anaphylaxis, and are associated with inflammation and allergic reactions. It has been speculated for some time that PAF inhibitors would have a role in the control of disease, and a number of PAF antagonists have been discovered and tested clinically for effectiveness. A series of macrocyclic compounds have been reported from Phoma spp., one isolated from a marine environment and the other from a MYCOsearch terrestrial collection. The phomactins were initially reported in 1991 (Sugano et d.,1991) and further metabolites (148-151)in 1995 (Sugano eta]., 1995). These compounds seemed to be relatively potent, with IC50 values of 2.3, 3.9, and 3.2 pM for PAF-induced platelet aggregation by E, F, and G, respectively, and ICs0 values for inhibition of PAF binding to the receptor of 5.19, 35.9, and 0.38 pM for E, F, and G, respectively. Related metabolites Sch-47918, 49026, 49027, and 49028 (152-155) were isolated by Chu et al. (1993) from a fungus from the MYCOsearch collection. These compounds gave a range of ICsOvalues from 3 to 30 pM.


53

(153) SCh 49026

,,+OH

@

0 H H

(154)Sch49027

OH

(156) ZG-1494a

STRUCTURES

152-156.

An alternative approach has been explored by West and colleagues screened for PAF biosynthetic inhibitors. PAF is biosynthesized via two systems. The first is the normal de novo pathway, and the second, the route used in disease response, is the remodeling pathway. The reason this group looked for biosynthetic inhibitors rather than antagonists is because PAF plays a role in normal metabolic function, and to block it may not be optimum for good health. From their screening program an inhibitor of PAF acetyltransferase, ZG-1494~~ (156) was discovered. The producing organism was Penicillium rubrum isolated from red pepper in Denmark. ZG-1494a selectively inhibited PAF acetyltransferase with an IC5,, of 40 pM. More(1996), who

54

CEDRIC PEARCE


55

over, the compound was shown to be sevenfold less inhibitory against the de novo system, with an ICs0 of 304 yM. In a further series of experiments to determine the antiinflammatory effect of ZG-l494a, this compound showed inhibition of PAF binding for PAF sites, pyrilamine binding to histamine HI receptors, and dexamethasone for glucocorticoid receptors, all with an ICs0 of 3 pM.These data suggest that ZG1494a may be a useful antiinflammatory agent. Blood coagulation has been implicated in a variety of conditions. Fungal metabolites that inhibit blood clotting and compounds that promote clot removal have been reported. Thus, a number of serine protease inhibiting asterriquinone (157-161) pigments were isolated from two Humicola fuscoatra, a Humicola grisea, a Botryotrichum sp., and an Aspergillus terreus, all of which had been isolated from soil samples collected in Mexico or Nevada (Mocek et al., 1996). The asterriquinones CT1-CT5 were specific inhibitors of certain serine proteases involved in blood coagulation. Factors VIIa, Xa/Va and Xa were inhibited, with ICs0 values between 3 and 135 yg/ml for those that were active, whereas thrombin and trypsin were very much less sensitive to these metabolites. Once blood clots have formed, the fibrinolytic system dissolves them over time. Plasmin, the primary degradation enzyme, is derived from plasminogen, which itself is activated by tissue-type activators, and this is accelerated on fibrin surfaces. Increasing the binding of plasminogen to fibrin is expected to enhance the whole system. A metabolite from Stachybotrys microspora has been isolated that increases the binding of plasminogen to fibrin (Shinohara et al., 1996). is a triprenylphenol shown to increase the binding of Staplabin (162) radiolabeled plasminogen to fibrin at concentrations between 0.3 and 0.6 mMto 110-270% of the control. The binding of labeled plasminogen to human U937 cells was increased approximately twofold by 0.37 nM staplabin. No further details of bioactivity were given in this preliminary communication. Platelet aggregation inhibitors have been found from extracts of Beauvaria bassiana (Kagamizono et al., 1995), the bioactive compound being a phenylalanine derivative, bassiatin (163). The producing culture was isolated hom a Chinese soil sample. Bassiatin inhibited rabbit platelet aggregation induced by ADP, collagen, and arachidonic acid, with ICs0 values of 0.19 mM, 0.38 mM, and 0.38 nM, respectively. Bassiatin did not inhibit ACAT at concentrations up to 10 yM. Cell adhesion and cell adhesion molecules have been implicated in various diseases, and this has created an interest in antiadhesion compounds. Hayashi et al. (1995) have isolated antiadhesant compounds from a Microsphaeropsis culture obtained from a soil sample. The two

56

CEDRIC PEARCE

OH

(163) Bassiatin

OH I

OH I

0

0

HO H3C

0U

C

H

s

(164) MacrospheliieA

(165) MacrosphelideB

0

H (166) Eporactaene

STRUCTURES 162-1 6 6.

macrosphelides (164 and 165)were tested in an adhesion assay using HL-60 cells that bind to human umbilical vein endothelial cells, and were shown to inhibit this process, with values of 3.5 and 36 pM for A and B, respectively. These compounds did not show cytotoxicity against a panel of human cell lines, and no acute toxicity was observed in mice at 200 mg/kg. Macrosphelide B was weakly antimicrobial, but the A component showed no activity against any of the bacteria or fungi used. The authors suggest it would be interesting to test these compounds against metastasis and tumor invasion. has been Finally, a very interesting compound, epolactaene reported from a Penicillium species isolated from a ocean sediment

(m),


57

sample (Kakeya et af., 1995). Epolactaene, which was produced in a complex medium containing glucose, starch, soybean meal, polypeptone, meat extract, yeast extract, potassium phosphate, magnesium sulfate, and 0.2% sodium chloride, causes changes in the morphology of SH-SY5Y cells, a neuroblastoma cell line. In control experiments, less than 5% of cells produced neurites from the cell bodies, but in the presence of 2.5-10 pg/ml of epolactaene 74% of the cells produced neurites. No further results were reported.

XI. Agriculturally Active Compounds

There is a long history of using natural products for agricultural and animal applications: for example, avermectins for antiparasitic compounds, and kasugamicin for rice blast disease. Natural products have been shown to be ideal agrichemicals in a number of ways, especially given their high potency and specificity. Arthrobotrys oligospora was first shown to trap nematodes in 1888 (Zopf, 1888), and subsequent study has demonstrated that this may be useful in the control of nematodes. Some chemical analyses of metabolites from Arthrobotrys oligospora have been reported, but only recently have nematocidal compounds been discovered. Thus, Anderson et a f . (1995) isolated a series of related compounds (167-172), including oligosporon, oligosporol A and B, 4’,5’-dihydro-oligosporon, hydroxyoligosporon, and lO’,ll’-epoxyoligosporon,from cultures of an Australian isolate of Arthrobotrys oligospora. Three of these compounds-oligosporon, oligosporol A, and oligosporol B-had been previously isolated (Stadler et af., 1993c) from another strain of Arthrobotrys ofigospora, although in this latter paper activity against Caenorhabditis elegans was not demonstrated. In the work by Anderson et al. (1995), the compounds were tested against an intestinal parasitic nematode, Haemonchus contortus, with oligosporon and 4’,5’-dihydro-oligosporon having LDS0 values of 25 and 50-100 &ml, respectively. The authors propose that in nature these compounds may play a role in the interactions of the fungus and its prey, as well as showing the potential for protection against infestations of nematodes. In a series of papers from the Kitasato Institute in Japan, a number of novel anticoccidial fungal and actinomycetes products have been reported. Fudecalone (173) was isolated from a Penicilfium sp. obtained from a soil sample collected in Shizuoka (Tabata et al., 1995b). This is a relatively simple terpene-type metabolite, structurally related to the plant products corymobtins and clerodane, and produced in a complex

58

CEDRIC PEARCE

0 (167) Oligosporin

0 (168) Oligosporol A

0 (169) Oligospord B

STRUCTURES 167-169.

medium containing starch, glycerol, soybean meal, yeast, potassium chloride, potassium phosphate, magnesium sulfate, and calcium carbonate. Fudecalone completely inhibited monensin-resistant Eimeria tenella at 16 pM, but no activity was observed against Gram-positive and -negative bacteria, or fungi. The bicyclic arohynapenes 174 (Masuma et al., 1994; Tabata et al., 1995a) were also isolated from a Penicillium sp. FO-2295 and shown to be active against Eimeria tenella. Of the four metabolites reported, arohynapene D was active against monensin-resistant organisms, with a minimum effective concentration of 0.51 pA4, and showed cytotoxicity against the BHK-21 cell line used at 1.0 pM. A screening program for ascomycete and basidiomycetes metabolites with activity against nematodes led to the discovery of a number of active compounds from Lachnum papyraceum. These include lachnumycorrhizin A chloromycorrhizin mon (175),lachnumol A A (m), and dechloromycorrhizin A (179) (Stadler et a]., 1993a,b).

(m),

(m),

59


(170) 4,5diydrc-oligospwon

(171) hydroxyoliispron

0 (172) 10,l I-epoxyoligospron

STRUCTURES 170-1 74.

Noting that these fermentation products contained chlorine atoms, these authors have investigated the effects of including other halogens in the production medium (Stadler et al., 1995a,b). In a medium in which the usual calcium chloride was replaced with calcium bromide, the fungus grew poorly and produced no significant secondary metabolites. However, when calcium chloride was used together with either calcium bromide or calcium fluoride, growth and appearance of the culture was similar to that of the usual fermentation. Although the fluoride-containing medium showed no difference in secondary metabolite profile, the inclusion of calcium bromide led to several significant

60

CEDRIC PEARCE 0

OH

CI

(175) Lachnumon

(176) Lachnumol A

0 (177) MycorrhizinA R,=H. R&l (178) Chloromyarrhizin A R j = R+I (179) dechlorormyarrhizinA Rl=R+-I

(169) Ghydroxymdlein R=H (181) 4-chloro-6,Edihydroxy-2-fnethylisochroman-l-one R=CI

OH

(182) 4-bromc-6,Edihydm~3-me~ylisochroman-l-one R=CI

CH30 R'

H

(183) Ehydroxy-Smethoxy-3-methylisochroman-1 one

R=H

(184) 4-chlorc-Ehydroxy-6-methoxy-3methylisochroman-l-one R=CI

(185) 4-chlorc-5.6,&trihydroxy-3-1nethyii~hroman-l -one

STRUCTURES 175-185.

changes. Thus, lachnumon, chloromycorrhizin, and dechloromycorrhizin were undetectable, while lachnumol A and mycorrhizin were produced as very minor components. Instead, five new metabolites were produced and a sixth, originally present as a very minor component, was produced at concentrations high enough for isolation and characterization. These compounds were the previously known 6-hydroxymellein (180),which had been isolated from a number of sources


61

including Gilmaniella humicola, and the following compounds previously unknown as natural products; 4-chloro-6-hydroxymellein 4-bromo-6-hydroxymellein (E), 6-methoxymellein 4-chloro-6(185). These methoxymellein W),and 4-chloro-6,7-dihydroxymellein compounds were weakly nematocidal, as well as showing some cytotoxicity, phytotoxicity, and antimicrobial activity. From fermentation and biosynthetic perspectives these results are very interesting. In the biosynthesis of mycorrhizins and similar compounds in Gilmaniella humicola (Chexal et al., 1979), it has been proposed that 6-hydroxymellein is an intermediate that is decarboxylated and prenylated. The lauchnumols and lachnumons are probably derived from a similar pathway, which in the presence of calcium bromide is inhibited. This leads to buildup of the intermediate 6-hydroxymellein, which is a substrate for more unusual halogenation, hydroxylation, and methylation reactions. Similar observations have been made on the effects of enzyme inhibitors on the metabolism and final products from actinomyces (Pearce, 1995; Pearce et al., 1991,1995, 1995), and this appears to be an area for further application by those interested in discovering novel metabolites from known organisms. Since calcium bromide appeared to inhibit the formation of derivatives of lachnumon or mycorrhizin, and because halogenation steps frequently occur towards the end of the biosynthetic route, it was reasoned that, if this was added after the end-products had started to appear, there may be a different effect on the final metabolites produced. When calcium bromide was added to 10-day-old fermentations, Lachnum papyrcreceurn produced eight novel metabolites, four of whichlachnumon B 1 (=), lachnumon B2 (=), mycorrhizin B 1 (=), and brominated (Stadler et al., 1995c,d,e).The mycorrhizin B2 (=)-were nonhalogenated compounds 1’Z)-dechloromycorrhizin A (190) and papyracons A, B, and C (191-193)are related structurally to mycorrhizins. All eight compounds had activity against Caenorhabditis elegans, a limited spectrum of fungi, were cytotoxic, and to varying degrees were antibacterial. Although more potent derivatives were not produced, this type of study is invaluable for understanding both the fermentation and biosynthetic processes, and for providing new compounds for additional structure-activity relationship studies. A number of interesting phytotoxins, some which are potential herbicides, are known to be produced by fungi, including tentoxin from. Alternaria alternata (Lax and Shepherd, 1988), various compounds from plant pathogenic fungi (Sakamura et al., 1988), cyclopenin and cyclopenol, 3,7-dimethyl-8-hydroxy-6-methoxyisochroman, and cinerain (Cutler et al., 1988), for example. The interest-

(m),

(m),

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0

OH

II

OH

1

0 (192) papyrachonB Rj=OH, R+ (193) papyrachonC Rj=H. R@H

/

o

(194)Come*slin R=CH3 (195) Hydroxymmexislin R = C H P H

STRUCTURES 186-195.

ingly named cornexistin (194) was reported from Paecilomyces variotii Bainier (Nakajima et d., 1991). This compound had activity against grass and broadleaf species. Further work on the producing strain led to the isolation of a second metabolite, 14-hydroxycornexistin (195) (Fields et al., 1996),which was discovered in the fermentation broth of P variotii as a very active metabolite. Subsequent testing against a variety of broadleaf and grass weeds showed that cornexistin and hydroxycornexistin display similar herbicidal activity, with the latter compound being particularly good against broadleaf weeds. In a second report, a Chrysosporium sp. that had been isolated from a soil sample collected in Wisconsin was found to produce dihydrobisdechlorogeoa new compound, and the known bisdechlorogeodin (197) din

(m),

63


H

&

W

o

\

H

3

G

-

w0

\ COOCH3

OH

OH

(196) Dihydro-bisdechlorogeodin

0 COOCH3

(197) (-)-bisdechlorogeodin

STRUCTURES 196,197.

