The Alkaloids: Chemistry and Pharmacology, Volume 40

THE ALKALOIDS Chemistry and Pharmacology VOLUME 40

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THE ALKALOIDS Chemistry and Pharmacology Edited by Arnold Brossi National Institutes of Health Bethesda, Maryland

VOLUME 40

Academic Press, Inc. Harcourt Brace Jovanouich, Publishers

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This book is printed on acid-free paper.

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Copyright 0 1991 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.

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Library of Congress Catalog Card Number:

ISBN 0-12-469540-X

(alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 91

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CONTENTS

CONTRIBUTORS ...................................................................................... PREFACE...............................................................................................

vii ix

Chapter 1. Plant Biotechnology for the Production of Alkaloids: Present Status and Prospects ROBERT VERPOORTE, ROBERTVAN DER HEIJDEN, WALTERM. VANGULIK,AND HENSJ. G . TEN HOOPEN

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI.

Introduction ....................................... Strategies to and Extracellular Aspects .. Large-Scale Production ................................................................... Nicotine ........................................................... .... Tropane Alkaloids .......................................................................... Isoquinoline Alkaloids ............... Cinchona Alkaloids ........................................................................ Indole Alkaloids ....................,....................................................... Caffeine ............................................. Steroidal Alkaloids ......................................................................... ........................... ,...................................... Concluding Remarks ....... References ............................................................

2 9 20 44 52 72 104 109 154 157 161 163

Chapter 2. Alkaloids from Mushrooms AND WIESJ/AW z.ANTKOWIAK R6ZA ANTKOWIAK

I. Introduction ..................................................................................

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

190 194 225 253 275 28 1 289 305 307 324

CUMULATIVE INDEXOF TITLES ................................................................

34 1

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

349

11. Physiologically Active Principles of the Genus Arnanita ........................

111. Indole Alkaloids ..... ,......................... ,............. IV. Pyridine Alkaloids ... ................................................... .............................. V. Hydrazine Alkaloids ................................

........................................................ .................. References

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

V

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’contributions begin.

R 6 z ANTKOWIAK ~ (189), Faculty of Chemistry, Adam Mickiewicz University, Poznan, Poland WIES~AW Z. ANTKOWIAK (189), Faculty of Chemistry, Adam Mickiewicz University, PoznBn, Poland HENSJ. G. TEN HOOPEN(l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Department of Biochemical Engineering, Delft University of Technology, 2628 BC Delft, The Netherlands ROBERTVAN DER HEIJDEN (l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Center for Bio-Pharmaceutical Sciences, Division of Pharmacognosy, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands WALTERM. VAN GULIK(l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Department of Biochemical Engineering, Delft University of Technology, 2628 BC Delft, The Netherlands ( l ) , Biotechnology Delft Leiden, Projectgroup Plant ROBERT VERPOORTE Cell Biotechnology, Center for Bio-Pharmaceutical Sciences, Division of Pharmacognosy,Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands

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PREFACE

Modern biotechnological methods of producing potentially useful alkaloids, particularly plant cell culturing, is an area of fundamental importance and has been receiving a lot of attention. An analysis of the state of the art and where it is headed is discussed here by a well-known group from The Netherlands in “Plant Biotechnology for the Production of Alkaloids: Present Status and Prospects.” Muscarine from the mushroom, Amanita muscaria, has become a valuable biochemical tool for measuring peripheral effects on cholinergic receptors (see Volume 23 of this series). Each year Phalloidin from the mushroom, Amanita phalloides, causes several deaths in Europe from mushroom poisoning and has been the subject of a book by Theodor Wieland. In this volume, the subjects of alkaloids and mushrooms are brought together in a paper written by a team of experts from Poland in “Alkaloids from Mushrooms.” Arnold Brossi National Institutes of Health

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-CHAPTER

1-

PLANT BIOTECHNOLOGY FOR THE PRODUCTION OF ALKALOIDS: PRESENT STATUS AND PROSPECTS* ROBERTVERPOORTE AND ROBERT VAN

DER

HEIJDEN

Biotechnology Devt Leiden Projectgroup Plant Cell Biotechnology Center f o r Bio-Pharmaceutical Sciences Division of Pharmacognosy Gorlaeus Laboratories 2300 R A Leiden. The Netherlands

WALTERM.

VAN

GULIK,AND HENSJ. G. TEN HOOPEN

Biotechnology Devt Leiden Projectgroup Plant Cell Biotechnology Department of Biochemical Engineering Devt University of Technology 2628 BC Delft, The Netherlands

I. Introduction ......................................... A. Production by Means of Genetically Engineered Microo ................ B. Production by Means of Plant Cell Cultures C. Bioconversion of Available Precursors. ... D. Production by Genetically Engineered Plan E. Production of Novel Compounds.. 11. Strategies to Improve Product Yield: Cellular and Extracellular Aspects.. .. A. Screening and Selection. .......................... B. Culture Conditions ............................... C. Alkaloid Storage Compartments .......................... D. Elicitation.. ..................................... E. Feeding of Precursors and Bioconversions. .......................... F. Immobilization. ........................ .................... G. Permeabilization ................................. H. Differentiation and Culture Type ................................... I. Genetic Approaches and Genetic Modification ................... J. Combination of Treatments: Toward High Productivity ...............

2 6 6 7 8 9 9 10

12 14 16

16 17 17 18 19 19

* The following abbreviations are used in the text: ABA: abscisic acid; BAP: 6-benzylaminopurine; DW: dry weight; 2,4-D: 2,4-dichlorophenoxyaceticacid; FW: fresh weight; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; MS: Murashige and Skoog medium; NAA: naphtaleneacetic acid. 1

THE ALKALOIDS, VOL. 40 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ROBERT VERPOORTE ET AL.

111. Large-Scale Production. . . . . . . . . . . .

A. Economic Feasibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Technological Aspects ................ IV. Nicotine ......... . ....... . . ........ ....... . ..... . ..... . .... . . . . A. Secondary Metabolites in Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Screening, Selection, and Stability C. Effects of Growth Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Large-Scale Suspension Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Root Cultures.. . . . . . . . . . . . . . . . . . . . F. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Tropane Alkaloids ........................ A. Plant Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Production of Tropane Alkaloids by Cell Cultures . . . . . . . . . . . . . . . . . . . . VI. Isoquinoline Alkaloids. . . . . . . . . . . . . . . . . . . A. Ipecacuanha Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Colchicine ........................ C. Cephalotaxus s . ............................ D. Bisbenzylisoquinoline Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Opium Alkaloids F. Sanguinarine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII. Cinchona

............................................ ...................... logy ..............................................

C. Bioconversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Conclusions .......................... VIII. Indole Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Catharanthus Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rauuolfia Alkaloids C. Miscellaneous Indol ........... IX. Caffeine ...... . ..... . . .... . .... . . ..... . . . .... . . ... . . .... . .... .. . .... X. Steroidal Alkaloids ............... B. Miscellaneous Steroid Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 22 25 44 44 47 48 48 50 51 52 52 65 72 72 74 74 75 77 85 94 104 104 105 108 108 109 109 142 149 154 157 158 161 161 163

I. Introduction Plants produce a variety of alkaloids. Table I presents some of the major classes and an estimation of the number of representatives (N. R. Farnsworth, personal communication). About 30 alkaloids have great commercial interest, mostly because of their use as medicines, flavorings, or poisons, sometimes as important tools in pharmacological studies. In all cases the total amounts produced worldwide are rather limited. For example, by volume, possibly the largest production involves the alkaloids quinine and quinidine. For their isolation 5000-10,000 metric tons of Cin-

1.

3

PLANT BIOTECHNOLOGY

TABLE I NUMBER OF UNIQUE STRUCTURES IN MAJORALKALOIDS CLASSES IN NAPRALERT DATABASE^ Class

Number

Indole Isoquinoline Protoalkaloids Quinoline Pyrrolidine/piperidine Diterpene Quinolizidine Pyrrolizidine Steroidal Tropane Pyridine Indolizidine Sesquiterpene Homospermidine/spermidine

4125 4045 1147 718 714 642 57 I 562 440 300 24 1 170 132 129 15,947

October, 1988. Totals include alkaloids isolated from plant, animal, and marine sources. N. R. Farnsworth, personal communication.

chona bark is processed yearly, yielding 300-500 metric tons of quinine and quinidine (I). The alkaloid ajmalicine, used as an antihypertensive, has a yearly market volume of about 3600 kg (2), for which probably 200-300 tons of roots of Catharanthus roseus is needed. Compared to laboratory-scale isolations, these seem impressive amounts, but compared to agricultural crops they are only very small volumes. Several compounds of interest are isolated from plants which need several years to develop. For example, Cinchona trees need 10-12 years before they can be harvested, and Coptisjaponica rhizomes, a source of barberine, require 5-6 years of growth before harvesting. In many cases little plant breeding has been done to improve yields of the alkaloids, and in some cases one simply relies on collection of plant material from the wild (e.g., tubocurarine is isolated from curare, which is collected by Indians from the liana Chondrodendron tornentosurn). Alkaloids are consequently valuable chemicals. Table I1 gives prices per gram of some commonly used alkaloids. As no single source was available for bulk prices of these chemicals, we used the price list of a large supplier of fine chemicals. These prices are per gram, higher than the bulk prices, but they give at least some indication of the values of these alkaloids. In this review we shall give a survey of the state of the art of using plant

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ROBERT VERPOORTE ET A L .

TABLE I1 PRICES,SOURCES, AND PRODUCT YIELDSBY MEANSOF ALTERNATIVE BIOTECHOLOGICAL METHODS OF SOME COMMONLY USED ALKALOIDS

Alkaloid Ajmalicine Vinblastine Vincristine Ajmaline Reserpine Rescinnamine Vincamine Strychnine Brucine Yohimbine

Coronaridine Quinine Quinidine Ellipticine 9-Hy droxyellipticine

Camptothecine Emetine Physostigmine Pilocarpine Caffeine Theobromine Atropine

Scopolamine

Cocaine Berberine Sanguinarine Berbamine Tu bocurarine

Plant source Catharanthus roseus Catharanthus roseus Catharanthus roseus Rauvolfia species Rauvolfia species Rauvolfia species Vinca species Strychnos species Strychnos species Rauvolfia species, Corynanthe species Tabernaemontana species Cinchona species Cinchona species Ochrosia elliptica Ochrosia elliptica Camptotheca acuminata Cephaelis ipecacuanha Physostigma venenosum Pilocarpus microphyllus Coffea, Camellia Theobromu Atropa belladonna, Datura species, Hyoscyamus species Duboisia species, Hyoscyamus species Erythroxylon coca Coptis juponica, Berberis Eschscholtzia, Papaver Menispermaceae Chondrodendron tomentosum

Price (DMlgram) 56.00 15,800.00 37,800.00 15.50 12.50 27.50 29.50 1.45 1S O 6.50

Production by cell culturesa Source

Yield

C. roseus SC C. roseus ShC C. roseus ShC RauvolJa SC Rauvolfia SC No data available Vinca minor SC No reports on PCTC No reports on PCTC No data available

0.2 g/liter Traces Traces 0.04 g/liter 0.002 g/liter

Tabernaemontana species SC Cinchona ShC Cinchona ShC 0. elliptica SC

Traces

C. acuminata SC

0.00025% DW

39.50

Cephaelis RC

0.3-0.5% DW

79.00

No reports on PCTC

25.50

No reports on PCTC

980.00 0.75 1.40 3940.00 4680.00 720.00

3.3 g/liter

0.01-0.1% DW 0.01-0.1% DW 0.005% DW

Coffea SC Coffea SC Atropa HR Datura HR

0.48 g/liter

26.00

Duboisia HR Hyoscyamus HR

0.08 glliter 0.4% DW

17.00 16.50

No reports on PCTC C. juponica SC Thalictrum SC Eschscholtzia SC Papaver SC Stephania RC No reports on PCTC

7 g/liter 0.87 g/liter 0.16 g/liter 0.25 g/liter 0.55% DW

0.12 0.95 5.20

72.00 265.00 126.00

0.1-0.2 g/liter 0.1-0.2 g/liter

(continued)

5

1. PLANT BIOTECHNOLOGY TABLE I1 (Continued)

Alkaloid Papaverine Noscapine Narceine Morphine Codeine Nicotine Colchicine Hamngtonine Aconitine Conessine Solasodine Shikonin'

Plant source Papaver somniferum Papaver somniferum Papaver somniferum Papaver somniferum Papaver somniferum Nicotiana species Colchicum autumnale Cephalotaxus harringtonia Aconitum species Holarrhena antidysenterica Solanum species Lithospermum erythrorhizon

Price (DM/gram) 0.43 13.50 16.00 510.00 25.50 1.55 75.00 NAb 1120.00 NA 170.00

NA

Production by cell cultures" Source

Yield

P . somniferum SC P . somniferum SC P . somniferum SC P . somniferum SC P . somniferum SC Nicotiana SC C . autumnale CC

Traces Traces Traces Traces Traces 0.36 g/liter 1.5%DW

C . harringtonia CC

Traces

No reports on PCTC Holarrhena CC

0.001%FW

Solanum SC

O.Ol-i% DW

L . erythrorhizon SC

4 g/liter

Results as reported in the literature. PCTC, Plant cell and tissue culture; CC, callus culture; SC, cell suspension cultures; RC, root cultures; HR, hairy root cultures; ShC, shoot cultures; DW, dry weight; FW, fresh weight. NA, Not available. Shikonin is not an alkaloid, but because it is the first product from a plant cell biotechnological production, it is included in the table to enable comparison with the alkaloids.

cell cultures for the large-scale production of alkaloids. Strategies followed to obtain high production (Section 11) and aspects of technology involved in the large-scale culture of plant cells and the economy of such processes (Section 111) are discussed briefly. Different classes of alkaloids are then discussed separately, with emphasis on production, be it by de nouo biosynthesis or bioconversion of added precursors by plant cells. Patents concerning the production of various alkaloids are also listed. We confine ourselves only to alkaloids derived from higher plants which are presently produced on an industrial scale by extraction of plant materials. Some classes of alkaloids for which production in cell cultures has been studied extensively are thus omitted, for example quinolizidine (lupine alkaloids), pyrrolizidine (Senecio and Symphytum alkaloids), and acridone alkaloids (Ruta alkaloids). For these classes of alkaloids we refer to recent authorative reviews (3-5). For some widely used alkaloids, such as pilocarpine, physostigmine, cocaine, strychnine, and tubocurarine, no studies have been published yet on the plant cell tissue and organ culture (see Table 11). It is obvious that with the rapid developments in biotechnology, alternative biotechnological production methods of plant-derived fine chemicals,

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like the alkaloids, became of interest. Several possibilities can be considered for applying biotechnology, namely, production of plant compounds by genetically engineered microorganisms; production by means of plant cell cultures; bioconversion of readily available precursors, by using genetically engineered microorganisms, plant cell cultures, or isolated plant enzymes; production by means of genetically engineered plants or plant cell cultures; and production of novel compounds. Besides the possibilities for production of known compounds, biotechnology can also be used to produce new compounds. We shall consider these possibilities in more detail. A. PRODUCTION BY MEANSOF GENETICALLY ENGINEERED MICROORGANISMS Genetic engineering of microorganisms is feasible nowadays. One can thus consider the possibilities of transferring the production of a plant secondary metabolite into a microorganism. To be able to do so one has to know the biosynthetic pathway of the compound concerned; one must identify the enzymes involved and the genes coding for the enzymes. As most plant secondary metabolites result from pathways involving a large number of steps (10-20 is quite normal), at least as many genes are involved. In fact, only a few secondary metabolite pathways are completely known at the level of enzymes, for example, the flavonoid pathway and the biosynthesis of some isoquinoline alkaloids. Consequently only very few genes from secondary metabolism are known (e.g. some of the key genes from the flavonoid pathway). In the case of alkaloids only a few isolated steps from the biosynthetic pathways have been studied to the level of the genes, for example, strictosidine synthase, a key enzyme in indole alkaloid biosynthesis from RauuolJia (see below) (6,7) and tryptophan decarboxylase, another regulated enzyme from indole alkaloid biosynthesis (8,9). Even if all the genes were known, transferring a large number of genes to a microorganism is not feasible, particularly as the enzymes produced have to act in a concerted way. Furthermore, in plants secondary metabolism is often compartmentalized on the subcellular or even cellular level. This will be impossible to realize in microorganisms. BY MEANSOF PLANTCELLCULTURES B. PRODUCTION

As genetic engineering of microorganisms does not seem to be a feasible approach, one should exploit the genetic information of the plant cell itself. Plant cells are totipotent, which means that each cell carries all the genetic

1. PLANT BIOTECHNOLOGY

7

information for all plant functions, including the biosynthesis of secondary metabolites. In theory it is thus possible to have in uitro cultured plant cells produce secondary metabolites. Below, in the review on the state of the art of plant cell biotechnology for the production of various commercially interesting alkaloids, it will become clear that this is only partly true. Table I1 summarizes the results for most of the alkaloids discussed here. Secondary metabolism is a form of differentiation, but cells grown in uitro are rapidly dividing, undifferentiated cells. Only at the end of the growth phase of batch-cultured cells may some form of differentiation occur, connected with the production of secondary metabolites. A plant produces a wide variety of secondary metabolites, all with different, mostly unknown functions. In in uitro cultured cells those compounds which defend the plant against microorganisms, namely, phytoalexins, are often easily formed. For example, Cinchona cell cultures produce large amounts of anthraquinones, but the alkaloids of interest, the quinolines, are produced in trace amounts only. Similarly Papauer cell cultures produce sanguinarine and closely related alkaloids, but no morphinane alkaloids. The various strategies followed to obtain high producing cell lines will be briefly discussed separately (see Section 11).The economics of a plant cell culture production process are discussed below (see Section 111). For cell lines that do not produce, it will be necessary to learn more about the regulation of secondary metabolism in order to eventually be able to use genetic engineering for improving production (see below).

C. BIOCONVERSION OF AVAILABLE PRECURSORS Based on knowledge of a biosynthetic pathway one can select certain steps which could be of interest for bioconversion of (a) readily available precursor(s). This could, for example, be stereospecific reactions, like the reduction of quinidinone in quinine or quinidine and the epoxidation of atropine to scopolamine. For the bioconversion one can consider using plant cells [e.g., the production of L-dopa from tyrosine by immobilized cells of Mucunapruriens (lo)]or isolated enzymes from the plant itself. An interesting example of the latter is the (S)-tetrahydroprotoberberineoxidase (STOX) enzyme, which oxidizes (S)-reticuline but not its stereoisomer (11). This feature can be used in the production of (R)-reticuline from a racemic mixture (see below). Immobilized strictosidine synthase has been successfully used to couple secologanin and tryptamine. The gene for this enzyme has been isolated from Rauuolfia (6) and cloned in Escherichia coli, in which it is expressed, resulting in the biosynthesis of active enzyme (7). The cultured bacteria produced 20 times more enzyme

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ROBERT VERPOORTE ET A L .

per liter than a plant cell suspension. The genetically engineered microorganism can thus be used for the large-scale production of this intermediate for indole alkaloid biosynthesis, using tryptamine and secologanin as precursors. Strictosidine, with its two secondary nitrogens, two aldehyde groups, a double bond, and an ester group, is an ideal synthon for the (bio)synthesis of a variety of new compounds which could be studied for biological activity (12). The first small steps have been made, but the field of bioconversion still contains numerous possibilities yet to be explored. Cloning of plant genes into microorganisms could be of interest, particularly in the case that cofactors are required. Bioconversions with plant enzymes seem to offer great potential for biotechnological applications.

D. PRODUCTION BY GENETICALLY ENGINEERED PLANTSOR PLANT CELLCULTURES The difficulties of low producing plant cell cultures have already been mentioned. By unraveling the biosynthetic pathways and the regulation thereof on the level of enzymes and genes, it might become possible to identify genes which could be subject for genetic engineering. Various possibilities can be envisioned: combining genes of secondary metabolism with other promoter genes; adding further copies of an already present gene to increase enzyme production; suppressing genes by antisense DNA (e.g., blocking competitive pathways or blocking catabolism); introducing part of a pathway into another plant that is already capable of performing part of the biosynthesis. The latter approach seems particularly interesting. One could consider transferring a pathway from a slowly growing plant into a plant which grows rapidly and is suitable for agriculture (e.g., transferring the final steps of Cinchona alkaloid biosynthesis into Catharanthus roseus). This would mean that the alkaloids could be produced in an annual crop, which is more easily tuned to the demand for the alkaloid. Recently we have been able to introduce the tryptophan decarboxylase gene from Catharanthus roseus into tobacco, resulting in plants producing significant amounts of tryptamine (9), thus again proving that genetic engineering of secondary metabolism in plants and plant cells is feasible nowadays. For all applications of genetic engineering, however, one has to know the mode of regulation of secondary metabolism at the level of a number of enzymes and genes. For the near future this will be a major challenge; at present knowledge is very limited.

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9

E. PRODUCTION OF NOVELCOMPOUNDS So far only the production of known compounds has been discussed. However, plant biotechnology also offers possibilities for new compounds. A number of plants have been studied phytochemically, sometimes in combination with assays for certain types of biological activity. This has resulted in discovery of numerous compounds with interesting biological activities. Many of the plants studied were collected in remote areas, and the large-scale production of the compounds isolated would be very difficult. Plant cell cultures do offer interesting perspectives, and they could be used to produce on a large scale compounds first found in the plant. Alternatively, one can screen cell cultures for new biologically active compounds. Such an approach has shown to be fruitful (Z3-16). Among others two alkaloids, pericine and apparicine, with activity in the central nervous system (CNS) have been isolated from cell cultures of Picrulima nitida (44). In connection, one might also think about the addition of elicitors to cell cultures; this would lead to the production of antimicrobial compounds (phytoalexins) which could be of interest for futher development as antibiotics. [For a review of new compounds isolated from plant cell cultures, the reader is referred to Ruyter and Stockigt

un.1

Another approach could be to use genetic engineering to introduce a further step in a biosynthetic pathway, leading to (for the plant) new compounds. This approach has, for example, been used to introduce new flower colors (18).It could also be of interest in improving the resistance of plants against microorganisms or predators. However, more insight into the role of alkaloids in plant survival in native ecosystems is needed for this.

11. Strategies to Improve Product Yield: Cellular and Extracellular Aspects

The production of alkaloids in plant cell cultures is a result of an enormously complex set of interactions between cellular and extracellular compartments. The extracellular compartment should at least offer possibilities for survival of the cellular compartment, but often cell growth and cell differentiation are prerequisites. The cellular compartment, however, is continuously changing the extracellular compartment by uptake of nutri-

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ROBERT VERPOORTE E T AL.

ents and excretion of metabolites. The changed environment of the cells will in turn affect the cellular compartment, and so on. A plant cell culture is thus a dynamic system in which the smallest change can have large, even fatal, consequences. On the other hand, such a system offers many opportunities for manipulation to improve product yields. For alkaloid production the extracellular compartment should supply such conditions, so that the cellular compartment is able to express its secondary metabolism; furthermore one of the compartments should provide secondary product storage facilities. Many studies have dealt with characterization of the extracellular “production-induction’’ conditions. Most of the studies, however, describe only the beginning and the end of the story: for example, omitting component Y from the medium will increase alkaloid formation x times. Little insight into the processes in between or, in general, into the regulation of alkaloid formation is, at present, available. Application of recent developments in enzymology and molecular biology offer great opportunities to fill the gap in our knowledge on the regulation of secondary metabolism. In this section some of the strategies used to improve alkaloid yields in plant cell cultures are discussed.

AND SELECTION A. SCREENING

For development of an alkaloid production system the cellular compartment is the basis of the system. Based on phytochemical or chemotaxonomical data a plant species is selected. From plants of that species, cell and tissue cultures can be initiated from various types of tissue, for example, leaf, anther, or root. From this tissue several types of cultures can be initiated, for example, callus, suspension, shoot, and root. Thus, from the very beginning of the production process one already has to deal with a large number of variables. It is clear that every cell line has its own unique characteristics and that certain strategies for production improvement will only work for that specific cell line. To obtain a high producing cell line, one has to perform a screening or selection procedure. Unlike screening, selection is an active process which deliberately favors only the survival of the wanted variant while the wild-type cells do not survive. This definition was given by Berlin and Sasse (19), who wrote a detailed review of screening and selection procedures. Screening of plant material can be performed at different levels: species, specimens, organ, cell culture, and even single cells or protoplasts. At lower levels of organization selection procedures can be performed, for example, on callus and suspension cultures.

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PLANT BIOTECHNOLOGY

11

1. Screening

Various screening procedures have been performed, resulting in cell lines with higher alkaloid contents than that of the parent plant or culture [e.g., for ajmalicine (20) and berberine (21)l. In a comparison of 458 cell lines of C . roseus cv. Roseus, which were all initiated from excised anthers and grown under identical conditions, several different production profiles, with respect to the presence of different types of indole alkaloids, were obtained. Productivity varied from nonproducing (32% of the cell lines) up to 1.5% total alkaloids per cell dry weight (22). This example illustrates the neccesity of screening of various cell lines. Visual screening, facilitated by the color of the alkaloid, yielded highly productive cell lines of berberine (21).The strong fluorescent properties of serpentine allowed the determination of its concentration in individual cells by flow cytometry, and subsequent sorting of the cells with high contents yielded a highly productive cell line (23).The same technique was used for berberine-containing cells (24). 2 . Selection

Selection pressure on a plant cell population can be applied by the addition of selective chemicals to the medium and/or by the creation of selective growth conditions. Selective chemical agents are mainly specific enzyme inhibitors, with which selection pressure is directed to obtain cells with increased (overproduced) enzyme activity. 4-Methyltryptophan was used for the selection of C . roseus cells with high tryptophan decarboxylase (TDC) activity (25).The selected cells contained, besides significantly higher TDC activity, higher levels of tryptamine. However, only one cell line produced higher levels of ajmalicine then the wild-type culture. Using the same selective agent, Berlin et al. (26)obtained a 4-methyltryptophantolerant cell line of Peganum harmala, which produced increased levels of serotonin and, compared to the wild-type culture, similar amounts of P-carboline. Selection with 5-methyltryptophan resulted in C . roseus cells with increased levels of tryptophan; however, it showed no effect on tryptamine and alkaloid levels (27). Nicotinic acid has been used succesfully for the selection of high nicotine-producing hairy root cultures of Nicotiana rustica (28).Attempts to use toxic end products such as vinblastine (29) and quinoline alkaloids (30) as selective agents for obtaining alkaloid-producing cells of Catharanthus and Cinchona, respectively, were not successful. Selection by changed environmental conditions were used for obtaining photomixotrophic and photoautotrophic cells. This yielded leaflike cells

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ROBERT VERPOORTE ET A L .

with well-developed chloroplasts. Lupine cells of this type were able to produce sparteine and lupanine (31).However, photoautotrophic cells of C . roseus did not produce vindoline (32).

B. CULTURECONDITIONS The environment of the selected plant cells should provide optimum conditions for the cells to express their genetic information concerning secondary metabolite formation, resulting in optimum levels of secondary product. Optimization of environmental conditions is a matter of trial and error because fundamental knowledge on the regulation of alkaloid formation is lacking. Conditions which have been reported to influence the productivity of the culture are, among others, the composition of the culture medium, light, temperature, bioreactor type, and aeration. Several methods have been used to determine systematically the influence of the different parameters on growth and production in a limited number of experiments (33-35). 1. Medium Composition Optimization of medium composition has led to the development of several media (20,36)that induce increased production of indole alkaloids. Some important factors concern the concentration and type of the carbon and nitrogen sources, the phosphate concentration, and the type and concentration of growth regulators. Induction and production media are used in a two-stage process: in the first stage biomass is produced, and the second stage allows alkaloid production. Morris, however, selected conditions for a Catharanthus roseus culture in which high alkaloid accumulation could be combined with high biomass accumulation (37). The initial pH of culture media is generally adjusted to 5.5-6. So far, little is known about the influence of the initial pH of the culture medium on productivity. For Catharanthus roseus cultures contradictory results have been reported: no effect at an initial pH of 5.5, 6.0, or 6.5 (38) but higher productions at pH 5.5 (39) and at pH 7.0 (40), with the results seeming to be cell line specific. In cultures of Hyoscyamus muticus a 7-fold increase in alkaloid production was found on media with an initial pH of 3.5 (41).More alkaloid was released by Nicotiana rustica hairy roots when grown on media with lower initial pH values (i.e., pH 5-5.5); buffering of the media with 50 mM MES decreased growth and total alkaloid production (42). Increased alkaloid production induced by increased osmotic strength of the culture media was detected in Catharanthus roseus suspension cul-

1.

PLANT BIOTECHNOLOGY

13

tures (43,44) and hairy root cultures of Hyoscyamus muticus (45).Osmotic agents that have been used include mannitol(43), NaF (46),and NaCl(47). Increased osmotic stress inhibited cell division and increased the rate of alkaloid production (43). 2. Light Light was found to stimulate serpentine production in Catharanthus roseus cultures (48);an inhibitory effect on alkaloid production was observed in cultures of Cinchona ledgeriana (49),Nicotiana spp. (50,51),and Scopolia parviJEora (52). In a light-grown Agrobacterium tumefacienstransformed culture of C. ledgerianu, 5- to 10-fold lower levels of tryptophan decarboxylase and strictosidine synthase were detected as compared to dark-grown cultures (53). The induction of serpentine production in light-grown Catharanthus roseus cultures probably resulted from a combined effect of light and medium composition: the induction by light was highest in the presence of low concentrations 2,4-dichlorophenoxyacetic acid (2,4-D), phosphate, and mineral nitrogen (48). The influence of light on product formation has been recently discussed (54).In large-scale fermentations the enhancement of product formation by light will be difficult because of technical and economical constraints. 3. Temperature Culture temperature influences both growth rates and productivity. By lowering the culture temperature of C. roseus cells from 27 to 16"C, a strong reduction in growth rate and a strong increase in alkaloid content was observed (55); however, the alkaloid yield per liter medium was not changed substantially. At a culture temperature of 38°C the alkaloid content was strongly reduced. The influence of culture temperature on the growth and productivity of C. roseus cells was also studied by Morris (56,57). For both biomass and alkaloid production the yield curves showed sharp maxima. By changing the temperature the serpentine/ajmalicine ratio could be influenced. Owing to temperature differences in the climate chambers in winter and summer seasons, even seasonal variation might occur (5637). An increased culture temperature may stimulate excretion of products, for example, as has been found for a transformed root culture of Datura stramonium. A 7- to 10-fold higher content of atropine in the medium was obtained at a culture temperature of 30°C compared to a temperature of 25°C (58). 4. Cell Density Cell density affects productivity directly: the more producing cells per liter, the more product per liter. High cell densities require specific pre-

14

ROBERT VERPOORTE ET A L .

cautions with respect to oxygen and nutrient supply (59). By adjusting aeration (oxygen added to the aeration gas) and stirring, Coptis japonica cells were cultured at densities of up to 75 g/dm3 (dry weight) in a culture tank fitted with a hollow paddle-type stirrer (60). Large-scale cultivation of transformed root cultures need specific requirements for growth and harvesting; a 500-liter droplet (or mist-phase) reactor in which the roots are immobilized in a matrix has been developed (61).

5. Gas Composition Aeration of suspension cultures grown in bioreactors needs to be optimized with respect to dissolved oxygen and carbon dioxide levels. High aeration rates may lead to excessive (toxic) concentrations of dissolved oxygen, increased shear forces, and subcritical levels of key volatiles such as gaseous hormones (ethylene) and carbon dioxide (62). Carbon dioxide enrichment of the aeration mixture increased biomass yields (63,64).Van Gulik (65) has recently reviewed this subject in detail. In shake flasks the dissolved oxygen and carbon dioxide levels are strongly determined by the type of closure of the flask. A cotton wool plug is highly permeable. Two layers of aluminum foil rapidly result in oxygenlimited growth of the culture. Silicon foam stoppers combine a good and reproducible gas exchange with low evaporation (66), which allows growth-curve determinations in a single flask by measuring the loss of weight caused by dissimilation (67). 6. Shear Stress

Shear stress caused by impellers in stirred tank fermentors proved to be less detrimental to suspension cultured cells than was expected from first reports (68). Several cell lines were resistent to impeller speeds of 1000 rpm (66,69-72). The various aspects of large-scale plant cell culture have been reviewed recently (73) (see also below). C. ALKALOID STORAGE COMPARTMENTS In plants the site of alkaloid biosynthesis is often separated from the site of storage; for example, tropane alkaloids are produced in the root and stored in the leaves. This means that the alkaloids need to be excreted from the biosynthetically active cells and then transported to and taken up by the storage cells. In undifferentiated tissue like cells in suspension culture this transport mechanism is likely to be seriously affected; this might result in low productivity. Uptake of alkaloids by isolated vacuoles and transport mechanisms over membranes have been studied in detail. Carrier-mediated active transport

1.

PLANT BIOTECHNOLOGY

15

over membranes (and thus a specific uptake) has been postulated for indole alkaloids (74), isoquinoline alkaloids ( 7 3 , quinolizidine alkaloids (76), and pyrrolizidine alkaloids (77). On the other hand, a passive transport mechanism, in which the neutral alkaloids freely diffuse through the membrane and the protonated alkaloids are stored in the acidic vacuole (ion-trap mechanism), has been advocated for indole alkaloids (78-81), quinoline alkaloids (82),and nicotine (83)in cells of C. roseus, Cinchona ledgerianu, and Acer pseudoplantanus, respectively. Ajmalicine diffuses across the tonoplast, driven by the pH gradient between cytosol and vacuole. It is subsequently trapped inside the vacuole in the form of serpentine, which is formed via oxidation of ajmalicine by the basic vacuolar peroxidases (81). Little is known about transport in the opposite direction, that is, excretion. For the ion-trap mechanism the pH difference over the membrane is the driving force for transport. A changed pH gradient caused by, for example, a low extracellular pH, might thus cause excretion. Excretion of alkaloids at low pH has been reported for Nicotiana, Cinchona (42), and Catharanthus cultures (84). Caffeine, an alkaloid with few basic properties, proved to be freely diffusible between cells and medium of a Coffea suspension culture (85). A cell line of Thalictrum minus was reported to excrete large amounts of berberine, a quaternary alkaloid (86); the spontaneous excretion could be inhibited by ATPase inhibitors, indicating an active (energy-requiring) transport mechanism. In some cases nature can be helped by release-promoting techniques such as immobilization, permeabilization, pH cycling ( 8 4 , electroporation (87), and elicitation (see below). Storage capacity in a cell culture is available inside the cells (e.g., vacuoles) or in the extracellular compartment. The storage facilities of the latter can be remarkedly improved by the addition of liquid organic phases, resins, or other sorbents to the medium. Addition of XAD-7 to Catharanthus roseus cultures resulted in increased yields of indole alkaloids (88). Addition of a dimethylsiloxane polymer to Eschscholtzia californica cultures improved yields of benzophenanthridine alkaloids (89), and increased yields of nicotine and anabasine were obtained by adding of XAD-2 and XAD-4 resins to Nicotiana transformed roots (90,91). The internal storage capacity is limited by the number of storage cells. Excreted alkaloids which are dissolved in the medium are exposed to catalytic activities in the medium (e.g., peroxidases) and can thus be degraded; as was demonstrated for quinolizidine alkaloids in lupine cell cultures (92). A role for peroxidases in product degradation has been recently demonstrated for indole alkaloids produced by suspension cultures of Tabernaemontana. Reduction of peroxidase activity by removal of Ca2+ ions from the medium resulted in the formation of the alkaloid

16

ROBERT VERPOORTE ET AL.

apparicine, which is not isolated from cultures with normal Ca2+ concentrations in the medium and thus high peroxidase activity. Apparicine is rapidly degraded after exposure to peroxidases (93). D. ELICITATION Elicitors are compounds which are able to induce the biosynthesis of phytoalexins or, in general, the biosynthesis of stress metabolites. Various elicitor preparations have been used, ranging from homogenates of microorganisms to metal ions. Homogenates of microorganisms may contain actual elicitor molecules, such as oligosaccharides or fatty acid derivatives. Other elicitors, like cellulase, metal ions, and UV light, release endogenous elicitors from the plant cell wall. The microbial and endogeneous elicitors are able to bind (largely unknown) receptors. Following receptor binding a set reactions is induced, in which ethylene, CAMP, and Ca2+play a role and which eventually leads to de nouo biosynthesis of the phytoalexins. This subject has been recently reviewed (94). There are only a limited number of alkaloids whose production can be induced by elicitors; the effects are cell line specific and often transient. Elicitors have been found for the induction of biosynthesis of, for example, indole alkaloids [ajmalicine in C. roseus cultures (95)], benzophenanthridine alkaloids [sanguinarine in Papaver cultures (96)], acridone and furanoquinoline alkaloids [in Ruta gruueolens cultures (97)], and protoberberine alkaloids [berberine in Thalictrum rugosum cultures (9691. When the alkaloids are excreted in the medium after elicitation, the biomass can be recycled and reelicitated. Successful reelicitation procedures have been developed for the production of sanguinarine and ajmalicine in Papaver and Catharanthus cell cultures, respectively (99). E. FEEDING OF PRECURSORS AND BIOCONVERSIONS Production can be increased by addition of precursors to the culture media, in which cases the precursors are not metabolized in the medium and, after uptake, appear in the right compartment of the plant cell. For C. roseus cultures, for example, it was found that increased indole alkaloid production was obtained after feeding with L-tryptophan, tryptamine, secologanin, loganin, loganic acid, or shikimic acid (20). Cell cultures have also been used for biotransformations, for example, the conversion of (-)-codeinone to (-)-codeine in Papauer sornniferum cultures (ZOO). For the tropane alkaloids a large number of precursor feeding and biotransformation studies with cultures of various solanaceous plants have been performed (see below).

1. PLANT BIOTECHNOLOGY

17

Bioconversion rates can be optimized by using immobilized cells, cellfree preparations, or immobilized (purified) enzymes. Furuya el al. (101) reported the reduction of codeinone to be more efficient with immobilized P. somniferum cells than with suspended cells. Strictosidine could be produced in large quantities using immobilized strictosidine synthase (102). The potential for this use of plant cell cultures is enormous; however, the number of successful applications is still limited.

F. IMMOBILIZATION Fixation of plant cells in a matrix of, for example, polyurethane foam or entrapment of the cells in calcium alginate beads provides an artificial surrounding for the cells, which protects them from hydrodynamic stress. high cell densities inside the matrix also allow cell to cell contact and communication. Inside the immobilized matrix nutrient and product gradients may occur. Furthermore, immobilized biomass is easily separated from the medium, which is useful in production and biotransformation systems. Immobilization of plant cells has been reviewed (103,104).

Large-scale immobilized alkaloid production systems have been described for C. roseus and Thalictrum rugosum, using glass fiber mats (105). Cinchona pubescens cells were efficiently immobilized in a semirigid matrix of polyurethane foam (106,107). Polyurethane foam was also used for the immobilization of P. somniferum cells; the cells were used for the biotransformation of codeinone to codeine (108). Cells of the same species were surface immobilized on a fabric of loosely woven polyester fibers (72). By elicitation and product adsorption on a hydrophobic resin, sanguinarine yields of 130 mg/g dry weight were obtained. Immobilization of Coffeu cells in calcium alginate resulted in a 13-fold increase of purine alkaloid production (109,110). G. PERMEABILIZATION

Efficient downstream processing of products is facilitated when the product can be recovered from the medium. Besides by elicitation and immobilization, excretion of the product can be obtained by permeabilization of the cells. Various strategies have been used: chemical permeabilization, electrical permeabilization, ultrasonic permeabilization, and iontophoretic permeabilization (111). Chemical permeabilization comprises the use of, among others, organic solvents as DMSO and chloroform and surface active chemicals (as Triton X-100).Results with the various tech-

18

ROBERT VERPOORTE E T AL.

niques used are always achieved at the cost of cell viability, and this might hamper further applications (111). H. DIFFERENTIATION AND CULTURE TYPE The formation in tissue cultures of several types of indole alkaloids was shown to be inseparably connected with morphological differentiation of the cells. For Catharanthus, a time course study has been made of the formation of alkaloids in seedlings, during the first stages of development (112). It was demonstrated that the formation of vindoline, and thus the synthesis of vinblastine, was connected to morphological differentiation. This explains why C . roseus suspension cultures do not produce vinblastine. Recently, a similar experiment was performed with Cinchona seedlings (113). In Cinchona seedlings alkaloid formation is abundantly expressed, but in suspension cultures productivity is very low, if any. Also, the formation of Zboga indole alkaloids in Tabernaernontana tissue cultures proved to be dependent on morphological differentiation (114). On the other hand, cell cultures are capable of producing alkaloids which had not yet been detected in the plant. In a recent review it was reported that a total of 85 novel compounds, including 23 alkaloids, have so far been isolated from 30 different plant cultures (17). Propagation of differentiated tissues as root and shoot cultures offer an alternative when the desired product is not formed in suspension cultured cells. In shoot cultures of C . roseus up to 2.6 pg of 3',4'-anhydrovinblastine per gram fresh weight was detected (115); shoot organ cultures of Cinchona ledgerianu produced 3.5 mg of alkaloids (quinine and quinidine) per gram tissue (116). Root cultures of this species were also able to produce these alkaloids; the productivity was increased 5 times by feeding tryptophan, a precursor of the alkaloids, to the culture (117). Alkaloids were also detected in root cultures of Hyoscyarnus niger (118). For most alkaloid-producing plants hairy root cultures, obtained via infection of the plant material with Agrobacteriurn rhizogenes, have been initiated: Hyoscyarnus, Datura, Atropa, Nicotiana, Catharanthus, Cinchona, and Peganurn. In general the alkaloid contents found in hairy roots are similar to those found in normal plant roots. An interesting aspect of hairy root cultures is that with A. rhizogenes other new genes can also be introduced, for example, genes connected with secondary metabolite production. Because of their rapid growth and the potential of genetic engineering, hairy roots may become superior producers compared to the plant. Hairy roots, or transformed cells, have been shown to be less sensitive to optimization procedures such as medium optimization (26) and elicitation (119).

1.

PLANT BIOTECHNOLOGY

19

I. GENETIC APPROACHES AND GENETIC MODIFICATION Several techniques are now available to change the genetic information in plant cells: transformation with Agrobacterium spp., direct DNA injection, and protoplast fusion. Novel techniques in molecular biology have found rapid application in plant cell biotechnology, as, for example, the use of antisense DNA (9,120,121). Knowledge concerning the genes involved in alkaloid biosynthesis can be exploited in several ways to obtain higher yields. Rate-limiting steps in the biosynthesis may be overcome by increasing the concentration of the enzyme. For this approach knowledge of the rate-limiting steps and characterization of the enzymes are prerequisites. Recently the number of characterized enzymes involved in the biosynthesis of alkaloids has increased exponentially. The pathways leading to berberine (121) and ajmaline (122,123) have been fully characterized. Substantial progress has been made with scopolamine biosynthesis (124-126) and early steps in indole alkaloid biosynthesis (6-9,127-130). After purification of an enzyme one is able to identify the gene encoding for this protein, which opens ways to genetic modification. Genes for various enzymes have been cloned, and several transgenic organisms have been obtained, for example, an E. coli strain producing strictosidine synthase (7) and tobacco plants containing tryptophan decarboxylase activity (9). In some cases microbial genes were also used for manipulation, for example, lysine decarboxylase (131), ornithine decarboxylase (132), and cytochrome P-450 (133). Further genetic strategies may include inhibition of competitive pathways which might lead to increased availability of precursors for alkaloid biosynthesis, or the opposite, the creation of a new branch on an existing pathway. Both strategies can be envisioned for both cell cultures and plants. An interesting option is introducing certain steps of a secondary pathway from one plant to another, with the aim to have the recipient plant produce a certain desired compound. This could, for example, lead to plants which could be harvested every year, instead of after 5-10 years. Introduction of the genes responsible for quinoline alkaloid biosynthesis from Cinchona into Catharanthus could be such an example.

J. COMBINATION OF TREATMENTS: TOWARD HIGHPRODUCTIVITY In the previous sections various treatments have been discussed which might lead to increased product yields. Many of the strategies have only limited value: they only can be used for specific cell lines, the effect is transient, or it causes cell death. At the moment these strategies are,

20

ROBERT VERPOORTE ET A L .

however, the only tools available to manipulate productivity. Most of the epigenetic treatments were reported for the first time more than a decade ago. At present much attention is paid to combined strategies, for example, elicitation of alginate-immobilized Catharanthus roseus cells led to a 45-fold increase of ajmalicine production (90 mg/liter), which was adsorbed from the medium by the resin XAD-7 (134). Combined treatments were also used for P. somniferum cultures (72), where immobilization, elicitation, and product adsorption have lead to high productivity of sanguinarine . For further development of an (economical interesting) production system one has to obtain a stable, with respect to production capacity and growth, cell line, which responds reproducibly to the selected treatments. So far few cell lines have been reported to be stable, but hairy root cultures in particular have a major advantage here (135). Many of the high producing cell lines obtained by various screening and selection procedures proved to be unstable: after a few months of subculture productivity returned to the same level as the wild-type culture. If stability cannot be obtained, a reliable backup system has to be developed, for example, by means of cryopreservation or other storage techniques. For a stable cell line growth and production can be optimized by changes in medium composition. When the product yield is still low even with medium modifications, improvement of product yields by a genetic strategy is the most rational, despite the fact that this may be a long and difficult task. An increase in concentration of a bottleneck enzyme will not necessarily lead to higher product levels as one has to increase the total substrate flow through the whole pathway. Elicitation or induction media might be of help for this. Furthermore storage facilities (and transport mechanisms) should be present in abundance and in such a form that the products are protected against catabolic processes. The latter aspect especially needs more attention as the major research effort is directed to aspects of biosynthesis. Alternative processes can consist of the production of precursors, on which the final steps are performed either by isolated (immobilized) enzymes or by chemical synthesis, or, alternatively, by bioconversion of readily available synthetic precursors with plant cells or isolated enzymes (see Section 111).

111. Large-Scale Production

The number of alkaloids which have been produced on an industrial scale through new biotechnological strategies is still rather small. There are three interrelated categories of problems hampering the development

1.

PLANT BIOTECHNOLOGY

21

of industrial plant biotechnology : biological problems, economical problems, and technological problems. Below we briefly discuss some aspects of these problems. Biological Problems Although there has been a huge effort in investigating plant cell cultures, our fundamental knowledge still is restricted. Essential problems with respect to industrial production of alkaloids are the following: biochemical pathways and their regulation are only partly known; the effects of environmental conditions on growth and production need more investigation; and productivity of selected or genetically modified plant material might be unstable. In section I1 these problemshave already been discussed as well as the strategies used to solve them. Economical Problems By conducting a feasibility study the bottlenecks in the economics of production processes employing plant cells in suspension culture can be assessed. Data from the few studies published on this subject have shown the need for further research. In general the productivity of plant cell cultures per amount of reactor volume per unit time has to be increased considerably in order to make most processes economically feasible. In this section these aspects are discussed in more detail. Technological Problems A general scheme for the development of an industrial process for alkaloid production is depicted in Fig. 1. On the basis of both fundamental research and feasibility studies the decision can be made whether an industrial production process is achievable. For the design of the process (production volume, process type, bioreactor size and type) detailed knowledge of both the kinetics of growth and product formation and physical properties (rheology, shear sensitivity) is essential. The design should be developed in interaction with the downstream processing possibilities. After a process design has been made, the behavior of the process on a large scale can be predicted by scale-up studies. On the basis of these studies a final process design can be made. Problems in the following fields have to be anticipated. Reliable data on the kinetics of growth and product formation as well as data on the physical properties of plant cell cultures are rarely available, especially on a large scale. Determination of the data are difficult and time consuming.

A. ECONOMIC FEASIBILITY A number of cost-price estimations for products of industrial plant cell biotechnology have been published. The main aim of such studies is, of

22

ROBERT VERPOORTE ET A L .

Fundaments

FIG. 1. General design strategy of a biotechnological process.

course, analysis of the economic feasibility of an industrial process, although they may also reveal specific bottlenecks hampering commercialization. Goldstein et al. (141) published a study concerning the production of compounds by plant cell biotechnology . They developed a comprehensive process design and calculated the cost-price of products on the basis of this design. In their study they used growth and production parameters reflecting the state of the art in 1979, as well as more optimistic figures. They assumed production volumes varying from 10 to 1000 tondyear. In a Dutch report (2) the results from the Goldstein study are compared with data form the natural product market (Fig. 2). It is obvious from Fig. 2 that only a few products can be considered as candidates for industrial production; moreover, the first product of plant cell biotechnology, shikonin, is either hardly profitable or the data used deviate from the real situation. Fowler and Stepan-Sarkissian (142) estimated total costs of about U.S. $35,00O/kgfor the production of 375 kg/year of serpentine. Drapeau et a f . (143) reported a cost estimate for commercial-scale production of ajmalicine-rich Catharanthus roseus biomass. At the current state of technology the cost would be approximately U.S. $3215/kg ajmalicine. They suggest the coproduction of catharanthine could relieve the cost of ajmalicine production.

1.

PLANT BIOTECHNOLOGY

23

The studies mentioned above are based on the classic biotechnological approach of producing a secondary metabolite, namely, a two-stage process. During the first stage an amount of biomass is produced under conditions favoring growth, and during the second stage the desired secondary metabolite is formed under conditions favoring product formation. In most cases the product is accumulated by the cells and therefore has to be extracted from the biomass. An alternative approach is the use of more advanced technology such as, for example, reuse of biomass. Reuse of biomass can be accomplished either with free cell systems or with immobilized cells. For this approach it is essential that the product be excreted into the culture medium. Van Gulik et al. (I&), in a feasibility study on the production of ajmalicine, compared the classic two-stage process with a method where the produced biomass is used for longer periods of time. The parameters used in this study are given in Table IV. The values for both growth and production parameters represent reasonable figures from the literature available at the time the calculation was performed. The lower biomass concentration in the case of production with release by permeabilization was supported by the fact that a satisfactory recovery of ajmalicine is only feasible when the volume of the biomass does not exceed 30% of the reactor volume. A higher biomass volume results in a larger amount of ajmalicine remaining inside the cells, which results in a lower recovery. For calculation of the fermentation equipment needed, the authors started with the yearly ajmalicine production required and then calculated backward to the total reactor volume needed for the production stage, followed by calculation of the reactor volume needed to produce the inoculum for the ajmalicine production reactors. The investment costs needed for the process were roughly estimated by multiplying the equipment costs with the so-called Lang factor, a factor commonly used for cost calculations in fermentation processes (145).The annual costs were estimated by depreciation of capital equipment over a 10-year period. The medium costs were calculated with glucose as the carbon source. The bulk of the energy needed for the process will be consumed by the compressors and agitators in the reactors (energy costs needed for sterilization were included in the medium costs). A summary of the results of these calculations are given in Table IV. Costs of downstream processing and labor are not included in the calculation. The results show clearly that the stage of product release is a weak point in the current process. This is caused by the necessity to work with a lower final biomass concentration to achieve satisfactory product release. The advantage of reusing the biomass is marginal because the price-determining factor is not biomass production

24

ROBERT VERPOORTE ET A L .

GOLD

0

ILCHICINE OlGOXlN

0

;IKONIN

10'

RESERPINE

0

PILOCARPINE

0

10

PYRETHRINS ATROPINE

0

OPIUN

0

10

I

I

I

1

10

100

,DIOSGENIN

1000

Market volume ( t o n / y e a r )

FIG. 2. Relationship between estimated market price and market volume of a number of different plant products. The upper line represents the relation between the cost-price of a product produced by means of plant cell biotechnology and yearly production, at the current state of the art. The lower line is calculated from more optimistic figures and therefore represents a future situation. Products situated between the lines might be interesting goals for plant cell biotechnology in the near future.