(Tanaka et al., 1996). These compounds were initially isolated because fermentations of this fungus in a medium containing starch, glycerol, soybean meal, yeast, potassium chloride, calcium carbonate, magnesium sulfate, and the phosphate trapping-agent allophane exhibited antibacterial activity. The antibacterial activity was shown to be due to bisdechlorogeodin, and this was also shown to be antagonized by the addition of casamino acids, or L-alanine, L-aspartic acid, or L-glutamic acid to the bioassay. This suggests that bisdechlorogeodin is an antimetabolite. In addition, both dihydrobisdechlorogeodin and bisdechlorogeodin showed herbicidal activity, although at what seems like high concentrations. It was further proposed that dihydrobisdechlorogeodin is transformed into bisdechlorogeodin intracellularly. A variety of approaches have been used to discover insecticidal fungal metabolites, including both random and more directed searches. As is pointed out in the fungicidal section, it is possible that fungi engage in chemical defense by producing compounds that exert an effect on either a predator or competitor. There is evidence that fungi may produce compounds that act as insect antifeedants, and this area has been reviewed extensively (Gloer, 199513). Briefly, it has been discovered that certain fungal survival structures, such as sclerotia, which may be exposed to insect predation, often contain compounds that are antiinsectans. For example, it has been observed that the beetle Carpophilus hemipterus avoids sclerotia of Aspergillus flavus while eating other parts of the same fungus. When A. flavus was grown on corn, analysis of the sclerotia led to the discovery of a series of novel indole Using a C. hemipterus diterpenoid compounds, called aflavines (198). feeding assay, it was shown that the most common aflavine completely inhibited feeding at concentrations in the diet far below those found in nature. The compounds were not present in other parts of the fungus,

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H (199) Aflavarin

(198) Aflavine R=H m OH WH3

STRUCTURES 198, 199.

nor were they expressed in any liquid medium tested. This very interesting observation again focuses on the method used for cultivation as being critical for full metabolic expression from cultures being investigated. During the A. flavus study, Gloer’s group discovered a number of other novel compounds including aflavarin (199)and p-aflatrem (200). A wider search for metabolites from the sclerotia of fungi related to A. flavus led to the discovery of novel bioactive compounds from Aspergillus nomius, Aspergillus leporis, Aspergillus tubingensis, Aspergillus niger, Aspergillus carbonarius, Aspergillus sulphureus, Aspergillus rnelleus, and Aspergillus alliaceus (Gloer 1995b). Further studies by Gloer and his co-workers into the metabolites of other fungi led to investigation of antiinsectan compounds from the sclerotia and sclerotioid ascostromata of Penicillium and Eupenicillium. A number of novel metabolites were discovered including the sherinines 201-203 and isopentenylpaxillin 204 from Eupenicillium shearii (Belofsky et ul., 1995). These compounds were potent antiinsectans against H. zeu and C. hepipterus. The ascostromata of Eupenicillium crustaceurn were shown to contain 0.3% by weight of the aflavines previously discovered from Aspergillus tubingensis. This is a very interesting observation because both fungi, although quite different taxonomically, do inhabit similar areas and seem to have evolved or otherwise acquired similar biosynthetic routes. Other Eupenicillia were shown to contain quite different metabolites, emphasizing the distinct differences between the fungi and highlighting the value of testing various sources when looking for novel bioactive compounds.


6

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66

0

I (205)Destruxin A4

(206)Destruxin A5 STRUCTURES 205, 206.

From the white fly entomopathogen Aschersonia sp., two new dehave been isolated (Krasnoff et al., struxins, A4 (205) and A5 (m), 1996). In these new destruxins, methylisoleucine replaces the more common methylvaline residue found in other destruxins. Destruxin A4 and A5 showed insecticidal activity against Drosophila melanogaster, with LC50 values of 4 1 and 52 ppm, respectively. A completely different approach to that used by Gloer's group has been used by Morino et al. (1995),who discovered the insecticidal agent NK3 74200 (207). These workers screened for metabolites containing purine or pyrimidine. A Talaromyces sp. that had been isolated from a soil sample was shown to produce NK374200, and subsequent to the isolation and purification of this compound, by testing in various assays, it was shown to have antimosquito larval activity. This compound was shown to have low cytotoxicity against HeLa cells and in mice. No other activity was reported. XII. Summary and Conclusions

There is clearly a lot to be learned from the study of fungal metabolites, and clearly there is a lot we do not know about why and how these metabolites accumulate. Throughout the development of organic chemistry, there has been an interest in natural products, and fungal metabolites have received considerable attention. With the introduction of


67

(207)NK374200 STRUCTURE 207.

novel approaches to detecting biologically active compounds, there has been somewhat of a renaissance in the field, and, coupled with relatively nontraditional approaches to isolating fungi, new metabolites are being discovered. The importance of the new screening techniques is highlighted by the discovery of compactin and mevinolin and related compounds, and by the discovery of the zaragozic acid/squalestatin group. Both these families of metabolites were found independently by a number of groups and were subsequently shown to be quite common products hom not rare fungi. Recent discoveries of metabolites with a variety of activities are included in this chapter, but it is obvious that there are common themes. For example, there is a preponderance of antifungal metabolites, an interesting observation in light of Gloer’s ideas about antagonism between fungi in nature, and there have been a significant number of metabolites reported that affect cholesterol metabolism. Again this is perhaps of some significance in the metabolism of ergosterol and related compounds in the fungi. Both of these observations, however, could also be a reflection upon the fact that they comprise a number of complex enzymatically controlled events and as such provide a large number of different opportunities for inhibition. This is perhaps the reason why antiviral fungal metabolites are less common, because there are fewer significant unique biochemical events to be inhibited. Commonly, when a novel metabolite is detected, it is one of a family of metabolites produced by the culture being studied. This is apparent in the discoveries discussed herein, as well as in previous reports. This illustrates another powerful feature of drug discovery from fungal (and

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other microorganism) sources. Analysis of the bioactivity associated with each metabolite allows for structure-activity relationships to be formulated, if in a preliminary state. The future for this area is promising. There is huge potential for novel chemistry from the untapped fungi yet to be isolated. The basic assumption is that biodiversity translates into chemidiversity, which, although true at the nucleic acid level, is not proven to be so in a practical sense. However, time will tell. One clear area for advances is in the methods used for culturing fungi in ways that engender production of these unusual metabolites so often associated with biological activity. Other issues to be dealt with include rapid characterization of products and swift yield improvement to obtain material for biological testing. In these trimmed down times of “lean and mean,” the challenges are there, but so are the sources of bioactive compounds. REFERENCES

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Zopf, W. (1888). Zur Kenntnis der Infektions-Krankheiten neiderer Tiere und Pflanzen. Nova Acta Leopold Carol 52, 314-376.

Old and New Synthetic Capacities of Baker’s Yeast P. D’ARRIGO,

G. PEDROCCHI-FANTONI AND S. SERVI

CNR Centro per lo Studio delle Sostanze Organiche Naturali Dipartimento di Chimica Politecnico di Milano 20231 Milano, Italy

I. Introduction 11. Reducing Capacities A. Enzymes from the Fatty Acid Synthetase Complex B. Carbonyl Groups in Heterocyclic Compounds C. Nitrogen-Reducible Functional Groups D. Sulfur-Containing Compounds 111. The Formation of G--C Bonds n! Oxidations: Getting the Other Enantiomer V. Hydrolytic Activities: Phosphate Esters VI. Lyase Activity VII. The Biogeneration of Aroma Compounds VIII. Conflicting Reports IX. Conclusions References

I. Introduction

It is well documented in the literature of the last decade that biocatalysts (microorganisms and isolated enzymes) have found widespread application as selective reagents for the solution of synthetic organic chemistry problems. Although the extraordinary specificity of enzymatic catalysts has been known for a long time, they have only recently been applied in the selective transformation of unnatural substrates, mainly in the production of enantiomerically pure chiral compounds. It is now widely recognized that biologically active compounds used as pharmaceuticals should not be applied as racemates (Servi, 1996). Together with the absolute need to develop environmentally acceptable reagents and processes, these reasons are responsible for the observed growth of biocatalysis. The nature of enzymatic catalysis and the significance of stereochemistry in biological activity was already present in the work of Emil Fischer. The introduction to his article “Bedeutung der Stereochemie fur die Physiologie” (Fischer, 1889) could very well find a place in a 81 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 44 Copyright B 1997 by Academic Press, Inc. All rights of reproduction in any form reserved. 0065-2164/97 $25.00

82

P. D’ARRIGO et (11.

modern Introduction to Biocatalysis. Due to the growing number of papers in all sectors of chemistry, the wide range of interdisciplinary journals in which works in biocatalysis are published, and the rapid growth of this discipline, accurate awareness of the actual progress in this specific scientific domain requires continuous attention and the use of current awareness electronic bulletins and databases. While these tools are of fantastic help in information retrieval, the knowledge and citations of the work accomplished during or before the sixties are rapidly declining for the simple reason that they are not yet included in even the most comprehensive databases. Yet, the study of the archaeology of biocatalysis, to paraphrase the title of similar observations on enzymology (Neidleman, 1990), will not only be useful for the relevant scientific information found there, but also for the stimulating (sometimes frustrating) opportunity to compare the scientific novelty and intellectual effort present in today’s rapid communication with that met in similar studies of 80 years ago. Review articles would also help to fill this gap. These considerations apply equally well to the particular kind of biocatalysis represented by Baker’s yeast biotransformations. Baker’s yeast has had special merits in introducing the use of biocatalysts in the practice of organic synthesis, and hundreds of articles have been devoted to research in which yeast is applied in the biotransformation of unnatural substrates. At the end of the nineteenth century, microorganisms in general and yeast in particular were used for their production of hydrolytic enzymes that were applied in the transformation of polysaccharides. The specific action of yeast on different substrates was fundamental in the identification of new enzymatic activities and in the determination of their specificity. In “Bedeutung der Stereochemie fiir die Physiologie,” Fischer (1889) affirms that “bei der zuvor erwiihnten Versuchen hatte ich Gelegenheit, die Enzyme der hefe genauer kennen zu lernen” [following those experiments I had the opportunity to know with more precision the enzymes from yeast]. The process of submitting new substrates to yeast activity, allowing identification of new enzymatic activities, is of great interest in that genetic manipulation affords the possibility of enhancing synthesis of the enzyme with production of useful new catalysts. Baker’s yeast’s incredible ability to respond to new substrates with new synthetic activities gives the opportunity to uncover novel exploitable synthetic capacities. Comprehensive review articles (Czsuk and Gliinzer, 1991; Servi, 1990) and practical procedures (Roberts, 1992) for Baker’s yeast biotransformations have been reported. In this chapter some of the most interesting biocatalytic capacities expressed by yeast on old and new substrates will be critically considered.

OLD AND NEW SYNTHETIC CAPACITIES OF BAKERS YEAST

83

I I . Reducing Capacities The reducing capacities of yeast have attracted the interest of modern organic chemists in the preparation of chiral secondary alcohols (Ward and Young, 1990). The observation that the addition of powdered sulfur in fermenting yeast resulted in the formation of H2S (Dumas, 1874) was probably the first published evidence of the reducing capacity of yeast. However, following the observation that proteins containing sulfhydryl groups produce hydrogen sulfide in the presence of sulfur, Heffter (1908) denied that yeast enzymes were at all involved in the reduction of sulfur. The attention given to discriminating between actual products from enzyme catalysis and artifacts was particularly intense. The reduction of carbonyl groups was first shown with aldehydes and then on ketones, nitro groups, and so on. Most of this work was reviewed by Neuberg (1949). Subsequently (Levene and Walti, 1943; Macleod et al., 1964; Guette and Spassky, 1972; Deol et al., 1976), the use of Baker’s yeast in carbonyl reduction has been considered a means of obtaining chiral secondary alcohols in enantiomerically pure form. This aspect was not considered previously because of the difficulty involved in assessing the absolute configuration and enantiomeric purity of chiral compounds. The method of reducing carbonyl groups for preparation of secondary alcohols has since become an established procedure. The stereochemistry of the reduction can be predicted since it follows the Prelog rule (Prelog, 1964). Microbial reduction with microorganisms is still preferred to the use of isolated oxidoreductases, since the regeneration of cofactor is still an unsolved practical problem. However, even optimized procedures are often not compatible with the strict economical requirements that must be met in industrial practice (Schmidt et al., 1992). Industrial preparation of trimegestone ( via l ) the Baker’s yeast catalyzed reduction of the corresponding 1,Z-diketone (2) (Fig. 1)has been reported (Crocq et al., 1997; Buendia et al., 1993). Using glycerol as the energy source, 240 g of yeast was used per g of substrate at a concentration of about 2 g/liter. The reaction was best performed at 4O0C in aerobic conditions. Despite the large fermentation volume, 99% pure hydroxyketone was recovered in 75% yield. The enantiomeric excess of the product was higher than 99%. Although other yeasts from culture collections proved to yield higher productivity, Baker’s yeast was preferred because of its lower price. The fact that yeast biomass is applied on an industrial scale in enantioselective reduction adds significance to this biocatalyst. Further applications of Baker’s yeast in industrial biotransformations will be mentioned.