1.

25

PLANT BIOTECHNOLOGY

TABLE I11 PARAMETERS USEDFOR ESTIMATION OF COSTSOF AJMALICINEPRODUCTION BY CELL CULTURES OF Catharanthus roseus Design basis Yearly production of ajmalicine Product loss during recovery and purification Effective yearly operation period Growth parameters Specific growth rate Initial biomass dry weight concentration Inoculation ratio Dry weight yield from glucose Maintenance energy requirement Maximum oxygen uptake rate Single use of biomass Final biomass dry weight concentration Final biomass fresh weight concentration Final ajmalicine content of the biomass after a 21-day production period Repeated use of biomass Specific productivity in case of spontaneous product release Final ajmalicine content of the biomass in case of forced release

3000 kg 20% 300 days 0.029 hr-’ 2.5 kg/m3 1:7 0.61 kg/kg 0.0066 kg/kg . hr 0.0154 kmol/m3 . hr 40 kg/m3 320 kg/m3 0.009 kg/ kg 2.36 x W5kg/kg . hr 0.009 kg/kg

but product formation. For the same reason, increasing the productivity increases the economy of the process substantially.

B. TECHNOLOGICAL ASPECTS 1. Growth and Production Kinetics

To design a production process to obtain alkaloids from plant cell cultures with a promising productivity on a laboratory scale, detailed knowledge of the kinetics of growth and product formation of the cell culture is essential. In order to obtain a quantitative description of the behavior of the cell culture as a function of the external conditions, this knowledge is embedded in a mathematical model. On the basis of the model a process design can be made after which the economic feasibility of the process can be assessed. After implementation of the process the model is subsequently used in process control and optimization. The number of publications dealing with quantitative research on the kinetics of growth and product formation of cultured plant cells has been very limited. For the scarce kinetic research that has been reported batch cultures were used, which were mostly grown

26

ROBERT VERPOORTE ET A L .

TABLE IV BREAKDOWN OF COSTSOF DIFFERENT PROCESSES FOR LARGE-SCALE PRODUCTION OF AJMALICINE BY CELLCULTURES OF Catharanthus roseus

Product extraction Gross reactor volume for product formation (m3) Largest reactor volume for biomass formation (m3) Volume of storage tanks

6 x 145

37

Product extraction (lox increased productivity) 6 X 15

Natural product release 6

X

250

Product release by permeabilization with DMSO 6

335

10

82

3.8

X

3 x 1.2

7 x 150

7 X 150

1 . 1 x lo6

5.4 x 106

5.4 x lo6

0.75 x lo6

0.1 x lo6

3.3

x lo6

0.75 x lo6

0.08 x lo6

1.5

X

4.5 x lo6 1500

3 x 10

(m3) Depreciation costs (U .S .$/year) Medium costs (U.S.$/ year) Energy costs (U.S.$/ year) Total costs (U.S.$/year) Costs (U.S.$/kg ajmalicine)

3 x 106

5.1

X

lo6"

lo6

1.5 x lo6

1.3 x lo6

10.2 x lo6

12 x lo6

430

3400

4000

Including DSMO.

in shake flasks. The use of batch cultures has the advantage that the culture system is relatively simple, while the time needed to conduct an experiment is relatively short. An important disadvantage is that in a batch culture almost every parameter changes in time, thus making it very difficult and sometimes even impossible to draw the right conclusions from an experiment. An alternative that does not suffer these disadvantages is the chemostat culture. In a chemostat the cells can be grown at a constant growth rate at constant concentrations of the various nutrients of the growth medium, thus providing the opportunity to change only one variable at a time. It should be stressed that the chemostat culture is not considered to be a system for alkaloid production but rather a tool to obtain reliable kinetic information. In a future production process the use of batch or batch-fed culture is more likely, owing to the greater flexibility of such a process, the lower complexity of the culture system, and, as a consequence, lower equipment costs. Aspects of the various culture types are discussed in more detail below.

1.

27

PLANT BIOTECHNOLOGY

a. Mathematical Modeling. The character of the mathematical description of a physical, chemical, biological, economical, or social process depends on the character of the process, the available knowledge, and the intended use of the model. Models can be subdivided depending on our knowledge about the process (black box, gray box models), the number of units involved (stochastic or deterministic models), the consideration of particles (continuum or corpuscular models), and the eventual subdivision of the units of the total process (structured or unstructured models). For detailed information on modeling one should consult the specialized literature (e.g., see Refs. 146-148). Although several types of models have been explored in the description of biological systems, the unstructured black box continuum models based on a linear equation for substrate consumption are still most frequently used for the description of fermentation processes. In these models the growth rate of the cells generally is assumed to be a function of the external concentration of the growth-limiting substrate C, according to Monod kinetics : dC,/dt =pmaxx C,/(C,

+ K,)

x C,

The conversion rate of the growth-limiting substrate is divided into a growth rate-dependent term and a growth rate-independent term according to the following linear equation: dC,/dt

=

-(l/Y,,

x dC,/dt

+ m,

x C,)

where C , is the biomass concentration (in kg/m3), t the time (sec), pmax the maximum specific growth rate (l/sec), K , the saturation constant on substrate (kg/m3), C , the substrate concentration (kg/m3), YSxthe net biomass yield on substrate (kg/kg), and m, the maintenance coefficient (l/sec). The growth rate-independent, or maintenance, energy needed is assumed to be supplied by combustion of substrate (149),biomass (150),or a combination of both (151). These models can be extended for product formation. In population biology deterministic or stochastic corpuscular models are used, describing the behavior of a population of single cells or organisms. If cell division is considered a random process and the distribution of cycle times is assumed to be described by a normal distribution, population size models of the form dN(t)/dt = p x N(t)

(3)

where N ( t ) denotes the number of cells at time t , are formulated. Reviews of population size models are given by Eisen (152) and Bertuzzi and Gandolfi (153). Kato and Nagai (154) described the growth of their batch cultures of

28

ROBERT VERPOORTE ET A L .

Nicotiana tabacum with a linear substrate consumption model [Eqs. (1) and (2)]. The model parameters obtained were compared with values reported for Penicillium chrysogenum, Escherichia coli, and Saccharomyces cereuisiae. They concluded that the net yield of N . tabacum biomass on substrate was relatively high whereas the maintenance energy requirement was relatively low compared to the reported values. These observations were explained by the fact that tobacco cells contain large amounts of stored carbohydrates (e.g., starch). Because the energy required for synthesis and maintenance of storage carbohydrates is relatively low this might be the cause of the observed high yield and low maintenance coefficients. Pareilleux and Chaubet (155,156) studied batch suspension cultures of apple cells and Medicago satiua cells, reporting comparable high yield and low maintenance coefficients. Bailey and Nicholson (157) employed in principle the same model structure. However, in order to describe also the changing fresh weight/dry weight ratio, product formation, and cell death and lysis, they extended the model. Two extra variables were added to allow the prediction of the extension phase and a more accurate prediction of the culture death phase caused by shear stress. Another two variables were introduced to describe product formation. In a later paper (158) the authors extended the model further to describe the influence of temperature. With this model an optimal temperature control strategy was predicted. Van Gulik (65,159) performed glucose-limited chemostat experiments with the aim of examining whether the growth of plant cells in suspension cultures could be described satisfactorily with the linear equation for substrate consumption combined with Monod kinetics [Eqs. (1) and (2)]. Cell suspension cultures of two different species were used, Catharanthus roseus and Nicotiana tabacum. For both species the specific substrate consumption rate under steady-state conditions showed a reasonable fit to the linear equation. To give an example, the C. roseus data are shown in Fig. 3. It was reported that the model was of limited value under nonsteady-state conditions. With the model only a poor description of the relationships between growth, substrate uptake, and specific respiration in batch culture could be obtained (Fig. 4).This was partly caused by the fact that during growth in batch culture large amounts of storage carbohydrates are often accumulated. In the case of C. roseus the total amount of stored carbohydrates reached a maximum of nearly 40% of dry biomass concentration, shortly before the depletion of glucose from the medium (Fig. 5 ) . In order to describe a varying biomass composition, however, a metabolically structured model is needed. De Gunst et al. (160-162) used the stochastic corpuscular approach to

1.

29

PLANT BIOTECHNOLOGY

0.05 I A

r

- 0.04

8

; I

0

0.03

E

0.02

Ll

0

v

v,

0.01

0-

0.00 0.00

0.01

0.02

FIG. 3. Relationship between specific glucose uptake rate (4s) and dilution rate (0)in glucose-limited chemostat cultures of Catharanthus roseus.

describe the growth of plant cells in suspension culture. In the proposed model the total cell population is assumed to consist of two subpopulations: dividing cells and nondividing, differentiating cells. The number of dividing cells is assumed to depend on the hormone concentration in the medium, whereas the duration of the cell cycle is a function of the substrate concentration. In Fig. 6 model predictions of the growth of Nicotiana tabacum cells, with respect to dividing, nondividing, and total cells, are shown. The authors compared only the predicted total cell number with experimental data. Although this model needs more experimental validation and the character of the controlling factors (substrate and hormone levels) is not proved, it has some interesting features. First, it corresponds with the observation that cell cultures may consist of different cell types. Second, there are several examples showing that production of alkaloids is a capacity of differentiated cells. Therefore, models describing the development of differentiated cells in a cell culture may be of great importance for the design of alkaloid production processes. b. Conclusions. When black box models, in which biomass is considered as one compartment with a constant composition, do not provide a satisfactory description of the process, one has to develop more sophisticated models. In general this will result in a subdivision of the biomass into more than one compartment. However, increasing the complexity of a model results in an increasing amount of parameters to be determined. The applicability of highly complicated structured models might therefore be doubted. In practice a compromise should be obtained; the complexity of

30

ROBERT VERPOORTE E T A L .

-

0.025

1.00

=5

f

0.80

3 0

0.60

~

-

‘1. 0.40

; I

:

0.020

E

0.010

‘1 . 0

0

0 u

B

h

0)

0.015

v

g

e4

0.20

0.005 ‘ 9

*.I9

0.000

0.00 0

120

360

240

0

120

240

360

Time (h)

Time (h)

0.025 I h

L:

-

z I

0.020

o

0.015

E

0.010

‘1. v

hl

I

C

0.005

0-

0.000

0

120

240

360

Time (h)

FIG. 4. Growth of Catharanthus roseus cells in batch culture. (A) Concentrations of glucose (squares) and biomass (circles), (B) oxygen consumption, and (C) carbon dioxide production are given as a function of culture time. Vertical bars represent the standard deviation of the mean. Solid curves represent the model predictions.

the model should be as low as possible while still providing acceptable accuracy. Furthermore, the structure of the model should allow experimental validation. In order to develop structured models for the description of growth and product formation of cultured plant cells, knowledge of the pathways of primary and secondary metabolism is essential. This knowledge is still limited, however, especially with respect to secondary metabolism. Corpuscular models are not common in process development. It is interesting, however, to realize that corpuscular models of the type developed by de Gunst er al. (160-162) do offer the possibility of describing cell differentiation.

1.

31

PLANT BIOTECHNOLOGY

c

1

.-0,

-.-.-

Q)

-0-

40

3 x

Starch Sucrose

I

73

30

-0-

v) v)

0

E

20

cc

10

0 .a

Glucose Fructose

-+-

Total

0

M

0 0

72

144

288

216

360

T i m e (h)

FIG. 5. Changes in the amounts of intracellular carbohydrates in Catharanthus roseus cells

1,

,*..............

-**

i0

do

\

I

60

I

80

I

-

100 Time (hours)

I

120

I

140

I

160

FIG. 6. Experimental data from the growth of a batch culture of Nicotiana tabacum cells (circles). The solid line represents the prediction of the total number of cells obtained by the corpuscular growth model, the dashed line represents the predicted number of dividing cells, and the dotted line represents the predicted number of nondividing cells.

32

ROBERT VERPOORTE ET A L .

2. Process Design and Bioreactor Type a. Process Design. Whereas a plant is a fully organized structure containing specialized cells forming different types of tissue, a cell suspension culture reflects the totally unorganized situation. Between these two extremes there are several other ways to cultivate plant cells. The commonly used methods to grow plant cells in culture are as follows (with an increasing degree of organization):

Cell suspension cultures Immobilized cells Surface immobilization (biofilm) Gel-entrapped cells Callus cultures Organ cultures Roots Shoots Hairy roots Cell suspension cultures offer some advantages over the more highly organized structures. In a cell suspension culture the transport of nutrients, oxygen, precursors, and/or elicitors to the cells is not hampered by the limited diffusion which may occur in relatively large tissue structures. Another important advantage is the fact that plant cell suspension cultures can be treated almost exactly like cultures of microorganisms. This opens the possibility of using, to a large extent, existing knowledge, equipment, and technology. An important disadvantage which is frequently mentioned in the literature is the low degree of cell differentiation in suspension cultures of plant cells. As product formation in plants largely occurs in differentiated tissue, it seems reasonable that the undifferentiated state might not favor the formation of secondary products. In some cases, for example, the production of ajmalicine in cultures of Catharanthus roseus, it is indeed shown that the product, originally formed in the roots of the plant, is better produced in hairy root cultures than in cell suspension cultures. The use of immobilized cells opens the possibility of keeping the produced biomass in the reactor and using it for prolonged periods of time, under the constraint that the product is excreted by the cells. A disadvantage is the possible occurrence of mass transport limitations inside the biomass beads or in the biofilm. Much of the technology to realize a large-scale process with immobilized cells has yet to be developed, and it might well turn out to be economically unfeasible in most cases.

1.

PLANT BIOTECHNOLOGY

33

Culturing organized tissue such as callus, root, or shoot cultures offers the advantage of some degree of cell differentiation, which is still relatively low in the case of callus cultures but high in the case of root or shoot cultures. The disadvantage in this case is also the necessity of developing new technology. Other important constraints determining the design of the process are the relation between growth and product formation (growthassociated or non-growth-associated), the potential of the plant material to be empolyed for de nouo synthesis or for biotransformation, and the occurrence of product release or accumulation. Fermentation processes can be carried out in batch, fed-batch, or continuous mode. These different process types can be roughly characterized as follows. The batch culture is the simplest process. Nutrients and inoculum are brought into the reactor, and after a period of time (which may be estimated through an on- or off-line measurement) the reactor contents are harvested and processed. Some characteristics of batch culture are limited process control, productivity loss as a result of cleaning and sterilization after each fermentation run, and large flexibility in the process. In fedbatch culture the nutrients are supplied to the reactor during the process. Compared to a batch culture, a fed-batch culture has the advantage of an additional control parameter, namely, nutrient feed rate. In a continuous culture a continuous supply of nutrients and a continuous harvest of reactor contents (medium or medium plus biomass) can be achieved. Some characteristics are better process control, more equipment needed, greater equipment costs, contamination of the process has greater consequences, and relatively low flexibility. Continuous culture without cell retention is only employable with growing biomass (nongrowing biomass will be washed out with the effluent flow). For alkaloid production this type of continuous culture is only suitable if alkaloid formation is growth associated, which greatly restricts application. A further constraint is that large cell aggregates or hairy roots are not easily transported, unless the size of the piping is large compared to the aggregate size. A continuous process with cell retention can be applied when product release can be achieved. Immobilization of the cells, either naturally or artificially, might be an advantage. In particular, biotransformations could be carried out in the continuous mode with biomass retention. From a theoretical point of view the continuous process is often considered as the most attractive choice. In practice, however, the number of continuous biotechnological processes is still very limited. Most processes appear to be performed in the batch or batch-fed mode. The main reasons are the greater flexibility of a batch or a fed-batch process, the lower complexity and thus lower risk of failures, and lower equipment costs.

34

ROBERT VERPOORTE ET A L . TABLE V LARGESCALE PLANTCELLCULTURES

Volume

(m3) 0.134 1.5 20 0.15 5 60

Type

Species

Ref.

Tank Tank, mixing by aeration Stirred Tank Stirred Tank Stirred Tank Stirred Tank

Various Nicotiana tabacum Nicotiana tabacum Lithospermum erythrorhizon Catharanthus roseus Various

136 137 138 59 139 140

b. Bioreactors. An important part of the equipment needed in a largescale production process using plant cell cultures is the bioreactor. There are a great ‘variety of bioreactors for laboratory-scale studies reported in literature. At the industrial scale, however, the variety of applied types of bioreactors are mainly limited to stirred tank reactors, airlift systems, bubble columns, and fluid bed columns. In the fermentation industry the stirred tank reactor is standard equipment. Substantial experience has been gained with this type of reactor. Until recently most workers considered stirred tank reactors to be of limited use in plant cell biotechnology. It was observed that plant cells could not withstand the high shear forces caused by the impeller (165). Therefore, the airlift reactor, which is thought to provide a much friendlier environment for the fragile cells, received much more attention. However, foaming and oxygen transfer, especially at high biomass concentrations, can become a serious problem in airlift reactors (166,167). The fact that since as early as 1960 some successful plant cell fermentations in stirred tank reactors were performed (Table V), however, indicated that it might not be considered impossible. Recently Meijer (66,69) and Scragg and co-workers (70,71) studied the shear sensitivity of plant cell cultures. They found that many plant cell cultures appear to be shear tolerant. The differences between shear-sensitive and shear-tolerant cell lines could not be explained, although there was evidence that well-growing “healthy” cell lines became shear tolerant through regular subculturing. To meet specific needs, some special bioreactor designs for plant biotechnological processes have been described in the literature, including the rolling drum reactor (163),mist reactor (61), hairy root bioreactor (61), and polyurethane foam reactor (164). The lack of experience with respect to the scale-up possibilities provided by these reactors might be a problem. However, particularly in the case of hairy root and organ cultures, a special reactor design might be necessary.

1. PLANT BIOTECHNOLOGY

35

3. Downstream Processing An essential part of a production process for alkaloids is product recovery. The alkaloids have to be separated from the broth and purified. Application of alkaloids requires a high degree of purity. The high added value of these products, however, permits the use of advanced separation techniques. In the literature there are not many papers on downstream processing. Most studies are concerned with various aspects of regulating the productivity of plant cell cultures; if the productivity reaches an economically interesting level, downstream processing will be studied in direct relation with the development of a commercial process. Results of that research will rarely be published. In a discussion on downstream processing of alkaloids produced by plant cell biotechnology , two quite different cases can be distinguished, namely, product stored in the biomass and product excreted by the biomass. The first case is comparable with the classic production of alkaloids from plant material, although specific problems could arise from the character of the cellular biomass. In the second case a variety of advanced separation techniques could be used. A typical example from plant cell biotechnology is the forced release of alkaloids. In the following sections product recovery from biomass as well as product release and product recovery from spent media are discussed. a. Recovery of Alkaloids from Biomass. The problem of product recovery from biomass can be dealt with in two ways: (1) the biomass could be processed comparably to plant material in a classic alkaloid production process, and (2) the biomass could be fractionated, and, after separation of solids and liquid, the liquid phase could be processed comparably to spent medium containing excreted alkaloids. In classic alkaloid recovery the alkaloids are extracted from the biomass. In general, most alkaloids are basic, and this property is commonly used in most purification methods. One may distinguish three types of extrac tant s . Water-insoluble solvents. Most alkaloids are commonly present in the plant as organic salts. In order to solubilize the alkaloids the crude biomass extract is made basic by the addition of, for example, potassium hydroxide, potassium carbonate, or ammonia. At the resulting high pH the alkaloids are mainly present in their neutral form. The neutral alkaloids are then easily extracted from the aqueous phase with a water-insoluble organic solvent, for example, dichloromethane or chloroform. Water-soluble soluents. The alkaloids are extracted from the biomass with an alcohol, for instance, methanol, ethanol, or 2-propanol. Most

36

ROBERT VERPOORTE ET AL.

alkaloids, both salts and free bases, are readily soluble in alcoholic solvents. AcidiJied water. First the alkaloids are extracted from the biomass with acidified water. At a low pH most alkaloids are protonated and readily soluble in an aqueous solution. Subsequently the extract is made basic, and the neutral alkaloids are extracted from the aqueous phase with an organic solvent. Plant material, especially seeds and leaves, often contain large amounts of fatty compounds. These have to be removed before alkaloid extraction and is most frequently done with petroleum ether. Petroleum ether has the advantage that most alkaloids are not removed with this solvent. Another way to remove fatty compounds and other impurities is acidification of the aqueous extract followed by extraction with an organic solvent. The alkaloids remain in the aqueous phase while the fatty compounds are removed by the organic solvent. The choice of acid for acidification is important as several anions will result in the formation of organic solventsoluble ion pairs with the alkaloids (e.g., chloride and acetate). After extraction with organic solvent, the aqueous phase is made basic, and the alkaloids are extracted with an organic solvent. In literature very little is published on the extraction of alkaloids on an industrial scale. The few papers available were published between 1950 and 1970 and concern the isolation of alkaloids from whole plants or plant parts (168,169). The extraction of catharanthine and vinblastine from C. roseus leaves on a pilot plant scale is described by Atta-ur-Rahman et a f . (170). Svoboda developed a method for the extraction of ajmalicine, vinblastine, and vincristine which has been used by Eli Lilly & Co. (169,171173). Supercritical fluid extraction is a method which is used for the extraction of caffeine from coffee beans. This method also seems of interest for further studies of other alkaloids. b. Forced Release of Alkaloids. For several reasons in a plant cell biotechnological production the release of product from the biomass could be of interest for process design. During cell line selection, product release can be considered as a possible selection criterion. A striking example of this approach concerns the production of berberine by means of Thafictrum cell cultures that excreted almost all the alkaloid produced (see below(523)). Also, during medium optimization product release could be an aim. Therefore it is essential to determine the product of interest not only in the biomass but also in the medium. This might seem obvious, but it is not always done. If spontaneous release of the product is not accomplished, however, there still exist ways to force release of the product. Knowledge of the mechanisms involved in product storage are therefore

1.

PLANT BIOTECHNOLOGY

37

essential. However, studies on product routing are scarce, and the results seem contradictory (see discussion in Section 11). Product release brought about by lowering the pH of the culture medium was shown in case of Cinchona ledgeriana (42,82,174), Nicotiana rustica (42), and Catharanthus roseus (175). Other successful approaches to stimulate product release have also been reported. Several studies have been carried out on the stimulation of product excretion by extraction of the culture broth with an inert organic solvent (176-181). Adsorption of alkaloids from the culture broth with adsorbents, mostly XAD resins, also showed promising results. In several cases product release is stimulated (42,90,181),and in some cases product formation is also increased (88,181). However, growth and/or product formation can be influenced negatively. This might be caused by adsorption of essential medium components by the added adsorbent (42,182,183). Product release has also been reported after immobilization of the biomass (181). The mechanism behind this release, however, is not yet understood. The mechanism of product release by membrane permeabilization with organic solvents, on the other hand, is clear. Positive results with this method have been reported by Brodelius and Nilsson (184). Recently, however, Brodelius (111) concluded that regrowth of the biomass after different permeabilization procedures has not yet been possible. Reuse of biomass has been an important objective in this kind of research. It is not always clear which mechanism is responsible for product release stimulated by a certain chemical compound. Meijer (69) showed product release through the addition of polyethylene glycol. It is difficult to distinguish between the various mechanisms, such as osmotic stress, permeabilization, or extraction. c. Recovery of Alkaloids from the Medium. For the recovery of alkaloids from the medium one can consider two possibilities: (1) liquidliquid extraction, that is, the classic alkaloid extraction procedure using an immiscible organic solvent to extract the alkaloid from the alkalinized medium, and (2) solid-phase extraction, in which the alkaloid is concentrated on a solid phase, for example, ion-exchange resins, C18 reversedphase chromatography materials, or polymer-type resins like XAD. The latter method is of particular interest for continuous removal of the alkaloid from the medium (see above), but it also, in processing the medium, has the advantage of avoiding the need for large-scale liquid-liquid extractions. To our knowledge no data have been published on such large-scale medium extractions. Further studies, also in connection with the processing of alkaloids from plant material, on solid-phase extractions seem of

38

ROBERT VERPOORTE E T A L .

interest, as such methods could probably reduce the use of toxic organic solvents. 4. Scaling Up of Plant Cell Cultures The majority of studies concerning the production of alkaloids by plant cells in suspension cultures are performed with shake flasks. Actual production processes will proceed at a much larger scale, and consequently there is a scaling-up problem. There are no straightforward guidelines to solve this problem because there is an interaction of the various mechanisms involved, for example, stirring speed will have a positive effect on oxygen transfer but might have a negative shear effect on plant cells. Consequently compromises have to be made. In the next section theoretical approaches to this problem are reviewed and applied. a. Theory. Several techniques have been developed to attack the scale-up problems as reviewed by Oosterhuis (185) and Kossen and Oosterhuis (147). The techniques for scale-up methodology are summarized below.

1. Trial and error 2. Rules of thumb 3. Scale-down approachhegime analysis 4. Dimensional analysidregime analysis 5. Semifundamental methods 6. Fundamental methods Trial and error is exactly the approach to be avoided, because it implies expensive trials and errors at a large scale. Dimensional analysis, semifundamental methods, and fundamental methods are based on considerable and wide-ranging quantitative knowledge of the mechanisms involved in the process. In plant biotechnology this knowledge is partly lacking. Therefore, a combination of rules of thumb and the scale-down approach with regime analysis is the methodology preferred. This approach, reviewed by Sweere (186), is shown schematically in Fig. 7. First, regime analysis must &veal which mechanisms are rate limiting. Whether there is one ruling mechanism (pure regime), or more (mixed regime), and whether there may be a change in ruling mechanism going from the small to the large scale must be determined. Second, experiments on a laboratory scale, aimed at process optimization, should be designed under the same ruling mechanism. Third, in the small-scale experimental setup the effects of the ruling mechanism can be studied. Finally, if the experiments reveal measures for process optimization, they should be translated to the production scale.

39

1. PLANT BIOTECHNOLOGY Production scale \

\

Regime

Application \

\

A

---------_-V

Simulation

\

\

’

\

Optimization Modeling

\

Laboratory scale

FIG. 7. Scale-down procedure for a biotechnological process.

The scale-down approach can be applied both in the case of an existing process and for a new process. In the latter case a process design is made for the new process from available process data, experimental evidence, and rules of thumb. Regime analysis is applied to this process design. Based on the results a further small-scale research strategy is developed, and eventually changes are made in the process design. In the following paragraphs this approach will be applied to production of an alkaloid from plant cells cultured in a bioreactor. Regime analysis can be performed by comparison of characteristic parameters of the mechanisms involved in the process. Here the characteristic time concept will be used. The characteristic time is a measure for the rate of a mechanism. A fast mechanism has a short characteristic time. Other terms used are relaxation time, process time, or time constant. A time constant is formally only defined for first-order linear processes. Not all mechanisms involved in a plant cell production process are first order, therefore the term characteristic time is used. The characteristic time is defined as the ratio of a capacity and a flow; for example, the characteristic time for oxygen transfer to the liquid phase in a aerated bioreactor to,Lbecomes to,^ = ( C o , d m - Co,L)lkLa ( C O , G-~ CO,L) ~ = 11kLa

(5)

where CO,Gis the oxygen concentration in the gas phase (in mol/m3),CO,L the oxygen concentration in the liquid phase (mol/m3),m the Henry coefficient, kLa the volumetric oxygen transfer coefficient (llsec),

40

ROBERT VERPOORTE ET A L .

(C0,Glrn - Co,L) the oxygen capacity of a (C0,Glrn - C O , ~the ) oxygen flow to a solution.

solution,

and kLa

When inferring the mechanisms involved in a process one should realize that mechanisms with characteristic time magnitudes larger or smaller than the process time can be ignored. In a microbial fermentation, for example, metabolic reactions in the cell are too fast (characteristic times less than lop4sec) and microbial selection is too slow (more than lo6 sec) to influence the process.

b. Process Design. In the following a hypothetical process for the production of an alkaloid is studied. The main process parameters are briefly summarized in Table VI. The process comprises two stages: (1) in the growth phase biomass is grown in a fed-batch manner in a fermentor cascade, and (2) in the production phase (stationary phase) production medium with a high glucose concentration is fed into the fermentor to induce product formation. After 21 days a product concentration of 2.5% of dry weight is reached. Data are partly based on growth kinetics research on Catharanthus roseus (65), values adopted from literature, and reasonable assumptions. From the data in Table VI the total volume of the production phase can be calculated to be 150 m3. To diminish the risk of loss of production owing to contamination or equipment failure and to increase process flexibility, it TABLE VI PROCESS PARAMETERS Parameter Design basis Production Product loss during downstream processing Operation period Growth parameters Specific growth rate Doubling time Initial biomass dry weight concentration Inoculation ratio Biomass yield coefficient on glucose Maintenance coefficient on substrate Biomass yield coefficient on oxygen Maintenance coefficient on oxygen Maximum oxygen uptake rate Maximum substrate uptake rate Final biomass dry weight Production parameters Final product concentration Production period

Measure kg/year %

day slyear

Value 500 20 300

c,,/ce, C,,lC,,lhr C,,/mol mol/C,,lhr mol/m3/hr mol/m3/hr kg/m3

0.018 38.5 2.5 12.5 0.65 0.0074 2.1 0.0073 10.6 23.4 20

kgk days

0.025 21

hr-' hr kg/m3 %

41

1. PLANT BIOTECHNOLOGY TABLE VII DIMENSIONS OF INDUSTRIAL-SCALE FERMENTOR AND GEOMETRICALLY DOWN-SCALED LABORATORY FERMENTOR Scale

Gross volume (m3) Net volume (m3) Height (m) Diameter (m) Liquid height (ungassed) (m) Number of impellers Impeller diameter (m) Impeller blade width (m) Baffle diameter (m)

Industrial

Laboratory

25 20 5.44 2.42 4.35 3

0.005 0.004 0.316 0.141 0.253 3 0.045 0.009 0.013

0.77

0.16 0.22

is adequate to perform the production phase in, for example, six bioreactors of 25 m3 each. Therefore the growth phase has to be performed in six parallel three-stage fermentor cascades of 0.063, 1.25, and 25 m3. In Table VII the dimensions of such a 25-m3 bioreactor and a geometrically downscaled 5-liter fermenter are given. Assuming further the general process data given in Table VIII, the operating conditions can be calculated using a methodology for analyzing agitator performance and mass transfer in multiturbine production fermentors developed by Bader (187). The application of this approach provides a method for determining axial dissolved oxygen profiles under conditions of known mass transfer rates as a function of agitation-aeration characteristics. A stagewise approach is used which divides the fermentor into a series of mixing cells. The results of the calculations are presented in Table IX. In the design of the large-scale fermentor it was assumed that the volumetric oxygen transfer coefficient kLa must be sufficiently high to provide nonlimiting oxygen transfer under worst case conditions (at the end of the exponential growth phase).

TABLE VIII GENERALPROCESS DATA Parameter

Measure

Broth density Kinematic medium viscosity Process temperature Back pressure Gas flow/reactor volume/time

kg/m3 m-*/sec "C N/m2 m3/m3/sec

Value 1030 I x 25 5 x 104 0.005

42

ROBERT VERPOORTE E T AL.

TABLE I X OPERATING CONDITIONS FOR INDUSTRIAL-AND LABORATORY-SCALE FERMENTORS FOR CONSTANTIMPELLER TIP SPEED( u ; ) AND POWERINPUT (PlV) v(m3) Parameter Gas flow (m3/sec) Superficial gas velocity (m/sec) H0Id-up Oxygen transfer coefficient (sec-I) Impeller speed (sec-I) Impeller tip speed (m/sec) Power input (W/m3)

25 0.1 0.02 0.043 0.0072 1.28 3.1 400

0.005 (Ui = C )

0.005 (P/V-C)

3.3 x 10-5 0.0014 0.039 0.03 21.9 3.1 7000

3.3 x 10-5 0.0014 0.016 0.004 8.6 1.2 400

c. Regime Analysis. For equations to calculate the various time constants in the hypothetical plant biotechnological process described above one should consult the literature (185). In Table X the results of the time constant calculations are presented. The growth and production time are the longest characteristic times in the process, thereby determining the total process time. By comparing the characteristic times of the 25-m3 fermentor, it can be concluded that there may be some problems in supplying oxygen to the plant cells. Oxygen transfer and oxygen consumption

TABLE X TIMECONSTANTS OF A 25-m3 FERMENTOR AND A DOWN-SCALE 5-LITER FERMENTOR FORCONSTANT TIP SPEED(ui) AND CONSTANT VOLUMETRIC POWERINPUT (PlV)

COMPARISON OF

Time constant

25

Mixing Oxygen transfer Oxygen consumption (growth) Oxygen consumption (production) Heat production Heat transfer Substrate consumption (growth) Biomass production Product formation

32 139 139 303 4800 4800 1500" 2 x 105 18 X lo'

a

Fed-batch process in which C , is maintained at 10 mollm'.

0.005 C)

(Ui =

2 33 139 303 1000 1000 1500"

2 x 105 18 x 105

0.005

(PlV-C) 8 250 139 303 4800 4800 1500" 2 x 105 18 x 105

1.

PLANT BIOTECHNOLOGY

43

times are equal on a large scale because design has been based on the critical oxygen transfer coefficient. However, mixing time is short compared to oxygen transfer time, so oxygen depletion is unlikely to occur in badly mixed regions of the fermentor. Heat transfer and heat production time are equal. The calculation is based on the assumption that all heat produced is exchanged. No temperature gradients are expected because the time of heat production is long compared to the mixing time. In the 5-liter fermentor for constant volumetric power input, oxygen limitation will occur because the oxygen transfer time is longer than the oxygen consumption time. When ui is constant the oxygen transfer time is much shorter than the oxygen consumption time, so growth will not be hampered by oxygen limitation. When the process is operated in a fedbatch manner, problems can occur when the substrate supply is limiting. From the characteristic time of substrate consumption, it can be concluded that in this case substrate limitation will occur after 25 min. It can be concluded that experiments to simulate the large-scale process cannot be performed on a 5-liter scale for constant volumetric power input. Under these conditions oxygen transfer will be the ruling mechanism for growth instead of maximum specific growth rate. For ui constant oxygen transfer is more than sufficient, so small-scale experiments should be preferably performed under this condition. d. Shear Effects. Characteristic times for shear effects on the cell population cannot be calculated because of various reasons: various effects, each dependent on the flow regimes in the fermentor, are acting simultaneously on the cells; and it is not possible to define a rate for the incompletely understood shear effects. An analysis of shear effects should therefore be based on comparison of the variety of mechanisms in relation to flow characteristics. This approach has been described by Cherry and Papoutsakis (188) for a system of animal cells on microcarriers. This methodology was also used by Meijer (69) to analyze a plant cell culture with the aim of selecting the optimal scale-down criterion for laboratoryscale shear studies.

e. Present Situation. Several large-scale plant cell cultures have been reported, both in airlift bioreactors and in stirred vessels (Table 111). One might conclude that the theoretical analysis given above is superfluous, given that successful large-scale plant cell fermentations have already been carried out. However, successful does not necessarily mean optimal. In a large-scale fermentor it is not at all easy to study factors that may eventually be limiting, although these limitations (e.g., in oxygen transfer or mixing) may have a deep impact on productivity and therefore the economics of a process. Further studies on the various aspects of large-

44

ROBERT VERPOORTE E T A L .

scale culturing of plant cells are thus of great importance to the eventual improvement of the economic feasibility of plant cell biotechnological production of plant secondary metabolites.

IV. Nicotine Tobacco is one of the major model systems in plant cell and tissue culture studies. Particularly in developing techniques for genetic engineering has it played a major role. Consequently numerous papers have described various aspects of tobacco plant biotechnology . For example, almost 200 entries for plant cell culture media for tobacco are given in the tabulation by George et al. (189). Some of the most commonly used media for plant cell cultures were, in fact, developed for tobacco callus cultures (190,191). Indeed, root cultures of several Nicotiana species were described as early as 1938 by White (192). Collins and Legg (193) reviewed the use of cell and tissue culture methods for the improvement of tobacco. Although the price of nicotine is very low, extensive studies have been made on the production of this alkaloid in cell cultures. Some patents have been filed (Table XI). Studies on the production of nicotine (1)and related alkaloids (2-4) in cell cultures are summarized in Table XII. A. SECONDARY METABOLITES IN CELLCULTURES From Table XI1 it is obvious that the alkaloid levels found in the cell and tissue cultures of Nicotiana species vary widely. The cultures do produce a variety of other secondary metabolites as well: sterols and triterpenes (245,246), ubiquinone (243, cinnamoyl putrescines (248-251), and the phytoalexin capsidiol and related sesquiterpenes (252-254). TABLE XI PATENTSCONCERNING PRODUCTION OF NICOTINE IN PLANTCELL AND TISSUECULTURES

I. Shio and S. Ota. Ajinomoto Co., Ltd. Jpn. Kokai, JP 480912187, 28 Nov 1973 Showa. JP 72-18357, 22 Feb 1972. Alkaloid production by tissue culture. Chem. Abstr. 80, 11839613. H. Smith and D. W. Pearson. Gallaher Ltd., Eur. Pat. Appl. EP 7244, 23 Jan 1980. GB 78-30381, 19 Jul 1978. Nicotine by culturing a Nicotiana strain. Chem. Abstv. 92, 194784b. A. S. Weaving, A. J. N. Bolt, D. J. Barlett, and S. W. Purkins. Imperial Group PLC. GB 2203022, 10-12-1988. Smoking material containing substrate of tobacco cells or plant material and cellulosic material with mixtures of lactose and glucose.

1.

45

PLANT BIOTECHNOLOGY

(1)Nicotine, R = CH3 (2) Nornicotine, R = H

(3) Anabasine

(4) Anatabine

TABLE XI1 OCCURRENCE OF NICOTINE IN PLANTCELLAND TISSUECULTURES Alkaloid Nicotine Nicotine Nicotine, nornicotine, anabasine Nicotine, anabasine Nicotine Nicotine Nicotine, anatabine Nicotine Nicotine, anatabine, anabasine Nicotine Nicotine Nicotine Nicotine Nicotine, nornicotine, anatabine, anabasine Nicotine Nicotine Nicotine Nicotine, nornicotine Nicotine Nicotine Nicotine

Plant species

Type of culture

Yield

Ref.

N. tabacum N. tabacum N . alata, N. glauca, N. paniculata, N . rustica, N . silvestris N . glauca

Roots Roots Roots

2.9% DW 0.2% DW

194,I 95

Roots

0.76% DW

I98

N . tabacum N . rustica N . tabacum

Callus Roots Callus

0.01-0.1% DW -

I99 200 201,202

N . tabacum N . tabacum

Callus Callus

0.26% DW 0.013% DW

203 204

N . tabacum

0 0.01% DW 0.5-1.1% DW 0.29% DW 1-3.4% DW

205

N . tabacum N . tabacum N . rustica N . tabacum

Callus Suspension Callus Callus Callus Callus

N . tabacum N . tabacum N . tabacum N . tabacum

Callus Callus Callus Callus

0.3-3.38% DW 0.04% FW 0.75% DW 0.004% DW

50,211-214 215 216-218 219

N . tabacum N . tabacum N . tabacum

Callus Suspension Callus Roots

0.17-0.75% DW 0.16% DW 3.2% DW

220 22 I 222

196 197

206,207 208 209 210

(continued)

46

ROBERT VERPOORTE ET A L .

TABLE XI1 (Continued) Alkaloid Nicotine Nicotine nornicotine, anatabine Nicotine anatabine, anabasine, myosmine, anatalline, nicotelline Nicotine Nicotine, nornicotine, anabasine Nicotine

Nicotine Nicotine Nicotine, nornicotine, anatabine Nicotine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine

Plant species

Type of culture

Yield

Ref.

N . tabacum N . tabacum

Suspension Callus

2.2% DW, 0.14 g/liter 0.16-0.7% DW

223 224-227

N . tabacum

Suspension

0.2% DW

228

N . tabacum, N . rustica N . tabacum

Suspension Callus

2.9% DW, 0.36 g/liter 1.6-3.75% DW

229 230-233

N . tabacum, N . rustica, N . debneyi, and somatic and sexual hybrids of these species N . tabacum N . tabacum N . tabacum

Suspension

0.007-0.041% DW, 0.007-0.018% DW, 0.005% DW

234

Callus Hairy roots Hairy roots

235-237 0.05% DW 90 1.5 mg/liter/day 0.03% (FW), 0.01 g/liter 238

N . rustica N . rustica

Hairy roots Hairy roots

0.065% FW 0.085% FW

239 28

N . tabacum

Hairy roots

0.12% FW

240

N . rustica

Hairy roots

0.04% FW

240

N . hesperis

Hairy roots

0.09% FW

240

N . africana

Hairy roots

0.12% FW

240

(continued)

47

1. PLANT BIOTECHNOLOGY TABLE XI1 (Continued) Alkaloid Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine Nicotine, nornicotine, anabasine Nicotine

Plant species

Type of culture

Yield

Ref.

N . umbricata

Hairy roots

0.1% FW

240

N . velutina

Hairy roots

0.14% FW

240

N . cavicola

Hairy roots

0.06% FW

240

N . rustica

Hairy roots

0.08% FW

241

N . hesperis

Hairy roots

0.1% FW

242

N . tabacum N . tabacum

Suspension Hairy roots

0.003% DW 0.11% FW

243 244

N . rustica

Hairy roots

0.08% FW

132

B. SCREENING, SELECTION, AND STABILITY After establishing a cell culture, alkaloid production may decrease during successive subculturing (208,2091, but stable, high producing cell lines can be obtained (299,208-220,222). Fluctuations in alkaloid production during successive subculturing may also occur (224).Shiio and Ohta (208) reported that during initial subcultures a considerable difference in alkaloid content exists for calli derived from different parts of the same tobacco plant, root calli being the best producers. For N. rustica such differences could not be observed (209), but a large variation in alkaloid content of single-cell clones derived from a callus cell line was noted. Based on this a selection program was conducted in order to obtain high producing cell lines. By a repeated single-cell cloning procedure high producing (1-3.4% alkaloid on a DW basis) N. tabacum cell lines were obtained (220). Ohta and co-workers (211,224) successfully selected a

48

ROBERT VERPOORTE ET AL.

stable high nicotine-producing (2.29% DW) tobacco callus line by screening at the level of calli Kinnersley and Dougall (215,220) found a strong correlation between alkaloid levels in the plant and those in the derived callus cultures. However, Roper et al. (229)could not find such a correlation. Great variation in the nicotine content of cultured cells of three Nicotiana species and their somatic and sexual hybrids have been found (234). Some of the cell lines showed stable production for a year, whereas others showed a change in nicotine production after a year of subculturing (100 subcultures). It was concluded that making sexual or somatic hybrids is an interesting way to obtain new cell lines (or plants) with improved characteristics such as nicotine production. Differentiation into root-type tissue results in increased alkaloid production (194-197,199,220,222,225,230,231), which is in agreement with the fact that the roots are the major site of nicotine biosynthesis in the plant (I 94,196). Plants regenerated from non-alkaloid-producing cultures did regain the capacity of alkaloid biosynthesis (207). Robins et al. (28) proposed the use of a biosynthetic precursor, nicotinic acid, as a selective agent for obtaining high nicotine-producing hairy root cultures of N. rustics. Nicotine did not work as a selective agent in the media, whereas nicotinamide proved to be toxic. C. EFFECTSOF GROWTHCONDITIONS A number of factors have been studied for their influence on nicotine production. Of these the negative effect of auxins, and in particular 2,4-D, on alkaloid production is worth mentioning (202-204,211,220, 225,226,229,255). In root cultures the addition of indoleacetic acid (IAA) also reduces alkaloid production (196). Light was reported to inhibit nicotine formation (50,255). In a green cell suspension, however, increased nicotine levels were found on illumination (229).Ikemeyer and Barz (243) reported that a photoautotrophic cell line of N. tabacurn did not produce nicotine, whereas a heterotrophic cell line did accumulate this alkaloid. Elicitation with a preparation of the fungus Phytophthora megasperma did not affect the nicotine levels of these cell lines. Addition of organic acids to the medium resulted in increased alkaloid formation in callus cultures (up to 3.25%) (230). For a review of the various cultural factors which influence secondary metabolism, the reader is referred to Mantel1 and Smith (255).

D. LARGE-SCALE SUSPENSION CULTURES From Table XI1 it is clear that most studies concerned callus cultures of Nicotiana species. For industrial applications however, suspension cul-

1. PLANT BIOTECHNOLOGY

49

tures are of more interest. The first report on a suspension culture in a bioreactor was by Kato et al. (256). Tobacco cells were cultured in a 30-liter jar fermentor in a semicontinuous mode. A biomass density of 20 g/liter was reached in batch cultures; the mean optimal growth rate during the exponential phase was a doubling time of 24 hr (specific growth rate, p , 0.69 day-'). In a semicontinuous culture the yields (dry weight) were 12-13 g/liter/day. The biomass was stirred by a disk turbine-type of impeller. Subsequently, a continuous culture in a 1500-literfermentor was reported (137). Mixing of the culture was achieved by aeration, and no agitator was present in the fermentor. The specific growth rate was less than that in a 15-liter fermentor (0.62 compared to 0.69 day-'). At a dilution rate, D , of 0.42 day-' and a sucrose concentration of 26 g/liter in the influent medium, maximum cell productivity was obtained (3.82 g/ liter/day). Although mixing of the culture broth in the 1500-literfermentor presented some problems, the authors considered the system to be suited for the production of tobacco raw material. Further optimization of the medium for batch cultures resulted in a further increase of the maximum specific growth rates from 0.80 to 0.96 day-' (257). In a two-stage continuous culture system using 60-liter fermentors, stirred by flat blade turbine impellers, Kato and co-workers (138,258)further improved the production of tobacco raw material; an average production of 6.9 g DW cell material/ liter/day was reported. In a batch culture on a 20,000-liter scale the cells showed a highest growth rate of 15 hr doubling time ( p = 1.09 day-') (138). Hashimoto et al. (221) reported the continuous culture of tobacco cells on a 20,000-liter scale, in a 66-day experiment. During steady state the biomass density was 16.5 g/liter (DW), the dilution rate was 0.35 day-', and a productivity of 5.82 g/liter/day was achieved. In this type of experiments biomass as a substitute for tobacco plant material was the major goal. No data on the nicotine production were given. A fed-batch fermentation on a 10-liter scale for the production of cinnamoyl putrescines by tobacco cells was reported by Schiel et al. (251). Flat blade turbines were used for stirring. Sahai and Shuler (259) described a multistage continuous culture of tobacco cells in chemostats of the Wilson type. Steady states under different conditions were maintained; concerning secondary metabolites, only the production of phenolics was measured. Mantel1 et al. (223) cultured tobacco cells in 5-liter Wilson-type batch fermentors. Mixing was by the aeration through air inlet tubes extended to the base of the vessel. By using media having a high sucrose (5%) and low phosphate concentration, nicotine levels of 2.2% (DW) could be reached, corresponding to 0.14 g/liter. Optimization of tobacco cell suspension cultures, with the aim of largescale production of nicotine, was studied by Roper et al. (229). A fast growing cell line with a high nicotine content was selected. Various combi-

50

ROBERT VERPOORTE E T A L .

nations of growth and production media were tested. Best results were obtained by a Murashige-Skoog (MS) medium containing 0.2 ppm naphtylacetic acid (NAA), 0.02 ppm kinetin, and casein hydrolysate (1 g/liter). The medium contained 4% sucrose; glucose was found to inhibit nicotine production. This medium was suited for both growth and production, and after 1 week of preculture on this medium the cells were subcultured on the same but fresh medium. After 21 days from the start of the preculture the nicotine content of the cells was 2.9% (DW), the cell yield was 1015 g/liter. The maximum yield obtained for this system was 0.36 glliter. With a mixotrophic green cell culture biomass yields of 14-27 g/liter were obtained, the nicotine production being 0.92 g/liter over a 2-week period. All these experiments were performed in shake flasks. When the cell lines were tested in fermentors different results were obtained. They did not grow well in a 20-liter airlift fermentor; in a 20-liter stirred fermentor good growth (doubling time 45 hr) was observed for one of the cell lines, but the nicotine production (0.006 glliter) was far below the 0.36 g/liter level observed for this cell line in shake flasks. Berlin and co-workers (250,251) reported on the production of cinnamoyl putrescines by tobacco cells in 70-liter fermentors, operated in the fed-batch mode. They also found considerable difference between results obtained in shake flasks versus fermentors. The large-scale culture of tobacco cells, claimed to contain 5-30% nicotine, for the preparation of a tobacco-smoking substitute have been patented (see Table XI). Hallsby and Shuler (260) reported the growth of tobacco cells immobilized in a membrane-type reactor using different flow patterns.

E. ROOTCULTURES As nicotine biosynthesis is connected with the roots, an obvious solution for obtaining high producing, stable systems is the transformation of tobacco cells with Agrobacterium rhizogenes, yielding the so-called hairy root cultures. Hamill et al. (238)first reported such cultures of N. rustica. These cultures did produce alkaloids, of which a substantial proportion was excreted into the medium. The alkaloid level was slightly higher than in 6- to 8-week-old plants. Via protoplasts single-cell clones were obtained from N. rustica hairy roots. Hairy root cultures regenerated from the clones showed variation in morphology, alkaloid formation, and T-DNA structure. Some clones showed increased alkaloid production (239).Also the accumulation ratio, that is, the amount of alkaloid released into the medium compared to the amount in the cells, varied largely. Parr and Hamill (240) compared alkaloid production of the transformed hairy root

1.

PLANT BIOTECHNOLOGY

51

cultures of a number of Nicotiana species with that of the plant roots. The biosynthetic capacity of the hairy root cultures was similar to that of the intact plant. Differences in accumulation ratios observed for the various hairy root cultures did not correlate with the ability of the plants to transport alkaloids from the roots to aerial parts. The N. rustica hairy roots were successfully grown in a packed-bed fermentor, yielding biomass densities (DW) of 10 g/liter (90). The growth rate was comparable with a cell suspension culture. The fermentor was operated as a batch fermentor for 11 days, after which is was run as a continuous culture. Nicotine was isolated from the medium during the continuous operation. The alkaloid production rate was estimated to be 1.54 mg/liter/day during this phase. To improve the release of alkaloids by the hairy roots, a continuous removal of the alkaloids from the medium with XAD-4 as an adsorbent was tested. Hairy roots cultured in flasks did not produce more alkaloids in the presence of sachets with XAD-4. However, the amount of alkaloid released into the medium increased; in other words, the accumulation ratio was affected by the adsorbent. The effect of addition of various precursors of nicotine such as nicotinic acid (28,195,198,211,222,228),ornithine (198,211,222,228), and putrescine (211,222,228) on alkaloid production has been studied. The results of these studies are of particular interest in connection with a better understanding of the regulation of the biosynthesis of nicotine. The recent and extensive studies in this field (227,230-233,235-237,241,242,244,261-263) [for a review, see Wagner (264)],particularly those concerning the enzymes involved in the biosynthetic pathway of nicotine, hold the promise that in the near future the genes regulating this pathway will be isolated. This will open the way for genetic modification as a tool to improve (or block) nicotine production in plants or plant cells. The feasibility of such an approach was recently proved by Hamill et al. (132).By overexpression of a yeast ornithine decarboxylase (ODC) in transgenic roots of N. rustica, the production of nicotine in the root culture could be enhanced about 2-fold. The yeast ODC gene was combined with the CaMV35S promoter and introduced in the plant using a binary vectorlAgrobacterium rhizogenes system.

F. CONCLUSION Despite the extensive studies of tobacco cell and tissue cultures, so far no stable and high producing, large-scale production system for nicotine has been achieved. The technology of growing tobacco cells on a large scale is available, but suitable cell lines remain to be developed. Although the hairy roots are fast growing, the large-scale production of such cultures

52

ROBERT VERPOORTE E T A L .

needs further study. The alkaloid production in these cultures probably needs to be enhanced before such a production becomes economically feasible. In the near future, genetic engineering might offer new perspectives in this area.