P. D’ARRIGO et al.

84

2

1 FIG.1.

A. ENZVMES FROM THE FATTY ACIDSYNTHETASE COMPLEX

Some of the most interesting enzymatic capacities found in yeast are expressed by the fatty acid synthetase complex responsible for in vivo synthesis of fatty acids (Walsh, 1979a). The dehydrogenases catalyzing the reduction of the 3-oxothioester groups and of the C=C double bond conjugated with the carbonyl group can be exploited synthetically on structurally analogous substrates. Both the 3-oxoester and the C=C double bond reduction are considered by organic chemists to be of special interest for the production of chiral hydroxyesters, valuable chiral synthons, and of building blocks chiral for the presence of a methyl-bearing carbon, respectively. At least two different oxidoreductases are found in yeast that are able to reduce 3-oxoesters (3) with opposite stereochemical preferences (Heidlas et a]., 1988) (Fig. 2). The production of enantiopure 3-hydroxyesters (4) is an important achievement due to the value of these and related compounds as chiral synthons (Sheldon, 1993). A number of structurally related 3-hydroxyesters (4) of (S)-absolute configuration (L-series) can be obtained with yeast and other microorganisms with different conditions and efficiency and high to very high enantiomeric excesses. Simple structural variations allow for alteration of the stereoselectivity of the reduction. When R = CH3 the enantiomer of opposite configuration is available either by depolymerization of natural polyhydroxybutyrate (Seebach et a]., 1982) or by oxidation of butanoic acid with Candida rugosa (Hasegawa et a]., 1981). In practice, both enantiomers of 3-hydroxybutyrate and valerate are available in enantiomerically pure form through biotransformation or fermentation. The preparation of a-alkyl-P-hydroxyesters in the form of enantiomerically pure single diastereomers represents a more challenging synthetic problem and is not easily

85


2

RM o R R

3

OR

4 FIG.2.

L-enzyme 1 L-enzyme 2

l6

anti (2S.3S)

FIG.3.

accomplished by bioreduction of the corresponding racemic ketone. Also in this case, the carbonyl group is usually reduced with high enantiospecificity irrespective of the configuration of the adjacent chiral center. Enzymes with different stereochemical preferences for 3-0x0esters and 2-alkyl-3-oxoestershave been isolated (Heidlas et a]., 1988; Shieh et al., 1985; Shieh and Sih, 1993; Sih et al., 1983; Nakamura et al., 1991). They are NADPH-dependent enzymes and are able to catalyze the reduction of oxoesters of different type. Figure 3 shows that the two substrates 5 and 6 are in rapid equilibrium through the enol form (2).The L-enzyme (I)binds preferentially to the S-ketoester (5),whereas the L-enzyme (2) preferentially binds the R-ketoester (6). In both cases, the hydride equivalent is delivered to the Re face of the carbonyl group to give the respective products (Shieh and Sih, 1993). Enantiomerically pure anti and syn hydroxyesters can be obtained when the purified enzymes are used. In yeast the two enzymes have been estimated to be approximately 1:l. The kCat/Kmratio for the two

86

P. D'ARRIGO et a].

FIG.4.

FIG.5.

enzymes should account for the stereochemical outcome of the transformation. In the case where R, = ally1 and R2 = ethyl, the result is a 7 7 2 3 antisyn ratio. Creative variations of the use of yeast on 0-ketoesters have allowed kinetic resolution of racemic secondary alcohols and amines through Baker's yeast reduction of the corresponding acetoacetyl derivatives with low to medium enantiomeric excesses. The recognition of the sign of chirality on a carbon far removed from the actual center involved in biotransformation is remarkable (Hudlicky et a]., 1991, 1992) (Fig. 4). In a similar experiment, a-alkyl-P-ketoesters of a chiral alcohol were reduced but with a purified L-a-alkyl-P-ketoester reductase, and the resolution of the alcohol was complete, as shown in Fig. 5 (Kawai et al., 1995b). The following example refers to a remarkable case of diastereo- and enantioselectivity displayed on the same substrate, allowing establishment of two chiral centers in one operation (Fig. 6). The yields are very high (40 and 80% of maximum obtainable), as are the diastereoisomeric and enantiomeric excesses (Eh and Kalessem, 1995). The preparation of chiral compounds with a tertiary carbon atom chiral for the substitution pattern in Fig. 7 can be effected through the asymmetric reduction of a triply substituted C=C double bond.


87

40% yield, de>98%, ee=93%

FIG.6 . X

I

FIG.7.

This reaction, which has no general examples in asymmetric hydrogenation with chiral catalysts, can give very good results if the double bond is activated with strongly polarizing groups. The ability to reduce C=C double bonds has been attributed to the enoate reductases (EC 1.3.1.31) of the fatty acid synthetase (Cornforth, 1959; Dugan et al., 1970). Their activity has been found in several microorganisms (Holland, 1992), and their biocatalytic properties have been studied extensively in Clostridia (Thanos et al., 1987). While the reducing capacity of Baker’s yeast is quite accessible, microorganisms of the Clostridium type are anaerobic bacteria very sensitive to dioxygen and not as easy to grow (Kuno et al., 1985; Bader and Simon, 1980). However, they have extraordinary reducing capacities with very high productivity numbers (10-100 times higher than the corresponding reactions with yeasts), allowing reduction of large amounts of substrate with very little biomass. They have high reducing potentials, unique among microorganisms, allowing them to reduce carboxylic acids to primary alcohols, activity seldom reported in the literature (Fronza et al., 1995), or, as in this case, the C=C double bond of a$-unsaturated acids or esters. Moreover, in their use in biocatalysis with resting cells, the limited amount of cofactor present in the cells can be efficiently regenerated with hydrogen gas in the presence of small amounts of artificial mediators (viologen) or with an electrochemical regeneration system. The use of Clostridium tyrobutyricum DSM 1460 and of Clostridium kluyveri DSM 555 has been fully described (Thanos et al., 1987). These enzymatic systems have not become so popular among chemists due to the initiation required in manipulation of material and methods not familiar to organic chemists. The capacity of these systems are described in detail in the literature (Simon et al., 1985). The C=C reduc-

88

€? D’ARRIGO et a1

X = H , D, CH,, CI, Br, CF,

A = CHO. CH,OH, CH(OCH,),. COOR, NO,

FIG.8 .

ing capacity is assigned to an enzyme that has its best activity in a typical range of pH (5-8) and is usually employed at pH 6. It is NADHdependent, and a number of regenerating systems have been proposed. Ki for aliphatic enoates and their reduced products are rather high (500 mM), and they are therefore suitable for preparative applications. Stereochemistry of double bond reduction occurs with trans hydrogen delivery, and the chirality of the product obtained has been studied. The stereochemical preference is opposite for E and Z double bonds. Low enantiomeric excesses are obtained if the Z-E conversion is favored in the reaction conditions. Substrates accepted by the enzymatic systems are restricted to the ones having a small X, usually -CH3 in most examples, but also halogen or ethyl. Enoate reductase from yeast is a similar enzyme in many respects, but it is not able to reduce a$-unsaturated acids or esters, unless X is a halogen. The activating groups of the substrates are -CHO or NOz. Allylic alcohols are substrates since they are usually partially transformed with yeast cells to the corresponding aldehydes by the alcohol dehydrogenases present (Servi, 1990). Kinetic constants and operating parameters are not as well defined as for the parent enzyme from Clostridium that has been purified and used in the absence of other enzymes. All the examples reported in the literature for yeast enoate reductase make use of whole-cell biocatalysts. The range of substrates submitted to reduction is quite large, and the yields and enantiomeric excesses of products are remarkable. The two different types of triply substituted olefins in Fig. 8 can be the substrates for the reduction, and they give as products methyl alkanols (or corresponding products) of two different chirality senses.

89


/substrate/

[substrat4

AoMe W

C 0

OMe

H

T

0

Colt

O

i

+ O H .

?OH

C0,Et

b u g 1

8

O

H

9

voH A \

0

OH

OH

11

10

14

15

17

FIG.9.

The synthetic application of the compounds obtained in homochiral form using this method is found in the preparation of natural products. Compounds 8 , 9 (Leuenberger et al., 1979), and lo (Fuganti et al., 1988b) in Fig. 9 are equivalent bifunctional chiral synthons that have been employed in the synthesis of a-tocopherol (Fuganti and


90

5OHCHO

0

0

20 l9

\slow

-& fast

CHO

OH

0

22

21 FIG.10.

Grasselli, 1979, 1982). Compounds 11, 12, 13, and 14,deriving from olefins of type B, have the opposite configuration of the chiral methyl, Ohta et bearing carbon (Gramatica et al., 1987, 1988; Sat0 et ~ l .1988; al., 1989). Compounds 15 and show that the stereochemistry of the newly formed chiral center depends on the configuration of the double bond (Utaka et al., 1987). Compounds 17 and 18 are intermediates in the synthesis of zeaxanthin and of another precursor of a similar carotenoid. The products are prepared with yeast at a multi-kg scale (Leuenberger, 1985). The mechanism of the reduction of allylic alcohols has been studied in detail in the case outlined in Fig. 10. The aldehyde 19 is rapidly reduced to the allylic alcohol 20, and its level stays low and constant for the entire reaction time. The aldehyde present at equilibrium conditions is then transformed into the saturated aldehyde 21 and then into the final product 23. This behavior is confirmed by the fact that allylic alcohols are not reduced in the absence of an alcohol dehydrogenase, that is, with a purified enoate reductase. The reduction shown in Fig. 10 has been scaled to hundred of grams for the preparation of C5 chiral synthons (Fuganti et ~ l .1992). , B. CARBONYL GROUPSIN HETEROCYCLIC COMPOUNDS

Masking and unmasking carbonyl groups is a common technique in organic synthesis for the temporary protection of such a reactive func-


23

27

28

25

91

24

26

FIG.11.

tionality. One way of doing so is to build a heterocyclic ring across the functional group to be protected and subsequently release it under the mildest possible conditions. Among the possible heterocyclic rings employed to mask functionalities are isoxazoles, isoxazolines, thiazoles, furane derivatives, thiophenes, and dithianes. Acetyl pyridines, isoxazoles, isoxazolines, and thiazoles are effectively reduced by fermenting yeast to the corresponding secondary alcohols, usually with good control of enantiomeric purity (Fig. 11). Compounds 23 and 24 are obtained in good yield and high enantiomeric excess, although of unknown absolute configuration (Bianchi et al., 1984). From the acetyl isoxazolines, the two diastereoisomers 25 and S resulting from enantioselective carbonyl reduction are obtained. The enantiomeric excess is strongly enhanced by using 2-propanol as an additive (Ticozzi and Zanarotti, 1988).The thiazolyl ketone 22 is easily reduced to the carbinol of S-configuration. This compound is considered an equivalent of lactaldehyde (Dondoni et al., 1988).It has been reported that other isoxazoles fused in a six-membered ring are reduced in fermenting yeast with cleavage of the N-0 bond (Fig. 12). This kind of ring opening usually occurs with hydrogen gas in the presence of metal catalysis. Very functionalized molecules often do not withstand

P. D'ARRIGO et al.

92

X = CH,,

FIG.12.

FIG.13.

these conditions. However, this method seems deprived of practical , application since product yields are very low (Easton et ~ l .1994). Sulfur heterocycles can be easily transformed into saturated carbon chains via catalytic reduction. The reduction with Baker's yeast of a series of sulfur heterocycles acting as protecting groups is reported in Fig. 13. In reactions 1-4 the reduction takes advantage of the higher enantioselectivity usually displayed by cyclic substrates when com-

OLD AND NEW SYNTHETIC CAPACITIES OF BAKER'S YEAST

93

FIG.14.

pared to open-chain ones. In this way, a-substituted-P-hydroxyesters of high enantiomeric excesses are obtained (Hoffmann et al., 1981). In the case reported in reaction 5, the preparation of methyl alkanols via the thiophene intermediate was chosen because of the higher enantiomeric excess and efficiency observed in the production of the product. It was also shown that a family of 5-substituted compounds could be prepared by a general methodology and that all could be reduced with similar efficiency. In this way, a broad series of 2-methylalkanols were made accessible in enantiomerically pure form (Hogberg et al., 1992). C. NITROGEN-REDUCIBLE FUNCTIONAL GROUPS

The reduction of the nitro group to the corresponding amine is an important transformation, especially when homochiral amines become accessible. Neuberg studied the conversion of nitrobenzene to aniline in fermenting yeast (Neuberg and Welde, 1914b). The product was isolated in high yields. In order to elucidate the mechanism of such a reduction, and considering unlikely the direct transformation of the nitro group, a number of other possible intermediates were prepared and added to fermenting yeast. Figure 1 4 summarizes the positive results obtained. According to these findings (Neuberg and Welde, 1914a), it was deduced that nitrobenzene is initially reduced to nitrosobenzene and then to phenylhydroxylamine before this compound is further transformed into aniline. However, the yield of each individual step was lower than for the total transformation of nitrobenzene-aniline. This fact remains without an explanation.

94


FIG.15.

In a subsequent work (Neuberg and Reinfurth, 1923), m-dinitrobenzene was also submitted to fermenting yeast and the product of partial reduction was obtained (m-nitroaniline). The reduction of substituted m-dinitrobenzene has been reinvestigated and the regioselectivity of the reaction has been studied (Davey et al., 1994). The products obtained in regioselective reduction are the consequence of combined steric and electronic effects (Fig. 15). In the same work, o-nitrobenzonitrile (29) was reduced, but the corresponding aniline, expected from previous reports, was present in only minor amounts, the main reaction product being 2-aminobenzamide (30).This product might result from the initial reduction of the aniline to the hydroxylamine derivative 31, cyclized in turn to the benzoisoxazolidine 32 and then reduced again to the isolated product 30 (Fig. 16). This mechanism would reveal that the hydroxylamino compound is an intermediate in the reduction of nitro compounds, as previously inferred by Neuberg. A similar mechanism could also be invoked in the Baker’s yeast reduction of the nitroalkene bearing a cyan0 group in position 2 reported in Fig. 17, which leads to the formation of 5-amino isoxazole (34)(Navarro-Ocana et al., 1996). Since several grams of biomass per gram of substrate are usually employed for these biotransformations (24 in the last example), the possibility of a chemical reduction due to the presence of a stoichiometric amount of low-molecular-weight reducing compounds like carbohydrates or mercaptans should always be considered. In the latter case, for instance, it is known that unsaturated p-nitro nitriles are converted into amino-isoxazoles using mercaptans as reducing agents (Colau and Viel, 1980). In some cases the absence of enzymatic catalysis is evident (Baik et al., 1994) from the fact that operational conditions are employed (pH 14, 80°C) in which yeast does not retain any enzymatic activity. If nitro groups are presented in a different context, namely in the absence of an


29

95

30

!