V. Tropane Alkaloids The tropane alkaloids represent from pharmaceutical point of view one of the most important groups of alkaloids, on the one hand because of the alkaloids atropine (5)* and scopolamine (6), both widely used in pharmacotherapy, and on the other hand because of cocaine, most known for is its abuse as a stimulant. The former two alkaloids are extracted from a variety of Solanaceae, and the latter alkaloid is isolated from the leaves ofErythroxylon coca. For the plant cell and tissue culture of the latter plant we have not been able to find any literature. For the Solanaceae, however, many studies have been published. Several genera of this family have been studied extensively, for example, Anisodus, Atropa, Datura, Duboisia, Hyoscyamus, and Scopolia. Of these Datura has widely been used as a model system for the development of various techniques in plant cell and tissue culture and for basic studies of cultured plant cells, without reference to alkaloid production. Scopolamine is a more costly derivative of atropine: it has an epoxide function (6). Therefore, many studies concern the bioconversion of atropine (I-hyoscyamine) to scopolamine. The isolation of the enzymes involved and the subsequent cloning of the genes open new avenues for the production of scopolamine, either by genetic engineering of plants or by introducing the gene(s) responsible into microorganisms which could then effectively perform the desired bioconversion. The industrial interest in the tropane alkaloids is well reflected in the large number of patents concerning the plant cell biotechnology of tropane alkaloids (Table XIII). The first reports of the occurrence of tropane

* We shall here use the name atropine, which stands for the racemic mixture of 1- and d-hyoscyamine, to avoid confusion, as in none of the publications concerning tropane alkaloids is the optical rotation of the alkaloid isolated mentioned, and even the co-occurrence of atropine and hyoscyamine in cell cultures has been reported (265-270)!

1.

53

PLANT BIOTECHNOLOGY

'*a

ofiH

H

0-c-c

II I

0 CHzOH (5) Atropine

(6) Scopolamine

alkaloids in cultured cells or tissues are quite old. James (271) reported the presence of alkaloids in meristems of some solanaceous plants. The first report on a suspension culture of Datura tumor tissues was in a patent of 1956 (see Table XIII). Telle and Gautheret (272) and Stienstra (273) reported the production of tropane alkaloids in cultured roots of Hyoscyamus niger and Datura stramonium, respectively. Reinouts van Haga (274,275) studied the biosynthesis of tropane alkaloids in root cultures of Atropa belladonna. Root callus cultures of Atropa belladonna were reported to contain atropine (276). In Table XIV through XVIII the occurrence of alkaloids in various types of cell and tissue cultures of Atropa, Datura, Duboisia, Hyoscyamus, and various other species in the family Solanaceae is summarized. From these data it is clear that the production of tropane alkaloids in cell suspension cultures is rather low. Only in root cultures has production similar to, or even higher than, the original plant been obtained. For this reason an extensive discussion on efforts to improve production in cell suspension cultures is not useful; instead, we briefly deal with the application of plant biotechnology for the improvement of the tropane alkaloid-producing plants. Finally, we discuss the bioconversion of added precursors.

A. PLANTBIOTECHNOLOGY

The cell and tissue culture of the major tropane alkaloid-producing species does not apparently offer any special problems. The regeneration of plantlets from callus and tissue cultures seems to be routine (286,307,309,323,325,332,350-352). Plants have also been regenerated from protoplasts of Atropa belladonna (353),Duboisia rnyoporoides (354), and Hyoscyamus muticus (355,356). Cryopreservation has been reported for Anisodus and Datura species (349,357).

54

ROBERT VERPOORTE ET AL. TABLE XI11 PATENTSCONCERNING TROPANE ALKALOIDS IN PLANTCELLAND TISSUECULTURE

1956 J. B. Routien and L. G. Nickell. U.S. Patent 2,747,334, 29-05-1946. Cultivation of plant tissue. 1974 American Cyanamid Co. D1-108-769. 26.07.72-D1-164695 (05-10-74). Cell culture production of plant metabolites by inducing redifferentiation of undifferentiated plant cell cultures ( e g , Datura). I980 Eisei KK. JP 024116, 29-02-1980. Isobutyroyl-tropine and valeryl-tropine preparation by culturing callus derived from Duboisia plant and extracting cultured cells. A. R. Saint-Firmin. U.S. Patent 4241536 30-12-1980. GB 76-46866 10-11-1976. Embryogenesis in uitro, induction of qualitative and quantitative changes in metabolites produced by plants and products (e.g., Hyoscyamus muticus). Chem. Abstr. 94, 171236b. I986 Y. Mano, N. Nabeshima, and H. Okawa. Sumitomo Chemical Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 61254195 A2 11-11-1986. JP 85-97326,7-05-1985. Tropane alkaloid production by tissue culture (hairy roots, Scopolia and Datura). Chem. Abstr. 107, 174434n. 1987 Seitaikinou Riyou. J62248429 A 29-10-1987, 86JP-089975;21-04-1986. Tissue cultivation of Duboisia plants by infecting callus, shoot, or adventitious root cultures with Agrobacterium rhizogenes. H. Ideno and C. Habara. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 62006675 A2, 13-1-1987. JP 85-143882,2-7-1985.Tropane alkaloid production by Duboisia tissue cultures. Chem. Abstr. 107,5796b. H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 62006674 A2 13-1-1987. JP 85-143881, 2-7-1985. Tropane alkaloid manufacture by Duboisia tissue culture. Chem. Abstr. 107, 17443513. Y. Yamamoto and R. Mizuguchi. Nippon Paint Co., Ltd. Jpn Kokai Tokkyo Koho. JP 62186790 A2, 15-9-1987. JP 86-30501, 13-2-1986. Manufacture of 15N-containing compounds by plant cell culture (Datura taluta) Chem. Abstr. 108,73743~. H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 62205792 A2, 10-91987. JP 86-47046,4-3-1986. Manufacture of alkaloids by genetically engineered Solanaceae plant cell culture (hairy roots, Atropa belladonna). Chem. Abstr. 108, 73775q. 1988 H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 63039595 A2,20-21988. JP 86-181532, 1-8-1986. Medicinal alkaloid production by plant tissue culture (hairy roots, Datura). Chem. Abstr. 108,220369r. H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 63039596 A2,20-21988. JP 86-181533, 1-8-1986. Manufacture of alkaloids by cultivating genetically engineered Solanaceae plants (hairy roots, Datura innoxia). Chem. Abstr. 109,53249~. H. Kamata and H. Saga. Lion Corp. 88JP-064650, 17-03-1988. EP-283051,21-09-1988. Alkaloid($ preparation from Solanaceous plants by transformation with Agrobacterium rhizogenes, culturing hairy roots produced, and continuous recovery from alkaloids (Hyoscyamus, Scopolia). (continued)

1. PLANT BIOTECHNOLOGY

55

TABLE XI11 (Continued) S. Takayama. Kyowa Hakko Kogyo Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63105689 A2, 10-5-1988. J P 86-252759, 23-10-1986. Scopolamine and its manufacture with plant tissue culture. Chem. Abstr. 109, 168983r. H. Ideno, Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. J P 63087991 A2, 19-4-1988. JP 86-230156, 30-9-1986. Scopolamine and hyoscyamine manufacture enhancement by tissue culture of tropane alkaloid-producing Solanaceae (Duboisia myoporoides). Chem. Abstr. 109,228787s. K. Shimomura, A. Yagi, and N. Okumura. National Institute of Hygeinic Sciences. Jpn. Kokai Tokkyo Koho. JP 63059897 A2, 15-3-1988. JP 86-203184, 29-8-1986. Therapeutic tropane alkaloid manufacture by plant tissue culture (Duboisia). Chem. Abstr. 110, 22332b. H. Ideno and H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63084497 A2, 15-4-1988.JP 86-230155, 30-9-1986. Therapeutic tropane alkaloid manufacture by plant tissue culture (Duboisia myoporoides). Chem. Abstr. 110,22334d. T. Emoto, H. Ideno, and T. Yoshioka. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63116691 A2,20-5-1988. JP 86-26-0768, 4-1 1-1986. A plant tissue culture method using a medium containing adsorbents (Duboisia). Chem. Abstr. 110, 37782~. H. Ideno, T. Emoto, and F. Ito. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167790 A2, 11-7-1988, JP 86-309270, 27-121986. Tropane alkaloids and their manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 110,210958j. Y. Mano, Y. Yamada, and H. Okawa. Sumitomo Chemical Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63226280 A2, 20-9-1988. JP 87-63092, 17-3-1987. Tropane alkaloids and their manufacture with plant tissue culture of Duboisia (hairy roots, Duboisia leichhardtii). Chem. Abstr. 110, 17182213. H. Ideno, T. Emoto, and F. Ito. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167790 A2, 11-7-1988. JP 86-309270, 27-121986. Tropane alkaloids and their manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 110,210958j. H. Kamata and S. Marumo. Nippon Kayaku Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63216491 A2, 8-9-1988. JP 87-49949, 6-3-1987. Manufacture of atropine and scopolamine from indoleacetic acid derivatives by plant tissue culture (Duboisia, Hyoscyamus, Scopolia, and Datura species). Chem. Abstr. 110,210979s. H. Ideno and H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167793 A2, 11-7-1988. JP 86-309271, 27-121986. Tropane alkaloid manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 111,22173~. Seitaikinou Riyou. 563129982 A 02-06-1988. 86JP-276787; 21-1 1-1986. Plant tissue culturing method, by culturing plant tissue in liquid medium containing surfactant and discharging out secondary metabolic products of plants from cells. Seitaikinou Riyou. 563226278 A 20-09-1988. 87JP-001118; 08-01-1987. Plant tissue culturing method - using culture medium containing one or more sulfoxide(s), polyhydric alcohol(s), organic acids, and steroids. Seitaikinou Riyou. 563226281 A 20-09-1988. 86JP-308544; 26-12-1986. Tissue culturing to release secondary metabolic products of plant from cells comprises using liquid medium containing sodium or potassium ions, or calcium or magnesium ions. (continued)

56

ROBERT VERPOORTE E T A L .

TABLE XI11 (Continued) 1989 T. Takahashi, Y. Kita, and K. Koide. Oji Paper Co. Ltd. Jpn Kokai Tokkyo Koho. JP 01 168294 A2, 3-7-1989. JP 87-323744, 23-12-1987. Manufacture of tropane alkaloids by plant tissue culture of Solanaceae (Hyoscyamus niger). Chem. Abstr. 112,34439~. S. Takayama and H. Tanaka. P.C.C. Technology, Inc. Jpn Kokai Tokkyo Koho. JP 01243991 A2, 28-9-1989. JP 88-69745, 25-3-1988. Plant metabolites manufacture with differentiated plant tissue (Atropa belladonna root cultures). Chem. Abstr. 112, 137584~. S . Kitani and H. Ideno. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 01124383 A2, 17-5-1989. JP 87-280893,9-11-1987. Tropane alkaloid enhanced manufacture with arylmethylbutanediacids in plant tissue culture (Duboisia myoporoides). Chem. Abstr. 112, 1567042. M. Sakai and H. Ideno. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 01273597 A2, 1-11-1989. JP 88-102735,27-4-1988. Manufacture of tropane alkaloids by plant tissue culture with adenosyl compounds or cyclic adenosine phosphates (Duboisia myoporoides). Chem. Abstr. 112, 15670q.

1. Atropa

Eapen et al. (286) studied the morphogenesis of haploid (obtained from anthers) and diploid tissue cultures of Atropa belladonna. The haploid tissue was found to regenerate more readily; the diploid tissue also lost its regenerative potential more rapidly on prolonged subculturing. The regenerated plants showed alkaloid production similar to plants grown from seeds. Alkaloid (atropine) production increased with increased differentiation. The loss of regenerative potential was in agreement with observations made by Rashid and Street on haploid suspension cultures (358).The suspension cultures showed a decrease in embryogenic potential on succesive subculturing, paralleling a decline in the proportion of haploid cells. Jung and Tepfer (269) regenerated plants from Atropa belladonna roots which were transformed with Agrobacteriurn rhizogenes. The transformed plants had similar alkaloid levels in the root as the nontransformed plants, but the leaves had a 3 times lower alkaloid content. These experiments thus show that genetic engineering is feasible for this species. 2 . Datura

Hiraoka and Tabata (307) were able to grow plants from cellular aggregates formed in cell suspension cultures of Datura innoxia. Most of the plants obtained were diploid (82%). Surprisingly only a few were aneuploid or polyploid, given that in the suspension culture only 32% of the cells were diploid. In the development of plants from the non-alkaloidproducing suspension cells, an increase in alkaloid production was ob-

1.

57

PLANT BIOTECHNOLOGY

TABLE XIV OCCURRENCE OF TROPANE ALKALOIDS IN Atropa PLANT CELLA N D TISSUECULTURES A1kaloid Atropine Atropine Atropine scopolamine, tropine, cuscohygrine Atropine Atropine Atropine, Choline Atropine, scopolamine, tropine Atropine Atropine Atropine Atropine, scopolamine, cuscohygrine, plus 14 tropane derivatives Atropine, scopolamine hydroxyhyoscyamime Atropine, scopolamine Atropine Atropine, scopolamine Atropine, scopolamine, cuscohygrine Atropine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine

Plant species

Type of culture

Yield

Ref.

A . belladonna A . belladonna A . belladonna

Roots Callus Roots

0.5% DW 0.05% DW 0.096% DW

274,275 2 76 277

A . belladonna

Suspension, aggregates Callus, suspension Roots Callus Callus Callus

-

278

0.45% DW 0.53% DW

279-283 284 285

0.0012% DW

286

A . belladonna A . belladonna A . belladonna A . belladonna

Callus Roots Shoots Roots Suspension

0.75% DW 0.5% DW 0.81% DW 0.0005% DW

287 288 289 290

A . belladonna

Roots

0.34% DW

291,292

A . belladonna

Hairy Roots

0.4% DW

293

A . belladonna A . belladonna

Immobilized roots Hairy roots

0.2% DW -

294 268

A . belladonna

Hairy roots

1.3% DW

269

A . belladonna A . belladonna, A . caucasica A . belladonna

Shoots Hairy roots

295 296

Callus

0.01% DW 0.2% DW, 0.12% DW 0.09%DW

A . belladonna

Roots

0.25% DW

298

A . belladonna A . belladonna A . belladonna A . belladonna

-

297

TABLE XV OCCURRENCE OF TROPANE ALKALOIDS I N DATURA PLANTCELLA N D TISSUECULTURES Alkaloid

Plant species

Alkaloids Atropine, scopolamine Atropine, scopolamine Scopolamine

D. stramonium D. tatula D. metel D. stramonium

Atropine, scopolamine Choline, pseudotropine, cuscohygrine Atropine, scopolamine Choline, scopolamine Atropine, choline Scopolamine

D. quercifolia D. innoxia D. metel D. stramonium D. stramonium D. tatula D . metel D. innoxia

Atropine Aposcopolamine Choline, atropine, scopolamine Atropine

D. D. D. D.

Atropine Atropine, scopolamine Atropine, tropine, scopolamine

D. innoxia D. innoxia D. innoxia, D. stramonium D. clorantha

metel meteloides metel metel

Type of culture Roots Roots Roots Callus suspension Callus Callus Callus Suspension Suspension Callus Callus Callus Shoots Callus Suspension Callus Callus suspension Callus Suspension Callus

Yield 0.06% DW -

0.03% DW, 0.003 g/ liter 0.02% DW 0.06% DW 0.006% DW -

0.18% DW 0.04% DW 0.01% DW 0.015% DW

Ref. 2 73 299,300 301 302

303 304 305 306 265 307

0.08% DW 0.01 1% DW

308 309 266 310

0.0016 g/liter 0.15,0.1, 0.15% DW

31 I 312 313

Atropine, scoplamine, h ydrox yh yoscyamine Atropine Atropine, scopolamine

VI

Scopolamine Atropine, scopolamine Atropine, scopolamine, apoatropine Atropine Atropine, scopolamine Scopolamine Atropine, scopolamine, plus 17 tropane derivatives

D. stramonium, D. innoxia, D. leichhardtii, D. fastuosa D. stramonium D. chlorantha D. ferox D. fastuosa D. innoxia D. metel D. meteloides D. quercifolia D. rosei D. sanguinea D. stramonium D. stramonium var. inermis D. stramonium var. stramonium D. strumonium var. tatula D. stramonium D. candida D. stramonium D. wrightii Datum F, hybrid D. strumonium, D. innoxia, D. lanosa, D. pruinosa D. kymatocarpa D. candidu

Roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Callus Hairy roots

0.42, 0.47, 0.19, 0.27% DW 0.6% DW, 0.1 g/liter 0.25% DW 0.23% DW 0.2% DW 0.22% DW 0.31% DW 0.08% DW 0.26% DW 0.17% DW 0.13% DW 0.31% DW 0.22% DW 0.19% DW 0.4% DW 0.56% DW 0.68% DW 0.18 giliter -

291,292 314 296

315 316 317 317 317 318 319

60

ROBERT VERPOORTE ET A L .

TABLE XVI OCCURRENCE OF TROPANE ALKALOIDS IN Duboisia PLANTCELLAND TISSUE CULTURES Alkaloid Atropine, scopolamine Atropine, scopolamine, hydroxyhyoscyamine, valtropine Nicotine, tropine esters Nicotine, anabasine Atropine, scopolamine, nicotine Atropine, scopolamine, nicotine, nornicotine, anabasine Atropine, scopolamine, nicotine Atropine, scopolamine Atropine, scopolamine, hydroxyhyoscyamine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine, nicotine

Plant species

Type of culture

Yield -

Ref. 320 32 1

D . myoporoides Duboisia hybrid

Callus Shoots

D. leichhardtii D. myoporoides D. leichhardtii

Callus Callus Roots

0.8% DW

322 323 324

D . myoporoides

Roots

0.02% DW

325

D . myoporoides, D . leichhardtii, D . hop woodii Duboisia hybrid D . leichhardtii

Roots

0.4, 1.69, 1% DW

326

Roots Roots

0.65% DW

32 7 291,292

D. myoporoides Duboisia hybrid D . leichhardtii D.leichhardtii

Hairy roots Hairy roots Hairy roots Roots

1% DW 0.13% DW 2.1% DW 0.1% DW

328 296 329 298

served with an increase in differentiation, and production of scopolamine (the major alkaloid in the plant) was associated with the formation of roots. Some of the plants obtained showed abnormal alkaloid metabolism; for example, in some plants hydrolysis of scopolamine was observed during drying of harvested leaves, and in another plant alkaloid production occurred only after flowering. Kibler and Neumann (312) reported that diploid plants of D . innoxia had higher alkaloid contents than plants obtained from haploid tissue. Androgenic diploid plants obtained from anther cultures of a Datum innoxia plant showed considerable variation in alkaloid content. High scopolamine-producingplants could be obtained in this way (352).Plantlets were regenerated from a Datum candida hybrid hairy root culture (326). For a more extensive review on the in uitro propagation of Datum, the reader is refered to Petri and Bajaj (357). 3 . Duboisia

Conditions for the tissue culture and plant regeneration of Duboisia myoporoides have been described (323). The alkaloid content was fol-

1. PLANT BIOTECHNOLOGY

61

lowed during the development of the plants (351). A clear difference was found between the alkaloid content of a seedling, which has most of the alkaloids in the leaves, and a regenerated plantlet, where most of the alkaloids are found in the roots, the leaves being devoid of alkaloids. During development of the regenerated plant various patterns of alkaloids were observed in the leaves. In the fully developed plant, however, the alkaloid distribution was similar to that in the mother plant. In a further study it was found that in the regenerated plants very low levels of atropine esterase (a tropane alkaloid-hydrolyzing enzyme) activity in the roots coincided with the absence of alkaloids in the leaves (359). Griffin (360) reviewed various aspects of Duboisia species, including plant cell culture work. He concluded that it ought to be possible to develop high producing strains of Duboisia by means of plant tissue culture methods in combination with a sensitive quantification method for scopolamine. Kitamura (361)reviewed the in uitro regeneration of Duboisia species.

4. Hyoscyamus Corduan (309) reported methods of obtaining haploid and homozygous diploid plants form anthers of Hyoscyamus niger. Wernicke and Kohlenbach (350) described a method to generate plants from cultures obtained from microspores. Regeneration of Hyoscyamus muticus from callus was reported by Grewal et al. (332).Wernicke et al. (355)was able to obtain plants from leaf protoplasts isolated from a haploid Hyoscyamus muticus. From in uitro cultures of anthers of Hyoscyamus niger and H . albus, plants could be obtained which produced scopolamine as the major alkaloid, like the mother plants; however, qualitative and quantitative differences existed in the total spectra of alkaloids present (267).Considerable somaclonal variation was found in cell cultures derived from protoplasts of Hyoscyamus muticus (339,341,343).The plants also show considerable variation in alkaloid content. High alkaloid-producing plants can be developed via selection, and plants with a scopolamine content of 4% have been obtained (342,362). Introduction of genes in Hyoscyamus muticus with Agrobacterium tumefaciens (363) and electroporation (364) has proved feasible. An extensive review of the in uitro propagation, plant breeding, and cultivation of Hyoscyamus species is given by Strauss (365).

5. Conclusion Summarizing, most methods for in uitro propagation and plant breeding in modern plant biotechnology have successfully been applied to the major tropane alkaloid-producing Solanaceae species. Even transformation with Agrobacterium has been applied extensively. So far, new genes, coding for certain desired traits, have not been introduced into these plants, but

TABLE XVII ALKALOIDS I N Hyoscyamus PLANT CELL AND TISSUE CULTURES OCCURRENCE OF TROPANE Alkaloid

8

Plant Species

Alkaloids Atropine, scopolamine, cuscohygrine Atropine Atropine, scopolamine

H. niger H. niger H . muticus H. niger

Atropine Atropine Scopolamine Atropine, scopolamine

H . niger H. muficus H . niger H. niger H . albus H . gyorffi H . pusillus H . muticus H. bohemicus

Type of Culture Roots Suspension Callus Suspension Roots Callus Hairy roots Hairy roots Callus Roots Callus Roots Callus Roots Callus Roots Callus Roots Callus Roots

Yield 0.075% DW 0.11% DW 0.09% DW, 0.01 g h t e r 0.3% DW

0.61% DW 0.55% DW <0.01% DW 0.2% DW 0.03% DW 1.2% DW 0.015% DW 0.4% DW <0.01% DW 0.2% DW <0.01% DW 0.2% DW <0.01% DW 0.2% DW

Ref. 2 72 330,331 332 333 334-336 267 337,338 337,338 292

H . canariensis Scopolamine Atropine, scopolamine, hydrox yhyoscyamine

H . muticus H . niger H . albus H. svorfJi H . pusillus H . muticus H . canariensis

Atropine

H . muticus

Atropine, scopolamine Atropine, scopolamine

H . muticus H . albus H . aureus, H . bohemicus, H . muticus, H . niger H . niger H . albus

8 Atropine 7p-Hydroxyhyoscyamine, 6p6-hydroxyhyoscyamine, scopolamine Atropine, scopolamine

H. niger, H . albus

Callus Roots Suspension Roots Roots Roots Roots Roots Roots Callus Roots Suspension, immobilized Suspension Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Roots

<0.01% DW

0.2% DW 0.016% DW 0.23% DW 0.5% DW 0.6% DW 0.12% DW 0.25% DW 0.2%DW <0.01% DW 0.2% DW 0.02% DW 0.6% DW 0.7% DW 0.1% DW 0.05% DW 0.4% DW 0.07% DW

339 291

340 341-343 296 296

-

0.09, 0.32% DW

315 344 298

64

ROBERT VERPOORTE E T A L .

TABLE XVIII OCCURRENCE OF TROPANE ALKALOIDS IN VARIOUS SOLANACEAE PLANTCELLAND TISSUECULTURES A1kaloid

Plant species

Type of culture

Atropine, scopolamine, apoatropine, tropine Scopolamine Atropine, scopolamine Atropine, scopolamine

Scopolia parvgora

Atropine, cuscohygrine Atropine, scopolamine Atropine Atropine Atropine, scopolamine

Calystegia sepium Scopolia carniolica Scopolia straminifolia Lycium barbarum Anisodus acutangulus

Anisodus acutangulus Anisodus acutangulus Scopolia japonica

Yield

Callus Roots Callus Suspension Hairy roots

0.007 DW 0.06% DW 0.02% DW 0.04% DW 1.3% DW

Roots, hairy roots Hairy roots Hairy roots Callus Callus Roots Shoots Suspension

0.25,0.3% DW 0. IS% DW 0.019% DW 0.74% DW 1.1% DW 0.23% DW 0.22% DW 0.05% DW

Ref.

this seems a feasible approach. Studies on the biosynthesis of atropine and scopolamine will result in the isolation of genes which could be candidates for introduction into the plant, for example, the genes responsible for the conversion of l-hyoscyamine to scopolamine, which could lead to plants with a high scopolamine content. In studies on the biosynthesis of these alkaloids at the level of the enzymes involved one has used root cultures (124-126,291,298,366-375). The first step in the conversion of l-hyoscyamine to scopolamine is catalyzed by the enzyme hyoscyamine 6P-hydroxylase (291) (Fig. 8). This enzyme has been isolated and purified

I- hyoscyamine

I

6p - hydroxyhyoscyamine

scopolamine

hyoscyamine 6p-hydroxylase

II 6p - hyd roxyhyoscyam I ne epox idase FIG. 8. Biosynthesis of scopolamine from l-hyoscyamine.

52 345 346 347 348 269 296 296 2 70 349

1.

PLANT BIOTECHNOLOGY

65

from root cultures of Hyoscyamus niger (124-126,367,370). The gene of the enzyme has recently been cloned (124,126).The next step, formation of the epoxide, occurs with retention of the oxygen (366).The responsible enzyme, 6P-hydroxyhyoscyamine epoxidase, has been characterized (371).These results mean that within the next few years transgenic plants containing the genes for these enzymes coupled with various promoters can be expected.

B. PRODUCTION OF TROPANE ALKALOIDS BY CELLCULTURES For the isolation of atropine, Atropa belladonna, Datura species (e.g., D . stramonium and D . innoxia), and Hyoscyamus species are presently being used, which have atropine as the major compound. Hyoscyamus muticus is of interest for industrial-scale isolation, having an average alkaloid content of about 0.035-2.15% (365) in the leaves, with only small amounts of other alkaloids. Duboisia species, small trees whose leaves contain scopolamine as the major alkaloid during a certain period of seasonal development, are the main source for the industrial isolation of scopolamine. The leaves of Hyoscyamus niger and Datura stramonium were widely used in pharmacy, they contain, besides atropine as the main alkaloid, considerable amounts of scopolamine, the ratio of atropine to scopolamine being, respectively, about 2 : 1 and 1.2 : 1. They contain, respectively, 0.1-0.16% (365) and 0.02-0.08% total alkaloid. In Atropa belladonna, atropine is the major alkaloid, with only small amounts of scopolamine; the alkaloid content of the roots is about 0.3-0.7%, that of the leaves 0.2-0.6%. 1. Cell Suspension Cultures For the in uitro production of tropane alkaloids by means of cell cultures numerous studies have been made (see Tables XIII-XVIII). In all cases only low levels of alkaloids, if any, were found in cell suspension cultures, the obvious goal for large-scale production. In some cases alkaloid levels were found to decrease during successive subculturing (297,324,330).Despite efforts to increase yields by varying medium composition and growth conditions, levels remained too low for industrial interest. We therefore do not review this work here. Generally only in case of differentiation of the cells into root-type tissue were increased levels of alkaloids found, whereas shoot cultures did not produce any larger amounts of alkaloids (52,118,277,278,290-292,313,324,325,330). This is in accord with the fact that alkaloid biosynthesis is thought to occur in the roots.

66

ROBERT VERPOORTE E T A L .

2. Hairy Root Cultures Differentiation into root-type tissues is apparently necessary for alkaloid production. Consequently the transformation of solanaceous plants with Agrobacterium rhizogenes, yielding so-called hairy roots which can easily be grown in vitro, was a logical step in efforts to produce tropane alkaloids in vitro (135,268,269,293,296,314-316,319,328,329, 338,347,348). In Table XIX results obtained with Agrobacterium rhizogenes-transformed hairy root cultures are summarized. Generally the Solanaceae cell and tissue cultures follow the alkaloid production pattern as found in the plant. For the production of atropine, Atropa belladonna, Datura stramonium, and Datura innoxia (see Tables XIV and XV, e.g., 291,292,296) cultures are particularly suited. For the production of scopolamine, Hyoscyamus species (see Table XVII, e.g. , 291,292,296,339,363, Duboisia species (see Table XVI, e.g., 291,292, 296), Scopolia species (e.g., 52,296,348), Anisodus acutangulus (349),and some Datura species (see Table XV, e.g., 291,292,296,316) are of interest. The production in Hyoscyamus hairy roots was reported to be stable during at least 10 subcultures (337). Hairy root cultures of Scopoliajaponica were selected for high alkaloid production. Media were optimized for growth and alkaloid production. One cell line, S1, produced scopolamine as the major alkaloid (0.5% of DW); the productivity was approximately 17 mg/liter in 4 weeks. Cell line S22 had atropine as the major alkaloid (1.3% of DW), from which a productivity of about 117 mg/liter in 4 weeks can be calculated (347,348).A similar productivity for atropine was found in Datura stramonium hairy roots (50-100 mg/liter in 4 weeks) (314,317). The Datura roots excreted about 5% of the atropine into the medium; in the stationary phase this increases to about 30% with a concomitant decrease in total alkaloids (317). Datura wrightii was found to produce about 180 mg/liter of atropine, a production which was reached after only 15 days, which is considerably faster than the 45 days after which maximum alkaloid production is achieved in D. stramonium (317). Jung and Tepfer (269) reported that growth of the transformed root cultures was considerably faster than that of normal root cultures; for Arropa belladonna growth rates increased by a factor of 24. In 2-liter fermentors cultures produced 0.02 g/liter/day of biomass (DW). For Calystegia sepium and Datura innoxia the biomass production was even higher: 0.8 and 1.3 g/liter/day, respectively, that is, comparable with plant cell suspension cultures. These cultures were also grown in 30-liter stirred fermentors. The fermentors contained a stainless steel basket to which the roots adhered. The Calystegia culture produced cuscohygrine (7)as major alkaloid (2.3 mg/liter/day) in the 2-liter fermentor. The Atropa belladonna hairy roots produced 0.95% atropine as major alkaloid, and cuscohygrine

TABLE XIX ALKALOID CONTENT OF Agrobacterium rhizogenes-TRANSFORMED HAIRYROOTSCULTURES Plant species Hyoscyamus muticus Hyoscyamus niger Scopolia japonica Atropa belladonna Datura stramonium Atropa belladonna Calystegia sepium Datura innoxia Duboisia myoporoides A. belladonna A. caucasica D . chlorantha D. ferox D. fastuosa D . innoxia D. mete1 D. meteloides D. quercifolia D . rosei D. sanguinea D . stramonium D . stramonium var. inermis D . stramonium var. stramonium D. stramonium var. tatula Duboisia hybrid H . albus H . aureus H . bohemicus H . muticus H . niger Scopolia carniolica Scopolia straminifolia Datura stramonium Hyoscyamus niger Duboisia leichhardtii Datura candida Datura wrightii Datura candida and D . aurea hybrid

Atropine (% DW)

Scopolamine (% DW) -

0.5% -

1.3% 0.3% 0.37% 0.95% Traces 0.86, 1.42% 0.01-0.2% 0.08-0.12% 0.24% 0.06-0.23% 0.13% 0.06-0.22% 0.12-0.31% 0.07-0.085 0.063-0.26% 0.15-0.17% 0.07-0.13% 0.04-0.31% 0.06-0.22%

0.4% -

0.5% 0.024% -

0.09% 1.3% 0.15% ND-0.02%b 0.0049-0.0 14% 0.0035% ND-0.0023% 0.071% ND-0.024% ND-0.0073% 0.008-0.064% ND-0.0025% 0.0015-0.001 9% 0.0006-0.00 14% ND-0.011% ND-0.0041%

Biomass yield (giliteriday DW)U -2 FW -0.16 FW -0.32 -0.12 -0.57 0.02 0.8 -0.57 -

Ref. 33 7 347 348 293 314,317 269

-

328 296 296 296 296 296 296 296 296 296 296 296 296 296

-

0.1 1-0.19%

ND-0.0033%

-

296

0.05-0.4%

ND-0.013%

-

296

0.023% 0.01-0.52% 0.13-0.66% 0.069% 0.046% 0.01-0.3 1% 0.01-0.15% 0.019% 0.07%

0.112% 0.00 13-0.047% O.OOO8-0.062% 0.036% 0.000 1% 0.0019-0.034% ND-0.005% ND 0.56% -

0.11% 0.1% FW 0.02% FW

0.57%

1.8%

0.05% FW

-0.57 -0.36 -0.14 -4 (FW) -5 (FW) -4 (FW)

296 296 296 296 296 296 296 296 315 329 316,319 317

Calculated as biomass increase (glliter) divided by the culture period (days), using in most cases the data as presented in the cited reference. Usually biomass growth is determined over a period of 4 weeks. ND, Not detectable.

68

ROBERT VERPOORTE ET A L .

CH3

CH3

(7) Cuscohygrine

was also present (0.28%). Under the best growing conditions for this culture 5.3 mg/liter/day of alkaloid was produced. Knopp et al. (296) established Agrobacterium rhizogenes-transformed root cultures of 24 tropane alkaloid-producing Solanaceae. For optimal alkaloid production clones has to be selected which combine good growth and high alkaloid production; for example, although Hyoscyamus niger has a high scopolamine content, because of poor growth its alkaloid production per liter (0.5 mg/liter) is lower than that for Atropa belladonna (3 mg/liter). Generally alkaloid production proved to fairly stable during subculturing. Scopolamine production in a line selected from 750 hairy root cultures of Duboisia leichhardtii could reach 78 mg/liter (329). The maximum percentage of scopolamine detected in one of the cell lines was 2.1% (DW). In some cell lines some scopolamine was also found in the medium; however, atropine levels in roots and media were low (0.07-0.82%). The growth index of the roots was found to depend on the inoculum size: the larger the inoculum, the lower the growth index. 3. Large-Scale Culture Although culturing of cell suspensions of the various tropane alkaloidproducing Solanaceae does not seem to offer any particular problems, only for Datura stramonium (304) and Datura innoxia (312) have large-scale cell suspension cultures in bioreactors been reported. Large-scale culture of hairy roots of Atropa belladonna, Datura innoxia, and Calystegia sepium in 2- and 30-liter stirred bioreactors was reported by Jung and Tepfer (269).The C . sepium culture produced 2.3 mg/liter/day of cuscohygrine in a 2-liter fermentor. Atropa belladonna hairy roots cultured in a 2-liter fermentor contained 0.95% (DW) of atropine as the major alkaloid; the biomass production was 0.02 g/liter/day , corresponding to an atropine production of 0.2 mg/liter/day. Immobilization of Datura innoxia cells in calcium alginate has been reported by Brodelius and Nilsson (184).The cells could be permeabilized with 10-25% DMSO. Cells of Atropa belladonna and Hyoscyamus muticus have been immobilized in polyurethane foam (340).Hamilton et al. (294)reported a novel membrane-based culture system for callus and root cultures of Atropa belladonna.

1. PLANT BIOTECHNOLOGY

69

4. Precursor Feeding and Bioconversions

Because of the low alkaloid production in Solanaceae cell cultures, a number of studies have been carried out on the influence of feeding precursors of the alkaloids to the cultures (Table XX). Tropine (8) (376,377) added to Datura innoxia root cultures was mainly converted to its acetyl derivative (9). Stohs (305)reported the conversion of tropine and tropic acid to atropine and scopolamine by suspension cultures of this plant. Tobacco and Datura tatula cell cultures were also found to convert these precursors to atropine and scopolamine. The cultures lost most of their catalytic potential on successive subculturing. This is in agreement with the studies of Romeike and co-workers (376-379) and Hiraoka et al. (380), who found the conversion of tropine to its acetyl derivative to be the major reaction. Hiraoka did find about a 4- to 5-fold increase in alkaloid content after feeding phenylpyruvate, tropic acid, or tropine and tropic acid at the end of the growth phase. Feeding N-methylputrescine, phenylalanine, and tropic acid were reported to increase alkaloid production in Hyoscyamus niger suspension cultures (336); however, the results were quite variable. Tropine feeding only slightly increased alkaloid production. Addition of putrescine resulted in increased scopolamine production in root cultures of Duboisia myoporoides, as did spermidine. A series of related polyamines were also active. Little effect on the production of atropine was found (381).

Romeike (382) reported that several cultures of Datura species grown on 2,4-D were unable to esterify tropine with tropic acid. Only a D . innoxia culture grown on NAA was capable of this bioconversion. Scopolia lurida, Hyoscyamus niger, and Atropa belladonna were incapable of any esterification of tropine. Hiraoka et al. (383) could not confirm these results with their Datura cell lines, but they found that D . innoxia is capable of acetylation of other compounds, such as pseudotropine, scopine, and scopoline, as well. A series of other plants tested, for example, Atropa belladonna and Scopolia japonica, were unable to carry out such an

OR

(8) Tropine, R = H (9) Acetyltropine, R = Ac

TABLE XX PRECURSOR FEEDINGA N D BIOCONVERSION WITH SOLANACEAE CELLA N D TISSUECULTURES ~~

~

~~

Species

Culture type

Compound

Product

Ref.

Datura stramonium Datura innoxia Datura stramonium, Datura tatula, Nicotiana tabacum Datura innoxia Datura metel Datura innoxia, Datura tatula Datura innoxia Datura innoxia, Datura metel, Datura stramonium Duboisia hybrid

Roots Roots Suspension

Phen y lalanine Tr opin e Tropine plus tropic acid

Atropine, scopolamine Acetyltropine Atropine, scopolamine

301 376,377,379 305

Roots Suspension Suspension

Atropine Tropine plus tropic acid Tropine plus tropic acid

Scopolamine Acet yltropine Acetyltropine

386 379 383

Callus Callus Callus Suspension Callus

Tropine plus tropic acid Tropine plus tropic acid Tropine plus tropic acid Tropine plus tropic acid Atropine

382 382 382 382 321

Hyoscyamus niger Datura innoxia

Roots Suspension

Duboisia leichhardtii Duboisia myoporoides Anisodus tanguticus

Roots Callus Shoots Suspension

Atropine Tropine plus tropic acid Pseudotropine Scopine Scopoline Atropine Tropine Tropine Atropine

Datura innoxia

Suspension

Tropic acid

Atropine Acet yltropine Acet yltropine Acet yltropine Scopolamine, 6hydroxyhyoscyamine Scopolamine Acet yltropine Acet ylpseudotropine Acet ylscopine Acet ylscopoline Scopolamine Acetyltropine Butropine 6-Hydroxyhyoscyamine, scopolamine Glucose esters

335 383

324 384 387 389

1. PLANT BIOTECHNOLOGY

71

esterification. Callus cultures of Duboisia myoporoides converted tropine to acetyltropine; shoot cultures of the same plant esterified tropine with endogenous isobutyric acid, yielding butropine (384). Feeding hygrine, tropinone, and tropic acid to suspension cultures of Atropa belladonna did not result in alkaloid production; these precursors were taken up by the cells but were metabolized (385). Root cultures of Datura innoxia were reported to convert atropine, added to the cultures, to scopolamine (386). Non-alkaloid-producing callus cultures of a Duboisia hybrid were shown to be able to convert atropine to scopolamine and 6-hydroxyhyoscyamine (321) (Fig. 8 ) . Root cultures of Hyoscyamus niger are capable of converting atropine to scopolamine, but the opposite reaction was not found (118). Suspension cultures were not capable of either reaction. Yamada and Endo (324) reported that Duboisia Zeichhardtii root cultures converted added atropine to scopolamine; in callus and callus-shoot cultures only very little conversion was observed. A non-alkaloid-producing Anisodus tanguticus cell suspension culture converted added atropine to 6-hydroxyhyoscyamine and scopolamine. The ratio of the two products depended on the time in the growth curve choosen for the feeding of atropine (387). The cell cultures also converted 6-hydroxyhyoscyamine to scopolamine (388).

The steps in the conversion of 1-hyoscyamine to scopolamine have extensively been studied by Hashimoto et al. The first step involves hydroxylation at the 6 position. The enzyme catalyzing this reaction, hyoscyamine 6P-hydroxylase, does not hydroxylate d-hyoscyamine (291). This enzyme activity could be detected in a series of root cultures of Atropa, Datura, Duboisia, and Hyoscyamus species. Indolebutyric acid (IBA), the auxin used to improve growth of the roots, suppressed enzyme activity in the cultures. Hyoscyamine 6P-hydroxylase has been isolated from root cultures of Hyoscyamus niger and further purified and characterized. The enzyme is capable of hydroxylating a number of substrates having tropine moieties, for example, homatropine, benztropine, and phenylacetyltropine; however, tropine itself was not hydroxylated (124,125,367,370). By feeding "0-labeled 6P-hydroxyhyoscyamine, obtained by hydroxylation with hyoscyamine hydroxylase from Hyoscyamus niger, to Duboisia myoporoides shoot cultures, Hashimoto et al. (366) proved that the conversion of this intermediate to scopolamine does not involve a dehydration step as the labeled oxygen was retained in scopolamine (Fig. 8). The responsible enzyme, 6p-hydroxyhyoscyamine epoxidase, has been isolated and characterized from root cultures of Hyoscyamus niger (371).

72

ROBERT VERPOORTE ET AL.

5. Conclusions

So far no biotechnological production of tropane alkaloids has been achieved. Cell suspension cultures do not produce tropane alkaloids at all or in minute amounts only. A major breakthrough was the development of Agrobacterium rhizogenes-transformed root cultures. However, these still have a productivity too low for industrial applications, typically in the range of 0.1-0.2 g/liter. The technological problems of large-scale culture of hairy roots also need further studies. On the other hand, based on the experience gained in studies on in uitro alkaloid production, considerable progress has been made in the field of enzymology and molecular biology of tropane alkaloid biosynthesis. The first genes have been isolated, and others will probably become available in the near future. Genetic engineering of Solanaceae plants does seem to be feasible. This opens some interesting possibilities for transgenic plants or plant cells, (0ver)producing the desired tropane alkaloids, for example, plants producing high levels of only scopolamine.

VI. Isoquinoline Alkaloids Isoquinoline alkaloids represent one of the largest groups of alkaloids. Many pharmaceutically important compounds belong to this group. The production of isoquinoline alkaloids by means of cell cultures has been reviewed by Riiffer (390).Extensive studies of the enzymes involved in the pathways leading to various classes of isoquinoline alkaloids have been made. Zenk (11,121) and Galneder and Zenk (391) have extensively reviewed this work. In the case of berberine the complete pathway has been elucidated at the enzyme level by Zenk and co-workers (11,121,391). Recently the cDNA for (S)-tetrahydroberberine oxidase, the enzyme responsible for the final step in the berberine pathway, has been isolated from Coptis japonica cells, sequenced, and expressed into Escherichia coli (392). By means of antibodies the presence of the protein could be confirmed; however, no enzyme activity could be detected. This is a further step on the long but promising road to the use of genetic engineering for altering isoquinoline alkaloid pathways. ALKALOIDS A. IPECACUANHA The ipecacuanha alkaloid emetine (10) and crude extracts of the ipecacuanha radix (derived from Cephaelis ipecacuanha) are widely used in

1.

PLANT BIOTECHNOLOGY

H N’

73

-

(10)Emetine

pharmacy. The world market was estimated to be about 10-100 metric tons of plant material per year (393).Despite this economic importance, work on cell and tissue culture of this plant is limited. The micropropagation of this tree has been reported by Ideda et a f . (394). Multiple shoots were obtained from node segments, using a Gamborg B5 medium containing 0.01 ppm NAA and 5 ppm 6-benzylaminopurine (BAP). For rooting of the shoots a B5 medium containing 3 ppm IAA gave the best results. Comparison of alkaloids contents in various parts of the regenerated plants with the mother plants showed that they were similar. Jha and Jha (395)produced shoot cultures from nodal explants of seedlings. Up to 12 axillary shoots were obtained per explant using MS medium containing 0.05 ppm NAA, 8 ppm kinetin, and 200 ppm adenine. Shoot cultures could also be obtained from shoot tips on a medium containing 0.1-0.25 ppm NAA and 8 ppm kinetin. Rooting was initiated with 2 ppm IBA. The micropropagated plants all had a uniform diploid chromosome number. Callus, root, and root suspension cultures of C. ipecacuanha were found to contain emetine and cephaeline (396). The root suspension culture had similar alkaloid levels as the mother plant. Highest yields were obtained in a MS medium containing 1 or 3 ppm IAA or 1 ppm NAA. Emetine levels were 0.3-0.5% of dry weight, and cephaeline was present at 1-1.3% of the dry weight of the suspended root culture. Callus cultures only contained trace amounts of alkaloids. The influence of various media constituents on growth and alkaloid production in callus cultures were studied by Jha et al. (397). 2,4-D and NAA were required for callus induction but had detrimental effects on growth. The best growth was observed using IBA and IAA. The highest alkaloid production was obtained on a Schenk and Hildebrandt medium containing 8 ppm IBA, 4 ppm IAA, and 4 ppm NAA. The cephaeline level was 0.93% and emetine 0.35%, based on dry weight.

74

cH30

ROBERT VERPOORTE ET A L .

CH3O

HAc

CH30

.

0

OCH3

(11) Colchicine

B. COLCHICINE Initiation of callus cultures from Colchicum autumnale was first reported by Hunault (398). The production of colchicine (11) and demecolcine by means of callus cultures of C . autumnale was patented by Hayashi et al. (399). Yields obtained were 15 and 0.3 mg/g dry weight, respectively. The influence of media composition on growth of calli, cell suspensions, and colchicine production has been studied in detail (400). Levels of alkaloids markedly decreased on media containing 2,4-D; cell growth was also inhibited by this synthetic auxin. A MS medium containing IBA (0.1 ppm) and kinetin (0.1 ppm) gave best results for growth and alkaloid production. The use of both nitrate and ammonia in the medium as the nitrogen source resulted in improved alkaloid production. Addition of 20-50 mM of sulfate resulted in about a 5-fold increase of colchicine production. Maximum yields of colchicine in callus cultures were 30-40 pg/g fresh weight. Further studies on feeding precursors to the cell cultures showed that addition of a combination of tyramine and p coumaric acid increased alkaloid levels (220 pglg fresh weight) in cell suspension cultures. Feeding demecolcine also resulted in increased colchicine levels in suspension cultured cells (up to 116 pg/g fresh weight) (401).

C. Cephalotaxus ALKALOIDS The slowly growing tree Cephalotaxus harringtonia is the source of cephalotaxine (12)and its esters harringtonine (13), deoxyharringtonine, isoharringtonine, and homoharringtonine. These compounds have shown significant antitumor activity. Production of these alkaloids by means of cell and tissue cultures has been patented (see Table XXI). Delfel and Rothfus (402) found also that callus cultures of this plant did produce the alkaloids, although at much lower levels (1-3% of levels found in the mature tree). About 60% of the alkaloids formed was cephalotaxine, and the esters represented 40%. Deoxyharringtonine could only be found

1.

PLANT BIOTECHNOLOGY

H

75

I OCH3

(12) Cephalotaxine, R = H (13) Harringtonine, R =

in the medium. One new alkaloid, homodeoxyharringtonine, was also detected in the callus cultures. Another strain of C . harringtonia produced a series of unknown alkaloids instead of the known compounds. Media variation, using a multifactorial aproach, did not result in the production of cephalotaxine-type alkaloids (403). Improvement in growth of callus cultures depended on a combination of media components (404). Misawa et al. (405,406)reported on cell suspension cultures of C . harringtonia. Cells grown on a MS medium containing 3 ppm NAA produced only trace amounts of harringtonine and homoharringtonine. Harringtonine, homoharringtonine, and cephalotaxine have been produced by means of callus cultures of Cephalotaxusfortunei (407).

D. BISBENZYLISOQUINOLINE ALKALOIDS The bisbenzylisoquinoline alkaloid tubocurarine would be an obvious goal for biotechnological production. However, to our knowledge no reports on cell cultures of Chondodendron tomentosum, the plant from

TABLE XXI PATENTS ON PRODUCTION OF Cephalotaxus ALKALOIDS BY MEANSOF PLANTCELL AND TISSUE CULTURES U S . SEC of Agriculture. US 4152-214. 07-10-1977-US-840423 (01-05-1979). Preparation of cephalotaxine and related antitumor alkaloid(s) by cultivation of Cephalotaxus harringtonia tissue in nutrient medium. Kyowa Hakko Kogyo KK. J5 7102-194. 17-12-1980-JP-177122(25-06-1982). Cephalotaxine and ester(s) production by sub-iectingplant cells to liquid culture.

76

ROBERT VERPOORTE E T A L .

BG, CH OCH3 30

H3CN,

:

\ /

\

CH3

/

CH O C H ~3~ H3C,

!

\

\

HO

/ N

(14) Aromoline

OH

g/

,

CH3

\

(15) Berbamine

which this alkaloid is isolated, have been published. Other bisbenzylisoquinoline alkaloids, however, have been found in plant cell cultures of other plant species. Akasu et al. (408) isolated aromoline (14) and berbamine (15)from callus cultures of Stephania cepharantha. Root cultures of this plant furthermore produced isotetrandrine, homoaromoline, and cycleanine (409). The two major alkaloids aromoline and berbamine were present in root cultures at 0.64 and 0.55% (of the dry weight), respectively, when grown on B5 medium containing lop6M IBA. Growth was very slow on this medium; the highest production per liter was obtained at a concentration of M IBA. On a modified B5 medium aromoline yields of 2.25% (DW) were found. In a B5 medium containing M IBA, lop6M gibberellin, and 3% sucrose the aromoline content was 1% of the dry weight and the berbamine content 0.5% (410). The high production of alkaloids remained stable for longer periods; however, the ratio aromoline to berbamine increased (411). Berberis cell cultures were also reported to produce bisbenzylisoquinoline alkaloids. Cassels et al. (412) screened 34 callus cell lines (33 species). In all cases the protoberberine alkaloid jatrorrhizine was the major component; in all but 12 cell lines bisbenzylisoquinoline alkaloids were found. High levels of berbamine were found in B. angulosa (0.8% of dry weight) and B. henryana (0.48%). Berbamunine (16) and the new alkaloid 2norberbamunine (17) were the major alkaloids in B. stolonifera cell lines

R

,N

i

\

OH

HO

(16) Berbamunine, R = CH3 (17) 2-Norberbamunine, R = H

‘CH3

77

1. PLANT BIOTECHNOLOGY

(respectively, 0.96 and 0.94% of DW in cell line V29). Furthermore the alkaloids aromoline and isotetrandrine could be identified in a number of cell lines. A cell suspension culture of cell line V29 of B. stolonifera produced about 0.25 g/liter of berbamunine; the highest level was reached in the stationary phase. E. OPIUMALKALOIDS Opium is the source of a series of alkaloids which are used as drugs in modern medicine: codeine (18), morphine (19), papaverine, noscapine, and narceine. Opium is the dried latex collected from the capsules of the opium poppy (Papaver somniferum). In 1986 the estimated legal world production of opium was more than 1000 metric tons. The demand of pure alkaloids was about 660 metric tons of codeine and 200 metric tons of morphine (through Ref. 413). The alkaloids are isolated from opium or poppy plant extracts, which yields an excess of morphine that is chemically converted to codeine. Thebaine (20), from which codeine can be obtained by chemical conversion, is particularly abundant in P . bracteatum. A review on the occurrence of alkaloids in two sections of the genus Papaver was given by Phillipson (414). Preininger (415) reviewed the chemotaxonomy of the genus. Because of their economic interest, biotechnological production of these alkaloids would be of great interest; particularly as it could contribute to reduce the illicit production of opium, the raw material for heroin manufacture. As a result, extensive studies have been made on the application of plant cell and tissue culture for the production of opium alkaloids. Furthermore bioconversion of certain semisynthetic products has been studied. A series of patents in this field has been filed (Table XXII). Various types of cultures of Papaver somniferum and P . bracteatum have been found to be excellent producers of sanguinarine. The production of this alkaloid is dealt with separately (see Section V1,F). Pathways leading to the major alkaloids are summarized in Fig. 9. Several reviews on RC

HC

(18) Codeine, R = CHs (19) Morphine, R = H

(20) Thebaine

78

ROBERT VERPOORTE ET A L .