I

31

32 FIG.16.

34

33 FIG.17.

aromatic ring, the group is not reduced. Thus, prochiral a$-unsaturated aliphatic nitro compounds are enantioselectively reduced to the chiral saturated compounds (Ohta et al., 1989), while y-nitro ketones are enantioselectively reduced to the corresponding (S)-4-nitroalcohols (Guarna et a].,1995). Other nitrogen-containing compounds have been transformed by other yeasts, including oximes and imines. It has been shown for instance that Torula yeasts are able to transform inorganic nitrates and nitrites into oximes and to further incorporate them into amino acids or their amides (Virtanen and Csaky, 1948). The reduction of aliphatic oximes has been reported. A mixture of E- and Z-stereoisomers of

96

P. D’AEWGO et 01.

L35

PhC ‘OOH

36

37 FIG.18.

38

39

R,, R, = H, Ph, Cyclohexyl R, = NHR, OH

FIG.19.

butanone oxime has been reduced to the corresponding amine of 58% ee. A similar result was observed by submitting to reduction the oxime ester 35 as a precursor of the oxime (Fig. 18). The oxime of benzoyl formic acid (36)of E-configuration was also reduced, although with low enantioselectivity. The (I?)-phenylglycine37 obtained was of only 20% ee. However, this reaction is of great potential interest. The involvement of a transaminase acting on the hydrolyzed oxime was excluded by an evaluation of the ratio of the compounds obtained (Fig. 19). From the aliphatic substrate, a small percentage of the hydrolysis product (Z-butanone) was isolated (Gibbs and Barnes, 1990). The authors considered the possibility that a specific hydrolase could be present in yeast. In fact, on other substrates and in different experimental conditions it was found that this hydrolytic activity is the prevalent or exclusive one. Indeed, a preparative method for the transformation of hydrazones and oximes of type 38 into the corresponding aldehydes and ketones 39 using Baker’s yeast has been proposed (Kamal and Reddy, 1992). The carbonyl compounds are obtained in yields higher


97

than 80%. 1 g of the hydrazone or the oxime was treated with 4 g of yeast in 70 ml of toluene and 6 ml of water. The reaction was complete within 15 h. The substantial differences between the two studies could be attributed either to substrate modification or to the experimental conditions (presence of solvent in the second case). No speculation about the nature of the catalysis was made. The N-oxides of pyridine, isoquinoline, morpholine, and others are reduced to the corresponding amino compounds in 40 to 70% yields in 5-6 days. 500 g of yeast per g of substrate were used (Takeshita and Yoshida, 1990). D. SULFUR-CONTAINING CO~OUNDS

Sulfur-containing compounds can undergo very different transformations by Baker's yeast, as shown from the following examples. Neuberg and Nord (1914) showed that thioacetaldehyde (40)in situ generated from the stable precursor 41can be reduced to ethylmercaptan ( 4 2 ) .Care was taken in showing that the reaction is not just an effect of the reducing capacities of carbohydrates. Higher homologues of thioaldehydes generated in situ by adding ammonia and hydrogen sulfide to the aldehyde were effectively reduced also using a cell-free enzymatic juice (Fig. 20). With other sulfur-containing substrates nonhomogeneous results have been observed. While diethyl disulfide was reported to be reduced to ethyl mercaptan (reaction 2) (Neuberg, 1949), some thiols, including benzyl mercaptans fermented under vigorously oxygenating conditions, were reported to be transformed into disulfides by yeast (reaction 3) (Rama Rao, 1992). However, other authors found that, under normal anaerobic fermenting conditions, benzyl mercaptan (43) was transformed in low yields into enanantiomerically pure (S)-benzyl thioglycerate (44). No trace of the disulfide mentioned in the preceding work (reaction 4) (Fronza et al., 1992a) was formed. In this example, selectivity was achieved by concomitant modulation of fermenting conditions and substrate modification. In the two equilibria between thiol and disulfide, it is likely that the catalyst is a thiol oxidase, which normally is active in the transformation of peptides and proteins containing sulfur functional groups. In the formation of disulfides, the concomitant chemical oxidation is occurring. The thioglycerate obtained in reaction 4 is probably formed in a mechanism in which benzyl mercaptans act as a nucleophile on an activated intermediate (a thioester, for instance) in the normal glycolytic pathway active in yeast metabolism. In fact, experiments with labeled glucose, fructose, and


98

I

H

41

2

42

40

vSH

\/s\s/\

3

4

___c

Ph-SH

OH

43

0

44

45

46

FIG.20.

mannose as nutrients have shown that the three carbon atoms found in (S)-benzylthioglycerate derive from the saccharides (Fronza et al., 199213). Moreover, experiments in D 2 0 show deuterium incorporation at various positions arising from the equilibrium between the three hexoses mentioned before and between dihydroxy acetone-P and glyceraldehyde 3-P. In an alternative mechanism, the thioglycerate could be formed by a reaction between benzyl mercaptan and hydroxypyruvaldehyde medi-

OLD AND NJZW SYNTHETIC CAPACITIES OF BAKERS YEAST

99

R = alkyl, aryl FIG.21.

ated by glyoxalase I in analogy with the behavior of glutathione with the same aldehyde. It is questionable, however, whether hydroxypyruvaldehyde is an intermediate in the alcoholic fermentation. Surprisingly, when racemic 2-methyl-3-phenylpropanethiol(45) (reaction 5) was used as the substrate under the same conditions as in reaction 4 of Fig. 20, (S)-2-methyl+phenylpropanethiol hemisuccinate (46)was obtained following partial kinetic resolution (40% ee) (Fuganti et al., 1991). In this case, the action of the nucleophilic thiol on an activated succinoyl unit probably mediated by succinate-thiokinase was invoked. The reactions of these sulfur derivatives are not particularly useful for preparative purposes. However, the results point out some exploitable enzymatic activities present in Baker’s yeast. It is worth noting, though, that performing reaction 4 of Fig. 20 in D 2 0 yielded isopropylidene glycerol selectively deuterated to a different extent at various positions depending on the monosaccharide used as the carbon source (glucose, fructose, or mannose) (Fronza et al., 1994). In none of the biotransformations described above involving mercaptans has the formation of products deriving from sulfur oxidation been observed. Specific strains of S. cerevisiae are reported as efficient biocatalysts in the oxidation of thio-analogues of fatty acids (Buist et al., 1990), of methyl-styryl sulfide (Fauve et al., 1991), and methyl tolyl sulfide (Beecher et al., 1995) (Fig. 21). Methyl thioethers of different structures have been oxidized to sulfoxides of R absolute configuration with the chloroperoxidase from Caldariomyces fumago (Colonna et al., 1990) and with the cyclohexanone monooxygenase from Acinetobacter calcoaceticus (Secundo et al., 1993). The Baker’s yeast reduction of the C=S bond in thioxoesters and thioketones has been the subject of investigation. The thiocarbonyl compounds analogues of P-ketoesters and ketones have been reduced. The thiols from the reduction of the thioxo group are accompanied by the alcohol from the reduction of the ketone formed by spontaneous

100

R

P. D'ARRIGO et al.

AO R

- - - C R

OR

OR'

FIG.22.

hydrolysis of the thio analogue. Comparison of the enantiomeric excess of the alcohol and of the thiol shows that the reduction of the thioxo group is less enantioselective than that of its keto analogue (Fig. 22). The effect of various additives used as inhibitors of oxidoreductases has been investigated. Increases in enantiomeric excesses are observed as in the case of the reduction of carbonyl groups. The ratio between substrate and yeast has been optimized with reference to the ee of products (Nielsen and Madsen, 1994). The conversion of thioureas and thiocarbamates to urea and carbamates has also been reported (Kamal et d.,1990).

I l l . The Formation of C-C

Bonds

The formation of carbon-carbon bonds is a fundamental operation in organic synthesis. If chiral centers are formed during C-C bond formation, stereochemical control is only observed when nonracemic chiral catalysts or reagents are involved. A valid complement to the asymmetric synthetic approach is represented by the use of biocatalysts, particularly aldolases and related enzymes, which find applications in the synthesis of rare carbohydrates (Wong et al., 1992). Although several aldolases, transketolases acyl-coA synthases, and other activities responsible for the formationhreaking of C--C bonds are present in yeast (Walsh, 1979b), they cannot generally be exploited in biocatalysis for biotransformation of unnatural substrates due to their narrow substrate specificity and the prevalent utilization of enzymes and natural substrates in the yeast metabolic cycles. Genetic engineering can provide a solution to the problem: fructose diphosphate (FruA) aldolase has been overexpressed in Saccharomyces cerevisiae and the whole cell organism utilized in biocatalysis using phenylacetaldehyde (Fig. 23) as the unnatural substrate (Compagno et al., 1993). Interestingly, the cells provide the cosubstrate (dihydroxyacetone phosphate, DHAP) and the phosphatase required to hydrolyze the initially formed phosphate ester (47).Although the product 9 is obtained in only low yields due to the concomitant utilization of the substrates by yeast, this system appears to be of great potential value. It can be compared with

OLD AND NEW SYNTHETIC CAPACITIES OF BAKER’S YEAST

/

101

Phosphatase

I

0

48 FIG.23.

multienzymatic systems where the same result is obtained by assembling the required enzymatic activities in a one-pot procedure, as in an artificial metabolism (Fessner, 1992). Other C-C bond-forming activities can be directly exploited in Baker’s yeast and constitute unique examples in biocatalysis of the utilization of whole-cell biocatalysts for this purpose. Although the biosynthesis of cholesterol and related compounds has been known for a long time, the efficient cyclization of polyenes to sterols has not been reported. The involvement of a single catalytic step from the open to the cyclized form has been postulated in lanosterol formation, but experiments were effected with minute amounts of protein from mammalian liver tissue. Oxido-sterol cyclase has been found in yeast. Efficient transformations of oxidosqualene (49) to lanosterol is possible if the biomass is sonicated in order to allow substrate diffusion probably by removing the obstructing outer cell membrane. Native Baker’s yeast can affect the transformation only marginally. Attention was specially devoted to the possibility of cyclizing unnatural substrates because of the relevant synthetic implication that this would, for instance, allow for preparation of remotely functionalized sterols like 50 in Fig. 24, a potent irreversible inhibitor of the 14-demethylase enzyme, with an important role in the biosynthesis of cholesterol (Kyler and Novak, 1992).


102

R = Methyl, Allyl, Ethinyl

52

51

FIG.24.

In this case, an efficient biomimetic cyclization was found before the equivalent enzymatic method was available (Johnson et a]., 1987). A similar enzymatic capacity has been observed from other sources (Abe et al., 1993; Xiao and Prestwich, 1991). The cyclization reaction would be of great interest in the cyclization of farnesyl pyrophosphate and its congeners to the bicyclic terpenes. The fact that such enzymes are not available prompted Kyler and Novak to design, on the basis of speculations of a hypothetical enzyme active site working model, intermediates that could eventually be cyclized by the yeast oxidosterol cyclase. In this way compound 51 was found to be a substrate for the enzyme to give a bicyclic product 52 convertible by desulfurization to the target bicyclic structure (Kyler and Novak, 1992). The success of this approach was only diminished by the low yields of the observed transformation (8%).

Pyruvate decarboxylase (PDC)is a thiamine pyrophosphate (TPP)-dependent enzyme catalyzing the decarboxylation of pyruvate to acetaldehyde or the transfer of a Cz unit of pyruvate onto acetaldehyde with formation of acetoin. The mechanism of the reaction is believed to occur through the initial formation of an adduct between TPP and pyruvate (Fig. 25). This adduct, depending on the organism and the environ-


\ +

103

/

\ N

HO OH

0-

I" TPP

+

OH

PDC R = CH,, X=OH. Y=H, R' = Ph BFD R = Ph, X=H, Y=OH, R = CH,

FIG.25.

mental conditions, can decarboxylate to give acetaldehyde (path a) or interact with another molecule of aldehyde (path b) to finally produce an acetoin that can be transformed by reducing enzymes to 2,3-butanediol. Neuberg found that added benzaldehyde in fermenting yeast entered path b as a second substrate giving rise to enantiomerically pure (R)-phenylacetyl carbinol (PAC) (Neuberg et al., 1923). The large-scale production of this compound has raised some interest, since L-ephedrine can be obtained by reductive amination. At present, PAC and L-ephedrine are manufactured by this method at BASF in Germany on a multi-ton scale. This process probably represents the largest industrial application of Baker's yeast on nonconventional substrates. The reaction efficiency is reduced by the concomitant reduction of benzaldehyde to benzyl alcohol. Various techniques have been devised to avoid the undesired side reaction. These include the slow addition of aldehyde to the fermenting mixture (Agarwal et aZ., 1987) or the use of alkyl pyridines as additives for the inhibition of the ADH responsible for aldehyde reduction (Gupta et ~ l .1979). , Why benzaldehyde is the preferred cosubstrate (after acetaldehyde) in the reaction with pyruvate is not known. Other aldehydes, including substituted benzaldehydes (Long et d.,1989; Ohta et QZ., 1989),furylacrolein (Fuganti et ~ l .1988a), , and cinnamaldehydes (Fuganti and Grasselli, 1977), are good substrates in parallel reactions. Higher reaction times usually allow production of enantiomerically pure vicinal diols (Fig. 26). The ones obtained from

104

P. D’ARRIGO et 01.

__c

OH

R = H. CH,

Epc synthesis

1

FIG.26.

cinnamaldehydes have found extensive application in the synthesis of enantiomerically pure compounds (EPCs) (Fuganti and Grasselli, 1985).

Incorporation of higher a-oxoacids has been explored, allowing for production of a higher homologue of the phenylacetyl carbinols, albeit with lower efficiency if compared to pyruvate (Fuganti et al., 1988b). Purified yeast pyruvate decarboxylase has been used with several aldehydes and pyruvate derivatives as C2-unitsas donors. The corresponding acyloins are usually obtained with lower yields if compared with the whole-cell system (Cardillo et al., 1991; Crout et al., 1991). Other decarboxylases with potential synthetic applications are found in other organisms. Benzoylformate decarboxylase (BFD) found in Pseudomonas putida and Acinetobacter calcoaceticus (Ward et al., 1992) operates with a mechanism similar to the one proposed for PDC. Benzoylformate is the first substrate instead of pyruvate, while the second substrate is acetaldehyde. The acyloin obtained in this case, IS)-2-hydroxypropiophenone, is isomeric with PAC obtained with PDC and benzaldehyde (Fig. 25). PDC may have application for production of acyloins containing a fixed acyl group with a variable aromatic group, while BFD may produce acyloins having a variable aliphatic group with a fixed aromatic component. A further C-C bond-forming reaction has been observed whose catalytic nature is not understood. Starting with a$-unsaturated ketones and esters in the presence of trifluoroethanol, fermenting yeast produces optically active fluorinated carbinols accompanied by the corre-


53

105

O

R = alkyl, OR'

J

R = OR'

54 FIG.27.

sponding allylic alcohols when a ketone is the substrate (Fig. 27). When the same transformation is applied to a#-unsaturated esters, the corresponding lactones are formed (Kitazume and Ishikawa, 1984). Enzymes catalyzing Michael-type addition to unsaturated ketones and esters with a broad substrate specificity would be of tremendous potential in biocatalysis, since there are apparently no general catalytic capacities of this kind. In another report on Baker's yeast applications, starting from cyanoacetone in fermenting yeast, the two stereoisomeric alkylated prodin Fig. 28 were obtained in high ee, and the results were ucts 55 and regarded by the authors as a consequence of the novel C-C bond-forming reaction catalyzed by Baker's yeast (Itoh et al., 1989). It was later shown that on similar active methylene compounds like 57 alkylation occurs through spontaneous aldol condensation between the acidic compound and the acetaldehyde presumably generated in situ by Baker's yeast oxidation of ethanol (Fuganti et al., 1990). The a$-unsaturated compound formed via dehydration of the aldol can as a then he reduced by yeast, affording the alkylated compound mixture of enantiomers, reflecting the stereochemistry of the alkene and the selectivity of the yeast reducing system. A similar mechanism was then proposed for the formation of the products obtained from cyanoacetone.

s

106

P. D’ARRIGO et a]. OH -

LcN - ‘y b.y.

OH

ACN

+

EtOH

55

56

cNx COOEt

CN

b.y.

v 57

EtOH

CN

/+

v

58

FIG.28.

IV. Oxidations: Getting the Other Enantiomer

Baker’s yeast is widely used as a reducing agent in organic synthesis for the preparation of enantiomerically pure chiral compounds. One enantiomer can often be prepared in a convenient way, while the other is usually not accessible with a similar procedure. This constitutes a serious drawback of the method, since in many applications of biocatalytic methods the goal is often to secure both enantiomers of a chiral drug for biological activity evaluation. When it is possible to obtain both enantiomers with the same biocatalyst, the advantage is obvious. The resolution of secondary alcohols with hydrolytic enzymes, mainly lipases, usually allows reaching that goal. Various techniques can be used to direct the enantioselectivity of carbonyl reduction by Baker’s yeast (the same concept is applicable to other microorganisms). Altering the surrounding of the carbonyl group (e.g., changing the substrate) is a strategy that can be followed and can be exemplified by the case of the reduction of P-ketoesters. The only variation that can be practical in this case is to modify the dimensions of the ester group. This technique has been successful in increasing the enantiomeric excess of alcohol obtained with the (S)-configuration.The rationale behind this behavior is well known: enzymes with opposite stereochemical requirements are


107

yeast

60 FIG.29.

present and acting at the same time on similar substrates. Increasing the differences in the groups flanking the carbonyl group, the object of the reduction, increases the differences in kinetics of the interaction of the two enzymes with the substrate, thus increasing selectivity. It has been shown that P-ketoesters reductases have very different k, and vma, thus explaining the shift in the enantiomeric excesses of the product when different concentration of substrate were used (Chen et al., 1983). If reductases of different stereochemical preferences are available, it is probably simpler to employ different microorganisms in order to obtain the opposite stereochemistry in carbonyl reduction. This is possible for this class of compounds since Baker’s yeast and Geotricum candidurn produce P-hydroxyesters of opposite chirality (Azerad and Buisson, 1992). In other cases, the use of selective inhibitors of enzymes with enantiomeric catalytic properties has been applied successfully. Figure 29 shows the application of this concept to the selective production of (Forni et the enantiomeric trifluoromethyl hydroxyketones 59 and

Ql.,

1994).

A further possibility for synthesizing the other enantiomer is to use the biocatalyst in the oxidation direction. Since enzymes catalyze reactions in both directions, it is conceivable that, while during the reduction of a prochiral carbonyl group 100% of the homochiral secondary alcohol can be obtained in theory, in a process that can be considered an asymmetric synthesis, the same enzymatic system, in catalyzing the oxidation in the reverse reaction, will act on the secondary alcohol with the same selectivity that gave the chiral alcohol in the reductive step. This means that the alcohol with the same chirality will be oxidized, leaving behind the other enantiomer in an enantiomerically enriched

P. D’ARRIGO et a].

108

61

62 0

OH

R = CH,, C,H,, n-C,H,,n-C,H,,

OH

Ph

FIG.30.

form. This process is a kinetic resolution and thus allows production of the other enantiomer, albeit at only 50% yield. Since the carbonyl compound thus obtained can be easily reduced to the racemic secondary alcohols by chemical means, in theory a cyclic succession of enzymatic oxidation and chemical reduction allows the entire transformation of the racemic ketone into the other enantiomer. This procedure was applied to the preparation of both enantiomeric forms of propylene glycol (PG) fiom 1-hydroxy-2-propanone (HP) (a) (Fig. 30). The Baker’s yeast reduction of HP gives (R)-PG in high yield and enantiomeric purity. This reaction was published in Organic Synthesis by Levene and Walti (1943). The same reduction is now effected on a large scale (Kometani et al., 1996). (R)-PG is actually used as a chiral building block for the preparation of (S)-ofloxacin (Kumobayashi et al., 1991). The R enantiomer in the racemic mixture is instead oxidized to HP, leaving behind (S)-PG (64). The ketone can then be reduced chemically, giving the racemic alcohol that can again be subjected to enantioselective oxidation. A series of higher homologues was prepared with the same methodology (Kometani et al., 1996). The oxidation was performed with fermenting yeast under aerobic conditions, and equilibrium was reached at 24 h. Kinetic studies of the reaction in the oxidative direction suggest that the activity responsible for the oxidation might be glycerol-DH. Lee and Whitesides (1986) used the GDH from bacterial sources for preparation via a similar oxidative resolution of (S)-1,2-butanediol. The Veschambre group reported the reduction of the diketone @ with Baker’s yeast, giving a mixture of the


66

65

109

67

bakefs yeast

FIG.31.

OH -

Ar

n

Ar = 2-furyl, 2-thieny1, phenyl

FIG.32.

hydroxyketone @ and the diol s7 (Besse et al., 1995). The diketone in Fig. 31 was shown to be the substrate for a yeast alcohol dehydrogenase (YADH), while the intermediate hydroxyketone, which is not the substrate for the same enzyme, was further reduced with GDH. From these results it was inferred that the two enzymes were actually involved in yeast reduction of the diketone (Besse et al., 1993). A similar oxidative method was proposed for the kinetic resolution of secondary alcohols of the type shown in Fig. 32. In this case, Baker’s yeast was employed as resting cells and the oxidation required several days to reach equilibrium conditions, in which the ketone/alcohol ratio varied according to the nature of the substrate. Very high enantiomeric excesses of the alcohols were obtained for conversions higher than 70%. Also in this case, of course, the alcohol with (I?)absolute configuration was obtained, opposite to the one obtained in the reduction (Fantin et al., 1993) In the example in Fig. 33, a mixture of ex0 and endo-bicycloheptenol (68)was oxidized by Baker’s yeast to give optically enriched bicycloheptenone (69) (fiom the oxidation of the endo alcohol) and the racemic exo-alcohol (70)unchanged (Dawson et d., 1983). The enantiomeric endo-alcohol was also recovered.

P. D’ARRIGO et ol.

110

68

69

70

FIG.33.

V. Hydrolytic Activities: Phosphate Esters

Proteases (Achstetter and Wolf, 1985), lipases (Schousboe, 1976), esterases (Parkkinen, 1980), phospholipases (Witt et al., 1984), amino acylases (Gliinzer et al., 1986, 1987a), and phosphatases (Trevelyan, 1966) have been recognized in yeast. Yeast glycosidases, in the hydrolysis of a-and P-glucosides, were first investigated by Emil Fischer, who developed the concept of lock-and-key enzyme-substrate interaction after these particular studies (Roberts et al., 1995). These enzymes are at work in many of the fermentative steps in which yeast is used in the food industry. Baker’s yeast has been used as an alternative to p-glucosidase in the synthesis of antirhine from secologanine, where the hydrolytic step is combined with a reductive one (Brown et al., 1991). The hydrolytic activities present, especially lipases or esterases, which can be undesirable, cause byproduct formation. In other cases, the unexpected hydrolytic activity is cooperating with a reductive step in the production of homochiral intermediates (Pedrocchi-Fantoni and Servi, 1991). Their use is justified In some specific applications, as in the resolution of racemic acetylenic alcohols-esters for which there are no efficient alternatives in the otherwise extremely versatile field of lipases (Gliinzer et al., 1987b). Peptide bond formation using immobilized viable Baker’s yeast in reversed micelles has been reported (Fadnavis et al., 1990). Good to excellent yields of peptide have been reported in the condensation reactions leading to a leucine enkephalin analogue (Fig. 34). Yeast was suspended in a reverse micellar medium consisting of aerosol/OT in iso-octane with a low water content. With controlled water activity, 80-90% peptide yields were obtained. Peptide bond formation competes with ester hydrolysis, which becomes important with long reaction times.

-

OLD AND NEW SYNTHETIC CAPACITIES OF BAKERS YEAST Ac-Phe-OMe + H-Leu-NH, Z-Ala-Phe-OMe + H-Leu-NH, Z-Gly-Gly-Phe-OMe + H-Leu-NH,

-

111

Ac-Phe-Leu-N H2 Z-Ala-Phe-Leu-N H, Z-Gly-Gly-Phe-Leu-NH,

FIG.34.

Enzymes responsible for the hydrolysis of phosphate esters are ubiquitous in living organisms. The number of specific phosphatases recognized in mammalian cells as well as in microorganisms probably exceeds that of any other enzymatic function. In Baker’s yeast the presence of phosphatases, associated with a number of metabolic functions, has been recognized. Many of these enzymes are linked with the metabolism of phospholipids and phosphopeptides. We have observed that some of the phosphate-hydrolyzing capacities present in Baker’s yeast can be useful in selective transformations in phospholipids. The action of fermenting Baker’s yeast transforms a mixture of phospholipids almost completely into glycerol, diglycerides, and inorganic phosphate. Toluene-induced autolysis of Baker’s yeast produces enzymes that catalyze extensive decomposition of the membrane phospholipids but leave substantial amounts of phosphatidylinositol (PI] unchanged. PI, which constitutes about 20% of all the phospholipids present in the yeast membrane, could then be recovered using a rather simplified procedure. Due to the separation difficulties usually encountered with phospholipid mixtures, this method was considered of interest for the preparation of PI (Trevelyan, 1966). The cell-free broth of the fermentation is actually deprived of phospholipase activity while still retaining acid and alkaline phosphatase activity. Therefore, in a crude mixture of phosphatidic acid (PA) containing soy phospholipids, there was selective hydrolysis of PA to inorganic phosphate and diglycerides, leaving the other components unchanged. This selectivity can be exploited in the purification of a mixture of phospholipids arising from a transphosphatidylation reaction on phosphatidylcholine (PC). Indeed, in the reaction of PC with a different alcoholic-bearing head group catalyzed by phospholipase D, a more or less significant portion of PA is formed due to a competing hydrolysis reaction (Fig. 35). Purification of the newly formed phosphatidic ester PX from PA is troublesome. Treatment with a phosphatidate phosphatase contained in the yeast broth allows selective hydrolysis of PA to diglycerides, easily removed by solvent partition (D’Arrigo, 1996).

112 P. D’ARRIGO et a].

OLD AND IWW SYNTHETIC CAPACITIES OF BAKERS YEAST OPO,H,

11 3

OPO,H,

H,O,PO

H,O,P 0

,..-

HO H0Q0p03H2 OH

OPO,H,

OPO,H,

72

71 FIG.36.

Whether the reaction is catalyzed by a specific phosphatase like phosphatidate phosphatase (Wu et al., 1993) or by other phosphatehydrolyzing enzymes has not been investigated. The broth exhibits phosphatase activity, with most phosphate esters submitted for hydrolysis, such as glucose 6-Pand p-nitrophenyl phosphate. Kinetic resolution of isopropylidene glycerol phosphate as either the barium or the calcium salt at alkaline pH with a cell-free fermentation broth at about 50% conversion gave products of low enantiomeric excess. A mammalian phosphatase had been used before in attempted resolution of glycerol in reverse hydrolysis conditions. However, phosphate ester obtained was isolated as a racemate (Pradines et al., 1986). Scollar et al. (1985)reported the resolution of phosphothreonine and phosphoserine using commercially available phosphatases from various sources. Yeast phosphatases have been exploited for hydrolysis of the natural (72) phytic acid 71 of Fig. 36 to ~-myo-inositol-1,2,6-tris-phosphate (Blum et a]., 1995). VI. Lyase Activity

Enzymes from the lyase group are not common in Baker’s yeast. However, some important reactions catalyzed by these enzymes from other microbial sources find important practical applications (Chibata et al., 1983, 1986).It has been found that addition of water onto the double bond of some a$-unsaturated aldehydes (73) generated optically active secondary alcohols (74) (Fronza et al., 1990) (Fig. 37). However, this reaction appears to be severely limited as far as variations in the substrate structure are concerned. Interestingly, it has been observed by other authors that a number of amines add efficiently onto cinnamic esters, giving S-amino acid esters

P. D'ARRIGO et al.

114

OH

0

R=H

ROWOH R

74

73 R' = H R" = COPh. CH,Ph

FIG.37.

of enantiomeric excesses ranging from 20 to 70% ee (Rama Rao et a]., 1990).

Other isolated cases of addition onto an activated double bond have been reported. VII. The Biogeneration of Aroma Compounds

There is current interest in the flavor industry in the generation of substantial quantities of products of relevant sensory properties, occurring in nature in trace amounts, to be used as food additives. In order to be considered as natural, these compounds must either be of extractive origin from natural sources or derive from natural products through manipulation while avoiding chemical reagents. Only biocatalytic or fermentative transformations are acceptable. Materials produced in this manner have enhanced commercial appeal and value since the label of natural products receives consumer acceptance (Fuganti et a]., 1993; Stofberg, 1986). Application of biocatalysis in this field is fairly active, and Baker's yeast has found industrial applications. Figure 38 shows two possible biocatalytic routes for the preparation of natural raspberry ketone, the impact flavor of the fruit. In the first preparation, betuloside, a glycoside from the bark of the birch Betula a h , is treated with a hydrolytic enzyme to give the secondary alcohol betuligenol. Oxidation catalyzed by the yeast C. boidinii allows production of the required ketone. In the second procedure, the unsaturated precursor obtained by basecatalyzed condensation of natural p-hydroxybenzaldehyde and acetone from fermentation is reduced to raspberry ketone, exploiting the wellknown selectivity of yeast in the reduction of a$-unsaturated ketones (Sakai et al., 1991; Kawai et al., 1995a). The ketone is produced industrially on a kilogram scale. (It might be of interest for the reader to know

OLD AND NEW SYNTHETIC CAPACITIES OF BAKER'S YEAST

115

116


75

76 FIG.39.

that the synthetic compound produced at about 20 tons per year has a price of 80 US$ per kg, while the biogenerated, but otherwise chemically identical material, is worth 4,000 US$ per kg.) A further example of the biogeneration of aroma compounds is given by the preparation of an important aroma found in fruits during ripen(I?)-&-decanolide0, ing. It can be prepared in its natural form by degradation of ricinoleic acid. Baker’s yeast was employed in the reduction of the corresponding 6-decenolide (75) (massoi lactone) isolated from the barks of the Massoi tree Cryptocaria massoia (Fronza et al., 1992b; van der Schaft et al., 1992) (Fig. 39).