TABLE XXII PATENTS CONCERNING BIOTECHNOLOGICAL PRODUCTION OF OPIUMALKALOIDS Sankyo K. K. J5 1151-316. 19-06-1975-JA-074961(25-12-1976).Thebaine production by culturing callus from plants of Papaver family, first in medium containing no plant hormone then in medium containing kinetin and/or coconut milk. IRAN MIN Science. US 41 14-314. 22-02-1977-US-770307 (19-09-1978). Fermentative production of thebaine by culture of cells from Papaver bracteaturn Lindl. Population Arya I1 on revised tobacco medium supplemented with agar. F. Constabel, W. G. W. Kurz, and W. H. J. Tam. GB2045243A. 25-02-1980-GB-8006252 (21-03-1979). Codeine production from opium poppy and using culture of cytodifferentiated polyploid cells. Synthelabo. 22-10-1980-FR-022538 (24-04-1982). In vitro metabolite production or biotransformation using plant cells is effected in an unstirred liquid medium without any support for fixation of the cellular tissue. T. Furuya. JP 56 61994. 27-5-1981. JP 79.138575, 26-10-1979. Production of opium alkaloids by tissue culture of Papauer. Chem. Abstr. 95, 112023~. T. Furuya. JS 9159-790-A. 03-03-1983-JP-034869 (10-09-1984). Codeine production using immobilized Papaver somniferum cells and codeinone as raw material can achieve nearly 100% conversion in 3 days.

the plant cell and tissue culture of Papaver have been published (413,416,417).

1. Plant Biotechnology For P . somniferum, which is grown from seeds, micropropagation is only of interest for plant breeding programs. On the other hand, embryogenesis is of interest for future development of synthetic seeds. For both the regeneration from cell and tissue culture is a prerequisite. The first mention of regenerated plants of some Papaveraceae plants from calluses was reported by Ikuta et al. (418). Poppy meristemoids from which different redifferentiated organs were obtained by treatment with different growth hormones were reported by Nessler and Mahlberg (419), Kamo et al. (420), Kutchan et al. (421), and Yoshikawa and Furuya (422). Nessler (423) accomplished the development of somatic embryoids from the meristemoids by removing growth hormones from the medium. The meristemoids developed in a cell suspension culture, grown on MS medium containing 2 ppm 2,4-D and 0.25 ppm kinetin. By placing the embryoids in the light on a solid medium, whole plants could be obtained. Light was needed for somatic embryogenesis (424). A similar procedure was reported by Schuchmann and Wellmann (425). Somatic embryogenesis was obtained by transferring cell cultures of P . somniferum or P . orientale from Gamborg B5 medium containing 2,4-D to a hormone-free medium. Yoshikawa and Furuya (426) regenerated plants from callus cultures of P . somniferum by transferring from a MS medium containing 1 ppm 2,4-D

CH3O

H

O

T OH \

ocH3

S - re t i c u I i ne

R-reticuline

1

1 C H30

C H3O 0 scou Ieri ne

salutoridine

codeinone

cheilanthifoline

stylopine

1

protopine

I

l-0

sanguinorine

FIG. 9. Biosynthetic routes to various isoquinoline alkaloids.

80

ROBERT VERPOORTE ET AL.

and 0.1 ppm kinetin to a medium containing 0.1-1 ppm kinetin under a 16 hr light-8 hr dark regimen. Papaver bracteatum can also be regenerated from callus cultures by conversion to hormone-free media and illumination (427). Czygan and Abou-Mandour (428) used MS medium containing 0.2 ppm IAA and 0.3 ppm kinetin for the growth of P . bracteatum callus. After transfer to hormone-free medium and subculturing in the light, regeneration was observed. The authors also reported the isolation of haploid and diploid (dihaploid?) cell cultures from anthers. Only the latter cell cultures resulted in stable plants. In general it can be concluded that somatic embryogenesis and regeneration of plants form cell cultures is feasible. This opens interesting perspectives with respect to breeding, for example, of high alkaloid-producing plants. Papaver also seems to be an interesting system for further studies of the development of synthetic seeds. 2. Production of Alkaloids by Means of Cell Cultures

a. Morphinan Alkaloids. Numerous papers have been published on plant cell and tissue culture of Papaver species. In only some cases have morphinan alkaloids been detected, and mostly in quite low levels (see Table XXIII). Only in two cases have significant amounts of alkaloids been reported: 0.15% codeine in a cell suspension culture of P . somniferum (429) and 4.7% in a callus and 5.7% in a cell suspension culture of P . somniferum (430). In the latter case all six major opium alkaloids were reported to be present in the cultures, morphine, narceine, and noscapine being the major alkaloids (respectively, 1.6, 1.9, and 1.8%). In callus cultures of P . somniferum it was found that growth hormones affect the total amount of morphinan alkaloids formed. The ratio between the two alkaloids formed, thebaine and codeine, was also dependent on the auxins added (420). Hodges and Rapoport (431) detected morphinan alkaloids by means of radioimmunoassay in callus cultures of P . somniferum. However, after repeated subculturing the callus cultures ceased production of morphinan alkaloids. Heinstein (432) reported a 10- to 20-fold increase in production of morphinan alkaloids in P . somniferum cell suspension cultures after elicitation with sterilized Verticillium dahlia or Fusarium moniliforme conidia, resulting in yields of approximately 4-5 mg/liter of both codeine and morphine. Kamimura and co-workers (433-435)reported extensively on the optimization of growth of callus and cell suspension cultures of P . bracteatum. The cultures contained low levels of thebaine; however, on prolonged subculturing the levels decreased considerably to only trace amounts of alkaloids. By subculturing a cell suspension on an auxin-free medium promoting aggregation, thebaine levels could be increased.

1. PLANT BIOTECHNOLOGY

81

TABLE XXIII PRODUCTION OF ALKALOIDS BY CELLCULTURES OF Papaver SPECIES~ Alkaloid

Plant species

Type of culture

Yield

Ref.

-

433,434 435 441 430,442

Thebaine Thebaine Thebaine Codeine, morphine, thebaine, papaverine, noscapine, narceine Morphine, thebaine, noscapine Codeine Noscapine, narceine Codeine, morphine, thebaine Codeine, morphine, thebaine, papaverine Codeine, morphine, thebaine Codeine, thebaine Codeine, morphine, thebaine Thebaine Thebaine

P . bracteatum P . bracteatum P . bracteatum P . somniferum

Callus Suspension Callus Callus, suspension

4.1, 5.1% DW

P . rhoeas

Callus

0.2,

Thebaine

P . bracteatum

Thebaine Codeine, thebaine Thebaine Thebaine Thebaine Morphine, codeine

443

0.6, 0.4% DW

P . somniferum P . somniferum

Suspension Suspension

P . somniferum

Suspension

P . somniferum

Callus

P . somniferum

Callus

P . somniferum

0.15% DW -

429 444 445

446

Callus

0.02, 0.09, 0.002, 0.0045% DW 0.0035, 0.0012 , 0.0045% FW 0.003% DW

-

Shoot

0.019% DW

420

-

Callus plus roots Meristemoids (nodules) Suspension, root, callus Shoot Callus, suspension, root Root Embryoids Callus, shoot, meristemoids Callus

0.002% DW 0.001% DW

420 420

P . somniferum P . bracteatum P . somniferum P . bracteatum

P . somniferum

43 I

420

438

0.007% DW

-

0.03% DW 0.2% DW 0.000005, 0.0005, 0.00005% FW

438 438 439 425 42 I

436

(continued)

82

ROBERT VERPOORTE E T A L .

TABLE XXIII (Continued) Alkaloid Codeine, morphine Codeine, morphine, thebaine Thebaine Noscapine Thebaine Thebaine Morphine Codeine, thebaine Codeine, thebaine Morphinanes Papaverine, codeine, morphine

Plant species

Type of culture

Yield

Ref.

P . somniferum

Suspension

P . somniferum

Green callus

P . bracteatum, P . somniferum P . somniferum P . somniferum P . bracteatum P . somniferum P . somniferum

Suspension

-

447

Callus Embryoids Callus Callus Callus

448 424 428 449 450

Suspension

0.005% DW 0.06% DW 0.09% DW 0.04, 0.06% DW -

Callus, suspension Immobilized cells

0.0000009, 0.00000004% FW -

463

P . somniferum

P . somniferum

0.15% DW, 0.004 g/liter; 0.14% DW, 0.005 g/liter 0.000001% DW

432 422

450

45 1

" See Table XXV for production of benzophenanthridine and protopine alkaloids

Hutin and co-workers (436,437)reported that usual alkaloid extraction procedures were unable to extract any alkaloid from cell cultures of P . somniferum. By refluxing with hydrochloric acid (1 M , 10 min) or formic acid (3 M , 4 min) morphine and codeine were extracted in detectable amounts. From the results with various types of cultures it can be concluded that the content of morphinan alkaloids increases with the degree of differentiation (e.g., 420422,424,438,425,463).On media inducing root (420,438)or embryo (420,424,425,438)formation in P . somniferum small amounts of morphinan alkaloids could be detected. In P . bracteatum morphinane alkaloids are also produced in root and embryo cultures obtained from non-morphinan-producing cell suspension cultures (438,439).Nessler and Mahlberg (419) reported that root and shoot cultures induced from callus cultures had cells similar to the laticifers in intact plants. Kutchan et al. (421)found a correlation between the occurrence of such laticifer-like cells and production of morphinan alkaloids (thebaine) during cytodifferentiation of cultured cells of P . bracteatum. The cytodifferentiation, characterized by the formation of shoots, was induced by removal of hormones from the culture medium. A correlation was also found in seedlings be-

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tween the appearance of morphinan alkaloids and laticifer cells (440). Differentiation of poppy cell cultures into shoot-forming meristemoids results in the formation of morphinan alkaloids as well (422). Again the presence of tracheary elements and laticifer cells in the calli were thought to be a prerequisite for morphinan alkaloid production. Czygan and AbouMandour (428)reported the occurrence of thebaine in P. bracteatum callus cultures. No laticifer cells could be detected, and no other form of differentiation could be observed. However, alkaloid levels were considerably lower than reported for differentiated cultures containing laticifers (see Table XXIII). Griffing et a / . (463)used a radioimmunoassay for the analysis of morphinans in P. somniferum hypocotyls, callus, and suspension cultures. Low amounts of alkaloid were detected in calli, even though they were found to retain some laticifer-like cells. Nonembryogenic cell suspension cultures did not contain morphinanes, whereas embryogenic cell suspensions did contain small amounts of these alkaloids. For a review of the media used for Papauer cell cultures, the reader is referred to Constabel (416) and Roberts (413). In conclusion, undifferentiated cell cultures of Papauer species do not produce morphinan alkaloids. On differentiation laticifers are formed; this is probably an important factor for the production of morphinan alkaloids, but other, still unidentified, factors also play a role. On the other hand, differentiation is not necessary for the production of sanguinarine (see Section V1,F). b. Nonmorphinan Alkaloids. The first report on callus cultures of P. somniferum was in 1963 (452);several unidentified alkaloids were detected in the extracts. Other early publications (453,454) reported the lack of morphinan alkaloids in callus cultures. Furuya and co-workers (418,455) identified a series of benzophenanthridine alkaloids in Papauer cell cultures. The production of sanguinarine in the Papauer cell cultures is discussed separatedly (see Section V1,F). Khanna and co-workers (430,442)reported the presence of noscapine, narceine, and papaverine in poppy cell cultures. Noscapine was also found by Jawadekar et al. (448). Furthermore protopine (418,455-457),cryptopine (438,455,456,458), and magnoflorine (418)have been identified in P. somniferum cell cultures. In cultures of P. bracteatum protopine and magnoflorine (418) as well as protopine and stylopine (434) have been identified. Morris and Fowler (444) identified noscapine and narceine as the major alkaloids in P. somniferum cell suspension cultures; no morphinan alkaloids could be detected. Lockwood (457)reported the presence of orientalidine and isothebaine in callus cultures of P. bracteatum; these two alkaloids have also been identified in cell cultures of P. somniferum. (459).

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Dopamine, an early intermediate in the pathway to isoquinoline alkaloids, has been found to be present in substantial amounts in cell cultures of P . bracteutum (421,460). Levels of up to 4 mg/g fresh weight were found, which is similar to levels in the latex. 3. Bioconversions The first reported biotransformation by means of cell cultures of P . somniferum was the conversion of thebaine to codeine (453).As P . somniferum cell cultures only produced alkaloids derived from (S)-reticuline (e.g., sanguinarine, Fig. 9) but none of the alkaloids derived from (R)reticuline, Furuya et al. (461) administered (R,S)-reticuline to the cell suspension cultures. After 3 days the alkaloids were isolated. Two alkaloids derived from (8)-reticuline were identified: cheilathifoline and scoulerine. A third alkaloid isolated was identified as pure (R)-reticuline. Thebaine, morphine, and codeine were not metabolized by these cell cultures. However, the cells were capable of stereospecifically reducing codeinone to codeine. Tam et ul. (462) also found the same bioconversion. Furthermore, the conversion of thebaine to neopine was reported. Codeine, neopine, papaverine, and D,L-laudanosoline were not metabolized. Enzymatic reduction of codeinone to codeine was also achieved with cell-free preparations of whole plants of both P . somniferum and P . bracteatum (464). Yeoman and co-workers (108,465) reported the use of in reticulate polyurethane immobilized P . somniferum cells for this bioconversion. Cell-free extracts of the whole plant were shown to be able to convert (R,S)-reticuline to salutaridine (466). The enzymatic conversion of codeine to morphine in isolated poppy capsules was reported by Hsu et al. (467). In addition, cultures of cells in an embryogenic state were able to convert codeine to morphine at a low rate, although the major metabolic products of codeine were N-oxides. (450). Furuya et al. (468) reported the immobilization of P . somniferum cells in calcium alginate. Cells remained viable for 6 months after immobilization. The cells were used in shake flasks and column bioreactors for the biotransformation of codeinone to codeine. The immobilized cells had a higher biotransformation ratio (70%) than suspended cells (61%). Most of the codeine formed was excreted into the medium (88%). The column bioreactor had a lower biotransformation ratio (42%). The cells in the bioreactor operated at 20°C and an aeration rate of 3.75 vvm (volume gas/volume brothlmin) remained catalytically active for 30 days. In a more detailed study on the influence of substrate transport in immobilized cells, it was concluded that limitation of oxygen inside the beads caused deactivation of the cells. However, the reaction rate of the system was not affected by the limitation of oxygen transfer (469). Immobilization of P .

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somniferum cells in reticulate polyurethane foam gave a conversion ratio (79%) for the conversion of codeinone to codeine (108) similar to that reported by Furuya et al. (468) for the calcium alginate bead procedure. The cells could not convert thebaine to codeine.

4. Conclusions So far no production of morphinan alkaloids of any significance has been achieved in large-scale cultures of Papauer species. Several approaches to increase production, such as elicitation and immobilization, have failed. A strategy using molecular biology to eventually increase alkaloid production by means of genetic engineering is hampered by the fact that no morphinan alkaloid production occurs in the cell cultures. Isolation of enzymes to identify the genes responsible for biosynthesis of the morphinan alkaloids has yet to be done, but this is a difficult task. The vast knowledge which is accumulating on the biosynthesis of other isoquinoline alkaloids (391) may contribute to a better understanding of the pathway leading to the morphinan alkaloids. Transformation of Papauer species with Agrobacterium rhizogenes has been proven to be feasible (470), thus opening the way for genetic engineering.

F. SANGUINARINE Although the occurrence of the benzophenanthridine alkaloid sanguinarine (21)in Papauer somniferum cell cultures was reported by Furuya et al. as early as 1972 (459, it was only in recent years that this has gained much interest. The P . somniferum cell cultures have been particularly studied for the production of morphinan-type alkaloids (see above). However, after the introduction of sanguinarine in toothpastes and mouthwashes, because of its antiplaque activity (471),Papauer cell cultures have become an interesting source of this alkaloid. This application of the quaternary benzophenanthridine alkaloid sanguinarine is connected with its antimicrobial activity. This application of the quaternary benzophenanthridine alkaloid sanguinarine is connected with its antimicrobial activity (472,473). A tuberculostatic activity (474), antifungal activity (473,

(21) Sanguinarine, R, + Rz = R3 + R4 = --CHz(26) Chelerythrine, R, = RZ = CH3, R3 + R4 =
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TABLE XXIV PATENTSCONCERNING SANGUINARINE PRODUCTION IN PLANTCELLCULTURES H. Bohm, J. Franke, and L. Lammel. Akademie de Wissenschaften der DDR. Ger. (East) DD 143270, 13-09-1980. Chelidoniurn majus alkaloids. Chem. Abst. 95, 39333~. T. Furuyo, Jpn. Kokai Tokkyo Koho JP 56/61994 [81/61994], 27-05-1981. Production of opium alkaloids by tissue culture of Papauer. Chem. Abstr. 95, 112023~. F. Constabel, W. G. W. Kurz, and U. Eilert. Canadian Patent Application No. 496,984, 5-12-1985. U.S. Patent Application No. 06/889247, 25-7-1986. European Patent Application No. 86309182.3, 25-1 1-1986. Danish Patent Application No. 5762/86, 1-121986. Japanese Patent Application No. 289735/86, 4-12-1986. Semicontinuous production and secretion of phytochemicals by plant cell culture with successive elicitation. Chem. Abstr. 107, 529422.

anti-yeast activity (476,477), and antimicrobial activity against various gram-positive and gram-negative bacteria (476) have been reported for sanguinarine. In recent years interesting plant cell biotechnological studies have been made on the production of sanguinarine by means of plant cell cultures. In Table XXIV the patents concerning the production of sanguinarine in cell cultures are presented. Table XXV summarizes the occurrence of sanguinarine and closely related benzophenanthridine alkaloids. 1. Cell Cultures of Pupuuer Species

a. Growth Conditions. In 1972 Furuya et ul. (455)reported the occurrence of sanguinarine (21)together with the related benzophenanthridine alkaloids dihydrosanguinarine (22), oxysanguinarine (23), and 6acetonyldihydrosanguinarine in callus cultures of Pupaver somniferum. The last mentioned alkaloid was an artifact caused by the isolation method, which included as a solvent acetone, which readily reacts with dihydrosanguinarine. Furthermore the callus culture contained protopine, cryptopine, and magnoflorine. At the time sanguinarine had not yet been isolated from the Pupuuer plant. Subsequent study of callus cultures and redifferentiated plantlets of a series of Papaveraceae species showed that all species studied produced benzophenanthridine alkaloids (see Table XXV), as well as protopine and magnoflorine. The P . bracteatum cell suspension cultures were found to produce the protoberberine-type al-

(22) Dihydrosanguinarine

(23) Oxysanguinarine

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kaloid orientalidine and sanguinarine (457). Kutchan et al. (421) reported that P . bracteatum callus cultures accumulated large amounts of dopamine (0.1-4 mg/g fresh weight) as well as smaller amounts of sanguinarine (10-500 plg fresh weight) and thebaine (0-6 pg/g fresh weight). These three alkaloids occurred in vacuoles of different densities (460).On culturing on hormone-free media, the accumulation of morphinan alkaloids increased with clear organogenesis of the calli. Similar observations were made for P . somniferum and P . orientale (425).After transfer to hormonefree media embryogenesis occurred, with a concomitant shift of alkaloid content from sanguinarine to morphinan-type alkaloids in the case of P . orientale. In P . somniferum the sanguinarine content increased considerably during embryogenesis; thebaine and protopine could also be found during the regeneration. Lockwood (459) reported on the alkaloid content of four Papauer species (see Table XXV). Sanguinarine was the major alkaloid in the cells (0.04-0.07% of dry weight). Other alkaloids found to be present were protopine, isothebaine, and orientalidine; these alkaloids were also found in the media. No morphinan-type alkaloids could be detected. However, after heat treatment (1 day at 36°C followed by 3 days at 5OC) of P . bracteatum cells, small amounts of thebaine could be detected in the medium. Sanguinarine biosynthesis was not affected by this treatment. Hook et al. (485)reported dihydrosanguinarine to be the major alkaloid in cell cultures derived form various strains of P . bracteatum. On a dry weight basis, 1% of this alkaloid was found in the cells, resulting in a yield of 0.178 g/liter culture. The alkaloid accumulation appeared to be growth associated. The occcurrence of dihydrosanguinarine in the cells was confirmed by means of observations with fluorescence microscopy, showing blue fluorescent crystals of the alkaloid in the vacuoles. Sanguinarine and oxysanguinarine were only found as minor compounds. Kutchan et al. (447) studied cytodifferentiation in relation to alkaloid production in P . bracteatum cell cultures. It was concluded that thebaine, sanguinarine, and dopamine accumulated in different sites. Thebaine was found in lacticifer cells; the other two alkaloids were mostly present in other cellular and subcellular compartments. b. Elicitation. The major breakthrough in sanguinarine production was the observation by Eilert et al. that fungal elicitors induced sanguinarine biosynthesis in P . somniferum cell cultures (96,489,490).Of a series of fungal elicitors tested, Botrytis proved to be the most potent inducer, resulting in sanguinarine levels of 2.9% of dry weight of the cells 79 hr after elicitation. No morphinan alkaloids could be detected. Considerable amounts of the alkaloid were excreted into the medium after elicitation (more than 50%). It was suggested that sanguinarine acts as a phytoalexin

TABLE XXV OCCURRENCE OF SANGUINARINE I N PLANTCELLAND TISSUE CULTURES Alkaloid

m 00

Sanguinarine, norsanguinarine, dihydrosanguinarine Sanguinarine, norsanguinarine, dihydrosanguinarine, oxysanguinarine

Sanguinarine Sanguinarine Sanguinarine, chelerythrine Sanguinarine Sanguinarine Sanguinarine, chelirubine, macarpine Sanguinarine

Plant species

Type of culture

Yield

Ref.

Papaver somniferum

Callus

-

455

Papaver somniferum P . setigum, P . rhoeas, P . bracteatum, P . orientale, Eschscholtzia californica, Chelidonium japonicum, Macleaya cordata, Dicentra peregrina, Corydalis incisa, C. pallida Macleaya microcarpa Papaver bracteatum Corydalis ophiocarpa

Callus

-

418

Callus Callus Callus

Macleaya microcarpa Papaver bracteatum Macleaya cordata

Suspension Suspension Suspension

Papaver somniferum, P . orientale

Suspension, after embryogenesis

478 457 479

0.05% FW

0.32% DW

480 42 1 481

425

Dihydrosanguinarine, dihydrochelirubine, dihydromacarpine, dihydrochelerythrine Sanguinarine

00 W

Sanguinarine Sanguinarine Sanguinarine, chelerythrine, chelirubine, chelilutine, macarpine Sanguinarine, dihydrosanguinarine, oxy sanguinarine Sanguinarine Sanguinarine, dihydrosanguinarine Sanguinarine, chelerythrine, macarpine

Eschscholtzia californica

Suspension

1.7% DW, 0.15 g/liter

482

Papaver somniferum, P. bracteatum, P. setigerum, P. nudicaule Papaver somniferum Fumaria capreolata Eschscholtzia californica

Suspension

0.05, 0.05, 0.04, 0.07%DW 2.9% DW, 0.2 g/liter 0.16 g/liter

459

Papaver bracteatum

Callus Suspension

1.11% DW 2% DW, 0.25 g/liter

485

Papaver bracteatum Papaver somniferum

Suspension Suspension

10% DW, 0.02 g/liter 3% DW, 0.2 g/liter

486 487

Eschscholtzia californica

Suspension

0.16% DW

488

Suspension Suspension Suspension

96 483 484

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in Papaver species. Cline and Coscia (486)further elaborated this hypothesis, using cell cultures of P . bracteutum. They found a significant activity of sanguinarine against some plant pathogenic microorganisms. Further experiments on the elicitation, using preparations derived from the fungi Dendryphion penicillatum (pathogenic for Pupaver species) and Verticillium duhliue (a general plant pathogen), were made. The presence of growth hormones in the medium did not effect the induction of sanguinarine. The sanguinarine production was about 0.3 mg/g fresh weight after 168 hr. Most of the sanguinarine was excreted into the medium. Thebaine production in cells grown on hormone-free medium was not influenced by the elicitors. The levels of dopamine (1-2 mg/g fresh weight) in the cells remained unaffected by induction of alkaloid biosynthesis. Dendryphion, the Papaver pathogen, was found to be resistant to sanguinarine, and this was thought to be due to metabolization of the alkaloid. Songstad et al. (491) found that elicitation with a Botrytis homogenate resulted in release of ethylene by P . somniferum cell cultures. However, addition of the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid or the ethylene-releasing agent ethephon did not result in induction of sanguinarine biosynthesis. The release of sanguinarine from the cells into the medium after elicitation was used as the basis for the design of an industrial process for production of the alkaloid (72,99,487,492,493).As the cells of P . somniferum remain viable after a 72-hr exposure to a fungal elicitor, it is possible to recycle the cells after induction of alkaloid biosynthesis. Because about 40-60% of the alkaloid is released to the medium, a simple change of medium is sufficient to collect the alkaloid and to recover viable cells. By a repeating sequence of elicitation and medium replenishment, a semicontinuous production process was obtained. Such a process was also developed for alkaloid production in Catharanthus roseus, Ruta gruveolens, and P . somniferum cell cultures using different elicitors (99). In case of the poppy cell cultures the process consisted of a 7-day biomass production phase, followed by elicitation with Botrytis, and subsequently every 48 hr the medium containing sanguinarine and dihydrosanguinarine (4- 15 mg/liter) was replaced. After the seventh replacement, cells were allowed to recover for 4-10 days, then elicitor was added again (99,493).The medium used was a standard Gamborg B5 medium containing 1 ppm 2,4-D, 0.1% casein hydrolysate, and 2% sucrose. Reelicitation after a 2-week period of medium replacement every 2 days resulted in increased alkaloid production: combined yields of 50,125, and 200 mg/liter total alkaloids were obtained in the collected medium after the first, second, and third successive elicitation, respectively. However, the growth rate decreased, and cells did not survive the fourth reelicitation (487,493). The timing of the elicitation is important, as optimum alkaloid production

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is observed after elecitation at the end of the growth phase. The inoculum density also plays a role. Inoculation at high density resulted in increased growth rates, but the cells showed more browning after elicitation and had lower alkaloid levels. The ratio of sanguinarine and dihydrosanguinarine differed through the elicitation cycles. Using acid hydrolysates of fungal cell walls as the elicitor resulted in less browning of the cells, and dihydrosanguinarine was the major alkaloid under such conditions. As the oxidation of the dihydro derivative to sanguinarine is easy to perform, the ratio of these alkaloids has little significance for the design of the industrial process. The reelicitation process was also studied using surface-immobilized cells (72,494). Papauer cells were grown on loosely wooven polyester fibers attached to a stainless steel frame in a stirred bioreactor. Reelicitation was possible, but only a small percentage of the alkaloids was excreted into the medium. By recycling the medium over an XAD-4 resin, the alkaloids could be collected from the medium, and release from the cells was increased considerably. After selection of a high producing cell line, a yield of 130 mg/g dry weight was reached.

2. Cell Cultures of Eschscholtzia Species The production of benzophenanthridine alkaloids in Eschscholtzia californica callus cultures was first reported by Ikuta et ul. (418) (see Table XXV). Besides sanguinarine and its oxy, dihydro, and nor derivatives, chelirubine (24) was identified. Furthermore protopine and magnoflorine were found to be present in the callus cultures. Berlin and co-workers (482) studied the production of alkaloids in cell suspension cultures. The naturally occurring dihydro derivatives of the benzophenanthridine alkaloids were found to be present in the cultures. These alkaloids are readily oxidized during the isolation procedure; therefore, the alkaloids were also determined quantitatively after mild oxidation with Cr03, yielding sanguinarine, chelirubine (24), macarpine (25), and chelerythrine (26). Dihydrochelirubine was found to be the major compound. On growth media the alkaloid production paralleled growth. In the stationary phase, however, the alkaloid level rapidly declined. High levels of sucrose (8%) increased alkaloid production (up to 0 . 1 2 g/liter). The best results were obtained with an induction medium reported for alkaloid production in Catharanthus roseus cell cultures (495); yields of 146 mg/liter were found, being 4- to 8-fold higher than in the plant. The hormone-free medium had a high sugar concentration (8%) and was low in phosphate. Elicitation of cell cultures of E. californica with Penicillium or Saccharornyces cell wall components resulted in production of a series of quaternary benzophenanthridine alkaloids [sanguinarine, chelerythrine, chelirubine, chelilutine (27), and macarpine, total yield 160 mg/liter] (484).

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(24) Chelirubine, R l + R2 = - O C H 2 0 - , R3 = OCH3,R4 = H (25) Macarpine, R I + R2 = 4 C H 2 ( f , R3 = R4 = OCH3 (27) Chelilutine, R, = R2 = R3 = OCH3, R4 = H

Phytophthora megasperma, an often used elicitor, and chitosan were not active on these cultures. On the other hand, polypeptide antibiotics, like polymyxin B, were found to be the most active elicitors. The yeast extract elicitor was tested on callus cultures of 190 different plant species from 25 families. The strongest effects were found in seven species and subspecies of Eschscholtzia, Chelidoniurn majus, Glaucium rubrum, G .fEava, Corydalis ophiocarpa, and Papaver somniferum, and in all cases the production of the quaternary phenanthridine alkaloids was induced, as easily recognized by the red coloration of the cultures. Collinge and Brodelius (488) studied the formation of benzophenanthridine alkaloids after induction with a yeast elicitor preparation in more detail. The levels of sanguinarine and chelerythrine reached their respective maxima of approximately 0.8 and 1.6 mg/g of dry weight about 6 and 8 hr after elicitation; when these start to decline macarpine levels increase (maximum of 1.1 mg/g dry weight after 60 hr). This parallels the biosynthetic sequence for the formation of macarpine (10,12-dimethoxysanguinarine), for which sanguinarine is a precursor (481). Significant amounts of the alkaloids are secreted into the medium (50-75%). The maximum effect of induction is observed for cells at the end of the growth phase or early stationary phase. Addition of fresh medium 24 hr after elicitation lead to a considerable increase in alkaloid production. The enzyme tyrosin decarboxylase was clearly induced by the elicitor, and the induction was much faster than in case of Thalictrum rugosum (see below) (496,497).As sanguinarine is not the major alkaloid in these cultures, the system is less suited for industrial production purposes. However, very rapid induction of biosynthesis and subsequent release to the medium are interesting features which should be further explored. Chitosan was also able to induce alkaloid production; Brodelius et al. (498) described a method to optimize the induction procedure with this elicitor, avoiding permeabilization of the cells. Byun et al. [see reference in Brodelius (111)l developed a two-phase system for the production of sanguinarine. A dimethylsiloxane polymer,

-

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an antifoam fluid, used in the second phase resulted in a 3-fold increase in alkaloid production. Elicitation in such a system increased the alkaloid production to 700 mg/liter. 3. Cell Cultures of Macleaya Species Experiments with Macleaya plant cell and tissue culture were reported as early as 60 years ago (see Ref. 499 for a review). In 1967 the occurrence of sanguinarine and allocryptopine in callus cultures of Macleaya cordata was noted (500,501). Koblitz et al. (478) reported on these alkaloids in M . microcarpa callus cultures. The colorless alkaloids allocryptopine and protopine were the major alkaloids, and the levels of the colored sanguinarine varied. Norsanguinarine, sanguinarine, dihydrosanguinarine, oxysanguinarine, and chelirubine were reported to be present in callus cultures of M. cordata. The major alkaloid norsanguinarine occurred only in trace amounts in plantlets; protopine and allocryptopine, on the other hand, were more abundant in the plantlets than in the callus cultures (418). By means of selection based on pigmentation, callus lines with high or low alkaloid production could be obtained (502). The colors of the cells were found to be due to either sanguinarine or carotenoids. Even cell lines colored by carotenoids were found to have increased alkaloid production. Yields of up to 1% of dry weight could be reached. The alkaloid contents of a cell line had some correlation with the percentage (ranging from 0 to 7.8%) of alkaloid-bearing cells. Such cells may contain intracellular crystals of sanguinarine, recognized by their typical fluorescence. In cell suspension cultures most of the alkaloids are excreted into the medium (480).

Lang and Kohlenbach (503) studied the differentiation of alkaloid cells in mesophyll protoplasts from M . microcarpa and M . cordata. In cell clusters which developed from protoplasts, cytodifferentiation into alkaloid cells and tracheary elements was observed. It was concluded that the alkaloid cells themselves produced the accumulated alkaloids. 4. Cell Cultures of Other Plant Species

Sanguinarine and related alkaloids were detected in a series of Papaveraceae plants (see Table XXV) (418). In the 11 species studied the alkaloid contents of callus cultures were very similar, and the redifferentiated plantlets had more characteristic alkaloid patterns. Further plants reported to produce sanguinarine among other compounds are Corydalis ophiocarpa (479) and Fumaria capreolata (483). None of these plants, however, has been further studied in connection with large-scale production of sanguinarine, probably because in most cases complex mixtures of alkaloids are formed.

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5. Conclusions The production of sanguinarine by means of plant cell cultures is feasible. The semicontinuous process with P. sornniferurn cells seems very interesting. However, to come to an industrially feasible process a considerable increase in alkaloid production will be necessary. In the literature a total production of 375 mg/liter culture medium is reported, obtained in a period of three cycles of 21 days each. The production per unit time is thus rather low , for example, compared with berberine production (504,505). Other plant cell cultures producing sanguinarine always produce a series of other alkaloids as well. The isolation of sanguinarine thus requires additional purification steps after the biotechnological production. Further screening and selection for high producing cell lines, or even genetic engineering, should be considered for further optimization of the systems described so far.

G. BERBERINE The quaternary isoquinoline alkaloid berberine (28) is widely used in Asia as a drug. Among other maladies, diarrhea, dysentry, cholera, and eye infections are indications for which berberine or plant extracts containing this alkaloid are applied, because of the antimicrobial activity of berberine (472,506). Berberine interacts with DNA, but this does not seem to be related to its antimicrobial activity (506). Berberine contracts also uterine muscle and is used to stop uterine bleeding. Furthermore berberine has an anti-inflammatory effect (507). In Japan berberine and in particular extracts of dried rhizomes of Coptisjaponica Makino var. dissecta (Yatabe) Nakai (Ranunculaceae) are widely used as a stomach tonic. The biosynthesis of protoberberine alkaloids, including berberine, has been extensively studied, and all the enzymes of the biosynthetic pathway have been characterized (11,121,391,508). Interestingly the pathway leading to berberine in Berberis was found to be different from that in Coptis and Thalictrurn (509). In the former species berberine is formed from columbamine, in the latter plant species from tetrahydroberberine (121). The production of isoquinoline alkaloids, including the protoberberine alkaloids, by plant cell cultures have been reviewed by Ruffer (390) and Ikuta (510). Table XXVI summarizes patents concerning the production of berberine by means of plant cell cultures. In Table XXVII a summary is given of the occurrence of berberine in some plant cell cultures. 1 . Cell Cultures of Berberis Species The first report on the occurrence of protoberberine alkaloids in plant cell cultures is that of Reinhard (511), concerning callus cultures of Ber-

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beris species. Hinz and Zenk (538) reported jatrorrhizine (29) as the major product in cell suspension cultures of several Berberis species, berberine being only a minor component. The yield of total protoberberine alkaloids was highest in case of Berberis stolonifera, namely, 1.72 g/liter. Breuling et al. (524) studied the culture of Berberis wilsoniae cells in 20-liter airlift bioreactors. The cell culture was capable of producing 3 g alkaloids per liter medium. Maximum berberine concentrations occurred during the lag phase. During the exponential growth phase and stationary phase columbamine and jatrorrhizine became the major constituents. Jatrorrhizine reached levels of 10% of the dry weight. High oxygen tension (50% saturation in the medium) and aeration rates were necessary to obtain maximum alkaloid production. A high producing B. wilsoniae cell culture has also been used as a source for the isolation of the enzyme (S)-tetrahydroprotoberberine oxidase (STOX) (539).This enzyme plays a role in several steps in the biosynthesis of isoquinoline alkaloids. Several methods were used for immobilization of the enzyme, and best results were obtained with controlled pore glass. In immobilized form STOX was found to be more stable (50 times better than the free enzyme). The immobilized STOX was used for the production of (R)-norreticuline from racemic norreticuline. In a cyclic process the racemic mixture was run through a column with immobilized STOX. Only the (S) form is oxidized, yielding the dehydronorreticuline. After passing through the column the reaction mixture is reduced with sodium borohydride, converting the dehydro compound to a racemic mixture of norreticuline. After the first cycle a RIS ratio of 75:25 was reached, and after six cycles the ratio was 97.5:2.5, which is close to the theoretical value (99.2:0.8). 2. Cell Cultures of Coptis Species Callus cultures of Coptis japonica were reported by Furuya et al. (512) as a source of berberine. In a subsequent study the presence of jatrorrhi-

(28) Berberine, R, + Rz = --CH2-, R3 = R4 = CH3 (29) Jatrorrhizine, R1 = H, RZ = R3 = R4 = CH3 (30) Coptisine, R1 + Rl = R3 + R4 = 4 H 2 (31) Palmatine, R1 = R2 = R3 = R4 = CH3

96

ROBERT VERPOORTE ET AL. TABLE XXVI PATENTS CONCERNING BERBERINE PRODUCTION IN PLANT CELL CULTURES

1972 T. Furuya and T. Ishii. Meiji Confectionary Co., Ltd. Japan. Kokai JP 47130897 [72/ 308971, 10-11-1972. Callus of Coptis japonica produced berberine. After extraction, berberine could be obtained pure by means of chromatography. Chem. Abstr. 78, 82201~. 1975 H. Kuroda and T. Ikekawa. Kanebo, Ltd. Japan Kokai JP 50/13519 [75/13519], 13-021975. Berberine and palmatine were produced by callus cultures of Phellodendron amurense. The alkaloids were purified by means of column chromatography on alumina. Chem. Abstr. 82, 167671~. 1976 A. Okamoto and Y. Sawa. Kanebo, Ltd. Japan Kokai JP 51112993 [76/12993], 31-01-1976. Callus cultures of Phellodendron amurense produced berberine and palmatine after light treatment in a medium containing only mineral salts. Yields of 0.65% of berberine chloride were claimed, which was about 3-4 times higher than untreated cultures. Chem. Abstr. 84, 176894t. 1977 Kanebo KK. J5 2003-816. 24-06-75-JA-078833 (12-01-77). Berberine type alkaloid production from cell-fused callus of plants, having stomachic, antifungal, antiinflammatory, and antitumor activities (Coptis, Phellodendron, and Hydrastis species). Sumitomo Chemical. KK 557144-992. 03-03-81-JP-030992 (07-09-82). Berberine alkaloid production by culturing calluses from Tinospora (Menispermaceae), separating alkaloid(s), mainly palmatine and/or jatrorrhizine. 1985 Mitsui Petrochem Ind KK. 56 0227-673. 26-04-84-JP-082952(12-11-85).Selection method for Thalictrum minus L. var. hypoleucum (T. thunbergii DC.) cultured cells producing enhanced amounts of berberine. Mitsui Petrochem Ind KK. 56 0227-691-A. 26-04-84-JP-082953(12-10-85). Manufacturing of berberine by immobilized cultured cells of Thalictrum minus L. var. hypoleucum Miq. (T. thunbergii DC.). M. Tabata, K. Nakagawa, A. Konagai, and H. Fukui. Mitsui Petrochemical Industries. Jpn. Kokai Tokkyo Koho JP 60/227690 A2 [85/227690], 12-11-1985. Production of berberine with Thalictrum minus plant cell cultures, yields of O.lg/liter claimed. Chem. Abstr. 104, 128223h. 1986 T. Suzuki and Y. Hara. Mitsui Petrochemical Industries. Jpn. Kokai Tokkyo Koho JP 61/285988 A2 [86/285988], 16-12-1986. Berberine containing plant cell cultures are screened for antimicrobial activity on Bacillus cereus, a berberine-sensitive microorganism. Chem. Abstr. 107,21936~. Y. Motoyama, N. Kihari, T. Ishida, and K. Kato. Seitai Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyu Kumiai. Jpn. Kokai Tokkyo Koho JP 62125991 A2 [87/25991], 3-021987. Extraction procedure for the isolation of berberine using alcohols in combination with HCI or HN03. Berberine could be crystallized from the crude extracts. Chem. Abstr. 107, 20966a.

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zine as a further major alkaloid in such cultures was described (514). Fukui et al. (517) reported the presence of berberine (7.4% DW), coptisine (30) (1.0% DW), palmatine (31) (3.1% DW), and jatrorrhizine (3.5% DW) in Coptis cell suspension cultures. In recent studies the occurrence of other alkaloids in the high berberine-producing cell cultures is not mentioned. The rather high level of alkaloids found in these cultures led to further studies on the production of berberine by means of plant cell cultures. Berberine is usually isolated from Coptis roots. This plant needs to grow 5-6 years to yield a rhizome weighing 1-2 g (dry weight) containing about 8% berberine, making them an expensive commodity (520). Plant cell cultures thus seems an interesting alternative. For a review of plant cell tissue and organ culture of Coptis with regard to plant breeding, the reader is referred to Ikuta and Itokawa (540). The first step in these studies was to select rapidly growing, high producing strains of Coptis cells. Yamada and Sat0 (516) described a method to obtain fast-growing cells from adventitious roots. A high degree of aeration in the culture flasks was found to stimulate growth and berberine production (see below), whereas light had an opposite effect. The alkaloid levels observed were similar to those found in the roots. In a subsequent study Sat0 and Yamada (22,521) reported screening for high producing strains. The screening was performed at the level of cell aggregates; selection pressure, achieved by adding tyrosine to the medium, was also used to obtain higher producing strains. It was found that repeated cloning was needed to obtain cell lines with a stable high production of berberine. The highest producing cell line originated from a strain which produced only moderate amounts of berberine; in other words, one does not necessarily have to start with high producing cell lines in the cloning procedure. Cytometric analysis of the berberine contents of cells from high and low berberine-producing lines showed that the percentage of alkaloidproducing cells was increased in high producing strains, with the level of alkaloids per individual cell being similar in high and low producing strains. A similar observation has been made for anthocyanins in Catharanthus roseus cell cultures (542). To obtain stable high producing cell lines three to four cloning steps were needed (537).By comparison of cell lines obtained from single protoplasts, Yamada and Mino (527) observed that instability of chromosomes and berberine production occurs; however, there did not seem to be a correlation between chromosome variation and alkaloid production. The cell culture conditions greatly influence production. Yamamoto and Tomimoro [see reference in Ikuta and Itokawa (540)l optimized the medium for growth and alkaloid production of callus cultures. Yamada and Sato (526)found that good aeration is an important factor. Cell cultures in

TABLE XXVII OCCURRENCE OF BERBERINE IN PLANT CELL AND TISSUECULTURES

%

Type of culture

Alkaloid

Plant species

Berberine Berberine Berberine Berberine, jatrorrhizine Berberine Berberine Berberine, coptisine, palmatine, jatrorrhizine Berberine Berberine, jatrorrhizine, palmatine, coptisine, columbamine Berberine, palmatine, jatrorrhizine, coptisine, columbamine Berberine

Berberis species Coptis japonica Phellodendron amurense Coptisjaponica Phellodendron amurense Coptis japonica Coptis japonica

Callus Callus Callus Callus Callus Suspension Suspension

Thalictrum minus Mahonia japonica

Callus Callus

Argemone mexicana

Berberine

Coptis japonica

Nandina domestica

-

Callus, suspension Suspension

Yield 0.06% DW

Ref. 511 512 513 514 515 516 517

0.9, 1% DW 0.65% DW 3.9% DW 7.4% DW, I .67 g/liter; 1.0, 3.1, 3.5% DW 0.67% DW -

518 519

-

519

0.03,0.05% DW

520

13.2% DW, 1.39 g/liter

21,521

W

w

Berberine Berberine Berberine, jatrorrhizine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine Berberine

Nandina domestica Thalictrum minus Berberis wilsoniae Thalictrum minus Thalictrum minus Coptis japonica Tinospora cordifolia Thalictrum rugosum Thalictrum minus Coptis japonica Thalictrum rugosum Thalictrum minus Thalictrum jlauum T. dipterocarpum Coptis japonica Coptis japonica Nandina domestica Thalictrum minus Coptis japonica Coptis japonicu

Callus Suspension Suspension Suspension Suspension Suspension Suspension Suspension Immobilized cells Suspension Suspension Suspension Suspension Suspension Suspension Suspension Callus Immobilized cells Suspension Suspension

0.6% DW 0.8 g/liter 3 giliter 0.8 g/liter 0.67 g/liter 5 4 % DW 0.035% DW 4.5% DW, 0.5 giliter 0.25 g/liter 7 g/liter 2% DW, 0.4 g/liter 0.875 g/liter 0.3 g/liter 0.4 g/liter I .66 g/liter 7% DW, 0.8 g/liter 0.55 g/liter 3.5 g/liter 1.5 g/liter

522 523 524 525 526 527 528 98 529 59 530,531 504 532 532 533 534 535 536 505 537

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flasks closed with aluminum foil, known to be subject to poor gas exchange (67), showed lower biomass yields and alkaloid production compared with cell cultures grown under cotton or silicone sponge plugs. The medium itself also influences production; White’s basal medium proved to be best for growth and alkaloid production. Best results were obtained with sucrose as the carbon source, 3% being the optimum concentration for growth and berberine production (21). For optimum production a single medium could be used, avoiding the need for a two-stage process often advocated for the production of secondary metabolites. A further increase in berberine yield could be obtained by using media with a high copper concentration (534). Using a LS medium, a 10-fold increase in copper sulfate concentration to 1 p M resulted in a 20-30% increase in berberine yield, without inhibition of cell growth. Hara et ul. (533) reported that addition of small amounts gibberillic acid (10-8-10-5 M) resulted in lower starch concentrations in the cells and a concomitant increase of berberine production of about 30%. In all media for Coptis cell cultures, 10 p M NAA and 0.01 p M BAP were included as growth hormones. Although product yields exceeding 1 g/liter could be obtained with cell suspension cultures of Coptis juponica, as described above, this was still not sufficient to come to an industrially feasible process. In order to further improve productivity, growth at high cell concentrations was studied. The constraints of such high density cultures were found to be achieving adequate agitation without cell destruction, maintaining a sufficient supply of oxygen, and providing a suitable supply of nutrients (59). In an ordinary batch culture a biomass density of 30 g dry weightlliter could be reached; by using a fed-batch mode this could be increased to 55 g/liter with an alkaloid yield of 3.5 g/liter (60). By adjusting aeration and stirring, Matsubara et al. (60) were able to further increase the density to 75 g/liter (dry weight), obtaining an alkaloid content in the cells of 10%. The 2.5-liter bioreactor used in the experiments was stirred with a hollow-paddle stirring wing, and oxygen-enriched air was supplied by a sparger at the bottom of the bioreactor. A critical element in high density cultures is the supply of the nutrients. This problem was solved by using a fed-batch type of culture or, even better, by a perfusion in which part of the medium is replaced continuously. In this way high density cultures were possible without loss of berberine production (542). Berberine yields of 7 g/liter have been claimed in such high density cultures using the copper-enriched LS medium (59); the highest yield for a product from plant cell cultures ever reported.

3. Cell Cultures of Thalictrum Species Coptis cells accumulate the alkaloids in the vacuoles, which means that the berberine must be extracted form the cells. However, for large-scale

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production it would be advantageous if the alkaloids could be extracted from the medium. Therefore, much attention has been paid to cell cultures of Thalictrum species (Ranunculaceae) (543).The first report on the occurrence of berberine in Thalictrum minus L. var. hypoleucum Miq. was from Ikuta and Itokawa (518). Nakagawa et al. (523) selected cell suspension cultures of this plant which produced considerable amounts of berberine, most of which was excreted into the medium (almost 90%). The alkaloid crystallized as its nitrate or chloride salt. Additional amounts of alkaloid could be recovered from the medium by passing it through a column of XAD-2, from which the alkaloid could be desorbed by washing with methanol. Yields were in the range of 400-800 mg/liter (12.1% of DW). Minor alkaloids identified in the medium were magnoflorine and thalifendine. In a subsequent study (526) it was found that growth and optimum alkaloid production could not be obtained in one medium. A two-stage process was suggested as the most effective, involving a growth medium containing only 2,4-D as growth hormone, followed by an alkaloid production medium containing a high concentration of NAA and BAP. The excretion (and uptake) of berberine by the Thalictrum cells was found to be an active, energy requiring, and selective process (86,544).About 90% of the berberine was excreted by the cells, whereas only 10% of the minor alkaloid magnoflorine was excreted. The high berberine production was found to be a stable trait of the Thalictrum cells (545).On the other hand, no correlation could be found between alkaloid content of the plant and cell lines derived therefrom, and in fact the highest berberine-producing cell line was obtained from a plant with a low alkaloid content. High production in a cell line did show a positive correlation with excretion. Depending on the cell line, figures of 23400% release of berberine into the medium were found. A study of alkaloid production in T. flauum and T. dipterocarpum showed that these species were capable of producing berberine. Only the latter species excreted the alkaloid during the exponential growth phase, but in the stationary phase berberine was taken up again. Suzuki et al. (546,547) reported a bioassay for the screening of high berberine-producing cell lines. Small cell aggregates (1 mm) from cell suspension cultures were plated on a solid medium, and after 2-3 weeks the clones obtained were cut into 3 mm segments. These were grown for 2 weeks on small agar cylinders. The clones were then subcultured again, and the agar pieces into which berberine was excreted by the cells were tested for their antimicrobial activity against Bacillus cereus. This was done by placing the agar cylinders on a solid medium which, after 18 hr at 25"C, was inoculated with bacteria. The inhibitory zones observed enabled an estimation of the excreted berberine. Cell lines of Thalictrum minus with a productivity exceeding 0.5 g/liter could be obtained in this way. However, the high productivity was not stable; after several subculture

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cycles the productivity returned to 0.1-0.2 g/liter, that is, the productivity before the selection procedure. Immobilization in calcium alginate beads allowed the continuous production of berberine (529). No growth occurred in the beads, and by optimizing the medium similar yields per unit volume of medium could be obtained as for cell suspension cultures. A bioreactor was described that was specifically devised for the production of berberine with the immobilized cells. A further improvement of this liquid-gas two-phase system, in which the beads are alternately soaked in the medium for 30 sec and exposed to air for 2 min, resulted in berberine yields of 875 mglliter. The bioreactor could be operated in a semicontinuous mode in which alkaloid production had a constant value of 50 mg/liter/day during the entire culture period of 60 days (504). An ample oxygen supply was shown to be crucial for high alkaloid production in both cell suspensions and immobilized cells (536). Based on this finding the size and cell density of the calcium alginate beads was optimized, resulting in a 1.5-fold increase of berberine yield. The production of berberine by Thalictrum rugosum cell suspension cultures was studied by Funk and co-workers (98,496,497),in particular after induction with a yeast-derived elicitor preparation. Untreated cultures produced approximately 0.2 g/liter berberine; after elicitation of a cell culture in the late exponential growth phase, productivity was around 0.5g/liter. The enzyme tyrosine decarboxylase, which channels tyrosine into the berberine biosynthetic pathway, was found to be induced by the elicitor in the late exponential and early stationary phase. A good correlation of activity of this enzyme with alkaloid biosynthesis was found (496,497,548).The use of an elicitor thus results in a considerable increase of production in the same culture time, To eventually come to commercially interesting levels, Funk et al. (98) suggested screening for high producing cell lines. Piehl et al. (530)reported growth and alkaloid production of T . rugosum cell suspension cultures in a novel membrane-stirred bioreactor. The bubble-free aeration through the porous membrane fibers in this type of bioreactor has the advantage of lack of foaming and flotation, problems which were observed with this cell line in a standard stirred fermentor. Even during long cultivation periods of more than 2 months, the membranes were not clogged by cell adhesion. In a 21-liter bioreactor a biomass density of 50 g/liter dry weight could be obtained. The major alkaloids produced by this cell line were columbamine and berberine; magnoflorine was only found during the stationary phase. Alkaloid yields were in the range of 0.3-0.4 g/liter. By transferring cultures to hormone-free media or to media containing NAA instead of 2,4-D, alkaloid production could be

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increased 20-40%, particularly magnoflorine production. Transfer of the cells to a phosphate-free medium led to increased alkaloid production, and the alkaloids were excreted into the medium (531). Older cells, in which the phosphate pools were depleted, showed the highest levels of excretion. In all cases, after several days on the phosphate-free medium reuptake of the alkaloids was observed. In contrast to T. minus cells, which excrete only protoberberine alkaloids but not magnoflorine, T . rugosum cells released all alkaloids. After this treatment regrowth of the cells could be obtained on the normal growth medium. Exploitation of alkaloid release for production purposes requires further studies with respect to timing of the induction of alkaloid biosynthesis, secretion of the alkaloids, and regrowth of the cells. Electroporation has been shown as a possible way to permeabilize T. rugosum cells; however, in obtaining complete release of the alkaloids, the cells lost their viability (549). The use of various chemical agents for permeabilization (chloroform, DMSO, Triton X- 100, hexadecyltrimethylammonium, phenethyl alcohol) gave similar results. At concentrations necessary to release berberine from the vacuole, the cells did not grow anymore after the treatment (87). Kin et al. (550)reported that light suppressed the secretion of berberine, whereas it increased alkaloid production and improved the growth of T. rugosum cell cultures. Alkaloid production was also increased by addition of ethephon, an ethylene precursor, to the cell cultures (551). 4. Conclusions

The development of plant cell biotechnological production of berberine by Coptis cells seems so far to be quite successful. Yields have been improved from a meager 2.4 mg of berberine hydrochloride isolated from 3.8 g of callus (0.06% of DW) (512)to 7 g/liter (10% of DW) (59),that is, an increase of more than 150 times the alkaloid levels in the cells. Several aspects can be pointed out that have contributed to this. First, all callus cultures induced do produce the desired product. Further, because of the yellow color of the alkaloid subsequent selection is easy to perform visually. Finally, the alkaloid production of cell cultures proved to be stable after repetitive selection. The other promising system, Thalictrum cell cultures, have the major advantage of excretion of the alkaloids. Selection has been quite successful, and cell lines with an alkaloid content 350 times higher than the original plant have been obtained (543). In this case stability of high producing cell lines might be a problem. On the other hand, the possibilities of induction of alkaloid production by elicitors offer further ways of improving production. As berberine is mainly found in the roots of both plant species

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mentioned, the use of Agrobacterium rhizogenes-transformed hairy root cultures for the production of berberine seems to be a further possibility which could be exploited.

VII. Cinchona Alkaloids

Of the various pharmaceuticals derived from plants, the Cinchona alkaloids are probably, by volume the largest market, with an estimated production of 300-500 metric tons a year of pure quinine (32)and quinidine (33).These alkaloids are extracted from the bark of Cinchona trees, which require about 10 years to mature before harvesting. Furthermore most of the plantations are in areas not easily accessible, often threatened by infections with Phytophthora cinnamomi. This leads to many uncertainties in planning of the production, and as a result alternative sources for the alkaloids are of interest. Various synthetic aproaches have been used (552) but are not of industrial interest. Therefore, interest in biotechnological approaches is large. Patents related to the production of quinoline alkaloids by means of plant cell cultures are summarized in Table XXVIII. A. PLANTBIOTECHNOLOGY Cinchona, as a seed-propagated outbreeder, exhibits much variation in characteristics, including alkaloid content of the bark (553,554). The micropropagation of high producing varieties is thus of great interest. Several authors reported methods for the succesful in uitro propagation (555-560). Hunter (553) reviewed earlier literature and reported a routine method for micropropagation. By culturing shoots from both shoot tips and nodal explants on a MS medium containing IBA, BAP, and gibberellin (1 ppm each) as growth hormones and phloroglucinol (100 ppm) to prevent

(32) Quinine, R = OCH,

(33) Quinidine, R = OCH,

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TABLE XXVIII PATENTAPPLICATIONS CONCERNING PLANTCELL,TISSUE,AND ORGAN OF Cinchona CULTURE Akad. Wissenschaft DDR. DD-205-184-A. 26-04-1982-DD-239293(21-12-1983). Cinchonu alkaloid production by cell or callus culture giving production free of plant material. H. Koblitz, D. Koblitz, H. P. Schmauder, D. Groger, and W. Inn. DDR Patentschrift 214,523 (1984). I n uitro Verfahren zur Bewurzelung von Pflanzen der Gattung Cinchona. I n uitro procedure for rooting of plants of the genus Cinchona ACF Chemiefarma NV. NL8700061 13-01-1987. Differentiated plant cell structures used for the production of alkaloids.

browning of the tissues, 10 or more explants could be obtained in 42 days from the starting plant material. Subculturing for more than 50 passages was possible. Plants were obtained by hormone dipping of the shoots and implantation in nonsterile peat; 83% of the implants gave roots. The rooting procedure lasted 6 weeks. The method was successfully tested on 14 seedling-derived plantlets and shoots of 4 different mature trees. Germplasm storage was feasible with shoot cultures by combining a low temperature (6 or 12°C) and a high osmolality (4% mannitol). Cryopreservation of callus was also feasible, but the risk of mutation makes this less suitable for germplasm storage. Storage of meristems at low temperature was not successful (553).

B. ALKALOID PRODUCTION BY CELLCULTURES 1. Growth Conditions Extensive studies have been made on production of quinoline alkaloids by means of cell cultures of Cinchona species, namely, C. ledgeriana, C. pubescens ( = C . succirubra), and C. robusta (for reviews, see Refs. 554,561-563). However, this has not lead to high producing cell suspension cultures. In Fig. 10, the approximate levels of alkaloids found in various types of cultures are summarized (564-573). Alkaloid production apparently increases with the degree of morphological differentiation. Root organ cultures (564,566) do produce alkaloids; however, the growth of such cultures is too slow for further biotechnological applications. Slowly growing cell aggregates have also been found to produce considerable amounts of alkaloids (570,571). Hoekstra et al. (573) found that by manipulating the growth hormones in the medium, compact globular structures (CGS) developed in Cinchona cell cultures. The CGS clearly showed a pattern of differentiation similar to that found in the bark of the tree, having a cambial zone, xylem, and phloem. As in the tree, the outer layers

106

ROBERT VERPOORTE ET A L .

I

Quinoline alkaloid coztent in mg/g dry weight

Differentiation

0.001

0.01

0.1

1.o

Fine cell suspensions1 - 1 Flne cell suspensions grown on solid media

4-

Callus culture Root organ suspension culture

Shoot culture

-

Cornpact globular structures

FIG. 10. Relationship between degree of differentiationand alkaloid productionfor Cinchona plant cell and tissue cultures.

of the CGS contained the alkaloids. Levels exceeding 1% of dry weight were observed in CGS. A disadvantage of this system is the slow growth. Payne et al. (49) reported alkaloid production in a Cinchona ledgerianu cell culture transformed with Agrobucterium tumefaciens. Although alkaloid production was 5-fold higher than in non-transformed cell lines, it is still too low (6.7 mg/liter) for commercial interest. White light was found to inhibit alkaloid production; green and red light resulted in production similar to that observed in dark-grown cultures. The growth rate of the transformed culture was rather low. Infection of shoots of C. ledgerianu with Agrobacterium rhizogenes resulted in a hairy root culture (574). Compared to hairy root cultures of other plants, however, the culture had a very low growth rate. The culture produced quinoline alkaloids, quinamine, as well as a number of other unidentified indole alkaloids. The amount of alkaloids formed was rather low: 50 pg/g fresh weight, 45 days after inoculation on fresh medium; this was lower than the A. tumefacienstransformed culture (270 pg/g FW). Although very low levels of alkaloids are produced by the Cinchona cell cultures, they are capable of producing considerable amounts of anthraquinones, particularly after elicitation with fungal elicitors. The anthraquinones are thought to act as phytoalexins in this plant genus (575). Anthraquinone production could be stimulated by adding polymeric adsorbents like Amberlite XAD-7 to the medium. A production rate of 20 mg/liter/day could be obtained in this way (576).

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2. Large-Scale Cultures

Several studies have been made of large-scale growth of Cinchona cell cultures. Schmauder and co-workers (577-579) reported the successful growth of C. pubescens cells in a 3.5-liter airlift fermentor. Strong acidification of the medium was observed in the bioreactor, and pH control in the fermentor resulted in strong growth inhibition. Growth of C. ledgerianu cells in a 7-liter airlift bioreactor was reported by Allan and Scragg (580,581). The growth rate in the bioreactor was higher than that in shake flasks (doubling time of 4.8 and 6.14 days), respectively. In addition, the lag phase was shorter in the bioreactor. The final dry weight obtained was 6.8 g/liter; the viability of the cells was high until1 the stationary phase was reached, when it dropped rapidly to less than 10%. The alkaloid production was very low: 0.009% quinine on a dry weight basis. In a study of the shear sensitivity of various plant cell cultures in a 3-liter fermentor stirred with a turbine impeller, it was found that at higher stirring rates (1000 rpm) the viability of cells decreased compared to the normal stirring rate of 250 rpm. Tobacco and Catharanthus roseus cells were not affected under these conditions (69). 3. Immobilized Cells and Excretion of Alkaloids Efficient immobilization of C. pubescens cells is possible in a semirigid matrix of polyurethane foam particles (107,162). The influence of immobilization on alkaloid production has not been studied. Should one be able to increase alkaloid production, it would be of interest to promote excretion of the alkaloids into the medium, in order to facilitate downstream processing. Parr et al. (82) reported that C. pubescens and C . ledgeriana cells are capable of taking up and releasing quinoline alkaloids in a nonselective way. Multiphasic kinetics were found, indicating that different mechanisms might be involved in the accumulation process. Ion trapping and alkaloid binding were among those mentioned as possible factors. Blom and van Vliet (582) found that the initial uptake rate of quinine and cinchonamine were linear over a wide range of concentrations, indicating a diffusion-based mechanism for uptake. Further experiments supported an ion-trap mechanism as the driving force for the accumulation of the alkaloids. Permeabilization of C. ledgeriana cells to promote release of alkaloids is possible with DMSO. However, for complete release of the alkaloids DMSO levels of 20% are necessary, and at these levels cells are severely damaged, hampering cell recovery after treatment (583).Finally, cell cultures might excrete up to 70% of the alkaloids into the medium (561).

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Addition of XAD-7 to the medium did not stimulate total alkaloid production, but most of the alkaloids produced were excreted (SO-90%) (42). Hairy root cultures released only about 1% of the alkaloids into the medium (574). C.

BIOCONVERSIONS

None of the precursors in the biosynthesis of quinoline alkaloids is available at a price lower than the alkaloids; thus, bioconversion is not of interest, except for one step. This step would be the stereospecific conversion of quinidinone (obtained by oxidation of quinine or quinidine) to quinidine (SR,9S)or quinine (SS,9R).This reaction offers the possibility of converting an excess of one of the naturally occurring stereoisomers to the other. It is now performed chemically as the demand for quinidine is higher than production from plant extracts. Screening of about 450 organisms by Ray et al. (584) resulted in one organism capable of the reduction of quinidinone to quinidine. The organism was identified as the yeast Hansenula anomala var. schneggii. The plant enzyme responsible for reduction of the ketone function was isolated by Isaac et al. (585). This cytosolic enzyme, cinchoninone: NADPH oxidoreductase, was present in two isoforms. They both catalyze reversible reactions; isoenzyme I was found to be specific for nonmethoxylated alkaloids, and isoenzyme I1 had a broader substrate specificity, being able to reduce methoxylated, hydroxylated, and unsubstituted quinolines. The ratio of the stereoisomeric alkaloids as found in the plant (SS, 9R : SR,9S) was not thought to be governed by this enzyme alone. The application of this enzyme for stereospecific reduction of the oxidized quinoline alkaloids needs further study. Feeding of tryptophan to Cinchona cell suspension cultures has been reported to result in considerable production of alkaloids (572,577,578). However, other authors reported that tryptophan inhibits the growth of the cell cultures and is converted, probably nonenzymatically , to norharmane and related compounds, and no increase in alkaloid production could be observed (586,587).

D. CONCLUSIONS Cinchona can be considered to be recalcitrant with regard to cell and tissue culture. Although it has been possible to obtain cell and tissue cultures of some Cinchona species, they often require special treatments; also, growth is usually slow, and viability of cells is rapidly lost. Moreover, the cell cultures are poor producers of alkaloids. In order to arrive at

1.

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biotechnological procedures for the production of the quinoline alkaloids, more must be learned about the regulation of alkaloid biosynthesis. This will lead to possible strategies for genetic engineering. For this one might think about influencing the regulation of biosynthesis in Cinchona itself. Another possiblity is the introduction of the final specific steps in the biosynthesis leading to quinoline alkaloids into another plant capable of producing strictosidine, the intermediate for a variety of indole alkaloids and the quinoline alkaloids. Transformation of Cinchona plants with Agrobacterium proved to be difficult as it does not activate the bacterial virulence genes (0. Goddijn, Biotechnology Delft Leiden, unpublished results). For genetic engineering of this plant further studies are needed.

VIII. Indole Alkaloids A number of terpenoid indole alkaloids have pharmaceutical interest. These alkaloids are isolated from plants belonging to the families Apocynaceae, Loganiaceae, and Rubiaceae. For the production of alkaloids by means of plant cell cultures, plants of the latter two families have proved to be rather recalcitrant (e.g., see Cinchona alkaloids). On the other hand, it has been reported by Pawelka and Stockigt that all apocynaceous cell suspensions they studied did produce terpenoid indole alkaloids (588). Here we confine ourselves to alkaloids which have direct commercial interest; the production of new, potentially interesting, compounds is not reviewed here. For this we refer the reader to reviews by Balsevich (589), van der Heijden et al. (tribe Tabernaemontaneae) (590), and Omar (Rhazya stricta) (591). A. Catharanthus ALKALOIDS Several important alkaloids have been isolated from Catharanthus roseus plants: ajmalicine (34) and serpentine (35)from the root, and the dimeric alkaloids vinblastine (36)and vincristine (37)from the leaves. Therefore, much research has been done on production of these alkaloids by means of plant cell culture. Catharanthus roseus is, in fact, one of the most widely studied plants for the production of secondary metabolites in cell culture systems. We here discuss the two types of alkaloids separately. Table XXIX summarizes patents concerning the production of alkaloids by means of cell cultures of C. roseus.

110

ROBERT VERPOORTE ET AL.

(34)Ajmalicine

(35) Serpentine

1. Ajmalicine and Serpentine

The pharmacological significance of ajmalicine stems from its use in the treatment of hypertension and obstructive circulatory diseases. Serpentine and ajmalicine can easily be transformed into each other by means of, respectively, simple reduction or oxidation. The yearly production of ajmalicine is estimated to be about 3600 kg (2).Ajmalicine and serpentine are the major alkaloids found in cell and tissue cultures of C. roseus. Both are representatives of corynanthean-type indole alkaliods, of which more than 25 different compounds are isolated from C. roseus cultures (592). Elucidation of the biosynthetic pathway leading from tryptamine and secologanin, the basic precursors of terpenoid indole alkaloids, to ajmalicine, lPepiajrnalicine, and tetrahydroalstonine was reviewed by Zenk (593)and Verpoorte (594).As both ajmalicine and serpentine are produced in cell cultures, most research has involved improving yields to commercially interesting levels. We here discuss various approaches to meet this goal.

a. Screening and Selection of High Producing Cell Lines. A rigorous study on the selection of high producing C. roseus cell lines was performed by Zenk and co-workers (20). First high producing plants were selected.

OH

oc-

CH3O

R (36)Vinblastine, R = CH3 (37)Vincristine, R = CHO

0

111

1. PLANT BIOTECHNOLOGY TABLE XXIX PATENTSCONCERNING Catharanthus ALKALOIDS IN PLANTCELLAND TISSUECULTURES~

1978 M. H. Zenk (Boehringer Mannheim, C3.m.b.H). Ger. Offen. 2,639,876 09-03-1978 Appl. 04-09-1976. Ajmalicine and serpentine from in vitro cultures. Chem. Abstr. 89, 3386d. 1981 P. Brodelius, M. H. Zenk, B. Deus, and K. Mosbach. Eur. Pat. Appl. 22,434 14-01-1981, Swed. Appl79/5,615,2706-79. Catalysts for the production and transformation of natural products having their origin in higher plants. Chem. Abstr. 94, 162739r. 1982 V. Petiard and D. Yvernel (Synthelabo SA). Fr. Demande FR 2,492,404 23-04-1982, Appl. 80/22,538,22-10-1980. Production or biotranstransformation of metabolites by plant cells in uitro. Chem. Abstr. 97,54020~. A. Rosevear and C. A. Lambe (U.K. Atomic Energy Authority). Eur. Pat. Appl. EP 62,457,13-10-1982, GB Appl. 81/10,421,02-04-1981. Biological production of chemical compounds (ajmalicine and serpentine are excreted in growth regulator-free medium, after placing the cell in a bag in this medium). Chem. Abstr. 98, 33079j. 1983 Y. Miura. Jpn Kokai Tokkyo Koho JP 58,201,982,25-11-1983, Appl. 82/85,228,19-5-1982. Production of vinblastine by a callus culture. Chem. Abstr. 100, 137404. 1984 U. Pfitzner and M. H. Zenk (A. Nattermann und Cie, G.m.b.H). Ger. Offen. DE 3,234,332,22-03-1984, Appl. 16-09-1982. Bond, stabilized, highly pure strictosidine synthase and its use in the synthesis of 3a (S)-strictosidine. Chem. Abstr. 101,3050f. 1986 F. Constabel, W. G. W. Kurz, and U. F. K. Eilert (Canadian Patent and Development, Ltd). Eur. Pat. Appl. EP 226,354,24-06-1987, CA appl, 496,984,05-12-1985. Semicontinuous production and secretion of phytochemicals by plant cell cultures with successive elicitation. Chem. Abstr. 107, 1529422. H. Kamata (Kyoura Hakko Kogyo Co, Ltd). Jpn Kokai Tokkyo Koho JP 61,274,694, 04-12-1986, appl, 85/118,644, 31-05-1985. Production of “Vinca” alkaloids (production of ajmalicine, vincristine, and catharanthine by A. rhizogenes-transformed cultures). Chem. Abstr. 107,234818~. A. Rosevear and S. D. Roe (U.K. Atomic Energy Authority). Ger. Offen. DE 3,616,357, 20-1 1-86,GB Appl. 85/12,438, 16-05-1985. Secondary metabolite manufacture using immobilized cells and affinity chromatography in a continuous flow process (continuous flow serpentine production by immobilized cells; the culture medium was recirculated through a C18cartridge). Chem. Abstr. 107,57438f. 1987 A. Rosevear, I. Hislop, C. A. Lambe, S. D. Roe, and A. H. Reading (U.K. Atomic Energy Authority). Br. U.K. Pat. Appl. GB 2,180,554, 1-4-1987, GB appl. 85/23,328, 20-09-1985. Biochemical reactor (production of serpentine in a flat biochemical reactor, i.e., a sheet of polyurethane foam). Chem. Abstr. 107, 13261111. J. I. Smith, N. J. Smart, and M. Misawa (Mitsui Petrochemical Ind., Ltd.). PCT Int. Appl. WO 88 00,968, 11-02-1988, U.S. Appl. 892,938,04-08-1986. Process for inducing secondary metabolite production in plant tissue culture and means thereof (use of osmotic stressors and plant growth regulators). Chem. Abstr. 109, 532508. (continued)

112

ROBERT VERPOORTE ET AL. TABLE XXIX (Continued)

Mitsui Petrochemical Ind., Ltd. Jpn Kokai Tokkyo Koho JP 63 119,690, 24-05-1988, U.S. Appl. 893,018,04-08-1986. A VLB preparation with peroxidase of C. roseus or other heme-containing catalysts in the presence of a peroxide. Chem. Abstr. 110,55994~. Nonalkaloid Patents 1985 H. Tanaka and M. Yajima (Asama Kasei KK). Jpn. Kokai Tokkyo Koho JP 60,180,594 14-09-1985, Appl. 84/334,440, 27-02-1984. Release induced by using high ionic strengths (extracellular release of 5’-phosphodiesterase from living plant cells). Chem. Abstr. 104, 18489%. 1987 M. Yokoyama (Shiseido Co., Ltd.) Jpn Kokai Tokkyo Koho JP 62,44,174, 26-02-1987, Appl. 85/185,438,23-08-1985. Arbutin manufacture by callus cultures: bioconversion of hydroquinone to arbutin by callus cultures. Chem. Abstr. 107, 17436%. M. Yokoyama Y. Fujinuma, and T. Asahara (Shiseido Co., Ltd.). Jpn. Kokai Tokkyo Koho JP 62,181,795, 10-08-1987, Appl. 86/25,177,07-02-1986. Alkylhydroquinone-P-Dglucoside manufacture with tissue cultures. Cliem. Abstr. 108,4705e. 1988 T. Matsumura, M. Kikuma, E. Fukuzaki, Y. Miyamoto, and Y. Hashimoto (Nitto Electric Industrial Co., Ltd.). Jpn Kokai Tokkyo Koho JP 63 87,989, 19-04-1988, Appl. 86/ 235,085,2-10-1986. Manufacture of vegetable oils rich in triglycerides by tissue cultures of C. roseus. Chem. Abstr. 110, 1134528 Several other patents concerning C. roseus, for example, various extraction methods, have been omitted.

From plants with an alkaloid content of more than 0.7% on a dry weight basis, callus cultures were initiated. From the callus cultures, cell suspension cultures were derived. Subsequently the cell line with the highest productivity was plated on alkaloid production medium containing 0.8% agar. The petri dishes were incubated for a period of 2 months to allow small cultures to develop. All calli from a randomly selected area were then collected and a portion analyzed by means of a specially developed radioimmunoassay (RIA). With the RIA technique specific and quantitative determination of 0.1 to 0.5 ng of ajmalicine and serpentine in crude tissue extracts was possible. The alkaloid content of the calli expressed as a percentage of biomass dry weight varied from 0 to 1.4% in the case of serpentine and from 0 to 0.8% in the case of ajmalicine. The calli with the highest alkaloid production were selected and transferred to liquid medium for the initiation of cell suspension cultures. In this way two high producing cell suspension cultures were obtained. On a specially developed production medium one strain reached a cell dry weight of 20 g/liter and a serpentine content of 162 mg/liter, whereas no ajmalicine was produced. The other strain reached a cell dry weight of 26 g/liter, a

1. PLANT BIOTECHNOLOGY

113

serpentine content of 77 mg/liter, and an ajmalicine content of 264 mg/ liter. Constabel et al. (595) analyzed 76 cell clones derived from one leaf of a C . roseus plant. Although this work shows the variation in alkaloid spectra of cell lines derived from a single leaf, the variation was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants. The majority of clones showed the occurrence of Corynanthe, Strychnos, and Aspidosperrna alkaloids. Of the 76 clones analyzed, 41 produced ajmalicine. Schallenberg and Berlin (27) selected several 5-methyltryptophanresistant cell lines from wild-type cells of different C . roseus cell suspension cultures. This resulted in increased tryptophan synthesis in all cell lines, and up to 30 times the normal levels of free tryptophan were detected. Increased tryptophan production did not, however, result in higher levels of tryptamine nor indole alkaloids. In an attempt to delineate the variability of serially cultured callus and cell suspension cultures derived from three cultivars of C . roseus obtained from highly uniform explants, Kurz et al. (596) screened several hundred cultures from anthers of buds at identical developmental stages. The total alkaloid content of the cell suspension cultures varied from 0 to 1.5% of dry weight. The relative amounts of alkaloids produced were found to be fairly constant for each culture and appeared to be cell line specific. Brown et al. (23)performed screening at the cell and protoplast level by using flow cytometry. Sorting rates of 1000 protoplasts/sec could be maintained, with about 70% remaining intact. In this way cells with a high content of the blue fluorescing serpentine were selected. Via addition of the fluorescent pH probe 9-aminoacridine a positive correlation was detected between vacuolar acidity and serpentine content. However, the authors mentioned several problems which were encountered with this method. Total cell fluorescence should be corrected for cell size if concentration is the quality under study. Second, net fluorescence is the product of fluorochrome concentration and quenching, either of which may cause the heterogeneity apparent in the population. Third, protoplasts retain only some of their serpentine content; therefore, flow cytometry with individual cells is preferable. b. Culture Medium Modifications. Plant growth regulators. As plant growth regulators play an important role in the regulation of cell division and differentiation, much attention has been paid to the influence of the concentration of various growth regulators on secondary metabolite production. A summary of the literature data on this subject is given in Table XXX.

TABLE XXX INFLUENCEOF PLANTGROWTH REGULATORS ON ALKALOID PRODUCTION IN Catharanthus roseus CELLCULTURES

Alkaloid produced

Biomass alkaloid content (mdg DW)

Total alkaloid yield (mg/liter)

Rate of production (mg/g/day)

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Catharanthine Ajmalicine Catharanthine

10.2 3.0 1.5" 0.3" 0.07" 0.07" 0.02" 0.02" 0.72" 0.98"

264.0 77.0 20.9 3.6 2.1 2.1 0.9 25.6 35

0.16 0.03 0.01 0.01 0.01 0.01 0.24 0.33

0.0 0.10

0.0 1.1

0.0 0.003

c L

P

Medium Zenk

Growth regulator 1 y M IAA, 6 y M BAP None

MS (599) + 8% sucrose MS + 8% sucrose

2 + M 2,4-D

MS

1.O mg/liter NAA, 0.1

MS B5 (600) + 2% sucrose

mg/liter kinetin 1.O mg/liter NAA, 0.1 mg/liter kinetin, 8.3 mg/liter ABA 1.O mg/liter 2,4-D, 0.1 mg/liter kinetin

Ajmalicine Serpentine

1

.o

Ref. 20 48 48 47 47

598

B5

+ 2% sucrose

None

MS

+ 2% sucrose

None

Sucrose, 8% MS

+ 2% sucrose

"(601) + 2% sucrose LS (191) + 2% sucrose B5 + 2% sucrose L + wl

+ 2% sucrose MS + 2% sucrose B5

None 0.1 mg/liter IAA, 2.0 mg/liter kinetin, 0.1 mg/liter 2,4-D 2.0 mglliter IAA, 0.2 mg/liter kinetin 0.18 mg/liter IAA, 1.1 mg/liter BAP 1.O mg/liter IAA, 0.1 mg/liter kinetin 1.O mg/liter NAA, 0.1 mg/liter kinetin 1 .O mglliter NAA, 0.1 mg/liter kinetin ~