VIII. Conflicting Reports

The idea of reproducibility in biocatalysis is not exactly the same as usually applied in organic synthesis, where a careful description of the experimental procedure must allow anyone to duplicate the experiments. The numerous parameters controlling the behavior of a microorganism are so complex that it is sometimes difficult to repeat the same performance even with the same strain. It is therefore to be anticipated with Baker’s yeast as the biocatalyst that there will be a certain range of uncertainty as far as isolated yields of products and enantiomeric excesses of chiral compounds obtained are concerned. In his pioneering work with yeast, Fischer affirmed that “Fiir die meisten Versuche mit Hefenenzyme ... kann man sich einer guten Brauereihefe bedienen, sicherer aber ist es, eine Reinkultur anzuwenden, wie sie heutzutage kauflich sind. Ich habe mich fiir alle entscheidenden Versuche einer Saccharomyces cerevisiae Typus Frohberg bedient” [For most of the experiments with enzymes from yeast one can use a good Brewer’s yeast, but it is more reliable to use a single-strain culture as they are available nowadays. For all my different experiments I have used a Saccharomyces cerevisiae type Frohbergl (Fischer, 1889). In order to give reproducible results it would suffice to


11 7

always describe a control experiment by using standard commercially available dried yeast (Sigma type 11). Experimental procedures for the preparation of various 3-hydroxyesters have been collected and the author’s procedures have been repeated by the editors using this kind of dried yeast. For all the reported transformations, good agreement between the controlled procedure and the one reported with local yeast has been observed (Roberts, 1992). Recently the NAD(P)H content in living cells in yeasts of different origin has been measured and a method for rapid evaluation of the reducing capacities of a Baker’s yeast brand has been proposed (Pereira, 1995). Sometimes, however, discrepancies in the results observed from different research groups on identical or very similar substrates are difficult to explain. A recent report concerns the cycloaddition of nitriloxide (77)to ethyl cinnamates (78) as catalyzed by Baker’s yeast (Fig. 40). In one article, the effect of added P-cyclodextrin on the regioselectivity of the cycloaddition was studied. It was concluded that the reaction was catalyzed by yeast and that the addition of P-cyclodextrin could completely reverse the regioselectivity of the reactions (Rama Rao et al., 1990, 1992). The isoxazolines 79 and 80 thus obtained were optically active. The authors stated that the reaction did not occur in the absence of yeast. The reaction of nitrile oxide with ethyl cinnamate was reinvestigated by other authors (Easton et al., 1995). They showed that yeast is not required for cycloaddition to take place and that the effect of P-cyclodextrin on regioselectivity was only marginal. That cycloaddition reaction had been reported before in the literature (Christ1and Huisgen, 1968). IX. Conclusions

Applications of Baker’s yeast to organic synthesis usually exploit old enzymes for new transformations. The continuous variation of the structural features of substrates presented to Baker’s yeast for biotransformation point to interesting new possible applications. In some cases, the real effect of Baker’s yeast catalysis is not understood. The nature of the studies published in organic chemistry journals indicates that Baker’s yeast is used for enantioselective reductions of carbonyl groups by researchers who only occasionally apply biocatalysis in organic synthesis. This confirms the tutorial function as well as the role as an asymmetric reducing agent that Baker’s yeast catalysis has had, particularly in the last 15 years. The attempted correlation of transformations

118 P. D'ARRIGO et a].

+


119

observed with known enzymatic activities widens the scope of the study of Baker’s yeast as a biocatalyst. The genetic modification of Baker’s yeast allows production of a series of microorganisms with a similar appealing quality as the wild-type organisms, but much enhanced in productivity and selectivity and of great potential for applications in organic synthesis.

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Investigation of the Carbon- and Sulfur-Oxidizing Capabilities of Microorganisms by Active-Site Modeling HERBERT L. HOLLAND Department of Chemistry Brock University St. Catharines, Ontario L2S 3A2, Canada

I. Introduction A. Oxygenase Enzymes in Microbial Biotransformations B. Rationalization and Predictability of Oxygenase-Catalyzed Reactions C. Models for Microbial Biotransformations 11. Models for Microbial Hydroxylations A. “Active-Site” Models B. Relationship of Hydroxylase “Active-Site” Models to Enzyme Structure C. Hydroxylation and Phylogeny D. Hydroxylation and Biosynthesis 111. Models for Sulfoxidation Reactions A. “Active-Site” Models B. Sulfoxidation as a Model for Other Processes C. Relationship of Sulfoxidase “Active-Site” Models to Enzyme Structure IV. Summary and Prognosis References

I. Introduction

A. OXYGENASE ENZYMESIN MICROBIAL BIOTRANSFORMATIONS

Microbial biotransformation has a long and distinguished history as a tool for the selective manipulation of organic molecules for synthetic purposes, ranging from an extensive literature on the biotransformation of natural products such as steroids (Charney and Herzog, 1967; Smith, 1974; Mahato and Majumdar, 1993), alkaloids (Rosazza and Duffel, 1986), terpenes (Lamare and Furstoss, 1990), and antibiotics (Sebek, 1980),to applications for the production of low-molecular-weight chiral molecules useful as starting materials in organic synthesis (Davies et a]., 1989).

The range of reactions that can be carried out by microbial methodology is large, covering most of the standard reactions of organic chemistry (Drauz and Waldmann, 1995). Of these, however, the most dramatic transformations are those reactions such as hydroxylation at 125 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 44 Copyright 0 1997 by Academic Press, Inc. All rights of reproduction i n any form reserved. OO65-2164/97 $25.OO

HERBERT L. HOLLAND

126 H

H

H

R X R

R- X -C HRz

OH

R X R

A

RXH

+

RzCO

(X=N, 0,orS)

FIG.1. Oxygenase-catalyzed biotransformation reactions.

unactivated carbon for which no analogous chemical procedure is known. Such reactions are carried out by the oxygenases, enzymes that require the participation of molecular oxygen in their reaction cycle and that catalyze the introduction of oxygen atoms directly into the substrate. These enzymes are responsible for a wide range of transforma-

CARBON- AND SULFUR-OXIDIZING CAPABILITIES

127

tions, summarized in Fig. 1,that comprise some of the more remarkable reactions of the biotransformation repertoire. For practical purposes, the reactions illustrated in Fig. 1 are most frequently carried out by whole-cell biotransformations using growing or resting cultures of fungi or bacteria. This protocol is dictated by the nature of the enzymes themselves, which for the most part are relatively intractable membrane-bound species that do not lend themselves to simple isolation. In addition, the oxygenase enzymes have extensive cofactor requirements that preclude their economic use in preparative applications. These limitations are particularly relevant in the case of the hydroxylase enzymes, which are generally assumed to be cytochrome P-450-dependent systems with all of the complex cofactor and electron transport requirements that that entails, and the bacterial dioxygenase systems that convert aromatic substrates to cis-dihydrodiol products (Holland, 1992). Those isolated microbial oxygenase preparations that have been described are limited in terms of their practical application to flavin-dependent monooxygenases, particularly BaeyerVilliger monooxygenases such as the cyclohexanone monooxygenase (CMO) of Acinetobacter (Ottolina et al., 1995) and the BVMO enzymes from Pseudomonas putida (Alphand et al., 1996). Many distinct oxygenase enzymes are capable of performing several of the reactions of Fig. 1. The cyt.P-450 monooxygenases are reported to be capable of hydroxylation of both aliphatic and aromatic carbon, epoxidation of olefins, heteroatom dealkylation (via hydroxylation a to oxygen, sulfur, or nitrogen), and oxidation at both sulfur and nitrogen (Holland, 1992). The flavin-dependent monooxygenases are capable of converting phenols to catechols, Baeyer-Villiger oxidation, and oxidation of sulfide to sulfoxide and of amines to N-oxides (Walsh, 1980), while the dioxygenases, once thought to be selective for dihydrodiol formation, are now known to carry out hydroxylation, desaturation, and sulfoxidation reactions in addition to their role in the direct oxidation of aromatic rings (Gibson et al., 1995; Lee et al., 1995). B, RATIONALIZATION AND PREDICTABILITYOF OXYGENASE-CATALYZED REACTIONS

This chapter will consider the various models that have been developed to interpret and predict the outcome of microbial hydroxylation and sulfoxidation reactions. Given the wide range of potentially useful reactions presented in Fig. 1 and the existence of many other useful whole-cell biotransformation reactions, it is not surprising that there

HERBERT L. HOLLAND

128

have been attempts to organize the wealth of empirical data available on microbial biotransformations into a predictively useful format. The rationale behind this type of analysis is twofold: first, the need to be able to predict the regio- and stereochemical outcome of biotransformation of a new substrate by a microorganism whose biocatalytic reactions on other substrates are known; and, second, the need to be able to predict the outcome of biotransformation of a given substrate by a hitherto uninvestigated microorganism. These disparate objectives are related in that their achievement lies ultimately in a knowledge of the natures and activities of the enzymes present in the relevant microorganisms. Considerable progress in this regard has been made in the areas of microbial hydrolytic and oxidoreductase enzyme activities, but in view of the difficulty in isolation and characterization of oxygenases of microbial origin, it is only comparatively recently that serious attempts have been made to explore the fundamental principles that lie behind the regio- and stereoselective nature of oxidative microbial biotransformations. This latter work has developed both as a result of an increasing amount of data having emerged on mammalian oxygenases and as a consequence of hard-won data finally becoming available on the structure and mechanism of a limited number of microbial oxygenase enzymes. As the fundamental approach to the problem of predictivity in wholecell biotransformations is common regardless of the nature of the enzymes concerned, Section 1.C will consider the models that have already been developed for microbial biotransformations other than the hydroxylation and sulfoxidation reactions that are the subject of this chapter.

c.

MODELSFOR MICROBULBIOTRANSFORMATIONS

1. Ester Hydrolysis

Microbial hydrolysis of esters, attributable to lipase activity, is a ubiquitous biotransformation. The reaction frequently proceeds with a degree of enantioselectivity in susceptible substrates, and examination of the hydrolysis of a large number racemic acetates by Rhizopus nigricans led Ziffer and co-workers (Kawai et al., 1981; Ziffer et al., 1983; Charton and Ziffer, 1987) to propose that the enantiomer shown in Fig. 2 is the more rapidly hydrolyzed. This model can also be applied to the hydrolysis of esters of cyclic carbinols (Kasai et al., 1984, 1985). The enantioselectivity of this reaction is highly dependent on the nature of the substituent groups and clearly has its basis in the substrate selectiv-


H

129

OCOCH3

SAL

S = small substituent, L = large substituent

FIG. 2. Structure of the enantiomer of acetate esters more rapidly hydrolyzed by Rhizopus nigricans.

S = small substituent, L = large substituent FIG.3. Prelog’s rule for the reduction of prochiral ketones.

ity of the lipases of R. nigricans. The availability of isolated lipase preparations has meant that enantioselective ester hydrolysis has relied less heavily on whole-cell biotransformations but has also reinforced the value of models such as that of Fig. 2 as their basis in enzyme structure and function becomes more apparent (Cygler et al., 1994). 2.

Carbonyl Reduction

Arising from a series of studies originally based on the fungus Curvularia falcata, Prelog (1984) proposed the model of Fig. 3 to account for the enantioface-selective reduction of simple acyclic and cyclic carbony1 compounds to chiral alcohols. Prelog recognized, however, that this rule did not apply to all systems, and that, as it was based on enzyme selectivity, could be complicated by the fact that many microorganisms contain several oxidoreductases that may operate on a common substrate with different selectivities. Although reduction of prochiral carbonyl compounds according to Prelog’s rule is observed for the majority of whole-cell-catalyzed examples, and many isolated microbial oxidoreductases do indeed operate according to the model of Fig. 3, there are several enzymes that express the opposite selectivity (Faber, 1992), and at least one whole-cell system (Yarrowia lipolytica) that reduces both acyclic and cyclic methyl ketones in an antiprelog sense (Fantin et al., 1996). An extension of this model to account for the yeast-catalyzed diastereoselective reduction of a-substituted-p-dicarbonyl compounds such as P-ketoesters and p-diketones was developed by Vanmiddlesworth

130

HERBERT L. HOLLAND

s

S = small substituent, L = large substituent FIG.4. Model for predicting diastereoselectivity in yeast reductions.

and Sih (1987). Their model, shown in Fig. 4, accounts for the regioand diastereoselectivity shown in a large number of such reactions by proposing a selective substrate binding based on the relative sizes of the a substituents, followed by diastereoface-selective delivery of a reducing equivalent from the enzyme’s cofactor. An extension of this model in which an internal hydrogen bond holds the ester group (“L”) away from the incoming reducing equivalent deals specifically with reductions of a-hydroxy-P-ketoesters (Sato et al., 1986). The more complex stereochemical possibilities offered by reductions of racemic bi- and polycyclic ketones have been dealt with by Nakazaki et al. (1980), who proposed a quadrant rule for reductions of such compounds by C u m l a r i a lunata and Rhodotorula rubra. Their analysis, illustrated in Fig. 5, is based on an optimal fit of substrate into four restrictive quadrants, with the relative ease of fit in the order UR > UL > LL > LR, followed by delivery of the reducing equivalent from the -z direction. Their analysis was able to account for the product distribution obtained hom microbial reduction of such ketones as (f)-norbornanone, (*)-twistanone, and (&)-4-protoadamantanone, although the relationship of the model to the selectivities of the various oxidoreductases present in C. lunata and R. rubra was not explored.


131

S = small substituent L = large substituent

+X

FIG.5. Quadrant rule for reductions of complex ketones by C. lunata and R. rubra.