a

~~~

The fresh weightldry weight ratio is assumed to equal 15.0.

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine

0.31 0.40 0.40 1.5 0.0 1.2 0.010 0.40

2.9 3.8 4.0 15 0.0 4.8 0.15 6.0

0.058 0.044 0.048 0.078 0.0 0.01 0.003 0.013

598

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine

0.19 0.40 1.2 3.0 0.80 2.3 1.7 2.4 1.5 5.6

1.7 3.6 15 37 12 33 20 29 18 67

0.003 0.02 0.057 0.26 0.028 0.12 0.12 0.22 0.34 0.52

598

598 598 598

598 598 598 598

116

ROBERT VERPOORTE ET A L .

Zenk e f al. (20)obtained the highest serpentine production in cultures by the addition of 1 p M IAA and 5 p M BAP to a specially developed induction medium. Knobloch et al. (48) showed that the addition of 2 p M 2,4-D to MS medium supplemented with 80 g/liter sucrose reduced ajmalicine production by a factor 23. Courtois and Guern (55) reported that the serpentine content of cultures increased from 0.005 to 4% on a dry weight basis after lowering the 2,4-D concentration of the B5 medium from 1.O to 0.01 pM or when 2,4-D was replaced by 0.5 pM NAA. Majerus and Pareilleux (597) observed a 6-fold increase in ajmalicine and serpentine content after a second transfer of the cells to B5 medium devoid of 2,4-D. After a third transfer to this medium, however, the cells died after a short period of poor growth. Morris (598) also examined the effect of 2,4-D on alkaloid production. The serpentine concentration on transfer to the production medium devised by Zenk et al. (20) which was supplemented with various 2,4-D concentrations (0.01,O. 1 , and 1.0 mg/liter) was followed. It appeared that serpentine yield decreased, whereas the specific growth rate increased with increasing 2,4-D concentrations. However, serpentine yield was always higher than observed on Gamborg B5 medium containing l .O mg/liter 2,4-D and a sucrose concentration of 5.0%. This indicates that, as observed by other authors, 2,4-D inhibits alkaloid production. A second conclusion that can be drawn from these experiments is that inhibition by 2,4-D may not be the only factor suppressing alkaloid formation in cells cultivated on B5 medium. Another eight induction media claimed to stimulate alkaloid production were tested. The final conclusion drawn by Morris is that the major factor which appears to control alkaloid production in these media is the level of 2,4-D. A stimulating effect of the addition of abscisic acid (ABA) on ajmalicine and catharanthine production in nine different cell suspension cultures of C. roseus is reported by Smith et al. (47). The response depended on the cell line, the concentration and source of the ABA, and the growth phase at which the cells were treated. Maximum ajmalicine production rate was 10 mg/liter/day after the addition of ABA to the culture. Carbon source. A number of studies have been conducted on the influence of the carbon source (kind of carbon source and concentration) on cell growth and alkaloid production (see Table XXXI). In order to enhance the productivity of high producing cell suspension cultures of C. roseus further, Zenk et al. (20) increased the sucrose concentration of their induction medium from 50 up to 80 and 90 g/liter. This resulted in alkaloid yields of 140 and 153%, respectively, of the yield obtained at a sucrose concentration of 50 g/liter. The biomass yield decreased, however, to 80 and 70%, respectively, of the value obtained at a sucrose concentration of

1.

117

PLANT BIOTECHNOLOGY

TABLE XXXI INFLUENCE OF CARBON SOURCE ON ALKALOID PRODUCTION IN Catharanthus roseus CELLCULTURES

Medium Zenk

Water

Water

4% glucose 2% sucrose 5% sucrose 0.23 M sucrose 0.23 M glucose 0.23 M maltose 0.23 M fructose 0.23 M galactose 0.23 M lactose

10.2 3.0 3.0 2.8 3.6 0.4 0.4 0.5 0.3 0.3 0.2

264.0 77.0 60.0 50.0 80.0 -

4% sucrose 6% sucrose 8% sucrose 10% sucrose 12% sucrose

Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine

0.2 0.3 0.6 0.5 0.1

-

2% sucrose

Ajmalicine Serpentine Ajmalicine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine

0.01 0.0025 0.1 0.01 0.14 0.30 0.35 0.35 0.35 5.24 9.02 9.24 8.05

0.28 0.10 2.8 0.39

6% sucrose B5

MS

Alkaloid produced

Total alkaloid yield (mg/liter)

Ajmalicine Serpentine Serpentine Serpentine Serpentine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine

5% sucrose

MS

B5

Carbon source

Biomass alkaloid content (mg/g DW)

2% sucrose 4% sucrose 6% sucrose 8% sucrose 10% sucrose 2% sucrose 4% sucrose 6% sucrose 8% sucrose

-

1.1

5.1 7.2 7.9 7.0 44.7 145.2 149.7 163.0

Rate of production (mg/g/day) 0.17 0.18 0.20 -

Ref. 20 602

36

-

-

36

-

-

0.0005 0.0001 0.005 0.0005 -

604

598

0.21 0.29 0.17 0.16

605

50 g/liter. Literature data on the influence of the carbon source on ajmalicine and serpentine production are given in Table XXXI. Doller (602) examined the effect of both glucose and sucrose at concentrations between 20 and 60 g/liter. The highest serpentine production occurred when sucrose was used at a concentration of 50 g/liter. Knobloch and Berlin (36) examined the influence of both the concentration and the

118

ROBERT VERPOORTE E T AL.

composition of the carbon source on the growth and the production of ajmalicine, serpentine, and total phenolics of a cell suspension culture of C. roseus. Following a 1 : 10 dilution of a cell suspension culture with a 0.23 M sugar solution, alkaloid production was comparable when either glucose, sucrose, or maltose was used. The use of fructose, lactose, or galactose resulted in significantly lower alkaloid production. The highest alkaloid production was found when the culture was diluted 1 : 10 with an 8% sucrose solution. The influence of phosphate and sucrose on the production of serpentine under steady-state conditions was examined by Balague and Wilson (603). It was found that alkaloid synthesis continued during phosphate but not during sucrose limitation. This interesting result indicates that alkaloids can be produced by growing C . roseus cultures, depending on the growthlimiting nutrient. The course of tryptamine, ajmalicine, and serpentine accumulation in C. roseus cells grown on B5 medium supplemented with either 20 or 60 g/liter sucrose was followed by Merillon et al. (604). Tryptamine accumulation was comparable at both sucrose concentrations and had a shape similar to the growth curve. Ajmalicine and serpentine accumulations were low at a sucrose concentration of 20 g/liter. The accumulation of both alkaloids was enhanced greatly when the sucrose concentration was increased to 60 g/liter. Production occurred, however, mainly at the stationary phase, after approximately 12 days of culture. Comparable results were reported by Morris (598).It was shown that the standard Gamborg B5 maintenance medium resulted in only low yields of serpentine, whereas no ajmalicine was produced. The cultures were maintained in continuous diffuse light. When the sucrose concentration of the medium was increased to 60 g/liter, a 3-fold increase in serpentine yield was observed. A further increase of the sucrose concentration did not enhance serpentine production. At sucrose concentrations of 100 g/liter, biomass yield decreased. On transfer of the cells from the maintenance medium to the production medium devised by Zenk er al. (20) ajmalicine was accumulated in excess of serpentine, but toward the end of the culture period (22-48 days) ajmalicine levels declined to zero while serpentine continued to accumulate during the entire culture period. It should be noted that the choice of parameters used to measure the influence of the nature and concentration of the carbon source on alkaloid production is of great importance. This was illustrated by Scragg er al. (605) when they determined three different parameters to characterize serpentine production of cell suspension cultures of C. roseus grown on MS medium supplemented with 20, 40, 60, and 80 g/liter of sucrose. The parameters were maximum serpentine content of the biomass (mg/g dry

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weight), maximum serpentine content of the culture broth (mg/liter), and serpentine productivity (mg/liter/day). The serpentine content of the biomass was highest at a sucrose concentration of 60 g/liter, the serpentine content of the culture broth was highest at a sucrose concentration of 80 glliter, and the productivity was highest at a concentration of 40 g/liter sucrose. Growth and biomass yield can also be expressed using different parameters, for example, dry weight, fresh weight, and cell number. In this case as well the choice of parameter might influence results. The conclusion can be drawn that the production of ajmalicine and serpentine can be enhanced by increasing the concentration of the carbon source by a factor 2 or 3 relative to standard growth media. This might result in an other medium component becoming the growth-limiting factor. The effect of the composition of the carbon source on alkaloid production, however, remains less clear. Phosphate and other major medium salts. The concentrations of phosphate and other medium salts have also been varied in order to increase alkaloid production in C. roseus cultures (Tables XXXII and XXXIII). Knobloch and Berlin (36) examined the influence of the concentration of various medium salts (e.g., nitrate, ammonium, and phosphate) on the growth and the production of ajmalicine, serpentine, and total phenolics in a cell suspension culture of C. roseus. The highest alkaloid production was obtained when the culture was diluted 1 : 10 with an 8% sucrose solution. When the culture was diluted with an 8% sucrose solution supplemented with either the phosphorus source of the growth medium (1.2 mM KH2P04) or the nitrogen source [18.9 mM KN03 and 20 mM (NH4)N03], alkaloid production was greatly reduced, whereas the addition of calcium or magnesium salts had no effect. Likewise (48) the effect of omission of the phosphorus and/or nitrogen source of the MS medium supplemented with 80 g/liter sucrose was studied. Phosphate removal had a negative effect on alkaloid production. When, in addition, the nitrogen source was also omitted, the serpentine content of the biomass was higher than in the control culture. Biomass yield and total alkaloid content per liter of culture broth were, however, lower. The omission of both ammonium and nitrate from the medium also had a negative effect on alkaloid production. Total alkaloid production as well as specific ajmalicine production were highest when both the phosphorus and the nitrogen source were present in the culture medium. In a later paper the influence of phosphate was further examined (606).It was reported that the phosphate present in the culture medium was taken up completely within 48 hr. At initial phosphate concentrations of 0 to 1.O mM the accumulation ajmalicine and serpentine was negatively correlated with the phosphate concentration of the medium. In this concentra-

120

ROBERT VERPOORTE ET A L .

TABLE XXXII INFLUENCE OF PHOSPHATE CONCENTRATION ON ALKALOID PRODUCTION IN Catharanthus roseus CELLCULTURES

Medium 8% sucrose

Induction medium

MS + 0.5% sucrose MS + 0.9% sucrose B5 + 3% sucrose

Phosphate concentration (mmol/liter)

Alkaloid produced

0.0 1.2 0.05 0.10 0.20 0.50 1.o 2.0 1.25b

Ajmalicine Ajmalicine Ajmalicine and Serpentine

Biomass alkaloid content (mg/g DW)

Rate of production (mg/g/day)

Serpentine

7.2" 0.6" 7.5" 5.1" 4.5" 1.1" 0.6" 0.4" 0.0

0.72 0.06 0.75 0.51 0.45 0.11 0.06 0.04 0.0

0.25'

Serpentine

0.11

0.018

0.0

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine

0.030 0.003 0.023 0.004 0.022 0.003 0.017 0.004 0.012 0.006 0.008 0.005 0.002 0.004

0.0043 0.0004 0.0033 0.0006 0.0031 0.0004 0.0024 0.0006 0.0017 0.0009 0.001 1 0.0007 0.0003 0.0006

0.025 0.050 0.10 0.25 0.50 1.1

* The fresh weight/dry weight ratio is assumed to equal 15.0. Chemostat culture, sucrose limited. Chemostat culture, phosphate limited.

tion region the activity of the enzyme tryptophan carboxylase was not, however, influenced by the phosphate concentration. The authors stress that the influence of phosphate must be related to the intracellular phosphate concentration because the entire phosphate pool of the medium is taken up by the cells within 48 hr. The influence of phosphate and sucrose on the production of serpentine in steady-state chemostat cultures was examined by Balague and Wilson (603). Alkaloid synthesis continued during phosphate but not during su-

Ref. 36

606

603

597

1.

121

PLANT BIOTECHNOLOGY

TABLE XXXIII INFLUENCE OF VARIOUS MEDIUM SALTSON ALKALOID PRODUCTION I N Catharanthus roseus CELLCULTURES

Medium

Medium modification

Biomass alkaloid content (mg/g DW)

Total alkaloid yield (mg/liter)

Rate of production (mg/g/day)

Ref.

Ajmalicine

7.2"

-

0.072

36

Ajmalicine

6.5"

-

0.065

Ajmalicine

8.0"

-

0.080

Ajmalicine

0.75"

-

0.008

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine

3.5" 0.6" 0.8" 0.5" 2.6" 2.0" 3.6" 5.3"

Alkaloid produced

21 4 1

0.8 9.2 7.1 7.2 10.5

1.5 0.04 0.06 0.04 0.19 0.14 0.26 0.39

' The fresh weight/dry weight ratio is assumed to equal 15.0.

crose limitation, indicating that alkaloids can be produced by growing C . roseus cultures, depending on the growth-limiting nutrient. Majerus and Pareilleux (597) determined the influence of the initial phosphate concentration of Gamborg B5 medium supplemented with 30 g/liter sucrose on the alkaloid content of the cells after 7 days of cultivation. Decreasing the phosphate concentration of the original medium resulted in a lower tryptamine and higher ajmalicine content of the biomass, whereas the serpentine content remained unchanged. It was reported that the biomass yield showed a linear increase with initial phosphate concentration of the medium at concentrations between 0 and 700 pM. From the work reviewed above it appears that a decrease in the phosphate concentration of the medium has in most cases a positive effect on alkaloid production in C. roseus cell suspension cultures. The precise effect, however, remains unclear because in none of the experiments described was a time course of alkaloid production given. Another question which has to be solved is the precise mechanism behind the observed

48

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ROBERT VERPOORTE ET AL.

effects. Is there a direct influence of intracellular phosphate concentration on the rate of secondary metabolite production, or is there a much more complex interrelation between primary and secondary metabolism? These and other questions remain to be solved in future research. c. Precursor Feeding. When the biosynthetic pathway to a certain desired alkaloid is known, it is possible to increase the productivity by the addition of precursors. As tryptamine and secologanin are the basic constituents of terpenoid indole alkaloids these two compounds or their precursors have frequently been added to C. roseus cultures in order to enhance alkaloid production. Zenk er al. (20)added different precursors to the induction medium and studied the effect on growth and the accumulation of ajmalicine and serpentine. Three groups of precursors were used: precursors of the indole portion of the alkaloid molecule, those of the monoterpene portion, and general or more distant precursors. The addition of 500 mg/liter Ltryptophan resulted in a 1.75-fold increase in biomass yield and a 2.8-fold increase in alkaloid production. The stimulating effect of L-tryptophan, however, appeared to depend greatly on the cell line. When L-tryptophan was added to a cell line derived from the same parental line as the one which showed enhanced serpentine production, alkaloid production declined markedly. Among the monoterpenoid precursors, loganin in particular had a stimulating effect on alkaloid production, but biomass yield was only slightly increased. Doller (602) studied the effect of the addition of both L-tryptophan and tryptamine to MS medium supplemented with 50 g/liter sucrose at the beginning of the incubation. Nine different concentrations between 10 and 1000 mg/liter were used. In all experiments both the growth and the yield of serpentine compared to the control were inhibited. The next experiment was the addition of 500 mg/liter L-tryptophan after 0 , 6 , 12, and 18 days of culture. This resulted in an enhanced cell yield after 12 and 18 days of culture, but an inhibition of alkaloid yield was noted. Deus and Zenk (607) also added L-tryptophan or tryptamine to an induction medium. The effect was studied using several cell lines derived from a single parental line. Whether lower or higher alkaloid production compared to the control resulted, for both precursors, greatly depended on the cell line used. MCrillon et al. (608) added 1.8 mM L-tryptophan or 0.124 mM secologanin or a combination of the precursors to 10-day-old cultures grown on different media. In none of the cultures was the biomass concentration significantly affected after a 20-day culture period. The addition of secologanin enhanced both serpentine and ajmalicine yields. The addition of

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L-tryptophan enhanced the yield of tryptamine, whereas the yield of serpentine was not affected. Judging from the literature it seems unlikely that the biosynthesis of tryptophan or tryptamine are the rate-limiting steps in the formation of serpentine and ajmalicine in cell suspension cultures of C. roseus. The fact that addition of secologanin in particular has a positive effect on alkaloid production indicates that the terpenoid pathway might be a better candidate for the rate-limiting step. d. Elicitors and Bioregulators. Another possible way to stimulate secondary metabolite production in cell suspension cultures of C. roseus is the use of elicitors or bioregulators (Table XXXIV). Lee et al. (609) determined the effect of five different carotenoid inducers on growth and alkaloid production. Of the five compounds tested 1,1-dimethylpiperidine, 2-diethylaminoethyl-2,4-dichlorophenylether, and 2-diethylaminoethyl-Pnapthylether at concentrations of 5 ppm increased total alkaloid production up to about 20% with concomitant increases in ajmalicine and catharanthine. The same compounds tested by Lee et al. were also used by Kutney et al. (610). They report that 1,l-dimethylpiperidine and 2diethylaminoethyl-3,4-dimethylphenyletherwere able to raise the ajmalicine content to 9 mg/g biomass dry weight after 6 days of incubation compared with 2.5 mg/g for the control culture. Smith et al. (611) studied the effect of vanadyl sulfate on catharanthine and ajmalicine production in various cell lines of C. roseus. After 5 days of culture 50 mg/liter vanadyl sulfate was added. The amounts of catharanthine and ajmalicine were determined after 3 days of incubation with the bioregulator. Alkaloid production was stimulated in most cases, but the exact effect depended greatly on the cell line used. The effect of increasing the osmolarity of the medium on growth and serpentine production using mannitol was examined by Rudge and Morris (612).The addition of 0.6 M mannitol resulted in a decrease in cell number, a reduced cell volume, and an increased serpentine content of the cells. From the results presented, however, it is not clear whether net serpentine production was affected by this treatment. Eilert et al. (95) reported the treatment of five different cell lines of C. roseus with homogenates of various fungi as well as with chemically defined phytoalexin elicitors. It was found that a Pythium aphanidermatum homogenate concentration of 5% and a Rhodotorula rubra homogenate concentration of 0.5% affected alkaloid yields. The response of the cells to elicitor treatment revealed a temporary increase of alkaloid content in the cells and in the culture medium 12-24 hr following addition.

TABLE XXXIV INFLUENCE OF ELICITORS AND BIOREGULATORS ON ALKALOID PRODUCTION IN Catharanthus roseus CELLCULTURES

Compound added"

Alkaloid produced

None 1 (5 ppm) 2 (5 ppm) 3 ( 5 ppm) 4 ( 5 ppm) 5 (5 ppm) None 1 (2 ppm) 5 (2 ppm) None 6 (50 mglliter) None

Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine

Medium

SH (613)

B5

MS + 3% sucrose MS + 2% sucrose

9 B5

MS + 8% sucrose

None 7 8 None 10

Biomass alkaloid content ( m g k DW)

Total alkaloid yield (mg/liter)

0.008 0.017 0.021 0.028 0.026 0.014 0.25 0.9 0.9 0.07' 0.13' 0.3 I .9 0. I 2.2 0.008 0.16 0.21

-

-

2.5 12 9 1.7 3.1 40 70

Rate of production (mddday) 0.04 0.15 0.15 0.02 0.04 0.01 I 0.21 0.28 -

Ref. 609

610

611 612

95

134

a Key to elicitors: 1, 1 ,I-dimethylpiperidine; 2, 2-diethylaminoethyl-3, 4-dichlorophenylether; 3, 2-diethylaminoethyl-2.4-dichlorophenylether; 4, 2-diethylaminoethyl-P-naphthylether;5 , 2-diethylaminoethyl-3, 4-dimethylphenylether; 6 , vanadyl sulfate; 7, Pythium uphanidermatum homogenate; 8 , Rhodolorulu rubra homogenate; 9,0.6 M mannitol; 10, Phytophthoru cactorum. * The fresh weightidry weight ratio is assumed to equal 15.0.

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Asada and Shuler (134) reported that autoclaved cultures of the mold Phytophthora cactorum resulted in a 60% increase in ajmalicine production. The response time to elicitor addition was less than 11 hr.

e. Other Environmental Factors. p H . Initial pH values recommended for plant cell culture media are usually between 5 and 6. Because of the low buffering capacity of these media, the pH changes considerably during cultivation. Ten Hoopen et al. (614) found that during batch cultivation of C. roseus cell suspensions in 3-liter stirred fermentors the pH of the culture drops within 24 hr to a value of 4, after which a quick rise up to pH 5 was observed. At the end of the culture period the pH increased to a value between 6.5 and 7.0. Doller (602) studied the effect of various initial pH values of the culture medium on growth and serpentine production in C. roseus cell suspension cultures. Both growth rate and serpentine production were highest at initial pH values of 5.5. Renaudin (84) studied the influence of pH on the uptake of indole alkaloids by C. roseus cells. He found that alkaloid uptake was influenced by both the intra- and extracelluar pH as well as the pK, of the alkaloid under study. From these results the conclusion was drawn that alkaloid uptake was caused by an ion-trap mechanism. Recent studies by Blom et al. (80,81)showed that the accumulation of ajmalicine in vacuoles of C. roseus is due to an ion-trap mechanism. In the vacuole ajmalicine is converted to serpentine by basic peroxidases. Serpentine does not pass the tonoplast and is thus trapped in this way. No active transport of the alkaloids, as had been previously reported (74,75), could be detected. Pareilleux and Vinas (615) also attributed the changing ratio between intraand extracellular concentrations of serpentine and ajmalicine in a batch culture of C. roseus grown in a 3.5-liter stirred tank reactor to a changing culture pH. Majerus and Pareilleux (175) observed that a sudden drop in pH from 6.0 to 5.0 resulted in enhanced excretion of tryptamine and ajmalicine in a conthuous culture of immobilized C. roseus cells. From the literature it becomes clear that the pH of the culture influences alkaloid transport between cells and medium. Whether the external pH also affects alkaloid biosynthesis is still a matter of investigation. Temperature. Plant cell and tissue cultures are normally grown at temperatures between 25 and 27°C. However, several authors have studied the effect of cultivation temperature on growth and alkaloid formation of cell suspension cultures of C. roseus (Table XXXV). Courtois and Guern (55) conducted a series of experiments at temperatures between 16 and 40°C. The maximum growth rate of the cultures was highest at temperatures of 27,32, and 35°C. The cultures grown at 20°C or

126

ROBERT VERPOORTE ET A L .

TABLE XXXV INFLUENCEOF CULTURE TEMPERATURE ON ALKALOID PRODUCTION I N Catharanthus roseus CELLCULTURES

Medium MS + 2% sucrose

Rate of production (mg/g/day)

0.0 0.0 < 0.05 < 0.05 0.1 0.1 0.1 0.3 < 0.05 < 0.05 0.0 0.0 0.0 0.0 0.0 0.0 1.15

Culture temperature (“C)

Alkaloid produced

10

20

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Serpentine

0.0 0.0 0.08 0.12 0.40 0.50 0.78 0.04 0.04 0.05 0.0 0.0 0.0 0.0 0.0 0.0 2.26

0.0 0.0 2.5 2.0 17 15 9.0 30 2.5 2.0 0.0 0.0 0.0 0.0 0.0 0.0 20.7

25 30

Serpentine Serpentine

2.70 0.12

44.7 0.87

15 20 25 30 35 40 45

MS + 2% sucrose

Total alkaloid yield (mg/liter)

Biomass alkaloid content (mg/g DW)

Ref. 56,616

617

2.79 0.07

below showed a significantly reduced growth rate, whereas cell death occurred at a temperature of 40°C. Ajmalicine and serpentine contents of the cells were only determined from the cultures grown at temperatures of 16 and 27°C. Alkaloid determination was performed after 28 and 7 days of culture, respectively. At 16°C both ajmalicine and serpentine contents were highest. The specific serpentine production of the cultures grown at 16 and 27°C was 3.6 and 1.7 x lo-* pglcelllday, respecpglcelll tively, and specific ajmalicine production was 4.3 and 7.1 x day, respectively. Morris (616) showed the influence of culture temperature on the kinetics of growth and product formation- in batch-cultured C. roseus cells. The cells were cultivated at temperatures of 15,25, and 35°C. At a temperature of 15°C growth and alkaloid formation were suppressed although they remained coupled, as was the case at the maintenance temperature of 25°C. At a temperature of 35°C no serpentine or ajmalicine accumulation occurred. A later paper (56)described a more detailed study performed on

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the effect of culture temperature on growth and product formation in the same cell line of C . roseus. Culture temperature was varied between 10 and 45°C. The maximum specific growth rate of 0.033 hr-' was reached at a temperature of 35°C. It was found that the growth rate declined rapidly above 35 and below 25°C. Maximum serpentine yields reached a peak between 20 and 25°C and fell sharply above and below these temperatures. Ajmalicine showed a sharp peak of accumulation at 20°C. From these figures it can be inferred that the serpentine/ajmalicine accumulation ratio can be influenced greatly by culture temperature in a range between 20 and 30°C. Scragg et al. (627) also studied the influence of culture temperature on the growth and productivity of a cell suspension culture of C. roseus. Culture temperatures used were 20,25, and 30°C. Growth rate was maximal at 30"C, whereas specific serpentine production was highest at a temperature of 25°C. On the basis of the results presented by Morris (37,616), Bailey and Nicholson (157) developed a structured model for the description of the kinetics of growth and product formation in cell suspension cultures of C. roseus. It was shown that the model, which was calibrated using the data of Morris, gives a good description of the course of cell dry weight, cell fresh weight, intracellular product concentrations, and viability of the cells during growth in batch culture at different culture temperatures. In a later paper (158), the model was used to predict optimal temperature control in order to maximize productivity. In this way a 22% increase in final product yield is predicted. Unfortunately, the authors did not compare the model predictions with experimental results. Light. Light is not necessary for growth of plant cells or tissues in culture as plant cell cultures are able to grow heterotrophically on a medium containing a single carbon and energy source. However, light greatly influences plant cell metabolism. In callus cultures light can influence morphogenesis and differentiation (618). From literature reports it is known that light influences the ajmalicine/serpentine accumulation ratio (Table XXXVI). Knobloch et al. (48) found that cell suspension cultures of C. roseus grown in the light accumulated significantly higher amounts of serpentine and lower amounts of ajmalicine than dark-grown cells. The authors concluded that the influence of light on the ajmalicine/serpentine ratio was likely due to a stimulation of the conversion of ajmalicine to serpentine because the sum of both alkaloids remained unchanged. Drapeau et al. (243)obtained similar results. When the cultures were exposed to light for only 15 hr per day instead of 24 hr, the effect of light on serpentine content could not be explained only by conversion of ajmalicine to serpentine. In

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TABLE XXXVI INFLUENCE OF LIGHTON ALKALOID PRODUCTION I N Catharanthus roseus CELLCULTURES ~~~~~~~~~

Medium 8.0% sucrose

Light exposure

Alkaloid produced

24 hr dark

Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine Ajmalicine Serpentine

24 hr light 8.2% glucose 7.4% glucose 8.2% glucose

24 hr dark 15 hr light

24 hr light

Biomass alkaloid content (mg/g DW)

Total a1kaloid yield (mg/liter)

8.4" 0.30" 4.2"

5.3" 1.8 0.0 1

.o

6.0 2.0 0.8

7.0 0.0 4.0 24 8.0 3.0

Rate of production (mg/g/day) 0.60 0.02 0.30 0.38 0.15 0.0 0.05 0.20 0.15

0.06

" The fresh weight/dry weight ratio assumed to equal 15.0.

addition, catharanthine was also produced. Furthermore the authors report that in dark-grown cultures 80% of the total amount of alkaloids produced was excreted into the medium, whereas only 20% was excreted when the cultures were exposed to light. Scragg et al. (627) followed the production of serpentine in dark-grown C. roseus cultures during a large number of subcultures. The serpentine content was found to vary greatly as a function of subculture number (between 0 and 1.4 mg/g dry weight). When the cultures were grown for one culture period in the light this resulted in a significant increase in serpentine production. After a growth period of 14 days in the light the serpentine content of the biomass reached values between 2.4 and 4.0 mg/g dry weight. When the cultures were exposed to light during further subcultivation serpentine production decreased. From literature it becomes clear that light reduces ajmalicine formation while stimulating serpentine and catharanthine formation. It should be kept in mind, however, that exposure of plant cell cultures to light in large-scale culture vessels might be problematic or at least expensive. The applicability of light in a large-scale production process should therefore be doubted.

f. Cultures of Differentiated Cells. Because it is often observed that a certain degree of cell differentiation favors alkaloid production, several workers considered the use of organ cultures of plant cells. It seems reasonable that alkaloids which are normally produced in the root of the plant are best produced by root cultures, whereas alkaloids produced in

Ref. 48

143

1. PLANT BIOTECHNOLOGY

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the shoots are best produced by shoot cultures. An interesting possibility is the use of hairy root cultures, which are obtained by infection of plants or plant material with the soil bacterium Agrobacterium rhizogenes. Infection results in the transfer of a root-inducing or RI plasmid from the bacterium into the genome of the plant. The plant responds by the formation of hairy roots at the site of the infection. The hairy roots so obtained can subsequently be subcultivated under aseptic conditions. Several workers claimed a high growth rate of these hairy root cultures, combined with a high productivity. Pan- et al. (619) investigated alkaloid formation in transformed root cultures of C . roseus. In addition to ajmalicine (between 200 and 330 pglg fresh weight) and serpentine (between 50 and 100 pglg fresh weight), a low amount of vinblastine was also produced (between 0.003 and 0.05 pglg fresh weight). Growth of the cultures was slow, approximately 0.0032 hr-' calculated from the increase in fresh weight. Alkaloids were produced throughout the growth cycle. Endo et al. (115) compared the alkaloid contents of shoot, root, and cell suspension cultures with those of the intact plant. In all systems alkaloid levels were low (below 0.12%) (see Table XXXVII). Ajmalicine levels were highest in dark-grown shoot cultures (0.062%) and dark-grown suspension cultures (0.115%) whereas uindoline had its highest level in lightgrown shoot cultures (0.004%). g. Large-Scale Culture. In order to scale up ajmalicine production, studies were performed on the growth and alkaloid formation of C . roseus cultures in bioreactors. In the case of cell suspension cultures convenTABLE XXXVII PRODUCTION OF AJMALICINE IN DIFFERENT CULTURE SYSTEMS OF Catharanthus rosema

Medium

Culture system

B5

Shoots

B5 B5

Roots Suspension Intact plants Leaf Stem Root

a

Data from Endo ef a / . (115)

Light regime

Alkaloid produced

Biomass alkaloid content (mgk D W

Light Dark Dark Light Dark

Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine

0.10 0.62 0.30 0.22 1.15

Ajmalicine Ajmalicine Ajmalicine

0.03 0.01 0.34

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ROBERT VERPOORTE E T A L .

tional bioreactors such as stirred-tank and airlift fermentors can be used. With these systems plant cell cultures can be cultivated in the same way as cultures of microorganisms (see Table XXXVIII). Wagner and Vogelmann (165)showed that the reactor system used may have a considerable influence on yield and productivity for cell mass and secondary metabolites. The authors also reported that a cell suspension culture of C. roseus was disrupted completely after a 5-day culture period in a stirred fermentor, equipped with a flat-bladed turbine impeller with a diameter of 120 mm, operated at a speed of 28 rpm. In contrast, Scragg et al. (605)and Meijer (69)showed that their C. roseus cultures were less sensitive to hydrodynamic shear stress; the cultures could be cultivated in conventional turbine-stirred reactors at stirrer speeds up to 1000 rpm (stirrer diameters of 73 and 45 mm). TABLE XXXVIII LARGE-SCALE CULTURE OF Catharanthus roseus CELLSIN B~OREACTORS

Medium (Not given) Zenk (20) 8% glucose IM2 (127)

MS + 2% sucrose

MS + 2% sucrose MS + 6% sucrose

Bioreactor 10-liter airlift reactor 7.5-liter stirred tank 14-liter stirred tank Shake flask 25-liter stirred tank 750-liter stirred tank Shake flask 7-liter airlift 30-liter airlift Shake flask 7-liter airlift Shake flask Shake flask (fed-batch) 30-liter airlift 30-liter airlift (fed-batch)

Alkaloid produced

Biomass alkaloid content (mg/g DW)

Total alkaloid yield (mg/liter)

Serpentine

2.3

51

0.08

165

Ajmalicine

I .4

21

0.05

620

Ajmalicine

2

8

0.15

622

Ajmalicine Ajmalicine

1.3 0.8

30 18

0.09 0.05

139

Ajmalicine

0.5

15

0.03

Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine Serpentine

1.7 0.15 0.01 2.6 1.5 9.7 5.1

Serpentine Serpentine

2.1 1.4

17 1.2 0.09 21.2 16.3 23 1 135 29 18

Rate of production (mg/g/day)

Ref.

0.14 0.01 0.001

623

0.16

625

0.13 0.34 0.21

625

0.13 0.10

625

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Kurz et al. (620)reported that cultivation of selected high producing cell strains of C. roseus in a 7.5-liter stirred-tank bioreactor could further increase the productivity over that obtained in shake flasks. Growth kinetics of C. roseus cell suspensions in glucose-limited batch cultures in 14-liter turbine-stirred fermentors were studied by Drapeau et al. (621). Ajmalicine production was also followed on transfer of the culture to a 8% glucose solution. The maximum ajmalicine content of the cells in this medium was 2.5 mg/liter after 8 days of culture. In a later paper (622) a more detailed study was reported on the alkaloid production of C. roseus cells in 1Cliter stirred bioreactors. The course of ajmalicine, catharanthine, and serpentine accumulation after transfer of the culture to an 8% glucose solution was followed. From measurements of the oxygen uptake rate, the authors concluded that alkaloid accumulation occurred primarily during a 20-day transition period between growth-oriented and maintenance-oriented metabolism. Exposure of the cells to light during this period stimulated catharanthine accumulation and triggered a switch from ajmalicine to serpentine formation. Light suppressed the secretion of both ajmalicine and serpentine; without light nearly 80% of the alkaloids were found in the medium, whereas on light exposure less than 20% were secreted. Growth and product formation in C. roseus cultures in conventional stirred reactors of 25, 70, 300, 750, and 5000 liters equipped with flatbladed turbine impellers is described by Schiel and Berlin (139). The authors reported that, whereas the growth behavior in all reactors was comparable to growth in shake flasks, the inducibility of ajmalicine production decreased with increasing reactor volume. Possible explanations mentioned by the authors are increased shear stress at increasing fermentor volumes, different aeration regimes depending on the fermentor size, and different physiological states of the inocula used. The effect of scaleup on serpentine formation by C. roseus cultures grown in airlift reactors of 7, 30, and 100 liters working volume has been determined by Scragg et al. (623).The maximum specific growth rate of the cells in the bioreactors was reported to be higher compared to shake flask cultures. In contrast the accumulation of serpentine was low in all bioreactor cultures. The authors suggested that this might be attributed to the effect of ventilation. Owing to the better aeration of airlift reactors compared to shake flasks, higher concentrations of dissolved oxygen, lower concentrations of carbon dioxide, and lower concentrations of other volatile compounds such as the gaseous hormone ethylene occur, which might have influenced serpentine accumulation. In a later paper (624) it was shown that, in contrast to the abovementioned results, a newly selected C. roseus cell culture retained ser-

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pentine producing capacity in airlift bioreactors, but at lower levels than when the cells were grown in 250-ml shake flasks. Surprisingly, when the culture was grown in 2000-ml shake flasks the maximum serpentine concentration obtained was 18 times lower compared to the smaller flasks and 4 to 5 times lower compared to the bioreactors. The growth rate was also reduced in the 2000-ml flasks. For all bioreactor experiments, however, cells grown in 1000-ml shake flasks were used as inoculum (623,624).Thus, the question arises whether the poor and in some cases absent serpentine production in airlift bioreactors might have been caused by the way the inoculum was treated. Unfortunately the authors did not investigate the influence of the condition of the inoculum on the serpentine production characteristics of the C . roseus bioreactor cultures. In a third paper Scragg et al. (625) compared three different modes of bioreactor operation: batch, fed-batch, and draw-fill. The experiments were conducted using airlift fermentors of 7 and 30 liters working volume. Batch and fed-batch cultures were also performed in shake flasks. It appeared that serpentine production in batch cultures in shake flasks was highest. Batch cultures performed in airlift bioreactors showed a lower productivity. Fed-batch cultures showed a lower productivity compared with batch cultures, both in shake flasks and in airlift reactors. When the draw-fill method was used, serpentine production was negligible. Although suspension cultures are the easiest and most convenient way to cultivate plant cells, it might not be the most suitable choice to achieve the highest production of a desired secondary metabolite. It is often claimed that cell-cell contact, which is almost absent in a suspension culture, might favor secondary metabolite production. This can be achieved using immobilized cell reactors. A second advantage of immobilized cells might be that, if excretion of the product can be achieved, the produced biomass can be used for prolonged periods of time. For this purpose special bioreactor systems have been developed and studies have been performed on the alkaloid production in these systems. Literature data on the production of ajmalicine and serpentine in bioreactors with cell retention are given in Table XXXIX. A study on alkaloid production by nongrowing (owing to the absence of 2,4-D) cell suspension cultures of C . roseus in a continuous flow reactor with cell retention was performed by Pareilleux and Vinas (626). It was shown by the authors that part of the produced alkaloids were excreted into the culture medium. Although the continuous flow system seemed to be suitable for secondary metabolite production, the reported specific production rate was relatively low (0.012 mg ajmalicine/g DW/day). To obtain ajmalicine production 1.2 mM tryptamine had to be added to the influent medium. The duration of the continuous flow experiment was only

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2.4 residence times. From the results presented it is clear that a steady state with respect to the intra- and extracelluar concentrations of tryptamine, ajmalicine, and serpentine was not achieved. Majerus and Pareilleux (597) tried to enhance alkaloid formation in calcium alginate-entrapped C . roseus cells by the use of a limiting growth medium. The levels of 2,4-D and phosphate were lowered to concentrations of 45 nM and 100 p M , respectively. Using this medium a comparison was made of the biosynthetic capacity of free and entrapped cells. It was found that calcium alginate-entrapped cells showed a lower initial respiration rate and a lower biomass formation rate compared to freely suspended cells, which was very likely due to limited oxygen diffusion into the alginate beads. Although ajmalicine production in free cells leveled off after 300 hr of culture to a maximum content of 500 pglg dry weight, entrapped cells continued to produce until the end of the 600-hr culture period (maximum ajmalicine content was 1 mg/g DW). The tryptamine content of the cells was not significantly affected. Medium concentrations of ajmalicine of free cells were higher (70 pg/g DW) than in the case of entrapped cells (10 pg/g DW) and might be an indication of cell lysis in the suspension cultured cells. In a second paper (175) the production of ajmalicine by calcium alginateentrapped cells of C . roseus in a continuous flow reactor was described. The authors reported that, as was described earlier in their first paper on the subject, only a small amount of the produced alkaloid was excreted into the medium and that the greater part was trapped within the cells. The maximum productivity of the reactor was 73.3 pg/liter/day at a dilution rate of 0.72 day-'. Total alkaloid production of C . roseus cells immobilized on the surfaces of calcium alginate beads in a packed column bioreactor is described by Kargi (627). The alkaloid concentration of the medium and the biomass was determined by means of absorbance measurements at 280 nm. A calibration curve was established with the alkaloid ajmalicine. Total alkaloid content of medium and cells was claimed to be as high as 3.3% on dry weight basis, whereas the alkaloid content of suspension cultured cells was 0.4%. Another method to immobilize plant cells is entrapment in dialysis tubing. In a study conducted by Payne er al. (628) it appeared that no growth nor net alkaloid production took place in C . roseus cells entrapped in 12-cm-long sections of 7.6-mm-diameter dialysis tubing (molecular weight cutoff 50,000). However, cells which were removed from the dialysis tubing prior to transfer to production medium were observed to grow and to produce alkaloids. The authors attributed the absence of growth and alkaloid production of the entrapped cells to mass transfer limitations.

TABLE XXXIX LARGE-SCALE CULTUREOF Catharanthus roseus CELLSI N BIOREACTORS WITH CELL RETENTION

w

Medium B5 + 5% sucrose - auxin + tryptophan B5 + 3% sucrose + 45 nM 2,4-0 + 1WpM KHZP04 MS + 2% sucrose - auxin + BAP (I mg/liter)

Bioreactor system used

Cell culture

Dilution rate (per day)

Alkaloid Produced

Production rate mg/liter/day

mg/g/day

Ref.

Stirred tank (3 liters)

Suspension

0.12

Serpentine

-

0.012

626

Stirred flask (1 liter)

Calcium alginateentrapped cells

0.24 0.36 0.51 0.72

Ajmalicine

0.045 0.057 0.067 0.073

0.017 0.021 0.025 0.027

17.5

Packed column (50 ml)

Surface immobilized cells

-

Ajmalicine

-

627

10

BS + 2% sucrose

Zenk

Biofilm reactor

Shake flask

Zenk

Airlift reactor

Zenk

Stirred reactor

MS+3% sucrose

Shake flask

+

%

a

Polyester fiber. Glass fiber.

Biofilm 2 m m Biofilm 3 m m Biofilm 4 m m Biofilm 5 m m Biofilm 6 m m Suspension Surface immobilized cells" Surface immobilized cells" Surface immobilized cells" Surface immobilized cellsb

-

-

Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Ajmalicine Serpentine Ajmalicine Serpentine

-

-

0.014 0.030 0.13 0.21 0.15 0.0003 0.048 0.0006 0.013

-

-

633

630

-

Ajmalicine Serpentine

-

0.010 0.014

-

Total alkaloid fraction Ajmalicine

-

0.93

632

-

0.01

631

-

136

ROBERT VERPOORTE ET A L .

Therefore, a second immobilized cell system was investigated. In this system the cells were grown on cheesecloth filters in shake flasks. In this system also neither growth nor alkaloid production occurred. The third system described by the authors was a bubble column (1000 ml liquid volume) equipped with a cheesecloth filter at the bottom. This filter was used to facilitate the replacement of growth medium by production medium without removal of cells. It was also used as an air sparger. Both growth and total alkaloid production in this system were comparable to shake flasks. The scale-up potential of this system to larger volumes, however, remains questionable. A method to force C . roseus cells to excrete the produced alkaloids into the medium by iontophoresis is described by Pu et al. (629). The authors demonstrated that a direct current of 1-2 mA was sufficient to enable the release of the alkaloids ajmalicine and yohimbine. The reactor used consisted of a single porous hydrophilic ceramic tube with a pore size of 0.2 pm housed in a 1 1-cm-long glass tube 1.6 cm in internal diameter. Two platinum wires were used as electrodes, with one placed evenly in the shell region of the reactor as anode and the other placed in the ceramic tube as cathode. It was shown that the application of a direct current caused a release of ajmalicine of approximately 0.4 mg/liter/hr per gram dry cell mass. Passive diffusion of alkaloids from the cells was negligible. The increase of the direct current from 1 to 2 mA effectively doubled the release of ajmalicine. The possibilities of surface immobilization of C. roseus cells was investigated by Archambault et al. (630),Facchini and Dicosmo (631),and Rho et al. (632). Archambault et al. (630) used a fibrous polyester sheet and studied the effect of surface immobilization on alkaloid production. The cells were grown on the polyester matrix in shake flasks, a stirred vessel, or an airlift reactor. From the results presented it is clear that surface immobilization had a negative effect on alkaloid production in these experiments (Table XXXIX). Suspension cultures were found to produce significantly higher amounts of alkaloids than surface immobilized cells. Cultivation in bioreactors further diminished alkaloid production. Rho et al. (632) studied the physiological aspects of surface immobilization, using polyester fiber sheets mounted in a 2-liter stirred bioreactor. Nitrate and carbohydrate consumption rates as well as growth rate and biomass yield were reported to be equal or somewhat lower than for suspension cultured cells. The yield of total alkaloids was 13 mg/g dry weight after 14 days of culture. From this figure a specific alkaloid production rate of 0.93 mg/g/day can be calculated, which is relatively high. An immobilization procedure using a fiberglass substratum was described by Facchini and Dicosmo (631). By using different surface coatings

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a range of substratum surface tensions was obtained. Untreated glass, having the highest surface tension, was reported to give the best results with respect to production of the alkaloids ajmalicine, catharanthine, and tryptamine. When compared with suspension cultured cells, however, immobilization resulted in slower growth and decreased accumulation of all alkaloids. h. Economic Aspects. At present ajmalicine represents a market with an approximate volume of 3600 kg/year and a market price of $2000/kg. The amount of naturally grown C . roseus roots containing 1 kg of ajmalicine would cost approximately $619. Drapeau et al. (143) calculated the costs of production of an amount of C . roseus biomass, containing 1 kg of ajmalicine, by means of largescale plant cell culture. In this calculation a biomass ajmalicine content of 0.6% on dry weight basis was assumed. Considering a yearly production of 800 kg of ajmalicine, a price of U.S. $3215 for biomass containing 1 kg of ajmalicine was calculated. The authors concluded that the principal reason for the high cost of the plant cell culture process is not the low growth rate of the cells (0.35 day-'), but rather the low product formation rate (0.26 mg/g/day). The authors state that productivity will have to be increased by a factor of 40 to make the process competitive. In a later study van Gulik et al. (144) conducted a similar calculation, based on a yearly production of 3000 kg ajmalicine (see above). They assumed a product concentration of 0.9% on a dry weight basis and calculated a production price of U.S.$lSOO for an amount of biomass containing 1 kg of ajmalicine. A second calculation assumed a specific growth rate of the biomass twice as high and an ajmalicine content 10 times as high. This resulted in a price of U.S.$430 per amount of biomass containing 1 kg of ajmalicine, which is still too high to be competitive with the traditional process. These authors also consider the low productivity of the biomass to be the major factor hampering commercial production of ajmalicine by means of large scale plant cell culture. 2. Dimeric Indole Alkaloids, Vinblastine Type The therapeutic and economic importance of the alkaloids vinblastine

(36)and vincristine (37)has stimulated a major research effort on the plant cell biotechnology of Catharanthus roseus. This work, which has recently been extensively reviewed (592), has resulted in several patents (Table XXIX). Concomitantly, modern analytical procedures, for example, HPLC (635,633, GC-MS (636), immunoassays (638-641), and supercritical fluid chromatography/mass spectrometry (642),have been developed to high sensitivity and selectivity to study alkaloid formation in plant tissues.

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ROBERT VERPOORTE ET AL.

a. Production of Dimeric Indole Alkaloids in Cell Cultures. With the above-mentioned analytical methods the alkaloid contents of various tissues and cell cultures were determined (Table XL). Vinblastine (VLB) is a dimeric indole alkaloid, consisting of a catharanthine and a vindoline moiety (Fig. 11). In C . roseus plants catharanthine, vindoline, and 3’,4’anhydrovinblastine (AVLB) are the major alkaloids in the leaves , present in amounts of the order of milligrams per gram of leaves. Catharanthine is produced in relatively large amounts by suspension cultured cells of C. roseus: 230 mgtliter after 1 week of growth using a high inoculum density of 5-methyltryptophan-resistantcells (643). The formation of vindoline, however, is dependant on morphological differentiation (644,645).Vindoline, AVLB, and VLB have so far been isolated only from differentiated tissues, and the contents are apparently related to the degree of differentiation (Table XL). The highest levels were found in the leaf of the plant, the lowest in callus cultures (641).In Agrobacteriurn rhizogenestransformed roots of C. roseus, VLB could be detected at a level of only 0.05 pg/g dry weight (619). b. Bioconversions. As both vindoline and catharanthine are available from the plants, the enzymatic and chemical coupling of these moieties has been extensively studied. The enzymatic coupling mechanism is thought to be similar to the modified Polonovski reaction (646): oxidized catharanthine is coupled to vindoline to form a dimeric, highly instable dihydropyridinum (iminium) intermediate, which subsequently undergoes 1,4reduction, hydroxylation, and reduction to yield VLB (647,648) (Fig. 11). AVLB is formed by 1,2-reduction of the iminium intermediate, which can be chemically performed by sodium borohydride (649,650). Therefore, AVLB is not a biosynthetic precursor for VLB (648). The enzymatic coupling of catharanthine and vindoline can be performed with enzymes isolated from leaves, suspension cultures, or even with peroxidases, for example, horseradish peroxidase (651). From a suspension culture five isoenzymes of a peroxidase nature, involved in the coupling reaction, were partially purified (652). Cell-free production of AVLB from vindoline and catharanthine, with enzymes from suspension cultured cells, was performed with a maximum yield of 22% (653,654).A yield of 25% was observed using flavin adenine dinucleotide (FAD) and manganese chloride in a crude enzyme preparation (654). A yield of 26% was obtained using a purified enzyme fraction (40-70% ammonium sulfate saturation) and hydrogen peroxide (655). Using horseradish peroxidase, in combination with either hydrogen peroxide or flavin mononucleotide (FMN) and manganese chloride, yields between 40 and 50% were reported (651).Using other sources of peroxidases

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TABLE XL DISTRIBUTION OF CATHARANTHINE, VINDOLINE, 3',4'ANHYDROVINBLASTINE, AND VINBLASTINE IN Catharanthus roseus PLANTSAND in Vitro CULTURES Tissue Seedling Intact plant Leaf

Leaf

Leaf Stem Root Shoot culture

Multiple shoot Leaf tissue Stem tissue Unorganized tissue Root culture

Alkaloid contenta

Ref.

V, 54 p d g FW; C: 37 pg/g FW

634

V, 1.44 mg/g DW; C, 0.62 mg/g DW; AVLB, 9.1 pg/g FW; VLB, 6.4 pg/g FW VLB, 1.4 mglg DW V, 2.5 mg/g FW; C, 1.2 mg/g FW; AVLB, 0.3 mg/g FW AVLB, 2.3 mg/g DW V, 0.27 mg/g DW; C, 0.13 mg/g DW V, not detected; C, 0.43 mg/g DW V, 0.43 mg/g DW; C, 0.10 mg/g DW; AVLB, 2.6 pg/g DW; VLB, not detected VLB, 15 pg/g DW VLB, 25 pg/g DW VLB, 42 pglg DW VLB, 1 1 pg/g DW VLB, 5 p g / g D W

662

V, not detected; C, 0.041% DW

662

C, 51 pdg FW; VLB, 0.003 pg/g FW VLB, 0.33 pg/g DW V, not detected; C, 0.34-1 mglg DW C, 16.9 mglg DW

619

641 663

658

662 662 662

664 641

A . rhizogenes-

transformed root Callus culture Suspension culture Suspension culture

641 662

643

a V, Vindoline, C, catharanthine; AVLB, 3',4'-anhydrovinblastine; VLB, vinblastine.

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ROBERT VERPOORTE E T A L .

C02CH3

- CH3CR

CH3O e

C AT HA R A NT HI N E

H

3

R-CO2CH3 VlNDOLlNE

,

C H30

OC-CH3 bH3 R

/

0

IMlNlUM

CH3O

OC-CH3 CH3

I’

0

R C02CH3 ANHYDROVINBLASTINE

FIG. 1 1 . Coupling of the monomeric alkaloids catharanthineand vindoline to yield dimers of the vinblastine type.

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and hemoproteins, such as hemin and microperoxidase, AVLB was formed with a yield of 58% (656). The step was performed even more effectively by DiCosmo (105); using a coupled enzyme system consisting of glucose oxidase (for the in situ formation of optimal concentrations of hydrogen peroxide) and horseradish peroxidase, a yield of 70% was obtained. Recently, Misawa’s group presented a semisynthetic production of vinblastine, using a highly productive suspension culture for the formation of catharanthine (230 mg/liter) followed by a two-step chemical synthesis of VLB (643). Using ferric chloride as a catalyst, catharanthine and vindoline were coupled to AVLB in a 90% yield. The increased insight into the biosynthetical and chemical reactions in the dimerization process has led to a “five-step, one-pot” chemical synthesis of VLB from catharanthine and vindoline, with a yield of 40% (657). The extraction of AVLB from fresh leaves has been optimized (658). Treatment with sodium borohydride enhanced the yields, suggesting that the iminium intermediate was present in the aqueous layer. The maximum yield of AVLB was 0.23% of the dry weight. Although AVLB is not a true intermediate in the biosynthesis of VLB, it can be converted to VLB in cell-free systems derived from both leaves (659) and suspension cultures (653,660). In the presence of FeC13, AVLB is converted to VLB in 50% yield (643). A biotechnological production process for VLB by means of cell suspension culture, which is regarded to be most favorable system, is thus hampered by the inability of suspensions to produce the precursor vindoline. The developmental regulation of vindoline biosynthesis has been studied in detail; various enzyme activities were determined in germinating seeds and seedlings and in different types of tissue cultures (645). The activity of two enzymes in the late steps of vindoline biosynthesis, namely, S-adenosyl-L-methionine :16-methoxy-2,3,-dihydro-3-hydroxytabersonineN-methyltransferase (NMT) and acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (DAT), could be detected only in the hypocotyls and cotyledons of the seedlings; activity could not be detected in cell suspension cultures. It was previously found that part of the vindoline biosynthesis could be localized in the chloroplasts of leaf cells and is specifically associated with thylakoids (661). However, the ability of suspension cultures to form dimers is demonstrated by the cell-free experiments and the characterization of some coupling enzymes. Further insight into regulation of the biosynthesis of vindoline, especially at the gene level, is now essential. Initial investigations in that direction, namely, the isolation of DAT and tryptophan decarboxylase cDNA clones, have been performed (8,9,645).

142

ROBERT VERPOORTE E T A L .

3. Genetic Engineering

Despite the numerous studies of production of alkaloids by means of cell cultures of C.roseus, a commercially feasible production method has not yet been developed, Most of the studies had an rather empirical character, for example, numerous media modifications have been tested. Only in recent years have studies on the regulation of the alkaloid biosynthesis been started (for reviews of the enzymes involved, see Refs. 592-594 and 645). This has already resulted in cloning of several genes, namely, those coding for strictosidine synthase (SSS) [from plants (665) and cell cultures ( 9 ) ] and tryptophan decarboxylase (TDC) [from plants (8,645) and cell cultures ( 9 ) ](Fig. 12). The SSS gene has been sequenced (665).The TDC gene has been transferred into tobacco plants together with a strong constitutive promoter (CaMV 35s) (9,666).This resulted in the biosynthesis of active enzyme, and tryptophan present in the plants was found to be converted to tryptamine. The levels of tryptamine were in the range of approximately 0.1% on a fresh weight basis, which is similar to the overall levels of indole alkaloids in plants producing such compounds. Both the SSS gene and the TDC gene were found to be activated in C. roseus cell cultures in media devoid of auxins (9). Addition of auxins immediately repressed the translation of the genes. Introduction of the TDC gene into C. roseus cells resulted in increased tryptamine levels; introduction of the antisense gene considerably reduced alkaloid production (9). These first steps on the long way to unraveling the regulation of alkaloid biosynthesis at the molecular level show that the approach is feasible. Although it is an elaborate method, it may eventually lead to cell cultures capable of producing high levels of indole alkaloids.

4. Conclusions Cutharunthus roseus is probably the most extensively studied plant for secondary metabolite production in cell cultures. As such it is an excellent model system; however, the levels of alkaloids produced are still far below amounts necessary for a commercially feasible process. The technology for the large-scale culture of C. roseus cells is available. Future studies should thus focus on the regulation of alkaloid production, with the aim of eventually applying genetic engineering to increase alkaloid levels.

B. Ruuvolfiu ALKALOIDS Three alkaloids from Ruuvoljiu species have industrial interest: ajmaline (38), reserpine (39), and rescinnamine. Ajmaline is used for its antiarrhythmic activity, reserpine and rescinnamine for their antihyper-

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PLANT BIOTECHNOLOGY

Primary metabolism

/

L -TRYPTOPHAN

I

TRYPTOPHAN DECARBOXYLASE

H TRYPTAMINE

SYNTHASE

+

STRlCTOSlDlNE

0 GI u

SAM :

loganic acid methyl transferase

1 Monoterpene hydroxylase. NADPH. 02

t +OH

GERANIOL

Primary metabolism

FIG. 12. Early steps in terpenoid indole alkaloid biosynthesis.

'%OH

(38) Ajmaline

(39) Reserpine

143

144

ROBERT VERPOORTE ET A L .

tensive activity. The use of reserpine in 1982 in pharmacy-distributed drugs in the 12 IDC (Industrial Drugs and Chemicals) countries was 300 kg (2).The source of these alkaloids is Rauvoljia serpentina. The biosynthesis of ajmaline has been studied in detail by Stockigt and co-workers (667-677). Fig. 13 summarizes the biosynthetic pathway. Studies on the production of the pharmaceutically interesting alkaloids has lead to several patent applications (see Table XLI). Table XLII lists the alkaloids produced in cell cultures of the various species studied. 1. Plant Biotechnology