3 . Epoxide Hydrolysis

Microbial hydrolysis of epoxides has recently been developed as a powerful tool for the preparation of both chiral diols and epoxides. The enantioselectivity of this reaction has been exploited in the preparation of a number of synthetic targets (e.g., Archelas et al., 19931, and its interpretation in microbial systems is soundly based on an understanding of the mechanism and selectivity of the corresponding mammalian microsomal epoxide hydrolase (Wistuba and Schurig, 1992; Wistuba et al., 1992; Lacourciere and Armstrong, 1993). Models have been proposed to account for the complementary enantioselectivities shown by Aspergillus niger and Beauveria sulfurescens in their conversion of substituted epoxides to vicinal diols (PedragosaMoreau et al., 1996a,b). These models, shown in Fig. 6, predict the preferred regiochemistry of attack by water, together with the predominant enantioselectivity (indicated on the substrate), the latter being determined by the relative sizes of two hydrophobic binding pockets, labeled RB (right back) and RF (right front), present in the respective enzymes. The rate of epoxide opening by B. sulfurescens, involving nucleophilic attack at the benzylic position, is further subject to influence by the conformation of the phenyl ring in the RF pocket (Pedragosa-Moreauet al., 1996a).

Baeyer-Villiger Oxidations The selectivity of Baeyer-Villiger oxidation of cyclic ketones to lactones by Acinetobacter NCIMB 9871 has been the subject of investigation by several groups. Alphand and Furstoss (1992) proposed the 4.

132

HERBERT L. HOLLAND

Epoxide opening by Aspergillus niger

Epoxide opening by Beauveria sulfurescens

FIG.6. Models for the regio- and stereochemistry of epoxide hydrolysis by Aspergillus niger and Beauveria sulfurescens.

Flavin

Flavin FIG.7. Preferred orientations of the enantiomers of a bicyclo[4.2.0]ketone undergoing enantioselective Baeyer-Villiger oxidations by Acinetobacter.

model shown in Fig. 7 based on the whole-cell oxidation of racemic bicyclic ketones. In this analysis, the preferred regio- and enantioselectivity of substrate oxidation was proposed to be determined by preferred binding of the enantiomeric forms of the reaction intermediate as shown, followed by a mechanistically dictated antiperiplanar arrangement of the migrating C-C bond and 0-0 peroxidic bonds of the intermediate, giving rise to the observed products.


133

6B FIG.8. Positions of testosterone a or vinylogous to carbonyl subject to hydroxylation under stereoelectronic control.

This mechanistic requirement was later shown to be a dominant feature in the enantioselectivity of the same reaction when catalyzed by the enzyme cyclohexanone monooxygenase (CMO) isolated from Acinetobacter (Kelly et a]., 1995). The latter authors also pointed out that, as the configuration of the tetrahedral peroxidic intermediate is controlled by the diastereofacial selectivity of the addition of a flavin peroxide to the substrate, it may be the latter process, and not interaction of the substrate with the enzyme’s active site, that indirectly controls the enantioselectivity of oxidation of small substrates. However, in an analysis of the oxidation of over 40 different substrates by this enzyme, Ottolina and co-workers (1996) suggested that enzyme-substrate interactions can indeed be a determinant of the selectivity of this reaction, and have proposed an active-site model for the Acinetobacter CMO based on restrictive-space descriptors.

II. Models for Microbial Hydroxylations

A. “ACTIVE-SITE” MODELS 1. Steroid Hydroxylation

As the single most frequently studied group of substrates for microbial hydroxylations, steroids have been the subject of several attempts to develop models that can be used to predict the outcome of these reactions (Holland, 1982). Hydroxylations that occur at positions a or vinylogous to carbonyl groups, such as those at C-2 and C-6 of A4-3-ketosteroids, illustrated in Fig. 8, are controlled by a stereoelectronic requirement for the axial addition of an electrophilic oxidizing species to the appropriate enolic intermediate. This process has been shown to

134

HERBERT L. HOLLAND

co

* OH

4

7.5 FIG.9. Relatiye positions of hydroxylation and binding sites in Calonectria decora (dimensions in A).

be operative for the C-6P-hydroxylation of A4-3-ketosteroidsby Rhizopus arrhizus (Holland, 1984), and for the C-20 hydroxylation of testosterone and related substrates by Gnomonia fructicola (Holland et al., 1988). The sites of hydroxylation at other, unactivated positions of the steroid nucleus may be directed by the position of existing oxygen substituents, which presumably act as binding sites for the substrate in the active site of the hydroxylase enzyme. A spatial relationship between such binding and hydroxylation sites is shown in Fig. 9. This relationship, first derived for hydroxylation of steroids by Calonectria decora, was also found to be generally applicable to hydroxylations carried out by Rhizopus nigricans, R. arrhizus, Wojnowickia graminis, Ophiobolus herpotrichus, Daedalea rufescens, and Leptosporus fissilis, but not to steroid hydroxylations carried out by some other microorganisms such as Aspergillus ochraceus (Jones, 1973). In this model, both carbonyl and hydroxyl groups are capable of acting as binding sites: monofunctional ketones are typically dihydroxylated at two sites as shown, but diketones are bound at two sites and hydroxylated at the third. Other oxygen substituents such as enol ethers and acetals can also exert a direct influence on the position of hydroxylation (Evans et al., 1975),but halogen atoms are ineffective in this role (Bird et al., 1980). Application of this model is, however, complicated by the fact that two-point binding of a steroid in an active site may occur in up to four different orientations, as shown in Fig. 10 (Brannon et al., 1967). A single substrate may thus give rise to up to four different hydroxylation products related by the binding patterns shown in Fig. 10. These spatial relationships account for such hydroxylations as that of 19-nortestoterone ( at l C-16 ) by the 2P-hydroxylator Gnomonia fructi-


135

FIG.10. Possible orientations of a steroid as a result of two-site binding [e) with equivalent hydroxylation sites at C-2K-16 and C-1/12 shown (+).

cola (Holland et al., 1988), of 5a-androstan-7,17-dione (2) at C-2 by the C-16 hydroxylating fungus L. fissilis (Denny et al., 1980), hydroxylation of testosterone (3)at both C - l a and C-12p by Penicillium species ATCC 12556 (Tweit et a]., 1962), hydroxylations of gP,lOa-retrosteroids (4J at C-9a by the Ila-hydroxylator Rhizopus arrhizus (Favero et a]., 1979), and the frequent cooccurrence of C-iiaIC-7p and C-llP/C-l4a hydroxylations of androstanes and pregnanes. Disruption of this binding pattern (e.g., by removal of one of the substrate’s oxygen atoms) leads to a shift in the balance of competitive binding, expressed as a change in the ratio of regioisomeric products (Zakelj-Mavric and Belic, 1987). Application of this model to nonsteroid substrates has met with some success. It may account for the hydroxylations of the bicyclic substrates

136

HERBERT L. HOLLAND

FIG.11. Three-point model for the hydroxylation of kauranones by R. nigricans.

(I)R = H (3) R=CH3

o--I’I’

0

4

(4) R = C(O)CH,, 0 or OH

(5) C-4(5) saturated (6) C4(5) unsaturated

STRUCTURES 1-6. 5 and 6 by C. decora and R. nigricans at positions 7-9 A away from the oxygen substituent as shown (Bailey et al., 1977), and has been used to rationalize the hydroxylations of gibberellins at C-15 (Fraga et al., 1993) by R. stolonifer and of 17-norkauran-16-one (2) and ent-17-norkauran16-one (8) by R. nigricans (McCrindle et ~ l .1975) , using the relationships illustrated in Fig. 11. -

2. Terpene Hydroxylation

.

The previously discussed successful application of the steroid hydroxylation model to some terpene hydroxylations suggests that similar predictive relationships may exist between binding and hydroxylation sites for other terpene substrates. The importance of binding sites in terpene hydroxylations is clearly demonstrated by the observations that,


137

"OH (17)

FIG.12. Hydroxylation of 1,4-and 1,8-cineole by Bacillus cereus.

whereas myrcene (9 and related hydrocarbons are generally poor substrates for microbial hydroxylations, the derived cyclic sulfones (e.g., lo)are typically hydroxylated in good yield by a variety of microorganisms (Abraham and Arfmann, 1992; Abraham et al., 1992). This phenomenon is also observed for hydroxylations of the phenylcarbamate by Beauveria sulfurescens (Hu et derivatives of dihydroartemisinin (TI) al., 1991) and of geraniol(l2) by A. niger and B. sulfurescens (Zhang et al., 1991), where hydroxylations of the parent molecules proceed in poor yield or not at all. When specific binding sites are absent, hydroxylation of terpenes typically leads to the formation of a mixture of isomeric products.

138

HERBERT L. HOLLAND

OCONHPh (11) STRUCTURES 9-12.

12

STRUCTURE 18.

Hydroxylation of 1,4-cineole (13)by Bacillus cereus yields a mixture of 2-ex0 and 2-end0 alcohols (14and l5),which may arise by hydroxylation of two bound forms of 13 in a single active site, as shown in Fig. 1 2 (Liu et d., 1988).Hydroxylation of 1,8-cineole (16) by B. cereus can be explained by the same model, the sole product (17)arising from preferred binding of the substrate in the orientation illustrated in Fig. 1 2 , left (Liu and Rosazza, 1990). Hydroxylations of the sesquiterpene cedrol 18 by a range of microorganisms occur predominantly in the region of the molecule around C-3 and C-12, and this has been attributed to the role of the C-8 alcohol group in anchoring the substrate in the hydroxylase active site and directing the site of hydroxylation (Abraham et d . , 1987; Lamare et a]., 1987; Fraga et d.,1996).

139

CARBON- AND SULFUR-OXIDIZING CAPABILITIES Hydroxylating site

Hydroxylating site

Binding site

Binding site

Flavonoids

koflavonoids

FIG.13. Model for the hydroxylation of flavonoids and isoflavonoids.

With the exception of the norkauranone substrates illustrated in Fig. 11,there have been no attempts to rationalize microbial hydroxylations

of the higher terpenes. 3 . Hydroxylation of Aromatic Compounds

The microbial oxidation of aromatic compounds to produce phenols is a common biotransformation, but few models dealing with this reaction have been proposed. Fig. 13 presents the proposal of Ibrahim and Abul-Hajj (1990a,b) for the hydroxylation of flavonoids by a range of microorganisms. This proposal accounts for the predominance of hydroxylations at the 3' and 4' carbons in both the flavone and isoflavone series of substrates by the assumption that binding of the substrate oxygen functionalities to polar groups of the enzyme is nonspecific. The importance of these oxygen atoms in substrate binding was confirmed by a study of the microbial hydroxylations of mono- and di-deoxyflavonoids, which demonstrated that at least one oxygen atom in the substrate was necessary for hydroxylation to occur (Abul-Hajj et al., 1991).

A model for the biotransformation of aromatic substrates by Streptomyces griseus (Fig. 14) that encompasses phenol formation together with 0- and N-demethylation has been proposed by Sariaslani and Rosazza (1984). This model suggests the existence of both a nonpolar binding region (the n-binding site) and a polar binding site (the electrophilic region), and accounts for the conversion by S. griseus of alkaloidal substrates such as 19 to phenolic products (R = H + OH), to 0-demethylated products (R = OCH, + OH), and to N-demethylated products (via inverted binding to bring N-CH, to the oxidation site).

140

HERBERT L. HOLLAND

0 = oxidation site E = electrophilic region n

(19) R = OCH, or H

= n-binding site

FIG.14. Model for hydroxylation and 0- and N-demethylation by S. griseus (dimensions in A).

4 . Benzylic Hydroxylation

Mortierella isabellina ATCC 42613 performs the benzylic hydroxylation of a range of aromatic compounds, and for the majority of these substrates the model shown in Fig. 15 can be used to predict the outcome of their biotransformation by this fungus. An earlier version of this model was first proposed to account for the absolute stereochemistry of the hydroxylation of ethylbenzene by M. isabellina (Holland et al., 1993),and the model was later extended to cover the biotransformation of phenyl-substituted olefins to vicinal diols, a reaction proposed to proceed via epoxidation according to the parameters of Fig. 15 (Holland et al., 1994a). The model was recently refined following an analysis of the biotransformation of over 60 substrates and potential substrates by M. isabellina (Holland et al., 1997a). The model proposes a binding region selective for an aromatic ring (A) and an aliphatic binding region (B), both of which are of fixed dimensions defined by molecular modeling of acceptable substrates, together with a polar binding region P located at the rear of B. Figure 15 illustrates its application for the hydroxylation of simple phenyl alkanes and benzylcycloalkanes. The most efficient conversions in terms of yield and enantioselectivity are achieved when R is either of the optimal size for pocket B (e.g., cyclobutyl) or contains a polar substituent capable of interacting with P (e.g., conversion of chroman, 20, to (R)-4-chromanol (a) in >98% enantiomeric purity (Holland et al., 1991a). The model also predicts the regioselectivity observed by Baciocchi et ~ l(1995) . during hydroxylation of 22 by M. isabellina at the site indicated. The ethyl group is best accommodated in pocket B, with the methyl substituent at the rear of A; the alternative binding mode with the ethyl group in A and methyl substituent in B is less favorable.

141


/ 6

4

fl

4

A: aromatic binding pocket B: aliphatic binding region P: polar binding site [O]:oxidking site dimensions in Angstrom

Benzyk hydroxyktion of phenyl alkanes (R = akyl) a d benzyl cycloalkanes (R = cycloakyl) by removal of H* to generate (R)alcohols FIG.15. Model for the benzylic hydroxylase of Mortierella isabellina.

5. Hydroxylation of Miscellaneous Compounds

Hydroxylation of 2,2-dimethylcyclohexanone(23) to give the 4-(S)-alcoho1 by E. coli containing the cloned genes of a cytochrome P-450 camphor hydroxylase from Pseudomonas putida has been interpreted in terms of the active-site structure of this enzyme (Yamamoto et al., 1990). The proposed model, shown in Fig. 16, assumes binding of the substrate in a boat configuration analogous to that of the natural substrate, camphor, with hydroxylation occurring exclusively from the direction indicated.

142

HERBERT L. HOLLAND

(20) R = H (21) R = O H

(22)

STRUCTURES 20-22.

S’

Hydrophobic interactions with VAL-295

[OH]+

H

H

*Binding

to lYR-96

Hydroxyhtbn of camphor

Hydrophobic

& interactions . with VAL-295 [OH]-

( 2 3 ) 4 + &

H

H

*Binding

to lYR-96

Hydroxylation of 2,2-dimethylcyclohexmne FIG.16. Hydroxylation of 2,2-dimethylcyclohexanoneby a cloned cyt.P-450 enzyme.