Rauvoljia serpentina roots are the source used in industrial extraction of the alkaloids. The plant is propagated by root cuttings as it has low seed viability (697).The use of micropropagation is thus of interest, and several authors reported successful procedures for micropropagation (698-701). Mathur et al. (697) reported the micropropagation of colchicine-induced tetraploids. Multiple shoot formation was obtained, as reported by Roja et al. (688),on M S medium containing 1 ppm BAP and 0.1 ppm NAA. 2. Alkaloid Production by Cell Cultures: Ajmaline and Related Alkaloids The first report on callus cultures of R.serpentina dates from as early as 1962 (702). Growth conditions for callus cultures of several plants in the family Apocynaceae were established. Slow growth was noted, but no analysis of alkaloid production was made. Vollosovich et al. (703) compared alkaloid production in callus cultures of R. verticillata, R. caffra, R. canescens, and R. serpentina. The latter species was found to have the highest alkaloid production. Subsequently extensive studies were made for optimization of growth and alkaloid production. During growth shifting, percentages of diploid, triploid, tetraploid, and multiploid cells were noted. In addition to the karyotype heterogeneity, the alkaloid contents of cell lines derived from a single cell line also showed considerable variability. Cell suspension cultures were obtained producing 1.5% total alkaloids on dry weight basis; the indoline alkaloid production was 0.6%. This work has been reviewed by Kunakh and Alkhimova (704). Growth and alkaloid production in callus cultures of R. serpentina were extensively studied by Ohta and Yatazawa (683). 2,4-D was found to induce callus formation and to promote growth, but ajmaline levels in the calli decreased with an increase of 2,4-D in the medium. Kinetin was necessary for alkaloid production. Stockigt et al. (685) characterized the alkaloid content of a cell suspension culture of R. serpentina. Twelve alkaloids were isolated and identified

1. PLANT BIOTECHNOLOGY

CH300C

145

strictosidine

polyneuridine aldehyde

--TH

OH

OAc

\

vomi leni ne

CH3 a j ma1i ne

Hd

It O,,% ,,

Glucose

\\'

9

raucaffricine

FIG. 13. Intermediates in the biosynthesis of raucaffricine and ajmaline in Rauwolfia.

146

ROBERT VERPOORTE ET AL. TABLE XLI FOR BIOTECHNOLOGICAL PRODUCTION PATENT APPLICATIONS OF Rauvolfia ALKALOIDS

Lengd. Chem. Pharm. SU-679-625. 03-03-77-SU-481142 (18-08-1979). Rauvolfia tissue culture nutrient medium, containing additional ammonium sulfate and phosphate, potassium chloride, and calcium nitrate to increase medicinal alkaloid yield. Lengd. Chem. Pharm. In. SU-587-939. 20.05076-SU-364546 (17-01-1978). Ajmaline pharmaceutical preparation by extraction of Rauuolfia plant tissue culture with isopropanol, methanol, and benzene, using tartaric acid. American Cyanamid Co. Dl-108-769. 26-07-72-D1-164695 (05-10-1974). Cell culture production of plant metabolites by inducing redifferentiation of undifferentiated plant cell cultures (among others Rauvol&a for reserpine production). Sumitomo Chemical. Jpn. Kokai Tokkyo Koho. JP 55096096, 21-07-1980. JP 79-4275; 1601-1979. Enhancement of protoplast fusion in plant cell cultures (Rauvolfiaserpentina). Chem. Abstr. 93,218142q. Sekisui-Plastics. Jpn. Kokai Tokkyo Koho JP 62058993 A2 14-03-1987 JP 85-196108; 6-091985. Reserpine manufacturing by Rauuolfia callus culture (Rauvolfia vomitoria). Chem. Abstr. 107, 1326292. Sekisui-Plastics. Jpn. Kokai Tokkyo Koho. JP 62003794 A2 9-01-1987. JP 85-141504; 2906-1985. Rauvolfia alkaloid manufacture by tissue cultures (Rauvolfiaserpentina). Chem. Abstr. 107,5795a. Sekisui-Plastics. 56 0259-196: 07-10-1983 JP-18711I ; 21-12-1985. A process for preparing Rauuolfia alkaloids, e.g., reserpine, by tissue culture of Rauvolfia plants, using a culture medium containing 2,4-D and kinetin for callus proliferation and a culture medium containing naphthaleneacetic acid and cytokinins for callus growth deterioration. Chem. Abstr. 104, 147155d. K. Fujimoto, 0. Yamamoto, and M. Kashiwara. Jpn. Kokai Tokkyo Koho. JP 62 03,794 (87 03,794). Rauvolfia alkaloid production by tissue cultures of Rauvolfia serpentina. Chem. Abstr. 107,5795a.

by means of spectral data. The major alkaloid was vomilenine (57 mg/ liter), which had not been found in the plant itself but was known from R. vornitoriu. The cultured cells produced over 50-fold higher levels of this alkaloid than the plant. Further ajmaline-type alkaloids found were ajmaline (1.7 mg/liter), 17-O-acetylnorajmaline (0.8 mg/liter), 17-0acetylajmaline (0.2 mg/liter), and vinorine (0.4 mg/liter). The yohimbinetype of alkaloids were represented by reserpine (1.74 mg/liter) and yohimbine (0.2 mg/liter). Trace amounts of heteroyohimbine-type alkaloids (ajmalicine, 3-isoajmalicine, serpentine, alstonine) and sarpagine were also found in the cell culture. In a further study (689) a polar glycoalkaloid was reported to be the major product of the R. serpentinu cell culture. The alkaloid raucaffricine was isolated with rotation locular countercurrent chromatography (RLCC); the cell culture contained 1.6% on basis of dry weight of this alkaloid, which is considerably higher than levels in the plant R. cuffru from which it was first isolated. Further studies

1.

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TABLE XLII PRODUCTION OF ALKALOIDS I N PLANTCELL,TISSUE,AND ORGAN CULTURES OF Rauvolfia SPECIES Major alkaloid Reserpine Reserpine Ajmaline Ajmaline Vomilenine Vomilenine, ajmaline, reserpine Reserpine Ajmaline Raucaffricine Ajmaline, reserpine Ajmaline Ajmaline Reserpine Ajmaline Ajmaline, reserpine, vomilenine Raucaffricine

Ajmaline

Species

Type of culture

Alstonia constricta R . serpentina R . serpentina R . serpentina R . serpentina R . serpentina

Callus

R . yunnanensis R . serpentina R . serpentina R . serpentina

Callus Shoots Suspension Suspension

R . serpentina R . vomitoria R . serpentina R . serpentina R . serpentina

Plated cells Suspension Suspension Shoots Callus

R . serpentina, R . verticillata, R . mannii, R . caffra R . serpentina

Suspension

Callus Suspension Callus Callus Suspension

Shoots

Yield

Ref. 6 78

-

0.5% DW 0.02% DW 0.2% DW, 0.057 g/liter; 0.007% DW, 0.002 g/liter; 0.007% DW, 0.002 g/liter 0.081% DW 1.6% DW 0.012, 0.06% DW 0.1% DW 0.04 g/liter 0.1% DW 0.02% DW 0.3-1.6, 0.01, 0.5-0.9% DW 1.6 gfliter; 0.17% DW, 0.02 glliter; 0.08% DW, 0.01 g/liter; 0.18% DW, 0.05 g/liter 0.15% DW

679-681 682 683 684 685

686 687,688 689 690 691 691 692 693 694

695

696

of this alkaloid resulted in a revision of its structure; instead of galactose, glucose was found to be the glycosidic component of the alkaloid. Raucaffricine was thus identified as vomilenine-P-D-glucoside.The cell cultures contained an enzyme, raucaffricine 0-D-ghcosidase, which hydrolyzes raucaffricine to yield vomilenine. This alkaloid has a central role in the biosynthesis of ajmaline (Fig. 13). The enzyme could be determined in a series of Rauvoljia cell cultures; including those of R . verticillata, R . caffra, R . mannii, and R . serpentina. The presence of raucaffricine was clearly correlated with the occurrence of the enzyme (674). Further glucoalkaloids (acetylrauglucine, rauglucine, and acetylnorrauglucine) were identified as minor products of the cell cultures (705). As vomilenine has a number of reactive groups it is of interest as a

148

ROBERT VERPOORTE E T A L .

synthon in biomimetic syntheses. Therefore, further studies on the production of its glycoside raucaffricine have been made (695).Cell cultures of several Rauuoljia species were studied for their raucaffricine content (Table XLII). RauuolJia serpentina was found to contain the highest amount, and subsequent studies were conducted on this species. By using the alkaloid production medium as described by Zenk et al. (20)containing 10% sucrose and 0.25% magnesium sulfate, a raucafrricine yield of 1.6 g/liter could be obtained. The high production of this alkaloid was a stable trait over a period of 4 years. No cell line selection was performed. Vomilenine could be obtained in 85% yield from raucaffricine by incubation with a crude enzyme preparation from the cell culture under conditions of continuous extraction of the alkaloid with ethyl acetate. Arens et al. (691)reported a radioimmunoassay method for the quantitative analysis of ajmaline in plant material. Several plants were screened for their ajmaline content. The highest levels were found in R. uomitoria. Cell cultures of this plant were initiated and further optimized for ajmaline production. Tryptophan addition to the cell culture resulted only in one type of medium in an increase of alkaloid production (up to 0.04 glliter). The RIA method also proved to be suitable in screening R. serpentina cell clones for high production. Initial experiments showed variations in productivity over a factor 5 for cell clones obtained from one population. Cell line selection for improving alkaloid production thus seems promising. Kostin et al. (706) reported that total alkaloid extracts of R. serpentina cell cultures had a lower toxicity and a better antiarrhythmic activity than ajmaline. Roja et al. (688,693,696) reported the production of ajmaline (0.15% of DW) by means of shoot cultures ofR. serpentina. These cultures also produced ajmalidine and 3-epi-a-yohimbine. Growth and alkaloid production were stable over a period of 5 years. Westphal (140) reported on the large-scale culture of R. serpentina. In a semicontinuous mode cells were cultured on a 75,000-liter scale. However, no notable amounts of alkaloids could be detected. 3. Alkaloid Production by Cell Cultures: Reserpine and Related Alkaloids The R. serpentina cell cultures studied by Stockigt et al. (685)produced only small amounts of reserpine (39) (0.007% of DW, corresponding to 0.002 g/liter). The production of reserpine was not a stable trait of the cell culture; production gradually decreased during 8 months of subculturing. Reserpine production could be increased by selection of calli surviving stress induced by variations in the medium composition, namely, changing the phytohormones from 2,4-D and kinetin to NAA and BAP (690).After three selections reserpine levels reached 0.022% of the dry weight; the

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149

original stable cell line reached only 0.003%. The elevated reserpine production remained on subsequent subculturing on the original medium. Further optimization of the growth medium resulted in a stable production amounting to 0.03-0.06% of the dry weight. By selection of yellow-green fluorescent cell aggregates under UV light, cell lines with a high production of reserpine (0.1% of DW) could be obtained (692). 4. Enzyme Production by Cell Cultures: Strictosidine Synthase The enzyme strictosidine synthase (EC 4.3.3.2) is responsible for the stereospecific coupling of tryptamine and secologanin, yielding strictosidine (Fig. 12). This glucoalkaloid is the precursor for all terpenoid indole and related alkaloids, including among others the Cinchona quinoline alkaloids. Hampp and Zenk (707) isolated and purified this enzyme to homogeneity from a cell suspension culture of R . serpentina. The enzyme could successfully be immobilized on CNBr-activated Sepharose 4B, as was reported for this enzyme isolated from Catharanthus roseus (102,708). It proved to be more stable than the C. roseus enzyme; the half-life of the immobilized enzyme was 100 days at a temperature of 37°C. Purification of the enzyme opened the way for further molecular biology studies, resulting in the isolation of the cDNA clone for strictosidine synthase (6). Expression of the protein was obtained in Escherichia coli, and the protein was enzymatically active ( 7 ) . Cultures of the microorganism produced, per unit time, 20 times more enzyme per liter than the plant cell culture. C. MISCELLANEOUS INDOLEALKALOIDS 1. Vinca Alkaloids Vincamine (40) is derived from Vinca minor. The in uitro propagation of this plant has been studied (709). Axillary shoot proliferation was best with media containing BAP and NAA. As part of a program for the industrial production of this alkaloid plant cell cultures have been studied as a possible source (710). Petiard and co-workers (711,712) reported the initiation of cell cultures of V . minor; based on TLC analysis small amounts of

(40)Vincamine

150

ROBERT VERPOORTE E T A L .

TABLE XLIII FOR BIOTECHNOLOGICAL PRODUCTION OF Vinca ALKALOIDS PATENTAPPLICATIONS

Synthelabo. 04-02-1975 FR-003347 (05-08-1976). Alkaloid production by culturing Vinca minor cells in undifferentiated state in liquid medium. Synthelabo 22-10-1980-FR-022538(24-04-1982). In vitro metabolite production or biotransformation using plant cells is effected in an unstirred liquid medium without any support for fixation of the cellular tissue. P. N. Crespi, L. Garofono, A. Guicciardi, and A. Minghetti. Farmitalia Carlo Erba S.r.1. Ger. Offen. DE 390280 A l , 17-08-1989. Chem. Abstr. 112, 196670s.

vincamine were thought to be present in these cultures. Garnier et al. (713), however, could not confirm these results. Although small amounts of spots positive with Dragendorff’s reagent were detected on TLC plates, no vincamine could be observed in V . minor or V . major cv. variegata callus cultures. A lignan, lirioresinol B, was found to be the major compound in these cultures. Eilert et al. (714) identified strictosidine lactam as the main alkaloid produced in two cell lines of V . major cv. variegata. The high yielding cell line produced 0.5-1 mg/g dry weight. Transferring the cells to an alkaloid production medium resulted in a 6- to 8-fold increase in alkaloid levels combined with greening of the cells. Crespi et al. (715) patented the production of vincamine and epivincamine by means of cell cultures of V. minor. Yields of 3.3 and 0.9 g/liter, respectively, were claimed for the two alkaloids. Two other patents have been reported concerning the production of Vinca alkaloids (Table XLIII). 2 . Ochrosia Alkaloids

Ellipticine (41)and its 9-methoxy derivative (42) have great interest as antitumor drugs. These alkaloids are isolated from the leaves of Ochrosia elliptica, a shrub found on scattered islands in the Indian and Pacific Oceans. Plant cell cultures would thus be of interest as an alternative means of production of the antitumor alkaloids. For a review on the genus, its alkaloids, and cell culture, the reader is also refered to Chenieux et al. (716).

Kouadio and co-workers (717) reported the initiation of callus cultures of this plant. Best results were obtained with calli derived from leaves. Both ellipticine and its methoxy derivative were present in the calli at levels similar to those found in the leaves of the plant (60 and 150 pglg DW). About 5-10% of the total alkaloids could be isolated from the medium. Production of the alkaloids remained stable for a prolonged period. Weber et al. (718) could not detect alkaloids in calli derived from

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(41) Ellipticine, R = H (42) 9-Methoxyellipticine,R = OCH3

roots. On the other hand, calli derived from stems afforded ellipticine, 9-methoxyellipticine, and isoreserpiline. Cell suspension cultures of 0. elliptica were shown to produce the alkaloids ellipticine, 9-methoxyellipticine, as well as elliptinine, isoreserpiline, and reserpiline (719). The cell cultures were difficult to establish, however, owing to rapid browning followed by necrosis. The addition of adsorbents or antioxidants was not successful. By washing the cells every 2 days with fresh medium, growth of the cells progressively improved. Five cell lines obtained by this method produced about 50 pglg dry weight of ellipticine and 90 pg/g dry weight of 9-methoxyellipticine. The medium used was Gamborg B5 supplemented with 1 ppm 2,4-D, 0.5 ppm NAA, and 0.1 ppm kinetin. Alkaloids accumulated during the stationary phase. The production remained stable over more than 20 subcultures. Cloning of small cell aggregates showed that considerable variation occurred, which could be further exploited for obtaining high producing cell lines. Two cell lines were successfully grown in 2-liter bioreactors. Pawelka and Stockist (588) identified eight different indole alkaloids from cell suspension cultures of 0. elliptica. The pattern of the alkaloids found was quite different from the above-mentioned results reported by Kuoadio and co-workers (719). The alkaloids identified were tetrahydroalstonine, cathenamine, pleiocarpamine and two other methoxy-substituted heteroyohimbine alkaloids (Corynanthelheteroyohimbinetype), norfluorocurarine (Strychnos type), and apparicine and epchrosine (Aspidosperma type). The last mentioned alkaloid was a new compound (720) not known from intact plants. Plant cell cultures have also been used for making derivatives of ellipticine (721). Some selected strains of Choisya ternata were capable of converting ellipticine to its 5-formyl derivative. The bioconversion occurred only on solid media. 3. Tabersonine

For the industrial semisynthesis of vincamine, tabersonine is used. This alkaloid among others is found in the seeds of Amsonia tabernaemontana. Petiard et al. (722) reported callus cultures of this plant, but no further

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reports on alkaloid production in these cultures have appeared. Tabersonine was isolated from various other Apocynaceae plant cell cultures. Stockigt et al. (723) isolated it as one of the major alkaloids from Stemrnadenia tomentosa and Voacanga africana cell suspension cultures. In the latter culture tabersonine production could be increased to about 0.1 g/liter by inoculation on an alkaloid production medium (724). Voacanga thouarsii also proved to be a source for tabersonine (725,726) Furthermore, Rhazya stricta was found to produce tabersonine among a series of other indole alkaloids (727). 4. Dimeric Alkaloids

Because of the economic and pharmacological importance of the dimeric alkaloids of Catharanthus roseus (see above), extensive surveys have been made to locate other sources of the dimers. Cell cultures of various plants have also been studied for this purpose. Stockigt et al. (724) reported the first isolation of dimeric indole alkaloids from Voacanga africana. The alkaloids isolated, voafrine A and B (43 and 44),were dimers of tabersonine. The alkaloids were thought to be formed by an enzymatic reaction. Some microorganisms were also found to be capable of converting tabersonine to voafrine A and B. The cell cultures produced about 20 and 100 mglliter of voafrine B and tabersonine, respectively, after transfer to an alkaloid production medium. In a further study it was found that an enzyme from the leaves of Catharanthus roseus efficiently catalyzes the dimerization of tabersonine. This enzyme even produced small amounts of a trimer of tabersonine, which has a third 3”stabersonyl moiety bound to voafrine B in the 14’ position (728). The dimerization reaction of tabersonine is induced by an oxidation at C-3, yielding an iminium compound, which, similarly to vindoline, readily forms dimers. The first products formed by incubation of tabersonine with a crude enzyme extract from C. roseus were identified as 3-hydroxyvoafrine A and B (729).

CH3

’N‘

\

(43) Voafrine A (3’R) (44) Voafrine B (3’s)

1. PLANT BIOTECHNOLOGY

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Tabernaemontana cell cultures have also been studied as a source for dimeric indole alkaloids (for review, see Ref. 730). Although the plants produce considerable amounts of dimers, so far only small amounts of conodurine-type dimers and monogagaine could be found in callus cultures of T . elegans (732). Cell suspension cultures of this species produced various monomers, whereas only traces of yet unidentified dimers could be found (732).

5. Camptothecin The first mention of the production of the antitumor alkaloid camptothecin (45) by means of Camptotheca acuminata cell cultures was reported by Misawa (733).Sakato and Misawa studied the cell cultures of this plant in more detail, resulting in a cell suspension culture which produced 2.5 pg/g dry weight of the alkaloid (734,733, which is considerably lower than the intact plant. Patents concerning camptothecin production by means of plant cell cultures are summarized in Table XLIV. Camptothecin derivatives have been produced in microorganisms. Aspergillus flauus was reported to convert camptothecin to 10-hydroxycamptothecin (736).

(45) Camptothecin

TABLE XLIV OF CAMPTOTHECIN PATENTSCONCERNING BIOTECHNOLOGICAL PRODUCTION Kyowa Hakko Co. Ltd. 54 8028-691. 26-08-1971-JA-064706; 16-04-1973. Anticarcinogenic camptothecin preparation from Acuminata leaves by fermentation. H. Kashiki, E. Takahashi, and T. Takemoto. Kanebo, Ltd. Jpn. Kokai Tokkyo Koho. JP 62096088 A2 2-5-1987. JP 85-236574 (22-10-1985). Antitumor substances from Putterlickia or other plant tissue cultures. Chem. Abstr. 107, 381012. G. Zhu. Faming Zhuanli Shenqing Gongkai Shuomingshu. CN 85100520 A 13-3-1986. CN 85-100520, 1-4-1985. 10-Hydroxycamptothecin manufacture by Aspergillus frauus T-419. Chem. Abstr. 107,219478.

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6. Physostigmine

Despite the fact that physostigmine is an important pharmaceutical, no reports have appeared on efforts to produce this alkaloid by means of plant cell cultures. Daoust (737) reported the fermentative production of physostigmine by means of the microorganism Streptomyces griseofuscus. Iwasa et al. (738) employed Streptomyces pseudogriseolus for the production of physostigmine. Murao and Hayashi (739) isolated physostigmine and its N-8 nor derivative from Streptomyces SP. AH-4.

IX. Caffeine In connection with plant breeding programs, cell and tissue cultures of Coffea species have been studied extensively. We do not deal with this work here, however, and the reader is referred to Baumann and Frischknecht for a review (740,741). Although caffeine (46) can be easily isolated from coffee beans at low cost, a number of studies have been made on the production of caffeine in cell cultures of Coffea. The biotechnological aspects of the caffeine production by means of cell cultures has been reviewed by Prenosil et al. (109). In Table XLV the occurrence of caffeine in various cell cultures is summarized. The levels of caffeine and related purine alkaloids found in Coffea callus cultures are similar to those found in young leaves and ripe coffee beans (747,749), namely, concentrations of 1-2% on a dry weight basis. In suspension cultures somewhat lower levels have been found. The alkaloid content was highly variable from one cell line to another (0.030.7% DW) and also varied from one subculture to another (746). High producing cell lines can be obtained by selection of aggregated cells (740,741).The relationship between high productivity and large aggregates may, however, be lost during successive subculturing (741). The cell cultures produced also theobromine (47). The ratio caffeine to theobro-

CH3

(46)Caffeine, R = CH3 (47) Theobromine, R = H

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1 . PLANT BIOTECHNOLOGY TABLE XLV OCCURRENCE OF CAFFEINE AND OTHERPURINE ALKALOIDS IN PLANT CELLA N D TISSUECULTURES Alkaloid

Plant species

Caffeine, theobromine Caffeine Caffeine Caffeine Caffeine, theobromine Caffeine

Thea sinensis Coffea arabica Coffeaarabica Coffea arabica Coffea arabica Coffea arabica

Caffeine

Coffearobusta

Caffeine

Coffeacongensis

Theobromine

Coffea stenophylla

Theobromine Theobromine Theobromine Theobromine Theobromine Theobromine Theobromine Theobromine Theobromine

Coffea eugenoides Coffeahurnilis Coffeaarabusta Coffea liberica Coffeaabeokutae Psilantus mannii Theobroma cacao Paullinia cupana Thea sinensis

Caffeine Caffeine Caffeine, theobromine Caffeine, theobromine

Coffea arabica Coffea robusta Coffeaarabica Coffea arabica

Caffeine, theobromine Caffeine, theobromine Caffeine

Coffea arabica Coffea arabica Coffea arabica

Type of culture Callus Callus Suspension Callus Suspension Callus, suspension Callus, suspension Callus, suspension Callus, suspension Suspension Callus Callus Suspension Callus Suspension Suspension Callus Callus, suspension Callus Callus Suspension Suspension, immobilized cells Suspension Suspension Suspension

Yield 0.15% DW

-

0.038% DW 1-1.6% DW 0.03-0.7% DW 1% DW 0.03-0.7% D W 1.6, 0.04-1.5% DW 0.3. 0.4% DW

Ref. 742 743 744 745 746 747

Traces Traces 0.2% DW Traces Traces Traces Traces Traces 0.6% DW Traces, 0.01%DW 0.8% DW 1% DW 0.47 g/liter 0.03, 0.4 g/liter 0.04 g/liter 0.12 g/liter 1% DW, 0.48 g/liter

748 749 750 751

752 753 754

mine varied throughout the growth cycle of the Coffeu cell suspension culture, having a maximum at the end of the exponential growth phase. Theobromine represented about 25-50% of the total alkaloids (746). For optimal growth 2,4-D is the preferred auxin; by replacing this with NAA a 2- to 3-fold increase in alkaloid production was observed. Changing to IAA or no auxins at all resulted in decreased alkaloid production (741).

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Stress by high light intensity led to an increase in alkaloid production, particulary in low producing cell lines; high NaCl concentrations promoted increased alkaloid production in high producing aggregated cell lines (750). Addition of ethephon, an ethylene precursor, resulted in an 85% increase of purine alkaloids in a Coffeu cell suspension culture (753). In large part, the alkaloids are found in the medium (745,748,749). In fact, there is generally a free exchange of alkaloids between the medium and the cells, that is, the amount of alkaloids in the medium and cells is largely determined by the volume ratio of the two compartments (741). To some extent caffeine is accumulated intracellularly by complexing with chlorogenic acid (754,755). High levels of chlorogenic acid correlated with increased caffeine accumulation in the cells. In studies of plant cell cultures of various caffeine-producing plants it was found that low purine alkaloid production was correlated with a high catabolic activity for caffeine (747). Coffeu cells have been successfully cultured in a 14-liter stirred-tank fermentor, where the caffeine production was similar to that in flask experiments (109). Alkaloid levels increased rapidly to 0.032 g/liter after the carbon source was exhausted. Cell aggregates have been cultured in expanded bed reactors, and caffeine levels of about 0.07 g/liter were obtained (109,756). Cell aggregates have also been grown in a kind of membrane boat, floating in a liquid medium (109,757). The cells used in these experiments, however, did not produce caffeine. Immobilization of Coffeu cells in calcium alginate resulted in a 13-fold increase in purine alkaloid production (109,751). Production reached levels of 0.4 g/liter in 32 days. Pedersen et al. (758) also found increased alkaloid production for calcium alginate-immobilized Coffeu cells. Strategies for operating bioreactors for the production of caffeine have been discussed by Pedersen et ul. (752). As caffeine production appears at the end of the growth phase, that is, is not growth related, a strategy of periodic growth and production stages seems the most promising. The Coffea cells are capable of converting added theobromine to caffeine, and the highest bioconversion rates (- 1 mg/day/g DW) were found during the exponential growth phase (746,759). Ogutuga and Northcote (742) studied the production of caffeine and theobromine by callus cultures of Thea sinensis (Camellia sinensis). Caffeine contents of 0.15% (total in medium and callus per dry weight of callus) were found; considerable but variable amounts were excreted into the medium. The highest production was found for cells grown in the dark. Theobromine levels were much lower (0.02%), and most was found in the medium.

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X. Steroidal Alkaloids Several steroidal alkaloids have pharmaceutical interest, either because of their biological activity or as a raw material for the production of various steroids used in pharmacotherapy . Solanurn glycoalkaliods (48-53) are examples of the latter. Most of the work on the production of steroidal alkaloids concerns this type of compound.

(48) Solasonine, R = 4 a l a c t o s e - G l u c o s e

I

Rhamnose (49) Solasodine, R = H (50) Solamargine, R = --Galactose---Rhamnose

I

Rhamnose

(51)Solanidine, R = H (52) Solanine, R = 4 al act o s e- Gluc ose

I

Rhamnose (53) Chaconine, R = 4 a l a c t o s e R h a m n o s e

I

Rhamnose

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ROBERT VERPOORTE E T AL.

A. Solanum ALKALOIDS As can be seen from Table XLVI, results concerning production of glycoalkaloids in Solanum cell cultures are variable; some authors were unable to detect the glycoalkaloids in cell cultures (e.g., Ref. 760),others did find production of these compounds in various types of cell and tissue cultures. In fact, Zenk (761)showed that cell clones of Solanum laciniatum may vary significantly in solasonine (48) content, ranging from 0 to more than 3% dry weight, with a clear maximum in frequency of occurrence at about 0.2%. In most cases the alkaloid content was determined after hydrolysis of the glycoside, yielding the aglycone solasodine (49). The actual alkaloids present in the cell cultures were identified in only afew cases. Heble et al. (762) isolated solasonine from S. xanthocarpum; Kadkade and Madrid (765) identified solamargine (50) and solasonine (48) in S. acculeatissimum, and the aglycone was also isolated from this plant. Hosoda and Yatazawa (773)could not detect these two glycoalkaloids in S. laciniatum; instead,the major compound was a solasodine-based compound coupled with several sugars. Also, S. khasianum calli produced an unidentified glycoalkaloid as well as small amounts of solasonine and solanidine (51) (770). Zacharius and Osman (766) identified solanine (52) and chaconine (53) in S. tuberosum root-forming calluses, and a new glycoalkaloid, dehydrocommersonine, was isolated from S. chacoense. From the yields summarized in Table XLVI, it is clear that production of the glycoalkaloids is low in cell and tissue cultures, far too low to be of industrial interest. In fact a certain degree of organization leads to an increase of alkaloid production and in particular root formation was correlated with higher levels of glycoalkaloids (766,769,770,775, 783,784,786,787,792,796).Zacharius and Osman (766) reported that only root-forming calluses of S. tuberosum and S . chacoense were capable of producing glycoalkaloids. Several authors reported on the positive effect of light on the production of steroidal glycoalkaloids (785,786,793,796). Greening cultures in particular were found to have increased alkaloid levels (792,793). This could be connected with the fact that actively photosynthesizing chloroplasts are involved in steroidal alkaloid biosynthesis (792,793,801). Interestingly several authors noted an increased production of alkaloids in the presence of 2,4-D (763,781,796), whereas with other auxins in the media no production at all could be observed (763,781).The production of alkaloids is not restricted to a certain growth phase of cell suspension cultures (786,787,792). Several studies showed that alkaloid production in calli derived from different parts of the plant may vary (772,781,795). A selection of high

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1 . PLANT BIOTECHNOLOGY

TABLE XLVI OF Solanum STEROID ALKALOIDS IN PLANTCELLAND TISSUECULTURES OCCURRENCE Alkaloid" Solasonine Solasodine

Plant species

Type of culture

S . xanthocarpum S . xanthocarpum S . aviculare S . elaegnifolium S . khasianum S.nigrum S . acculeatissimum

Callus Suspension Suspension Callus Callus Callus Callus

S . chacoense S . tuberosum S . aviculare S . laciniatum S . khasianum

Callus roots Callus roots Suspension Callus Callus, callus roots

Solasodine Solasodine Solasodine glycoside Solasodine Solasodine Solasodine Solasodine Solasodine Solasodine Solasodine Soladulcidine Solasodine Glycoalkaloids

S . khasianum S . khasianum S . laciniatum S . laciniatum S . xanthocarpum S . jasminoides S . jasminoides S . verbascifolium S . verbascifolium S . aviculare S . dulcamara S . laciniatum S . nigrum

Callus Callus Callus Callus Callus roots Suspension Callus Suspension Callus Immobilized cells Callus Callus Callus, suspension

Glycoalkaloids

S . dulcamara S . nigrum

Solasodine

S. nigrum

Callus Callus Suspension Flat bed bioreactor Immobilized Callus, shoots

Solasodine

S . laciniatum

Callus, suspension

Solasodine Glycoalkaloids Solasodine Solasodine Solasodine, soladulcidine

S . laciniatum S . laciniatum S . xanthocarpum S . glaucophyllum S . dulcamara

Callus Suspension Callus Callus Callus, suspension, shoot

Solasodine, solasonine, solamargine Dehydrocommersonine Solanine, chaconine Solasodine Solasonine Solasonine, solanidine

Yield 0.003% DW 0.09% DW 0.032% DW 0.025% DW 0.004% DW -

0.026% DW 3% DW Traces, 5.2% DW 0.03% DW 0.07% DW 0.05% DW 0.03% DW 0.20% DW 0.19% DW 0.006% DW 0.005% DW

-

1.3, 1.8% DW 3% DW 1.2% DW 1% DW 1.5% DW 1.3% DW 0.14, 0.13% DW 0.12,0.05% DW 0.14% DW 0.2% DW 0.004% DW 0.02, 0.01, 0.16% DW

Ref. 762,763 764

765 766 766,767 768 76I 769,770 771 772 773 774 775 776 777 778 779 780 781 782 783

784

785 786,787 788 789 790 791 792

(continued)

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ROBERT VERPOORTE ET A L .

TABLE XLVI (Continued) Alkaloid"

Plant species

Type of culture

Solasodine

S.laciniatum

Callus, shoots

Solasodine Solasodine Solanine

S. glaucophyllum S.eleagnifolium S. tuberosum, S . dulcamara S. xanthocarpum

Callus Callus Callus

Solasodine Solasodine Solasodine

S. indicum, S. khasianum, S . xanthocarpum S. xanthocarpum

Suspension, immobilized cells Callus Suspension, immobilized cells

Yield 0.009,0.22% DW

-

0.21% DW 0.06,0.05% DW 0.045 mg/liter

0.003%DW 0.009% 0.012% 0.1% DW, 0.004 gl liter

Ref. 793 794 795,796 797 798

799

800

a In most studies the glycoalkaloids were first hydrolyzed, yielding solasodine, before quantitative analysis. The actual glycoalkaloids present were identified in only a few cases.

producing strains might therefore be useful. The stability of alkaloid production during prolonged subculturing was reported by Chandler and Dodds (786).Osman et al. (767) studied the metabolism of solanidine in S. tuberosum tuber tissue slices and cell suspension cultures. It was found that the aglycone is rapidly glycosylated, yielding the glycoalkaloids. The fact that these cell cultures were unable to produce glycoalkaloids (766) is thus most likely due to lack of biosynthesis of the aglycone solanidine. Continuous production of the glycoalkaloids by using immobilized cells was reported by Jirku et al. (780).The cells of S. auiculare were immobilized on a porous polyphenyleneoxide support by covalent binding with glutaraldehyde. During a 270-hr period the immobilized cells produced about 70-320 pg per gram dry weight of cells in 24 hr. The alkaloids were released into the medium, from which they were subsequently extracted. Subramani et al. (800) immobilized S . xanthocarpum cells in calcium alginate. The immobilized cells produced more alkaloids than the corresponding cell suspension. The highest production was obtained after immobilization of cells from a culture which had reached the stationary phase. Barnabas and David (798) also found S . xanthocarpum cells to display increased alkaloid production after immobilization; although the level of alkaloids in the immobilized cells was lower, more of the alkaloids were excreted to the medium than in suspended cells. Calcium alginateimmobilized cells of S. nigrum gave the highest alkaloid production in the light (1.3% of DW), and the alkaloids were not excreted into the medium

161

1. PLANT BIOTECHNOLOGY TABLE XLVII OF VARIOUS TYPESOF STEROID ALKALOIDS I N PLANTCELLAND OCCURRENCE TISSUECULTURES Alkaloid

Plant species

Type of culture

Yield

Ref.

Tomatine

Lycopersicum esculenturn Holarrhena antidysenterica Holarrhena jloribunda

Callus

0.0013%DW

802

Callus

-

803

Callus

0.001% FW

804

Conessine Conessine

(784). The cells were also grown in a flat bed bioreactor. In this system the cells are grown in a static system, where the medium is dripped on the cell culture. Growth was much lower than in suspension cultures, but alkaloid yields were slightly higher (1.2 versus 1% of DW). An interesting approach was reported by Gorelova et al. (789). Solanum laciniatum cells were cocultivated with the cyanobacterium Chlorogloepsis. Symbiosis resulted in increased glycoalkaloid content on nitrogen-free media. Feeding of cholesterol to S. aviculare cell suspensions resulted in an increase in solasodine content from 0.026 to 0.47% (768).

B. MISCELLANEOUS STEROID ALKALOIDS Aside from the steroid alkaloids from Solanurn, little work has been done on other groups of steroid alkaloids (Table XLVII). Roddick and Butcher (802) reported the isolation of tomatine from a callus culture of tomato; cell suspension cultures and several other callus cell lines did not contain this glycoalkaloid. Heble et al. (803) fed cholesterol to Holarrhena antidysenterica callus cultures. This sterol was among others incorporated into the alkaloid conessine. Callus cultures of Holarrhena Jloribunda were reported to produce small amounts of conessine as well as some unidentified alkaloids (total alkaloids were 0.001% of the fresh weight) (894).

XI. Concluding Remarks In this chapter we have reviewed the state of the art of plant cell biotechnology for the production of commercially important alkaloids. Plant cell and tissue culture methods play an important role in plant

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ROBERTVERPOORTEETAL.

breeding and improvement. For plants used for the isolation of alkaloids, these methods could be of great help in breeding varieties with increased alkaloid production. Considering plant cell cultures, large-scale culturing of plant cells is feasible. However, for none of the alkaloids reviewed has an industrial, large-scale production been realized. In most cases the production of the cell cultures was too low, resulting in a too high a price compared with already existing production methods. Berberine is probably the alkaloid closest to industrial-scale production; however, the market for this alkaloid is not very clear, as its use as pure compound is limited. Berberine is mostly used in traditional medicine in the form of plant extracts which can be produced locally at much lower price than the pure compound. Sanguinarine production has come close to being an economic process; however, the market for this compound has collapsed. As cell suspension cultures of alkaloid-producing plants have been studied exhaustively, little is to be expected from further empirical studies on the influence of growth conditions, selection, etc. Only insight into the regulation of secondary metabolism may give clues for increasing production to levels necessary for an industrial process. Because cell cultures are an excellent system for studying biosynthesis, the vast knowledge available from past studies provides a sound basis for further basic studies on secondary metabolism. Consequently, the more recent studies in plant cell biotechnology focus on biosynthetic pathways, namely, identification and characterization of the enzymes involved in secondary metabolism, in order to pave the way for cloning of the genes. With the genes in hand we may eventually be able to study regulation at the molecular level. The ultimate goal is genetic enigineering for improving (or changing) secondary metabolite production in plants or plant cell cultures. Initial results for certain steps in a biosynthetic route have already been reported (e.g., introduction of the tryptophan decarboxylase gene in tobacco and Catharanthus roseus). Future goals could be the creation of plants with a higher production of a certain compound by adding one or two genes responsible for a final step in a biosynthetic pathway (e.g., increased scopolamine yield, by increasing the activity of the enzymes responsible for the conversion of l-hyoscyamine to scopolamine). Another goal could be to produce a desired secondary metabolite in another plant with better agricultural properties by diverting a biosynthetic pathway from a common intermediate (e.g., terpenoid indole alkaloids derived from strictosidine). Compared to studies on secondary metabolism in microorganisms, the work with plants is hampered by the fact that no mutants are available which lack certain steps in a biosynthetic pathway. That means that in

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plants one has to go all the way from the intermediates in a pathway, through the enzymes, to the genes. On the other hand, with rapidly improving methods in molecular biology, the isolation of genes will become more and more routine. The methods for genetic engineering are also rapidly improving. To be able to utilize these tools to full potential, however, further studies on secondary metabolism are necessary. In this connection, knowledge on the role of secondary metabolites in the plant is also of interest. As secondary metabolites are probably involved in protection of the plants against diseases and predators, genetic modification of secondary metabolism might lead to plants which have an increased resistance.

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734. K. Sakato, H. Tanaka, N. Mukai, and M. Misawa, Agric. Biol. Chem. 38, 217 (1974). 735. K. Sakato and M. Misawa, Agric. B i d . Chem. 38,491 (1974). 736. G. Zhu. Faming Zhuanli Shenqing Gongkai Shuomingshu. CN 85100520 A 13-3-1986. CN 85-100520, 1-4-1985;Chem. Abstr. 107,21947g (1987). 737. D. R. Daoust, Merck and Co., Inc. U.S. U.S. Patent 3,734,832,22-5-1973. U.S. Patent 71-183649, 24-9-1971; Chem. Abstr. 79, 30496q (1973). 738. T. Iwasa, S. Harada, and Y. Sato, Takeda Chemical Industries, Ltd. Jpn Kokai Tokkyo Koho. J P 54062390, 19-5-1979. JP 77-127900, 24-10-1977; Chem. Abstr. 91, 106603t ( 1979). 739. S. Murao and H. Hayashi, Agric. B i d . Chem. 50,523 (1986). 740. T. W. Baumann and P. M. Frischknecht, in “Biotechnology in Agriculture and Forestry. Volume 4, Medicinal and Aromatic Plants I” (Y. P. S. Bajaj, ed.), p. 264 Springer-Verlag, Berlin and Heidelberg, 1988. 741. T. W. Baumann and P. M. Frischknecht, in “Cell Culture and Somatic Cell Genetics of Plants. Volume 5, Phytochemicals in Plant Cell Cultures” (F. Constabel and I. K. Vasil, eds.), p. 403. Academic Press, San Diego, California, 1988. 742. D. B. A. Ogutuga and D. H. Northcote, J. Exp. Bot. 21,258 (1970). 743. H. Keller, H. Wanner, and T . W. Baumann, Planra 108, 339 (1972). 744. E. Buckland and P. M. Townsley, Can. Inst. Food Sci. Technol. J. 8, 164 (1975). 745. P. M. Frischknecht, T. W. Baumann, and H. Wanner, Planta Med. 31,344 (1977). 746. P. M. Frischknecht and T. W. Baumann, Planta Med. 40,245 (1980). 747. T. W. Baumann and P. M. Frischknecht, in “Plant Tissue Culture 1982” (A. Fujiwara, ed.), p. 365. Maruzen, Tokyo, 1982. 748. T. Suzuki and G. R. Waller, in “Plant Tissue Culture 1982” (A. Fujiwara, ed.), p. 385. Maruzen, Tokyo, 1982. 749. G. R. Waller, C. D. MacVean, and T. Suzuki, Plant Cell Rep. 2, 109 (1983). 750. P. M. Frischknecht and T. W. Baumann, Phytochemistry 24,2255 (1985). 751. D. Haldimann and P. Brodelius, Phytochemistry 26, 1431 (1987). 752. H. Pedersen, G. H. Cho, R. Hamilton, and C. K. Chin, Ann. N . Y . Acad. Sci. 506, 163 (1987). 753. G . H. Cho, D. I. Kim, H. Pedersen, and C. K. Chin, Biotechnol. Prog. 4, 184 (1988). 754. T. W. Baumann and L. Rohrig, Phytochemistry 28,2667 (1989). 755. A. W. Kappeler and T. W. Baumann, Colloq. Sci. I n t . Cufk (Montreux),ASIC (Paris), 12th, 247 (1987). 756. M. Hegglin, J. E. Prenosil, and J. R. Bourne, Chimia 44, 26 (1990). 757. J. E. Prenosil, M. Hegglin, J. R. Bourne, and R. Hamilton, Ann. N . Y. Acad. Sci. 8,390 (1986). 758. H. Pedersen, G. H. Cho, D. Kim, D. Cazzulino, and C. K. Chin, Znt. Biofechnol.Symp. 8th 1,480 (1988). 759. T. W. Baumann, R. Koetz, and P. Morath, Plant Cell Rep. 2,33 (1983). 760. D. Vagujfalvi, M. Maroti, and P. Tetenyi, Phytochemistry 10, 1389 (1971). 761. M. H. Zenk, in “Frontiers of Plant Tissue Culture 1978” (T. A. Thorpe, ed.), p. 1. University of Calgary Offset Printing Services, Calgary, Alberta, 1978. 762. M. R. Heble, S. Narayanaswarni, and M. S. Chadha, Naturwissenschaften 55, 350 (1 968). 763. M. R. Heble, S. Narayanaswami, and M. S. Chadha, Phytochemistry 10,2393 (1971). 764. P. Khanna, A. Uddin, G. L. Sharma, S. K. Manot, and A. K. Rathore, Indian J. Exp. B i d . 14,694 (1976). 765. P. G.Kadkade and T. R. Madrid, Naturwissenschaften 64, 147 (1977). 766. R. M. Zacharius and S. F. Osman, Plant Sci. Lett. 10,283 (1977).

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767. S. F. Osman, R. M. Zacharius, and D. Naglak, Phytochernistry 19,2599 (1980). 768. P. Khanna, G. L . Sharma, A. K. Rathore, and S. K. Manot, Indian J. Exp. Biol. 15,1025 (1 977). 769. K. Kokate and S. S. Radwan, Planta Med. 33, 301 (1978). 770. K. Kotate and S. S. Radwan, Z. Naturforsch., C : Biosci. 34,634 (1979). 771. H. C. Chaturvedi, A. R. Chowdhury, and A. Uddin, IndianJ. Exp. Biol. 17, 107 (1979). 772. A. Uddin and H. C. Chaturvedi, Planta Med. 37,90 (1979). 773. N. Hosoda and M. Yatazawa, Agric. Biol. Chem. 43,821 (1979). 774. N. Hosoda, H. Ito, and M. Yatazawa, Agric. Biol. Chem. 43, 1745 (1979). 775. A. R. Chowdhury, R. N. Prasad, and A. Uddin, Q. J . Crude Drug Res. 17, 137 (1979). 776. S. C. Jain and S. L. Sahoo, Agric. Biol. Chem. 45, 2909 (1981). 777. S. C. Jain, P. Khanna, and S. Sahoo, J. Nut. Prod. 44, 125 (1981). 778. S. C. Jain and S. Sahoo, Pharmazie 36,715 (1981). 779. S. C. Jain and S. Sahoo, Chem. Pharm. Bull. 29, 1765 (1981). 780. V. Jirku, T. Macek, T. Vanek, V. Krumphanzl, and V. Kubanek, Biotechnol. Lett. 3, 447 (1981). 781. G. Willuhn and S. May, Planta Med. 46, 153 (1982). 782. E. Munteanu, V. Ciurdaru, and C. Bodea, St. Cerc. Biochim. 26, 160 (1983). 783. K. Lindsey and M. M. Yeoman, J. Exp. Bot. 34, 1055 (1983). 784. K . Lindsey and M. M. Yeoman, in “Plant Biotechnology” ( S . H. Mantell and H. Smith, eds.), p. 39. Cambridge Univ. Press, Cambridge, 1983. 785. P. N. Bhatt, D. P. Bhatt, and I. Sussex, Physiol. Plant 57, 159 (1983). 786. S. Chandler and J. H. Dodds, PIant Cell Rep. 2,69 (1983). 787. S. Chandler and J. H. Dodds, Plant Cell Rep. 2,205 (1983). 788. S. F. Chandler, Ann. Bot. 54,293 (1984). 789. 0. A. Gorelova, J. Rebarek, T. G. Korzhenevskaya, R. G. Butenko, and M. V. Gusev, Fiziol. Rust. (Moscow) 32, 1158 (1985); Chem. Abstr. 104,48786d (1986). 790. N. J. Barnabas and S. B. Davis, Geobios 13, 233 (1986); Chem. Abstr. 106, 47244e ( 1987). 791. S. C. Jain and S. Sahoo, Pharmazie 41,820 (1986). 792. A. Emke and U. Eilert, Plant Cell Rep. 5, 31 (1986). 793. A. J. Comer, Phytochemistry 26,2749 (1987). 794. S. L. Sahoo and S. C. Jain, Cell Chromosome Res. 10, 104 (1987); Chem. Abstr. 108, 164767e (1985). 795. H. M. Nigra, 0. H. Caso, and A. M. Giuletti, Plant Cell Rep. 6, 135 (1987). 796. H. M. Nigra, M. A. Alvarez, and A. M. Giulietti, Plant Cell Rep. 8,230 (1989). 797. P. Khanna, P. Kumar, and S. Singhvi, Indian J . Pharm. Sci. 50,38 (1988). 798. N. J. Barnabas and S. B. David, Biotechnol. Lett. 10,593 (1988). 799. N. J. Barnabas, R. Karamalawala, and S. B. David, Indian J. Exp. Biol. 27,664 (1989). 800. J. Subramani, P. N. Bhatt, and A. R. Mehta, Curr. Sci. 58,510 (1989). 801. N. K. Ramaswamy, A. G. Behere, and P. M. Nair, Eur. J. Biochem. 67,275 (1976). 802. J. G. Roddick and D. N. Butcher, Phytochemistry 11,2019 (1972). 803. M. R. Heble, S. Narayanaswamy, and M. S. Chadha, Phytochemistry 15, 1911 (1976). 804. L. Bouillard, J. Homes, and M. Vanhaelen, Phytochemistry 26,2265 (1987).

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-CHAPTER

2-

ALKALOIDS FROM MUSHROOMS* R 6 z ANTKOWIAK ~ AND WIESJLAW Z. ANTKOWIAK Faculty of Chemistry Adam Mickiewicz University Poznan, Poland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190 194 194 A. Muscarinic Exocyclic Amines. . . . . . . . . . . . . . . . . 208 216 225 226 A. Bufotenine 228 229 23 1 D. p-Carbolines . . . . . . . . . . . 233 .......... E. Ergot Alkaloids . . . . . . . . 253 .......... IV. Pyridine Alkaloids . . . . . . . . . 253 A. Orellanine.. . . . . . . ........ . . ...... ..... . . ... . .. 269 B. Necatorones . . . . 272 V. Hydrazine Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 275 ..................... 276 279 279 D. Xanthodermin. . . . . . . . . . . . . 280 280 28 1 28 1 .......................................... 283 283 286 287 VII. c Y - A m i n o A c i d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 .... 290 A. Active Principles of Clitocybe acromelalga . . . . . . . . . . . . 298 B. Coprine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Aniline Derivatives from Agaricus Species . . . . . . . . . . . . . . . . . . . . . . . . . . 299 303 11. Physiologically Active Principles of the Genus Amanita . . . . . . . . . . . . . . . . . .

* Dedicated to the memory of Professor Jerzy Suszko (1889-1972), the most distinguished Polish pioneer in natural products chemistry. 189

THE ALKALOIDS, VOL. 40 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

190

R6ZA ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

E. Connatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. P-Nitroaminoalanine VIII. Alkaloids of Miscellaneous Structures. ................................. A. Lepistine.. ......................................................

304 304 305 305

A. Cytochalasans ...................................................

307

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

324

I. Introduction

The popular usage of the word mushroom varies considerably. Usually the word is restricted to edible macrofungi with a cap borne on a stem, whereas large fungi with poisonous fruiting bodies are designated as toadstools. There is no sharp dividing line between edible and toxic macromycetes. Because the latter are especially interesting to chemists as a source of nitrogen-containing secondary metabolites, for the purpose of this chapter the word mushroom will be used as referring to that part of fungi whose size is sufficient for them to be recognized without help of lens. The position of mushrooms along with other common names of fungi in the taxonomic group system (1) was recently presented in an interesting graphic illustration by Koivikko and Savolainen (2) (Fig. 1). More detailed introduction to the taxonomy of fungi is included in several publications designed for amateur and professional mycologists. Examples of these are given in Ref. 3 and in Refs. I , 4, and 5 , respectively. The taxonomy used in this chapter is in accordance with the recommendations of the Commonwealth Mycological Institute given in Ref. I. Mushrooms found to be source of alkaloids occupy only a small taxonomic range. Generally, the species for which a particular alkaloid structure is characteristic are closely related and can be comprised within the next higher taxon, genus or family. The distribution of known natural producers of alkaloids in fungi kingdom is-summarized in Scheme 1. Only selected taxa are shown in Scheme 1 , with the figure in parentheses following the name of the taxon indicating the number of the next lower taxa derived from the taxon. Most of the genera shown include species (not depicted for clarity) which have been found to be toxic, though the cause of their toxicity may still be unknown. In contrast to the occurrence of

2.

ALKALOIDS FROM MUSHROOMS

191

DEUTEROMYCOTINA Z Y GOM YCOT INA

FIG. 1 . Relationship between major taxonomic and common names of fungi. (Reproduced from Ref. 2 by courtesy of Munksgaard International Publishers, Copenhagen.)

alkaloids, simple amines considered to be the primary metabolites are found in a large number of mushroom species (6, 7) and generally without any distinct pattern of distribution. The fungi world is a rich but only partly explored source of nitrogencontaining metabolities of various structures. However, apart from those which are essential for all forms of life, many secondary metabolites have been identified in the lower fungi. For this reason, and also because of their specificity, the large group of nitrogen-containing antibiotics of great importance in human therapy are beyond the scope of this chapter. Similarly, cytochalasans, a group of related alkaloids which have attracted special attention recently, are mentioned in the Addendum only (Section IX,A). The alkaloids which are the subject of this review are discussed according to their structural features; however, our intention is also to locate the alkaloid source of fungi. For this purpose the relationship in Scheme 1 is presented and in some cases in the text the fungus name is followed by a name in parentheses referring to higher taxa. Traditionally, names of English origin for alkaloids end in “-ine,” contrary to those formed in other languages. The latter are most often used in English without any changes, and this lack of consistency in the nomenclature is also reflected in this chapter.

192

5

193 0 *

.-C

SCHEME 1. Concise guide to selected taxonomic hierarchies in relation to various alkaloid structures

194

R 6 2 A ANTKOWIAK A N D WIESEAW 2. ANTKOWIAK

11. Physiologically Active Principles of the Genus Amunitu A. MUSCARINIC EXOCYCLIC AMINES One of the most beautiful and widespread mushrooms is undoubtedly the fly agaric, Amanita muscaria, which is found throughout the world outside of tropical zones. The trivial name, fly agaric, is derived from the insect-killing properties of its extracts. In the past the fruiting bodies soaked in milk were used as a fly poison. The symptoms of intoxication from this toadstool are generally associated with one of its toxic principles, muscarine. However, muscarine is not the main toxin of this species, as it is accompanied by its diastereoisomers and other flyicidal alkaloids of related structure: muscimol, ibotenic acid, and muscazone are present along with several other metabolities including quaternary amines like acetylcholine, muscaridine, and hercynine (Fig. 2).* In the late 1960s Eugster reviewed general studies of the active principles of A . muscaria (8, 9), and Wieland discussed the alkaloids along with other poisonous principles of the genus Amanita (10). 1. Muscarines Whereas ibotenic acid and muscimol have been shown to powerfully affect the central nervous system, the physiological activity of muscarine is concerned with the peripheral nervous system, the action strongly resembling that of acetylcholine. The binding affinity of muscarine with the acetylcholine receptor is so high that the compound is routinely used to study cholinergic pharmacology. Recently, the involvement of muscarine with second messengers such as inositol phospholipid metabolites has been demonstrated (11). Although the search for the toxic principles of A . muscaria started at the beginning of the 18OOs, attempts to obtain pure muscarine were not successful until 1957. In that year the outstanding scientist in muscarine chemistry, Eugster, in collaboration with Waser obtained muscarine ~ (12). The chloride in pure crystalline form (mp 182-183"C, [ a ]+8.1" structure of the alkaloid was determined a few years later by X-ray diffraction analysis (13). There are three centers of chirality in the.muscarine molecule, and thus four pairs of enantiomers are possible. All diastereoisomers, namely, (+)-(2S,3R,SS)-rnuscarine, (-)-2S,3R,SR,)-allornuscarine, (+)-(2S,3S,5S)-

* Although the names of the quaternary amines discussed here refer only to the cationic part of the molecule, they are commonly used, and occasionally also in this chapter, to describe a particular compound without mentioning the counterion of the salt.

2.

195

ALKALOIDS FROM MUSHROOMS

HO

HO

W L-(+)-Muscarine*

M

e

3

L-(+)-Epirnuscarine

(25,35,55)

(25,3R,55)

L-(-)-Allornuscarine

L-(+)-Epiallornuscarine

(25,3R,5R)

(25,35,5R)

=Me3 40+’Me3

OH

Acetylcholine

Muscirnol

‘

Muscaridine

Ibotenic acid

b

i

cooM e

Hercynine

Muscazone

FIG.2. Selected principles of Arnanita rnuscaria.

epimuscarine, and (+)-(2S,3S,5R)-epiallomuscarine, occur in nature. Each has been found, in different proportions, in A . muscaria and A . pantherina, and also in larger amounts mainly in numerous Inocybe and Clitocybe species (14-17). All were obtained by synthesis (18). The absolute configuration of the naturally occurring stereoisomers was established by Eugster. The exception is the epiallo isomer for which the (S) configuration at C-2 and consequently (3S,5R) has been assumed to comply with the demand that the biosynthetic pathway be equal (L series) for all diastereoisomer- (19,20).

196

R62A ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

According to earlier reports , difficulties in isolating the quaternary salts in a pure state and hence in detecting the diastereomers were surmounted by chromatographic separation of their nor-bases derived by pyrolysis (21) of the muscarine chlorides under reduced pressure at 200-240°C. In this way all muscarine diastereomers were found in A. muscaria, Znocybe patouillardi, and I . rimosa (14). Later, owing to impressive developments in instrumental analytical methods , detection and identification of the alkaloids as intact cations by recording secondary-ion mass spectra (SIMS), either of intact mushroom tissue or of ethanol extracts of defatted mushroom tissue, appeared to be possible (22). The total syntheses of (+)-muscarhe chloride, its optically active or racemic diastereoisomers, and analogs have been carried out independently in several laboratories. In most cases carbohydrates or, rarely, amino acids were used as chiral starting materials, enabling stereoselective synthesis. The different synthetic approaches as well as other aspects of muscarine chemistry, occurrence, and pharmacology were discussed in an excellent review by Wang and Joullie (18) in this treatise and earlier by Wilkinson (23). The cholinomimetic properties and attractive geometry of the relatively small muscarine molecules have created a challenge for many chemists, and these natural compounds and analogs have been the targets of several syntheses utilizing new methodologies. Most of the recently elaborated syntheses have been carried out on enantiomerically pure materials to yield natural alkaloid preparations. A simple synthesis of racemic allomuscarine via stereospecific intramolecular opening of the trans-substituted epoxide ring in butyl4,5-epoxy2-hydroxyhexanoate was reported by Chmielewski and Guzik (24,25) (Scheme 2). The furane ester intermediate was obtained in an ene reaction between butyl glyoxylate and 1-butene, followed by epoxidation of the

+ enantiomer

(‘)-Allomuscarine

SCHEME2. Synthesis of allomuscarine by Chmielewski and Guzik (24,25).

2.

197

ALKALOIDS FROM MUSHROOMS

double bond and intramolecular opening of the epoxide ring by the hydroxyl group catalyzed by stannic chloride. The resulting tetrahydrofurancarboxylate was successively treated with dimethylamide magnesium bromide and LiAlH4 to yield a nor-alkaloid, the quaternization of which to allomuscarine iodide was achieved with the high-pressure method elaborated by Jurczak er a1 (26). These results were followed by a report by Chmielewski er al. (27) of racemic epiallomuscarine and epimuscarine syntheses based on a strategy similar to the previous one (Scheme 3). The change in the stereochemical course of the syntheses was due to the replacement of the hydroxyl by an acetoxy group in the epoxide intermediate. The participation of the carbony1 oxygen in the epoxide ring opening forced the particular stereoselectivity, probably via an orthoester stage. Following the previously reported synthesis of racemic muscarine from methyl vinyl ketone (28), Amouroux er al. (29) prepared both L-(+)muscarine and its enantiomer in eight steps from D- and L-threonine, respectively, via the highly stereoselective iodocyclization of a y,bunsaturated 2,4-dichlorobenzylether, each being carried out separately (Scheme 4). A similar approach to tetrahydrofuran ring closure in the

(?)-Acetoxyepoxide (rel.config.ZA,&R,5R)

y

-

,v*, ~ , , , , 1 $ 1 ~ 3attack on C-4

(?)-Epimuscarine

"r"

CLLSnO;,, - +fico2Bu (2)-Acetoxyepoxide (rel.config.Z5,4R,SR)

-

attack on

C a H

SCHEME 3. Synthesis of epiallornuscarine and epimuscarine by Chmielewski et al. (27).

198

R 6 2 A ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK NH II

1.NaN02, HC1 2.Me1

@OH

1.DCBnOCCC13 2 . NaBH4

CI

NH2

7 KOH

F

O CI

H

(2R ,35) (25,3R)

U-Ihreoninc (2R,3S) L - l h r e o n i n e (25,3R)

ODCBn

-

ODCBn

. ...

OH

(25,3R,55) (ZR,35,5R)

(4R,5S) (4S,5R)

L-(+)-Muscarine

O-(-)-Muscarine

SCHEME4. Synthesis of L- and D-muscarine by Amouroux et al. (29).

synthesis of L-(+)-muscarine has been applied by Mulzer et al. (30) (Scheme 5). The synthetic application of the starting compound (R)-2,3-0isopropylideneglyceraldehyde has been reviewed (31). Studies of the synthesis of the D isomer led to the preparation of the butanediol ether (Scheme 6), the key intermediate of opposite configuration, from Dglucose. An interesting approach to the stereospecific synthesis of muscarines

R-enantiorner

erythro + threo

diastereomers

(7:3)

ODCBn HO

=

bH

TsCl

-

TsO

~

MeONa

- XBn0.''"

OH

( + ) -Mu s c a r i n e

SCHEME5. Synthesis of L-muscarine by Mulzer et al. (30).

2.

199

ALKALOIDS FROM MUSHROOMS

-

1.MeOCH2C1 2.AcOH.aq

QDCBn

NaI04

OH (DCEin = 2 , 6 - d i c h l o r o b e n z y l )

ODCBn

NaBH4

OH (2S,3R)

SCHEME 6. Mulzer et a / . approach to the synthesis of D-muscarine (30).