Hydroxylation of a series of substituted adamantanes (24) by various Absidia species leading predominantly to C-4=ial and C-3equatorial products has been explained by the model of Fig. 1 7 (Ridyard et al., 1996). The cooccurrence of hydroxylations at both these sites and the absence of C-4equatorialhydroxylated products requires an “end-on” approach (Fig. 17, right) of substrate to the heme-iron oxidizing center of the enzyme. The ratio of C-3 to C-4 hydroxylated products may then be determined by the nature of the interactions between the substituent R and the amino acid residues of the enzyme that lie perpendicular to the plane of the heme group.

143


JQ

H4eq

[OH]

(24)

&

H4ax

R

[OH] H3

H3

"side-on"approach leading to C-4 equatorial and C-3 products

"end-on"approach leading to C-4 axial and C-3 products

FIG.17. Models for the hydroxylation of substituted adamantanes by Absidia species.

C = cyclic system E = electron rich binding group L = lipophilic substituent [O] = site of hydroxylation

FIG.18. Original model for hydroxylation of cyclic compounds by B. sulfurescens.

6. Hydroxylations by Beauveria Sulhrescens

Beauveria sulfurescens ATCC 7159 (previously classified as Sporotrichum sulfurescens and Beauveria bassiana) is one of the fungi most frequently used for microbial hydroxylations. It has been successfully utilized for the hydroxylation of a range of natural products, synthetic cyclic amides, substituted aromatic compounds, and hydrocarbon substrates, and its application for the hydroxylation of amides was the subject of the first systematic investigation of the parameters that may influence the site and stereochemistry of microbial hydroxylation (Fonken et al., 1967). Their model for the hydroxylation of cyclic alcohols and amides is shown in Fig. 18 and proposes hydroxylation at a methylene group that is part of a ring system, C, to which is attached an electron-rich binding group, E, at an optimum distance of 5.5 A from

144

HERBERT L. HOLLAND

[C] = cyclic structure [B] = binding site for arnide oxygen [O] = site of hydroxylation FIG.19. Later model for hydroxylation of cyclic compounds by B. sulfurescens.

FIG.20. Quadrant rule for hydroxylations of cyclic amides by B. sulfurescens.

C. Group E may also carry a lipophilic substituent, L, which may or may not be part of C. This model was later refined (Fig. 19) to take into account the absolute stereochemistry of the hydroxylation reaction and the experimental observation that hydroxylations could occur over a range of 4-7 A from the binding center (Archelas et al., 1984; Srairi and Maurey, 1987; Fourneron et al., 1989). Hydroxylations of cyclic substrates by B. sulfurescens have also been analyzed in terms of the octant system illustrated in Fig. 20 for the definition of substrate-product relationships (Johnson et al., 1968; Archelas et al., 1984). In this analysis, the newly introduced hydroxyl


145

group is placed along the z coordinate of an xyz coordinate system, with the carbon undergoing hydroxylation located at the origin. The bulk of the substrate then preferentially occupies the rear quadrants labeled UL, UR, LL, and LR, in the order UR > UL > LL and LR. The models of Figs. 18-20 were developed specifically to explain the hydroxylation of cyclic amides and related substrates. They fail to satisfactorily account for such other B. sulfurescens-catalyzed hydroxylations as the formation of phenols (Vigne et al., 1986) and hydroxylation of hydrocarbons (Johnson et al., 1973). A more complete analysis of the hydroxylations performed by B. sulfurescens suggests the existence in this microorganism of at least three distinct types of hydroxylase enzymes, one specific for amides and related substrates, one for benzylic hydroxylations, and one for conversion of arenes to phenols (Holland and Zabic, 1996). 7. Dioxygenase-CatalyzedReactions

The microbial conversion of arenes to cis-dihydrodiols has been extensively employed as a synthetic source of the latter. This reaction, performed exclusively by prokaryotic microorganisms, is most frequently carried out using mutants of various Pseudomonas species, and a model that predicts both the regiochemistry and stereochemistry of oxidation of mono- and disubstituted monocyclic arenes by Pseudom o m s putida UV4 has been proposed (Boyd et al., 1995). This model proposes that the outcome of dioxygenation is controlled by the size of substituent groups on the aromatic ring for substituents comprising hydrogen, the halogen group (F, Br, C1, I), and methyl. In all cases with the exception of fluorobenzene, products are formed in high (>99%) enantiomeric purity, as depicted in Fig. 21. B.

RELATIONSHIP OF HYDROXYLASE “ACTIVE-SITE” MODELS TO ENZYME STRUCTURE

The various models discussed above have largely been empirically derived by a correlation of substrate-product relationships. Although often described as “active-site models,” this description is a potential misnomer in the absence of any definite information to the effect that the hydroxylations of a range of substrates by a microorganism are indeed performed by a single enzyme, and in the absence of any complicating factors such as selective transport phenomena. The relevance of these models to the real topography of an enzyme’s active site is therefore speculative, but there are nevertheless several features of the

146

HERBERT L. HOLLAND

OH

major

minor

Q

OH

OH

FIG.21. Models for the oxidation of substituted arenes by Pseudornonas putida UV4.

models that suggest their relevance to specific enzyme-substrate interactions. 1. Significance of Binding Sites

The importance of relatively polar electron-rich binding sites in the substrate in determining both the efficiency and regiochemistry of microbial hydroxylation is a recurring theme. The beneficial effect on the hydroxylation of otherwise nonpolar molecules of introducing polar substituents such as the sulfone or carbamate residue was discussed in Section II.A.2, and the directing influence of oxygen substituents in steroid hydroxylations, discussed in Section 1I.A.1, is well established. The importance of this phenomenon has also been established at the isolated enzyme level, where, for example, the o-hydroxylation of lauric acid derivatives 25 by cyt.P-450 4A1 is dependent on the position of heteroatom substitution in the substrate (Bambal and Hanzlik, 1996).


147

(25) STRUCTURE 25.

0

0

'

FIG.22. Relationship between hydroxylations at C-Zcc (0) and C-7p (*) in cyt.P-450 2a-5.

The subtle binding effects that control enzyme-substrate interactions, and hence the regio- and stereochemistry of product formation, in hydroxylase enzymes are illustrated by the observation that a single-site mutation of Phe-209 in cyt.P-450 2a-5 is capable of altering the regiospecificity of hydroxylation of dehydroepiandrosterone (26)by this enzyme (Iwasaki et al., 1995). Substitution of Phe-209 by leucine resulted in an enzyme with 2a-hydroxylase activity, whereas the major site of hydroxylation was shifted to C-7p on replacement of Phe-209 by valine. This shift may be attributable to an inversion of the binding mode, illustrated in Fig. 22, with hydroxylations at C-2a (0) and C-7p (*) of two orientations of the substrate being performed at a single oxidizing site. 2.

Comparisons with cyt.P-450

With the exception of the dioxygenase-catalyzed reactions referred to in Section II.A.8, the bulk of the microbial hydroxylation reactions discussed above are presumed to be carried out by membrane-bound cytochrome P-450-dependent monooxygenases, although the number of examples for which this has been unequivocally demonstrated is small (Yamamoto et al., 1990; Ahmed et al., 1996; Ridyard et al., 1996). In

148

HERBERT L. HOLLAND

view of the lack of detailed structural information available for microbial cyt.P-450, the structure of the atypical soluble cyt.P-450cAM has been used as a starting point for analysis of microbial hydroxylation reactions. This enzyme, for which high-resolution X-ray crystallographic data are available (Poulos et al., 1985), possesses a narrow access channel through which the substrate can pass to a protected active site, and binds its substrate camphor by specific hydrophobic contact interactions with nonpolar active-site residues such as valine or phenylalanine, and by polar interactions though the carbonyl group hydrogen bonding to Tyr-96 (see Fig. 16). It therefore exemplifies the combined features of polar binding and nonpolar size-restricted interactions that appear to be dominant for the microbial hydroxylations discussed above. Indirect information concerning the active-site environment of microbial hydroxylases may therefore be gleaned from a comparison of the known features of these enzymes with similar properties of cyt.P4 5 0 ~ NMR ~ ~ relaxation . studies on Cyt.P-450BM3 from Bacillus megaterium indicate a relatively large active-site region (Modi et al., 1995), unlike the highly restricted environment of the cyt.P-450cAMactive site, and a similar picture may be gleaned from site-directed mutagenesis (Furuya et a]., 1989) and from partial sequence alignments of cyt. P-45ocAM and other membrane-bound cyt.P-450, based on the assumption that areas of common sequence have similar three-dimensional structures (Nelson and Strobel, 1988). This analysis is consistent with the observation that the membrane-bound microbial hydroxylases have a wider substrate specificity than does cyt.P-450cAM but that there are sufficient similarities for the specific substrate binding properties of the latter (the specific combination of polar and nonpolar interactions) to be used as a guide for the regio- and stereoselectivity of hydroxylation by the former.

c. HYDROXYLATION AND PHYLOGENY An alternative to the structure-based approach to rationalization of hydroxylation reactions is the phylogenic approach, the systematic study of the hydroxylation of a single substrate by a wide range of microorganisms in an attempt to relate the regio- and stereochemistry of hydroxylation to the taxonomic classification of the microorganisms concerned. This approach has been systematically applied to hydroxylation of several terpenes (Abraham, 1994; Abraham et al., 1996). Bio-


STRUCTURES

149

27-29.

transformation of (+)-isopinocampheol (27) can lead to hydroxylations at C-1, -2, -5, -7, -8 and -9, whereas the (-)-enantiomer 28 is hydroxylated only at C-1, -2, -4, or -9 (Abraham, 1994), and, whereas the by a single microorganism showed some hydroxylation of 22 or enantioselectivity with respect to product distribution, the pattern of hydroxylations across a range of microorganisms, particularly for 27, showed a pronounced phylogenic dependency. Hydroxylation at C-1 was confined almost entirely to bacteria, whereas fungi, particularly the Zygomycotina, showed a predilection for hydroxylations at C-5 and C-7. Hydroxylations at C-2 were carried out by both fungal and bacterial biocatalysts. A similar analysis of the biotransformations of aristolenepoxide (29) has determined the phylogenic frequency of hydroxylations at C-3, -5, -8, -9, -10, -15, and -16 (Abraham et al., 1996). D. HYDROXYLATION AND BIOSYNTHESIS

The eclectic substrate specificity of many of the enzymes of secondary metabolism has led to the biosynthetic pathways of fungal secondary metabolites being used as a guide both for the selection of substrates suitable for biotransformation by those fungi and for the type of reactions that those substrates may undergo (Alam and Hanson, 1990). This technique has been successfully applied to hydroxylations carried out by Cephalosporium aphidicola of analogues of the biosynthetic intermediates of the diterpene aphidicolin 30, a natural product of C. aphidicola. Hydroxylations of the aphidicolin analogues 31-35 by C. aphidicola occurred at the positions indicated in a series of reactions clearly linked to the biosynthesis of 30 (Gordon et al., 1988, 1992; Hanson and Jarvis, 1994), and the sites of hydroxylation of the structurally isomeric diterpene stemodin S also show some analogies to the reactions of the aphidicolin biosynthetic pathway (Hanson et al., 1994).

150

HERBERT L. HOLLAND

HO HO(31) R=CH3 (32) R=CzHS

+OH.*,

J

(33)

i“”,, J

0::’ HO-.‘

(35)

Ill. Models for Sulfoxidation Reactions

A. “ACTIVE-SITE” MODELS 1. Aspergillus niger

Aspergillus niger was among the earliest microorganisms used for the conversion of prochiral sulfides to chiral sulfoxides, and a model (Fig.


151

FIG.23. Model for chiral sulfoxidation of phenyl and benzyl sulfides by Aspergillus niger.

23) was proposed to account for the enantioselectivity of the oxidations of a range of substituted phenyl and benzyl sulfides by this fungus (Auret et al., 1968). This model, based solely on the steric differences between R, and R2, predicts that the preferred direction of oxidation is B when R2 is larger than R,. However, the model is not universal, even for A. niger, and predicts only a general trend in the enantioselectivity of oxidation. The model of Fig. 23 has, however, been used to interpret the oxidations of phenyl and benzyl sulfides carried out by an isolated liver microsomal cyt.P-450 enzyme (Takata et al., 1980), which lends credibility to this approach for the rationalization of sulfoxidase enantioselectivity. 2. Mortierella isabellina ATCC 42613

The simple model of Fig. 23 has also been applied to the oxidation of phenyl and benzyl sulfides by M. isabellina ATCC 42613 by redefinition of R1 as an alkyl and R, as a phenyl or benzyl group (Holland et d., 1985). These reactions are, however, more accurately described by the model of Fig. 15, which has recently been extended to cover the enantioselective oxidation of prochiral sulfides (Holland et al., 1997a). Inhibition and induction studies have indicated that the same enzyme of M. isabellina is responsible for both the benzylic hydroxylation and sulfoxidation reactions (Holland et al., 1987), and Fig. 15 can thus be used to predict the structures of substrates that will be efficiently converted to (R)-sulfoxides by M. isabellina. These include aryl alkyl sulfides (e.g., p-bromophenyl methyl sulfide (37,e.e. 100%) or 3,5-dimethylphenyl methyl sulfide (38, e.e. 84%)), in which the aromatic binding pocket (A) of Fig. 15 is optimally occupied, or substrates such as phenyl n-propyl sulfide (39, e.e. 100%) that fill the aliphatic binding region (B). 3. Helminthosporium Species NRRL 4671

The fungus Helminthosporium species NRRL 4671 converts a wide range of prochiral sulfides to the corresponding chiral sulfoxides, the

152

HERBERT L. HOLLAND

(37)

STRUCTURES 37-39.

Hs: s m l hydrophobic pocket HL: large hydrophobic pocket PHP: polar hydrophobic pocket P: polar binding site LPP: lone pair pocket 0: oxidationcentre optimumP to 0 distance, 8 -10 /

Top view, dimensions in 8,

FIG.24. Model for chiral sulfoxidation of Helminthosporium species NRRL 4671.


153

os'n~c

US'" H2CH3

(40) (S) sulfoxidation, e.e. 84%

(41) ( R )sulfoxidation, e.e. 25%

(42) (k) sulfoxidation, e.e. 0%

(43)

(s)sulfoxidation,e.e. 45%

STRUCTURES 40-43.

majority of which have (S)-configuration at sulfur. Analysis of over 90 such biotransformations has resulted in the development of a model based on restrictive space descriptors that has been used to rationalize these reactions and also as a predictor of the outcome of Helminthosporium-catalyzed sulfoxidations (Holland et d., 1997b). This model, presented in Fig. 24, was developed from energy-minimized structures of compounds divided into groups of acceptable (>lo% yield) and unacceptable (

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