starting from a carbohydrate-type precursor was demonstrated by Bandzouzi and Chapleur (32) (Scheme 7). The method has been successfully applied to the preparation of (+)-muscarhe and (+)-epimuscarine (33). Chloromethylation of diisopropylidene-D-mannono-1,4-1actone with hexamethylphosphorous triamide-tetrachloromethane, followed by conversion of the resulting dichloroolefin to the unsaturated ketone and reduction with Raney nickel, gave two diastereomeric key intermediates with three-dimensional structures corresponding to those of L-epimuscarine and D-epiallomuscarine, respectively. The stereoisomer of the L series was transformed to both (+)-epimuscarine and (+)-muscarine. In the latter case, however, the synthesis required initial inversion of the configuration at C-3. Treatment of the key intermediate with triphenylphosphinediethyl azodicarboxylate (DEAD) and benzoic acid afforded the corresponding benzoate with net inversion of configuration at C-3 in 90% yield. In both cases the syntheses were completed by removing the isopropylidene protecting group. This was followed by a glycol cleavage and subsequent reduction with sodium borohydride. Separately the obtained diols were transformed in two steps to L-( +)-epimuscarine and L-( +)-muscarine using the method of Mubarak and Brown (34). Total syntheses of racemic muscarine and allomuscarine were achieved by Pirrung and DeAmicis (ZZ), who used a new approach to formation of the stereoselectively substituted tetrahydrofuran nucleus (Scheme 8). The key step in this procedure was the stereospecific photochemical ring expansion of a cyclobutanone derivative. The cycloaddition of silyl enol ether with methylchloroketene generated by zinc dechlorination of 2,2dichloropropionyl chloride followed by reductive removal of the halogen produced the methylsiloxycyclobutanone intermediate. Irradiation of the

200

R62A ANTKOWIAK AND WIESKAW Z. ANTKOWIAK

0-epiallo-isomer

L-epi-isomer

1.AcOH. aq 2.NaI04 3. NaBH4 4.NaOMe

OH J-QH

0 l.TsC1,PyH 2. NMe3

b

L-(+I-Muscarine

N

M

e TsO3

+

L-(+)-Epimuscarine

SCHEME 7. Bandzouzi and Chapleur synthesis of muscarine and epimuscarine ( 3 2 3 ) .

intermediate at -78°C provided a 2: 1 mixture of acetals in 55% yield. The acetals, having trans stereochemistry of the methyl and siloxy groups, were subjected to condensation with trimethylsilyl cyanide catalyzed by boron trifluoride etherate. Treatment of the diastereomeric nitriles after separation with borane-methyl sulfide followed by acid hydrolysis yielded

2.

201

ALKALOIDS FROM MUSHROOMS

OtBDMS

L

+

M e g ' CI 0

hv

QJBDMS

QJBDMS

QJBDMS 4

TMSCN BFjOEt2

QOMe

(1:l)

4.ion exchange

(+)-Muscarhe chloride

(+)-Allomuscarhe chloride

SCHEME 8. Synthesis of muscarine and allomuscarine by Pirrung and DeAmicis (11).

the deprotected primary amines. Quaternization of the amines with methyl iodide and 1,2,2,6,6-pentamethylpiperidine7and subsequent anion exchange, gave racemic muscarine and allomuscarine chlorides, respectively. An enantioselective synthesis of (+)-muscarine utilizing Rh(I1)catalyzed carbenoid C-H insertion cyclization of a-alkoxy diazoketones to furanones has recently been reported by Adams et al. (35)(Scheme 9). The cyclization reaction was found to favor cis-2,5-3(2H)-furanone, which has the right configuration for muscarine. The stereospecificity of the synthesis was achieved by using (R)-2-bromopropionic acid derived from D-alanine in which the bromide was displaced by sodium 2-benzyloxyethoxide with complete inversion of configuration. Diazomethylation followed by a cyclization catalyzed by a rhodium(I1) acetate dimer, and next by hydrogenolysis and reduction, afforded the muscarine precursor diol but contaminated by its diastereomer. The transformation of the main product (+)-muscarhe tosylate and next to the chloride by anion exchange is in line with the procedure of Mubarak and Brawn (34). A concise enantioselective synthesis of (+)-muscarhe from (R)benzyloxymethyloxirane was recently reported by Takano et al. (36) (Scheme 10). The starting oxirane was coupled with lithium acetylideethylenediamine, forming the hydroxyalkyne, which was converted to the acid-sensitive 2,3-dihydrofuran by methylation and subsequent treatment with base. The dihydrofuran derivative was hydroborated and oxidized in

202

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK NaN02

HZN y C HO Z H

HBr

-

(R)

qoZH EnOCH2CH20H NaH ,DMF

BF H

yCOzH

-

Y P O B n

(R)

0-Alanine

HGNzboBn

1.(COC1)2, DMF

1.H2/Pd(OH)2

[Rh(OAc),],

2 , CH2N2

O d O B n

CHZCZI

Z.N~HB(OAC)~

-

3.chromatogr.

0

H

(minor)

(major)

1

2.NMe3 3. anion exchange 1

L - ( + ) -Musca r in e chloride

SCHEME 9. Enantioselective synthesis of L-muscarine by Adams et al. (35).

Me1 ,BuLi

C+OB ,n OMSO

HC(

OH

L-(+)-Muscarine

H ,Cyo,Bn

- /'" Me

OH

iodide

SCHEME 10. Synthesis of L-muscarine by Takano et al. (36).

tEuOK

~

2.

ALKALOIDS FROM MUSHROOMS

203

a stereoselective manner to yield a (2S,3R,5S)-4-hydroxytetrahydrofuran intermediate accompanied by less than 5% of its epimer. The correct stereoisomer, after deprotection and consecutive substitution with tosyl, iodide, and trimethylamine, was converted to L-( +)-muscarine iodide in 38% overall yield.

2. Ibotenic Acid and Related Alkaloids On account of its high pharmacodynamic activity, ibotenic acid may be regarded as an essential component of the active principle of Amanita muscaria. This alkaloid, accompanied by related muscimol and muscazone, has also been found in other mushrooms in which the occurrence of muscarines has not been recorded. In the mid-1960s investigations carried out independently by four research groups led to the isolation and subsequent structure elucidation from Amanita muscaria, A . strobiliformis, and A . pantherina of three fly-killing and narcosis potentiating compounds: muscimol(37-40), ibotenic acid (40-44,and muscazone (43-47). At the same time tricholomic acid, another insecticidal constituent, was found in Tricholoma muscarium (48,49). The nomenclature commonly accepted for these alkaloids was proposed by Eugster and Takemoto (50). The neurotransmitter properties of ibotenic acid and muscimol were linked to the structures of alkaloids by the demonstration of their structural resemblance to some amino acids of the nervous system (Fig. 3). They reveal the powerful properties of glutamate and y-aminobutyric acid (GABA) agonists, respectively, and may be considered as conformationally restricted analogs of these amino acids, binding alternatively to their respective receptors (51). The key compound in studies of the molecular pharmacology of GABA agonists appeared to be dihydromuscimol, which, owing to its ability to adopt several conformations, almost perfectly mimics the “receptor-active conformation” of GABA. Attempts to synthesize the individual enantiomers of dihydromuscimol were reported by Brehm et al. (52). Initially the substance known as muscimol was also called pantherine (38,53)(@toxin, pyroibotenic acid, and agarine as well). Later, Onda proposed that pantherine, isolated from A . pantherina, might be represented by a peptide structure [see Ref. 49 in Eugster (8)].More recently, Onda identified pantherine as 5-aminomethyl-3-hydroxyisoxazole, the structure already attributed to muscimol, by means of spectroscopic experiments and a chemical correlation with synthetic compounds (54) (Scheme 11). In these studies pantherine was converted to acetoxypyrrolidone, and the product was compared with that obtained synthetically from ethyl 4-chloro-3-oxobutanoate. Additionally, 5-

204

R 6 2 A ANTKOWIAK AND WIESKAW Z . ANTKOWIAK

?-Aminob u t y r i c acid

WH3

-0

HO

aI cb io dt e n i c

% I

Muscirnol

coo-

cooTricholornic acid

Musca zone

FIG. 3. Ibotenic acid and muscimol as analogs of glutamic acid and GABA.

”

L

Panther ine ” (natural)

J

1

NaBH4

1.HC1

Et

OH

I

Acetoxypyrrolidone

Chlorooxobutanoate

Ac

CH(OEt)3

0

- ‘$-X-cl H2NOH

E t O w ‘ Et OEt

0HC1

___)

HY

HOE? OEt

Muscirnol (synthetic)

SCHEME1 1 . Chemical correlation between “pantherine” and muscimol.

2.

205

ALKALOIDS FROM MUSHROOMS

aminomethyl-3-hydroxyisoxazolewas obtained by synthesis from ethyl 4-chloro-3-0x0-butanoate.Acetalization with ethyl orthoformate, followed by conversion of the resulting acetal to the corresponding hydroxamic acid, and then cyclization and amination, yielded a product which was shown by direct comparison to be identical to the naturally produced pantherine. In spite of structural differences, muscimol (mp 156"C), ibotenic acid (mp 145"C, synth. 152"C), and muscazone (mp >190°C, dec.) are interrelated by simple chemical transformations. During attempts to remove its crystallization water, or when heated in an aqueous solution or dissolved in DMSO, DMF, or pyridine, or even after contact with cellulose during paper chromatography, ibotenic acid was shown to decompose readily with the loss of carbon dioxide and formation of muscimol. The ease of decarboxylation of ibotenic acid [in its early days also known as pramuscimol (43)]to muscimol led to a suggestion that the latter may not be a natural component ofA. muscaria (8).This supposition was not confirmed by Gore and Jordan (51), however, who elaborated a convenient microbore single column method for the quantitative determination of both ibotenic acid and muscimol using an amino acid analyzer. The method allows the evaluation of the level of the alkaloids at all stages of purification from fungal extracts, and it has been used for monitoring the chemical decarboxylation of ibotenic acid to muscimol. Ibotenic acid undergoes an interesting photoreaction comparable to a Lossen rearrangement of hydroxamic acid (Scheme 12). When irradiated in an aqueous solution with a low-pressure mercury lamp, it is transformed to muscazone with a yield of up to 40%. This occurs most probably via an unstable keto-lactam which then undergoes a [ 1,3]sigmatropic rearrangement (46). The optical inactivity of naturally occurring ibotenic acid may be easily explained by taking into account the acid properties of the proton attached

J$p.3

'0

coo-

SCHEME12. Formation of muscazone from ibotenic acid.

coo-

206

R6ZA ANTKOWIAK AND WIESKAW Z. ANTKOWIAK

SCHEME 13. Transformation of ibotenic acid on catalytic hydrogenation.

to the chiral carbon atom, the latter having been found to be subject to isotopic exchange in D20. Such properties of the appropriate proton in muscazone have not been observed. The racemic character of the natural muscazone seems to suggest that it is formed from racemic ibotenic acid in the course of biosynthesis. However, this suggestion has not been confirmed by the results of an experiment in which a dilute solution of ibotenic acid was exposed to daylight for several months. No muscazone was found even when the experiment was carried out in the presence of the red dye of the pileus added as a photosensitizer (8). There is a close structural affinity between ibotenic acid and its dihydro derivative, tricholomic acid (mp 207°C). Nevertheless the chemical interrelation between these compounds appeared to be difficult to prove because of the reactivity of the N - O bond which undergoes hydrogenolysis on mild catalytic hydrogenation of ibotenic acid (Scheme 13). The a-amino ketone formed in this way undergoes decarboxylation, dimerization, and reduction, becoming a trans derivative of piperazine (48).Tricholomic acid and its threo isomer have been synthesized from erythro- and threo-diethyl ~,~-3-hydroxyglutamate, respectively, by Iwasaki et al. (49). The first synthesis of ibotenic acid was published as early as 1965 by Gagneux et al. (53).3-Bromoisoxazole-5-carboxylicacid was used as the starting material and was transformed into the final product by the steps shown in Scheme 14. Later, a few other synthetic methods were reported (55,56). The procedure elaborated by Nakamura (57) (Scheme 15), the starting point being diethyl 3-chloroglutaconate, seems to be the most efficient. The intermediate ethyl 3-hydroxy-5-isoxazoleacetatewas also used, via anhydrous Curtius rearrangement of its hydrazide, for the preparation of muscimol. There is no direct experimental evidence relating to the biogenesis of ibotenic acid and its closely related derivatives. However, studies carried out on the biogenesis of muscarine with isotopically labeled precursors (58) led to significant conclusions regarding the formation of all the alkaloids discussed here (Scheme16). The incorporation and distribution of several I4C-labeled simple compounds into muscarine have been investigated using mycelial cultures of Clitocybe riuulosa. The highest specific radioactivity of muscarine chloride was observed in the case of pyruvic

2.

- '5 -

PhCH20H

Ny, COOH

OBn

~

$

,

"0

1. TsCl

1.NaCN ,NH3. liq

-

207

ALKALOIDS FROM MUSHROOMS

N

$

LAi1H4

-

0

OBn

2.EtOH,H+

COOEt

COOEt

1.HBr-AcOH 2.NH3.aq

* Ibotenic acid

SCHEME14. Synthesis of ibotenic acid by Gagneux et al. (53).

and glutamic acids. The carbon atoms of pyruvate were found to be incorporated into the muscarine molecule at positions 2 and 3 of the ring and the methyl at C-2. The carbons at positions 2 , 3 , and 4 of the glutamate were incorporated into the muscarine at positions 4 and 5 of the ring and the methylene at C-5, whereas the carboxyl carbons of the glutamate were lost during biosynthesis. It was therefore concluded that all of the discussed amino components of Amanita muscaria, including the (-)-(Z?)-4hydroxy-Zpyrrolidone (mp 153.5-155"C, [ a ]-44.5') ~ found by Eugster and co-workers (59), are derivatives of glutamate. In addition, the biosynthesis of muscarines takes place through condensation of pyruvic acid or (S)-lactic acid and a 3-ketoglutamic acid intermediate, with the subsequent loss of the two carboxyl groups. Extrapolation of these results also led to a reasonable explanation of the biogenesis of ibotenic acid and related alkaloids. However, since the

owooEt - : ! ~.H~NOH,OH-

E E tt O O

Z.EtOH,H+ Z.EtOH,H+

1.N2H4

OH

L

-

C CIl

OSOzPh

-0OEt N\O

OH N w O FOOEt O E t

1.HC1-AcOH

NBS

2.NH3.aq

(BzO)p,hv

SCHEME 15. Synthesis of ibotenic acid by Nakamura (57).

OH

208

R 6 Z A ANTKOWIAK A N D WIESKAW Z. ANTKOWIAK

0 II HOC

q : O o -

H NH3

-

+t

-

UOH

HO!

00-

0

H NH3

0

H (-)-(R)-4-Hydroxy2-pyrrolidone

L-Glutarnic a c i d

~ c ~ o o H 1 4 OH NH2

0

-Oo-:*H,O,

HO!&tOOH3C

Q?

(?)-Ibotenic

A-

,.' H NH3

k

H NH3

/

0

acid

\

(+)-(2S,3R,5S)-Muscarine (+)-(25,3S,5S)-Epirnuscarlne

coo-

(+)-(2S,3R,5R)-Allornuscarine

(-)-(2S,3S,5R)-Epiallomuscarine

Muscimol

(t)-Muscazone

SCHEME 16. Assumed biogenesis of ibotenic acid, muscimol, and muscazone.

experimental observations were based on Clitocybe rivulosa, which was not found to be a source of these alkaloids, the corresponding part of the conclusions remains only a hypothesis.

B. BETALAINE-TYPE ALKALOIDS Apart from the psychoactive principles from Amanita muscaria, the constitution of the pigments responsible for the striking red color of the caps of the toadstool has been a challenge to several chemists studying natural products. In 1930 Kogl and Erxleben (60) reported the isolation from the red skin of the fly agaric of a red crystalline compound with a proposed quinonoid structure which they called muscarufin. Later studies have not confirmed the presence of this principle in A . muscaria. The

2.

209

ALKALOIDS FROM MUSHROOMS

thorough investigations carried out more than 30 years later by Musso with his research team showed unequivocally that the pigments responsible for the toadstool color are related to the betalaine class of natural products. This subject was discussed and reviewed by the author (61,62)and treated more recently by Gill and Steglich (63) in their comprehensive article on the pigments of fungi. After this article had been prepared, a review of the betalaine alkaloids was published by Steglich and Strack in the previous volume (39) of The Alkaloids. The betalaine pigments were first recognized as being plant products isolated from the well-known source of red beet, Beta vulgaris, as well as from the species of the families Amaranthaceae, Aizoaceae, Cactaceae, and several others, all belonging to the order Centrospermae (64). The betalaine pigments are amino acid condensation products derived from betalamic acid and are divided into two classes, the orange-yellow betaxanthins and the red-violet betacyanins (Scheme 17). In the betaxanthins, the betalamic acid occurs as an imine derivative of a natural a-amino acid, the long-wavelength absorption near 480 nm being caused by the substituted 1,7-diazaheptamethin cation only. In the betacyanins

Kagl-Erxleben's

Betalamic

rnuscarufin

acid

1,7-Diaraheptarnethin c a t i o n

R

( f o r R'=H) (25,155)

Betaxanthins

Betanidine (R'=H)

Neobetanidine

Betacyanins (R'=carbohydrate residue)

(pentarnethyl d e r i v a -

SCHEME 17. Structural features of betalaines.

tive of)

210

R6ZA ANTKOWIAK AND WIESEAW 2. ANTKOWIAK

the chromophore is extended by conjugation with an aromatic group, producing a bathochromic shift of the absorption to around 540 nm. Furthermore, the betacyanins are glycosides of ( 2 S , 15S)-betanidine or its diastereomer (2S, 15R)-isobetanidine (65,66).Under diazomethane methylation, both stereoisomers gave neobetanidine (67). As a result of carefully repeated chromatographic experiments with different types of Sephadex, several unstable pigments were isolated (61,62)from the red skin of the fly agaric: the yellow muscaflavin (68),the orange musca-aurins I-VII (69),and the purple muscapurpurin (63).They all have electronic spectra characteristic of the betalaine chromophoric systems. For the sodium salts of these three groups of pigments the principal diagnostic A,, were determined as 420,475, and 540 nm, respectively. Muscaflavin, also available in greater abundance from Hygrocybe species (63,70),was found to be a dihydroazepine amino acid with a structure isomeric to betalamic acid. The proposal of such an unusual structure for a natural product was supported by biogenetic considerations and confirmed by a biomimetic synthesis (71) (Scheme 18). The idea of the synthesis, which followed from the biosynthetic proposal, is presented later. The biomimetic synthesis of a stable (+)-muscaflavin dimethyl ester contained the key intermediate step of pyrrolidine-catalyzed nucleophilic ring opening of the N-methoxypyridinium ion to an enamine followed by an acidcatalyzed cyclization. Treatment of the recyclized material with diazomethane gave a mixture of esters, with the pyrrole isomer as the major product. The latter could, however, be transformed in good yield into muscaflavin dimethyl ester by heating in a perchloric acid-formic acid solution followed by reesterification. Finally, the racemic dimethyl ester was resolved by chromatography on potato starch in a potassium citrate buffer at pH 7.0 (72), and the faster moving (+)-isomer was shown to be identical with the methylated natural product (62). Musca-aurins (or muscaurins) are betaxanthin-type pigments which when hydrolyzed yield betalamic acid together with one of the following amino acids: ibotenic acid, stizolobic acid, glutamic acid, aspartic acid, a-aminoadipic acid, glutamine, asparagine, leucine, valine, proline, or histidine. Musca-aurins I, 11, and VII have been isolated as single compounds, whereas musca-aurins 111, IV and V , VI correspond to mixtures of betalaines containing different acidic or neutral amino acids, respectively (Fig. 4). Some of these betalaines are found in higher plants. Of particular interest was the discovery of the first two pigments. Musca-aurin I, containing a residue derived from ibotenic acid, was found to be absent in the edible Amanita caesarea, which is also devoid of this hallucinogenic amino acid although it contains all of the remaining pig-

2.

1.MCPBA 2 .Me2S04

3. NaC104

-

21 1

ALKALOIDS FROM MUSHROOMS

H

s0 T+ @

THF, 0°C

-

OMe Me

c10;

~

M

e

0

O

~

-

~

c

O

MeO:%i12]

OH

0

A

OH

1.HBr,HC02H

2 .CH2N2

HC104,HC02H

A

O

M

OMe

0 (1.6%)

e

(21%)

"s

MeON

\

\

OMe

(11.5%)

SCHEME 18. Synthesis of muscaflavin by Musso and co-workers (71).

ments found in A . Muscaria (73). Musca-aurin I1 is similarly related to stizolobic acid, a known constituent of A . pantherina (74)and A . muscaria (69). This unusual amino acid, which is accompanied in nature by its isomer stizolobinic acid, was obtained synthetically by the condensation of diethyl acetylaminomalonate with ethyl 4-bromomethyl-2-oxo-2Hpyran-6-carboxylate (69,75) (Scheme 19). Further synthetic studies which

212

R62A ANTKOWIAK AND WIESEAW Z . ANTKOWIAK

Musca-aurin-I

Musca-aurin-I1

zo5

Musca-aurin-VII

0

OH

H 6.

II R

Vulgaxanthin-I (M-a.-II1,M-a.-IV)

(M-a.-V)

H$4 II [OH co;

II

H , IIh Z 1 2

R

R

&H ;. R Miraxanthin-I11 (M-a.-II1,M-a.-IV)

(M-a.-V)

Vulgaxanthin-I1 (M-a.-V,M-a.-VI)

(M-a.-III)

Indicaxanthin (M-a.-V,M-a.-VI)

(M-a.-V)

FIG. 4. Structures of musca-aurins,where R is a betalamic acid residue. The musca-aurin of which the pigment is composed is specified in parentheses.

,COOEt N -C H, A/C COOEt tj,

1.NaH, OMF

-

COOEt COOEt

cooHC1, AcOH

Stizolobic acid

SCHEME 19. Synthesis of stizolobic acid.

2.

ALKALOIDS FROM MUSHROOMS

213

led to the preparation of other possible natural products of potential importance for biosynthetic consideration like and (E)-2,3-dehydrostizolobic acid, cyclostizolobic acid, and isocyclostizolobic acid (Fig. 5 ) have been described recently (75). The structure of all musca-aurins has been confirmed by a partial synthesis using an appropriate amino acid and betalamic acid derived in situ from betanine in the presence of aqueous ammonia (Scheme 20). The transimination equilibrium was displaced toward the desired product by using a 5- to 10-fold excess of the amino acid. The progress of the reaction was monitored spectroscopically, following the change of absorption intensity at 540 and 475 nm (69). Muscapurpurin was found relatively recently by Musso [see Ref. 504 in Gill and Steglich (63)l to have the betalaine structure of extended conjugation which causes the purple color of the pigment (Fig. 6 ) . This result modified the earlier structural proposals (61) obtained after several years of investigations. The biosynthesis of betalaine pigments in fungi was generally accepted to follow the same pathway as that determined for higher plants (76,77). Successful incorporation studies using I4C- and 3H-labeled starting materials carried out in higher plants as well as in Amanita pantherina (78)and A. muscaria (61) have shown that the biosynthesis of the betalaines (76,79) and of stizolobic acid (77,78)involves tyrosine and its oxidated derivative L-3,4-dihydroxyphenylalanine (L-dopa) as the precursors. Several toadstools were found to be able to convert tyrosine to L-dopa ( 6 3 , and this metabolite, though rare in Basidiomycotina, has been detected in the fruiting bodies of some fungi: Agaricus bisporus (80),Strobilomycesjoccopus (Strobilomycetaceae, Boletales) (81), Hygrocybe conica, H . ouina (82), and Rhodocybe mundula (Entolomataceae) (63). In some mushrooms, such as Hygrocybe species, L-dopa was found to be responsible for the remarkable color change from grey to red and black arising from

(a-

(Z)-isomer ( E )-isomer Dimethyl N-acetylstizolobates

Cyclostizolobic acid

FIG. 5. Structures of compounds related to stizolobic acid.

D i m e t h y l N-acetyldehydroisocyclostizolobate

214

R~ZA ANTKOWIAK AND WIESJLAWz. ANTKOWIAK

+H

5-10 rnol eq.

SCHEME 20. Partial synthesis of musca-aurins.

melanin formation in sites of fungus injury (63,83)(Scheme 21). The chemistry of melanins was recently discussed by Crippa et al. in Vol. 36 in this treatise (84). On the basis of observations resulting from feeding experiments, especially those concerning the formation of labeled stizolobic and stizolobinic acids, as well as the mutual occurrence of those metabolites with muscaflavin and different betalaines in A. muscaria, a general mechanism of the metabolic formation of the pigments was proposed (61,63) (Scheme 22). This conclusion was supported by the established chemical relationship between dopa and muscaflavin in Hygrocybe species. The biotransformation of L-tyrosine via L-dopa involves the enzymatic cleavage of the catechol ring at the 4,5 or 2,3 bonds either side of the diol system giving rise to intermediates 1and 2, respectively, which can then undergo recyclization in different modes. This was supported by the results of Saito and Komamine (85), who isolated two enzymes, stizolobate synthase and stizolobinate synthase, from Stizolobium hassjoo seedlings. These enzymes catalyze the oxidative ring cleavage of 3,4-dihydroxypheny-

H OH

opl@:oi Muscapurpurin

FIG. 6

2.

215

ALKALOIDS FROM MUSHROOMS

Cyclodopa

L-Dopa

Melanin

SCHEME 21. Formation of melanin according to Steglich (63,83).

Hd"H2 H02C

H$"H2 HO2C

H&NH2 H02C

I;r, L-dopa

Stizolobic acid

-H20

-H20

t

Eetalamic acid

I

oxid.

oxid.

Stizolobinic acid

-H-O

t L

Muscaflavin

SCHEME 22. Biosynthesis of betalaines.

Muscapurpurlnic acid

216

R ~ Z A ANTKOWIAK AND WIESKAW

z. ANTKOWIAK

lalanine, subsequent recyclization, and dehydrogenation to stizolobic and stizolobinic acids. The intermediate 1 can cyclize (N, C-6 bond formation) to betalamic acid or to a lactone, stizolobic acid, via an intermediate hemiacetal. Similarly, intermediate 2 can be transformed into stizolobinic acid and alternatively into muscaflavin by N , C-7 ring closure. Additionally, muscapurpurinic acid can be formed from intermediate 2 by a dehydrative cyclization followed by hydrogenation. The subsequent condensation of betalamic acid with stizolobic acid or muscapurpurinic acid gives rise to musca-aurin I1 and muscapurpurin, respectively.

C. PEPTIDEALKALOIDS The characteristic toxins of the genus Amanita are cyclopeptide alkaloids, which are responsible for most fatal mushroom intoxications in human beings. These metabolites were isolated primarily from Amanita phalloides, the green death cap popular in Central and Eastern Europe, France, and Italy, but its occurrence has also been reported in Mexico and the United States, even in New York’s Central Park. In addition, these toxins have also been found in other Amanita as well as in Lepiota and relatively rare Galerina species, such as A . verna, A . virosa (known as the destroying angel or deadly agaric), A . bisporigera, A . tenuifolia, A . ocreata, A . suballiacea, L . brunneoincarnata, L . helveola, G . marginata, G . autumnalis, and G . venenata. The concentrations of peptide toxins in the toadstool species in which they have been recognized differ from species to species. Moreover, the individual components are not distributed in the same proportion in each species (8637). The first of the most significant results in the long-standing research on A . phalloides toxins, initiated at the beginning of nineteenth century, were recorded by Lynen and U. Wieland in 1938 (88) and by H. Wieland and Hallermayer in 1941 (89). The developments concerned the isolation in crystalline form of phalloidin and amanitin, respectively, the first known highly physiologically active principles of the toadstool. In subsequent years several other components with related structures were isolated using a rather complicated multistep procedure based on a partition chromatography on Sephadex G-25 with an organic-aqueous solvent mixture as the eluent. Later, Sephadex LH-20 with water was used as the most effective system for these purposes (90,91).In addition, several chromatographic methods for the detection of the deadly amanita toxins were developed (92), including reversed-phase high-performance liquid chromatography (HPLC) (93,94)and the sensitive and rapid high-performance thin-layer chromatography (HPTLC) (95-97).

2.

ALKALOIDS FROM MUSHROOMS

217

The history of the major achievements concerning the isolation, structure elucidation, chemistry, and biological activity of the cyclic peptide principles of A . phalloides was comprehensively covered mainly by T. Wieland, a man of great merit particularly in this branch of natural products research. He dealt with the subject in several review articles (10,87,94,98-101) and more recently in an excellent book on the peptides of poisonous Arnanita mushrooms (86). The number of references cited in the book exceeded 750, which gives a rough idea of the proportion of research carried out by chemists on this topic. The scope of the present chapter enables us only to summarize briefly the final results of these interesting studies. Based on toxicological properties and chemical features, the amanita peptide alkaloids can be divided into three groups: amatoxins (the amanitin family), the compounds actually solely responsible for the fatal intoxications; phallotoxins (the phalloidins), which are only detected in Arnanita species; and virotoxins, cyclopeptides with phalloidinlike activity recently found as additional principles of A . uivosa only (102). The last two groups do not lead to the lethal intoxications. The basic differences between the amatoxins and both phallotoxins and virotoxins lie in the mode and rapidity of their action. The phallotoxins and virotoxins act quickly, within 2-5 hr, whereas the action of the amatoxins is delayed (animals succumb within 2-8 days after toxin administration); however, the amatoxins are 10-20 times more toxic than phalloidin. Literature values concerning the amount of toxins in a single species differ depending on the part of the fruiting body examined, the place where the toadstools were collected, and the assay method applied. For example, the total amount of amatoxins in A . phalloides determined with a radioimmunoassay method (103) was 2.56 mg per gram of dry tissue, whereas for another collection of the same species 5.2 mg/g was reported (95) with a HPTLC-spectrophotometric assay. The average value corresponds approximately to 13 mg per 100 g of fresh mushrooms, which means that, since a lethal dose of a- and p-amanitins is about 0.1-0.2 mg/kg (LDSofor humans and mice), the toxin content of one medium-sized toadstool weighing 50-60 g may be sufficient to kill an adult human being. Amanitins can be differentiated analytically from phalloidins by a sensitive test reaction with cinnamaldehyde that yields a deep violet color in an atmosphere of hydrogen chloride. Also, the simple color test in which a trace of a toadstool impressed on a lignin-containing paper (e.g., newsprint) moistened with concentrated hydrochloric acid develops within 5-10 min a greenish-blue color when amanitines are present (86,104)may be of some analytical value. This reaction is also characteristic of other

218

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

substances containing a hydroxyindole ring, like bufotenine (brown-blue) and psilocybin (gray-blue), but gives negative results in the case of phallotoxins. Intoxications from toadstools containing amanita toxins, the pathological symptoms, the molecular mechanism of toxicity, diagnosis, and clinical therapy were discussed in several articles (e.g., 86,87,103).Biochemically, a-amanitin binds to RNA polymerase B and blocks the catalytic site of the enzyme, thus inhibiting the formation of messenger RNA and DNA transcription. The toxin easily penetrates into liver cells and within 15 min is attached to the nuclei, which are susceptible to the bonding owing to the rate of protein synthesis. A further biochemical effect of amanita peptide alkaloids is related to the metabolism of brain catecholamines, namely, a significant decrease in the concentration of brain noradrenaline (86,87). From a chemical point of view amatoxins and phallotoxins are bicyclic octapeptides and heptapeptides, respectively, whereas virotoxins are monocyclic heptapeptides (Figs. 7-9). All contain the indole nucleus of a

I

HN-CH-CO-NH-CH

I

oc

I

- CO-NH

-CHZ-CO

I

mRA NH

CH2

I

so I

H,,fr R5

I

H

I I

co

CH2

I

OC -CH -NH-CO-CH

I

I

/CH3

HC -CH

‘c2H5

- NH -CO - CH2-NH

H,C-COR3

R’ a-Amanitin

P- Amanitin y-Amanitin &-Amanitin Amanin Amanin amide Amanullin Amanullinic acid Proamanullin

RZ

R3

OH OH OH OH OH OH H H H

FIG. 7. Structures of natural amatoxins.

R4

R5

OH OH OH OH H H OH OH OH

OH OH OH OH OH OH OH OH H

2.

y

H~C-C-CO

219

ALKALOIDS FROM MUSHROOMS

y

-NH-CH I

I

I NH

- co -NH-!-CH~-

I I

co

I

CO

NH p 2

R4d

N

- CO -C -H I NHCO -$H

Phalloin (PHN) Phalloidin (PHD) Phallisin (PHS) Prophalloin (PPN) Phallacin (PCN) Phallacidin (PCD) Phallisacin (PSC)

R'

R2

CH3 CH3 CH3 CH3 CH(CH3)2 CH(CH3I2 CH(CH3Iz

CH3 CH3 CH3 CH3 OH OH OH

- NH-COI

R3 OH OH

OH OH C02H COZH C02H

R4

R5

OH OH OH H OH OH OH

CH3 CHzOH CHZOH CH3 CH3 CHzOH CHzOH

R6

FIG. 8. Structures of natural phallotoxins.

y H~C-C-CO-NHI NH I HO iiw

co

HOCH,

H 1

H

A

H IIIII OH

H2FoH

I

I OH

co

CH2 CH3X

I

- C-CH~-C-R

C-CO-NH

2

I

NH R ,1

N H H

1

c ///OH

I

1

HN-CO-~-NH-CO CH

HOs-\

CH3

Viroidin Deoxoviroidin Ala'-viroidin Ala'-deoxoviroidin Viroisin Deoxoviroisin

X

R'

R2

so2 so so2 so so2 so

CH(CH3)2 CH(CH3)2 CH3 CH3 CH(CH312 CH(CH3)z

CH3 CH3 CH3 CH3 CHzOH CHzOH

FIG. 9. Structures of virotoxins isolated from A. uirosa.

220

R~ZA ANTKOWIAK AND WIESJLAWz. ANTKOWIAK

tryptophan building block substituted in 2 position by a sulfur atom in various oxidation states. In amatoxins and phallotoxins the sulfur atom occurs as an (R)-sulfoxide and sulfide group, respectively, binding the cysteine and tryptophan residues to a structural element named tryptathionine. In the monocyclic virotoxins, the tryptathionine occurs in a cleaved form, and the sulfur atom is incorporated in a methylsulfoxide or methylsulfone substituent of the indole moiety. All amatoxins have a phenolic hydroxyl group at the 6 position of the indole nucleus, except amanin which has an unsubstituted tryptophan moiety like phalloidin. The amino acid sequences of virotoxin molecules appeared to be very similar to those of phallotoxins, the difference being that they contain D-serine instead of L-cysteine and two amino acids which are exceptional in nature: 2,3-truns3,4-dihydroxy-~-prolineand 2'-(methylsulfonyl)-~-tryptophan. The biological activity of amatoxins and phallotoxins was found to be strictly dependent on the tree-dimensional shapes of the peptide molecules. It was demonstrated that toxicity can be completely lost as a result of even a minor change of structure such as splitting of one of the peptide bonds or removing the sulfur bridge, which alters the conformation and thus the ability to bind to the protein receptor. The preferred threedimensional crystal structures of p-amanitin (105,106) and of other amanita peptides and analogs (107-109) were determined by X-ray analysis. It was found that the molecules assumed a cavity-shaped conformation in the p-amanitin crystal, the indole fragment being on the top of the convex surface. The three-dimensional structure is fairly rigid because of intra- and intermolecular hydrogen bonds, especially those with at least seven water molecules. The presence of so much solvent in the crystal suggests that the conformation determined in this way is probably similar to that occurring in an aqueous solution and thus is acceptable for considerations of biological interactions. This conclusion was confirmed by the results of 'H-, I3C-, and "N-NMR studies (110-113) in solution. Some of the naturally occurring amatoxins from the amanullin series do not contain a y-hydroxylated side chain of isoleucine and are nontoxic. Therefore, it has been concluded that, in contrast to the case for phallotoxins, the presence of such a group in the amatoxins is prerequisite for toxic activity. This type of hydroxyl group is located on the concave side of the 24-membered macrocycle in the three-dimensional molecular structure and is involved in binding to the RNA polymerase enzyme. The chemical effect of the amatoxin side chain and the contribution of every structural element to biological activity have been successfully investigated (107,114). In spite of the differences in structure and mode of action, the general shape of phallotoxins resembles that of amatoxins. As an example, spatial

2.

ALKALOIDS FROM MUSHROOMS

22 1

structure (111) generated from potential energy calculations and NMR data for phalloin (110) is presented in Fig. 10. The virotoxins are monocyclic peptides, and thus the conformations of the molecules are not rigid. The biological activity of the virotoxins is comparable to that of the phallotoxins, and the virotoxins also behave similarly to the phallotoxins on the molecular level as they bind to muscle actin. However, the flexibility of the monocyclic structure and the presence of two additional hydroxyl groups in the virotoxins suggest a different mode of interaction with actin. It is supposed that virotoxins may adopt a biologically active conformation by an induced-fit mechanism on contact with actin (102). Because of the similarities in toxicity and biological action, it is likely that the virotoxins are biosynthetically derived from the phallotoxins or from a common precursor molecule. A possible sequence of physiological reactions which convert phalloidin to viroidin (102) is outlined in Scheme 23. Intensive studies on the synthesis of amanita peptides, both total and partial via chemical modification, resulted in the preparation of some naturally occurring alkaloids, such as phalloin (115) and prophalloin (116)

FIG. 10. Calculated three-dimensional structure of phalloin. (From Ref. 221 by courtesy of VCH Verlagsgesellschaft.)

222

R62A ANTKOWIAK AND WIESEAW Z. ANTKOWIAK

Hydroxy

3

SCHEME 23. Possible metabolic transformation of phalloidin to viroidin. From Faulstich et al. (102).

as well as numerous analogs (117-119). Syntheses of bicyclic tryptathionine thioether, the smallest dipeptide phallotoxin model (119,120), and of phalloin ( I 15) exemplified the successful approaches and are illustrated in Schemes 24 and 25, respectively. In the former case L-tryptophan was first oxidized by either peroxy acid (121) or photochemically generated singlet oxygen (122) to ~-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b] indole-2-carboxylic acid (Hpi) and then condensed to Hpi-cysteine. This dipeptide, following treatment with trifluoracetic acid and heating of the resulting monocyclic derivative, yielded the bicyclic final product. In the synthesis of phalloin (Scheme 25), condensation of the S-chloride of Boc-alanyl-D-threonylcysteinylallohydroxyproline with alanyltryptophyl-y-hydroxyleucine lactone by the mixed-anhydride (MA) method gave the heptapeptide thioether, which yielded Boc-secophalloin lactone on cyclization. Removal of the Boc residue, followed by opening of the

2.

223

ALKALOIDS FROM MUSHROOMS

HOzC-CH-NHz I

J?

C02H

m

H H

7CHZ H

2

RCOjH

-

OH ___I

or lo2

N H

N H

CO2H

Hpj

I Hz N-C H-COz H Tryptathionine (thioether)

HN-CH

-

~

I

.co

A

-MeOH

NH -Boc,-Trt

HL(

CHZ I OC-CH

COzMe

TrtS Trt

=

NH

Bicyclic tryptathionine (thioether)

trityl

Boc = t e r t - b u t y l o x y c a r b o n y l

SCHEME24. Chemical synthesis of tryptathionine thioether.

H-Ala-NH-CH-CO-NH-

CH-CH2

'

I

CIS $.HZ H HO-aHyp-CO-CH-

-[

X

)-

OC-O

NkkDThr-Ala Boc

Ala-T;-NH-CH-CH2

I

'

X

oc-0

aHyp-Ala-DThr

'"& S

N

TFA,C1C02iBu D

0 - M e

I H -Ala-l

Ala-Trp-

I

2343 NH- CH-CH,-C, CO I OH I CH,

MA

I

-Boc

-AlaBoc Phalloin

SCHEME25. Overview of the chemical synthesis of phalloin.

224

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

lactone ring and a second cyclization, led to phalloin, which proved to be identical to the natural toxin in physical and toxicological respects (115). Besides the amatoxins, phallotoxins, and virotoxins in Amanita phalloides, two other groups of cyclic peptides have also been found: the nontoxic cycloamanides and the hemolytically active peptides. The latter are still poorly characterized, the best known representative being phallolysin, which was isolated in a homogeneous state by Faulstich et al. (121). The hemolytic activity of phallolysin results in the release of hemoglobin from erythrocytes into the blood plasma. This was demonstrated to be caused by insertion of the peptide into the cell membrane to form transmembrane ion channels which disturb the distribution of sodium and potassium ions. The toxicity of phallolysin even exceeded that of aamanitin several times [intravenous (i.v.) for mice LDso 120 pg/kg), and death occurs by acute hemolysis within a few minutes after injection. Nevertheless, it has been assumed that phallolysin, owing to its instability in acids and at temperatures higher than 65"C, cannot contribute to human intoxication by A . phalloides (86). In contrast to the case for phallolysin, the chemistry of cycloamanides is well developed. Among the numerous biologically inactive natural cycloamanides and synthetically obtained analogs (122),antamanide (123)attracted the most attention because of its unique antiphallotoxin (and, in a sense, antiamatoxin) properties. A peptide dosage of 0.5 mglkg is sufficient to fully protect mice from death by phalloidin when injected 1 hr before 5.0 mg/kg (more than two times the LDso value) of the toxin is administered. Like other members of this group, antamanide (also called antiamanita peptide, AA) is soluble in acetone and nearly insoluble in water. It can be extracted from the toadstool on partition between water and ethyl acetate into the organic solvent. Antamanide is a monocyclic decapeptide consisting of only four different L-amino acids, namely, alanine, valine, phenylalanine, and proline, in a molar ratio of 1:1:4:4(Fig. 11). The amino acid sequence was proved by systematic structural studies of the trifluoroacetylated products resulting from partial methanolysis of the peptide (124). The structure of antamanide was confirmed by total systheses based on several approaches ( 1 2 5 4 2 8 ) . The molecular geometry in different solvents and in the presence or absence of metal ions has been thoroughly investigated by X-ray (129)and NMR (130,131)methods. It was found that antamanide and some of its analogs are capable of complex formations in water-free solvents with alkali and alkaline earth metal ions. The complexes with ions like Na+ and Ca2+,with a radius of about 1 A, appeared to be especially stable. The antiphalloidin activity of antamanide relies on its high affinity for the

2.

ALKALOIDS FROM MUSHROOMS

225

Ph Ph

Ph

FIG. 11. Structure of antamanide.

same liver membrane proteins which are responsible for the transport of phallotoxins into liver cells (132,133). It appeared that antamanide also inhibits the inward transport of amatoxins (134). However, owing to the simultaneously reduced excretion of the latter into bile, which results in its accumulation in the cell, the protecting effect of antamanide in Arnanita poisoning is not to be expected (86). Membrane transport of amatoxins could also be inhibited by taurocholate, phalloidin, prednisolone, and silybin (a water-soluble component of natural silymarin), but not by penicillin G or thioctic acid (134). Interestingly, the latter two drugs together with silybin are successfully used for detoxification in clinical chemotherapy, acting by blocking the absorption of toxins and potentiating the mechanism of removal (103). 111. Indole Alkaloids

Mushrooms of the Hymenomycetes provide a good source of secondary metabolites having the indole moiety incorporated into their molecular structures, as in the case of Arnanita peptide alkaloids. Several of the indole alkaloids reveal strong physiological activity and have been found to be an important pharmaceutical material. The simplest unsubstituted indole and its methyl derivative, scatol, were found in some Lepiota and Tricholoma species. The probable biogenetic precursor of all indole metabolites is the amino acid tryptophan, widely distributed in fungi, or its derivative tryptamine, found in some species of Coprinus, Inocybe, Panaeolus, Sacrodon, and Boletus ( 7 ) . Investigation of a strong biological activity characteristic of an animal hormone function revealed the presence 5-hydroxytryptamine (serotonin), which was found in large amounts in some Panaeolus and Amanita species.

226

R62A ANTKOWIAK AND WIESKAW Z . ANTKOWIAK

A. BUFOTENINE Bufotenine, 5-hydroxy-N-dimethyltryptamine,a hallucinogenic metabolite previously known as a constituent of skin secretions of several toads of the species Bufo (135,136), was first isolated from mushrooms by Wieland er al. (137) from the false death cap, Amanita mappa ( A . cirrina) and called mappin. Later it was also found in other Amanita spp.: A . porphyrea (138), A . tormentella (139), and, in small amounts identified by paper chromatography, A . muscaria and A . pantherina (137). Interest in the biological properties of serotonin and its N-methyl derivatives stimulated studies of these systems. An interesting total synthesis of bufotenine starting from 3-nitro-5-hydroxybenzaldehydeconsists of achievements of two research groups (136,140) (Scheme 26). Other reported synthetic procedures have been summarized by Stoll et al. (136). The biosynthesis of bufotenine has not been entirely elucidated experimentally. Some biochemical observations, however, prompted Witkop (141) even at early stages of investigation to make the accepted (137) suggestion of the possibility of direct hydroxylation of the benzene ring in tryptophan prior to any reaction on the pyrrole part of the molecule. Since the hydroxyamino acid was found not only in toad secretions but later also in the mushrooms, it may be the precursor of serotonin and hence of the N-dimethyl derivative, bufotenine. 5-Hydroxytryptophan was first found

BzoaCHo MeN02

\

BzOaCH=CH-NOz

~

NOz

H

NO2

"'"a "'w CHzCOOH

-

1.CH2N2

CHzCON3

2'N2H4

CHZCONMez ~

BZOq)J$

1.LiA1H4 2.H2/Pd

\

H

\

3.HNOZ

H

H

HNMe2

Fe,AcOH

-

H

CHzCHzNMe2

H Bufotenine

SCHEME 26. Total synthesis of bufotenine.

2. COOH

COOH

NMe2

---

---c

H Tryptophan

227

ALKALOIDS FROM MUSHROOMS

H

H

5-Hydroxytryptophan

Serotonin

Bufotenine

SCHEME 27. Biosynthesis of bufotenine.

together with serotonin in Panaeolus campanulatus (142) and next in Amanita citrina (243), were bufotenine is present in the amount of 0.7-1.5% of dry tissue. This may indicate the possibility of enzymatic decarboxylation of the hydroxyamino acid followed by N-methylation to bufotenine in the course of fungal metabolism (Scheme 27). It is interesting that two closely related metabolic tricyclic derivatives of bufotenine, namely, bufothionine (244)and dehydrobufotenine (143, have been identified in skin secretions of toads but not, as yet, in mushrooms. The biosynthetic interconversions of these metabolites including the cyclization process (Scheme 28) were not elucidated (101) until recently. By quantitative chromatographic analysis [gas chromatography (GC) and TLC using either chemical or enzymatic methods of detection], Stijve (143) was able to demonstrate the presence in Amanita citrina mushrooms of various European origins of 0.03-0.06% of another nitrogen-containing principle, bufotenine N-oxide. It is a rare but not the only case of the occurrence in mushrooms of an alkaloid in the form of its N-oxide. Apart from orellanine, which is discussed later, examples of structures containing such a functional group are found in metabolites of fungi other than mushrooms, for example, the family of aspergillic acids (246) and the closely related di-N-oxide pulcherriminic acid (247) isolated from the mold Aspergillus flavus and the yeast Candida pulcherrima, respectively.

Bufotenine

Oehydrobufotenine

Bufothionine

SCHEME 28. Interconversion of alkaloids isolated from Bufo species.

228

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

B. PSILOCIN AND PSILOCYBIN Isomeric to bufotenine metabolites having the 4-hydroxylated indole ring system, psilocin and its phosphoric acid ester, psilocybin, were first isolated from Psilocybe mexicana and their structure determined by Hofmann et al. (148). The Mexican magic fungus Teonanacatl has been eaten for centuries by native Indian priests in religious or tribal ceremonies to induce illusions and hallucinations. The psychotropic action of these constituents is similar to that of mescaline and LSD. The active principles have subsequently been reported from more than 30 species of the genus Psilocybe and several species of other Agaricales, mainly in the genera Pluteus, Panaeolus, Stropharia, Znocybe, and Conocybe (149-152), collected in various countries. In some species the demethylated psilocybin analogs baeocystin and norbaeocystin have also been found, (153-155). Because of the potent hallucinogenic properties of psilocybin and psilocin, numerous studies relating to isolation and identification of the alkaloids in different mushrooms and by different methods have been reported (156159). The structures of psilocin and psilocybin were confirmed by synthesis (148,160) starting from 4-benzyloxyindole (Scheme 29). The biosynthesis of psilocybin has not yet been fully clarified, although some details of the pathway seem to be unquestionable. First, Brack et al. (161) found that psilocybin was biosynthesized from tryptophan, and then Agurell et al. (162) demonstrated that Psilocybe cubensis effectively incorporated tryptamine into psilocybin as well. In later studies, Agurell and Nilsson (163,164, using several precursors specifically labeled with I4C and 3H, showed that tryptamine and psilocin are “apparent interme-

Psilocin

H Psilocybin

SCHEME29. Synthesis of psilocin and psilocybin.

2. N H7

Q f -

229

ALKALOIDS FROM MUSHROOMS

H

- WNCCH3-

t OP03H2

IH

IH

1“

lH

-

&ifH2

H

H Norbaeocvstine

Baeocystine

Psilocybin

SCHEME 30. Metabolic grid proposed by Repke et al. (166).

diates” although N-meth~ltryptamine-~H especially was also clearly incorporated into psilocybin. It must be mentioned that 4-hydroxytryptamine-2’-14Cwas incorporated into psilocybin as well, but the introduction of this compound led to the formation of other minor products not normally detectable in cultures of this mushroom. In addition, the authors demonstrated that, in contrast to serotonin biosynthesis with the initial hydroxylation of tryptophan at C-5, 4-hydroxytryptophan is not the precursor of psilocybin, and the biosynthesis of this metabolite involves the decarboxylation of tryptophan to tryptamine in the first step. These observations led Agurell and Nilsson to suggest that psilocybin is formed in Psilocybe cubensis according the sequence tryptophan -+ tryptamine += N-methyltryptamine + N,N-dimethyltryptamine + psilocin + psilocybin. The authors also considered the possibility of phosphorylation occurring prior to methylation. The latter possibility was reconsidered more recently by Stijve (155, 165). According to his observations, psilocybin in Psilocybe semilanceata and in five Znocybe species was accompanied by its precursor baeocystine, which may indicate that during biosynthesis phosphorylation precedes methylation in conformity with the sequence tryptophan + tryptamine + 4-hydroxytryptamine += norbaeocystine + baeocystine -+

230

R6ZA ANTKOWIAK AND WIESEAW Z . ANTKOWIAK

psilocybin. This conclusion, however, would eliminate psilocin as the immediate precursor of psilocybin, which had been demonstrated previously by feeding experiments. The apparently conflicting opinions concerning the biosynthetic pathway seem to support the idea proposed by Repke et al. (166) that, owing to the lack of specificity of the enzymes involved, a “metabolic grid” can exist instead of a simple linear reaction sequence (Scheme 30). C. INDOLEPIGMENTS The pigment indigo (indigotin), the oldest known coloring substance originally obtained from various plant species, has been also found in mushrooms. This metabolite was isolated from Schizophyllum commune (Clavariaceae) (167,168), Agaricus campester (169), and Auriculariopsis ampla (Corticiaceae, Aphyllophorales) (63). In the case of S. commune the fungus was grown in a synthetic medium which contained thiamin with ammonium ions as source of nitrogen. The pigment appeared as a suspension in the culture fluid as well as in the mycelium. Contrary to the form found in plant sources, in the mushrooms indigo was not found as a glucoside. In the first two species the blue dye was found to be accompanied by the red indirubin and a yellow isatin (170). A simple synthesis starting from 2,4-dioxo-3,3-dichloro- 1,2,3,4-tetrahydroquinoline was reported (171) (Scheme 31). Several other examples of indole pigments with bisindolylmaleimide structures have been isolated from the slime mold Arcyria denudata (Trichiales, Myxomycetes, Myxomycota). The chemistry and physical

Indigo

Indirubin

Isatin

..

..

Arcyriarubin 6

SCHEME 31. Indole pigments form fungi.

2.

ALKALOIDS FROM MUSHROOMS

23 1

properties of this new group of natural pigments were recently reviewed by Gill and Steglich (63), and the synthesis of the simplest members (e.g., arcyriarubin B) has been reported (172). D. P-CARBOLINES The P-carboline skeleton, occasionally hydroxylated at the 6 position appears to be quite popular in nature, and alkaloids of this type have also been found in mushrooms. First, as the result of a thorough examination of the composition of Amanita muscaria metabolites carried out by Eugster, the occurrence in this species of 1,2,3,4-tetrahydro-l-methy1-/3-carboline3-carboxylic acid was established (59). This compound (mp 296-29SoC, [ a ]-~1lSo), known also as tetrahydroharman-3-carboxylicacid, was earlier (173) identified as a component of casein acidic hydrolysis products and was prepared synthetically by condensation of tryptophan with acetaldehyde or with pyruvic acid followed by decarboxylation of the 1,3dicarboxylic acid intermediate (Scheme 32). The P-carboline system alkylated at C-1 occurs frequently in the indole alkaloid series and in some cases in the 1,2,3,4-tetrahydro form, as shown above. The simplest homolog with a methyl substituent is called harman (Fig. 12). It has been found in several plant families and also in the higher fungus Coriolus maximus (174). Recently, particular interest in 1substituted P-carboline derivatives has also resulted from the antiviral activity against herpes simplex virus type 1 (HSV-1) discovered in eudistomins alkaloids (e.g., eudistomin S; Fig. 12), isolated from the tunicate organism Eudistoma olivaceum (175). Intensive studies of biologically active compounds from higher fungi recently carried out by the Steglich team (176) led to the isolation of some new alkaloids of the P-carboline type (Fig. 13). Investigation of the methanolic or acetone extract of a common toadstool, Cortinarius infractus, revealed the presence of infractin, 6-hydroxyinfractin, and the corresponding acids p-carboline- 1-propionic acid and 6-hydroxy-P-carboline-1propionic acid, as well as 4,5-dihydrocanthin-6-onealong with its 9hydroxy derivative, and the bitter principle infractopicrin (177). The strucI

n

SCHEME 32. Synthesis of tetrahydroharman-3-carboxylic acid.

U

232

R6ZA ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

Harman

Eudistomin 5

FIG. 12

tures of these compounds were determined by analysis of UV, IR, NMR, and mass spectra (177) and were confirmed by synthesis (176). The total synthesis of ethyl p-carboline-1-propionatefrom tryptamine and succinic anhydride was reported by Szantay’s team (178). The syntheses of hydroxyinfractin and infractopicrin carried out by Steglich’s group are illustrated in Schemes 33 and 34, respectively (176). The molecular skeleton of infractopicrin resembles that of vincamine, a representative alkaloid which lowers blood pressure, isolated from plants of the genus Catharanthus (Vinca). There is, however, no evidence of a biogenetic relationship of these alkaloids, and the absence of an angular ethyl group in infractopicrin and in the accompanying metabolites suggests rather different biosynthetic pathways in both cases (177). Studies on the canthin-6-one alkaloids of various natural origins were recently reviewed by Ohmoto and Koike in Volume 36 of this treatise (179). Although a great number of alkaloids having a p-carboline structure have been found in nature, very few reports have been published about their biogenesis, and these are concerned only with the plant material. From among the alkaloids mentioned above only harman (180) and 43dihydrocanthin-6-one (181) were demonstrated in feeding experiments with 3H- and I4C-labeled precursors to be derived biosynthetically from tryptophan. In the case of harman, tryptamine is probably condensed with pyruvic acid to give 1-methyl-1,2,3,4-tetrahydro-~-carboline-l-carboxylic

Infractin (R=H,

R’=Me)

4,5-Dihydrocanthin-6-one

Infractopicrin

(R=H)

FIG. 13. Alkaloids from Cortinarius infractus.

2.

9

ALKALOIDS FROM MUSHROOMS

233

BzlO

BzlO w i : B z '

"t.

PhH,reflux

-

Bz'

reflux HC1,THF

-

Me02 C

C02Me

O

o/'

W

-

HO MeONa

.

SCHEME 33. Synthesis of hydroxyinfractin.

acid as the key intermediate (Scheme 35). This compound was found to be well incorporated and converted via harmalan to harman along with another metabolite, eleagnine, in Eleagnus angustifolia and Passz3ora edulis plants (180). E. ERGOT ALKALOIDS The class of indole alkaloids encompasses another important group of metabolites called ergot alkaloids, though their occurrence is most often associated with other fungi besides mushrooms. In fact, the lower fungi appear to be an interesting source of many metabolites known for their high physiological activity, including the antibiotics from Penicillium, Aspergillus, and Streptomyces species (Actinomycetes). (Although the latter, called ray fungi, are in fact filamentous bacteria, they have sometimes been classified as Fungi Imperfecti.) In contrast to the beneficial properties of antibiotics, however, there are at least two different groups of fungi metabolites, the aflatoxins and ergot alkaloids, which have exerted

234

R6ZA ANTKOWIAK AND WIESJLAW 2. ANTKOWIAK

I C HO

1.LOA

2.BrCH2C02t-Bu

c

OMe

-

-

Me2NNH2

NMez 03

tBuOzC

OMe

-

CHO t B u O ; ! C h O M e

c

-

HBr-aq,reflux

SCHEME 34. Synthesis of infractopicrin.

a particularly painful influence on the health and economy of human beings throughout the centuries. Aflatoxins (182) are nitrogen-free mycotoxins; in 1960 they caused the previously unknown Turkey-X disease in England which resulted in the death of about 100,000 poults following the consumption of groundnut-derived feed contaminated by Aspergillus fluuus. On the other hand, one of the recurring calamities of the Middle Ages known as holy fire, St. Anthony’s fire, and in more recent time as ergotism is caused by contamination of wheat by the parasitic neurotoxin-producing fungus ergot (Clu-

2.

235

ALKALOIDS FROM MUSHROOMS

&COOH

Me

1.R=C02H 2. R = H

Eleagnine

Harmalan

Harman

SCHEME35. Biosynthesis of harman.

viceps purpurea). During recent decades, several outstanding achievements in mycotoxin chemistry have been recorded, and lately an entire issue of Tetrahedron (1989 No.@, has been devoted to the topic. Ergot is probably the oldest known source of mycotoxins. However, in accordance with the statement of Paracelsus (1493-1541), “The right dose differentiates a poison and a remedy,” the ergot alkaloids are also one of the most important groups of indole alkaloids from a pharmaceutical point of view. The chemistry and pharmacology of ergot alkaloids were reviewed in several articles and books, as well as in this treatise, recently by Ninomiya and Kiguchi (183). These alkaloids were discussed mainly in regard to biosynthetic approaches by Floss (184), Cordell (185), and Stadler (186). For many years the distribution of ergot alkaloids was considered to be limited to Claviceps species, especially C . purpurea and cultures of C. paspalum which appeared to be the most effective in the production of alkaloids on an industrial scale. During the last three decades, however, ergot alkaloids has been found in several other fungi in the Clavicipitaceae such as Balansia spp. and Epichloe typhina (187)and also in Aspergillus and Penicillium species as well as even in higher plants of the family Convolvulaceae. The molecules of all naturally occurring ergot alkaloids have a central tetracyclic ring system, known as the ergoline nucleus, or its seco analog with the D ring opened. As a rule and with a very few exceptions, the N-6 position is methylated, a double bond is present in ring D between carbons 8 and 9 or 9 and 10, and there is an additional carbon atom attached to C-8 which may be in a different oxidation state. In a majority of the alkaloids the carbon is part of a substituted amide group of the amide of lysergic

236

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

acid, which can be recognized as a fundamental building block of many ergot alkaloids. The oxidation state of the carbon attached to C-8 and the nature of its substitution are significant criteria for classification of all known ergoline alkaloids into three groups: the clavine alkaloids, the simple lysergic acid' alkyl amides, and the peptide derivatives of lysergic acid amide. 1. Clavine Alkaloids The clavine group is characterized by C-17 (attached to C-8) being at a lower oxidation state, as examplified by the structures shown in Fig. 14. Although most clavines contain only the carbon atoms of the 6,8-

&* \

H2C

H

H Ergoline

Agroclavine

Lysergoi

OH

Penniciavine

Isofumigaclavine A

Elymoclavine-O-p-0-fructoside

Chanociavine-I

Rugulovasine A

FIG. 14. Examples of clavine alkaloids.

2.

ALKALOIDS FROM MUSHROOMS

237

dimethylergoline system, a few exceptions have been recorded, as, for example, the long-known elymoclavine-O-P-D-fructoside and the elymoclavine-O-~-~-fructofuranosyl-(2+ 1 )-O-P-D-fructofuranoside recently isolated by Flieger et al. (188) from saprophytic cultures of Clauiceps strain. 2. Simple Derivatives of Ergine Free lysergic acid has never been found in large quantities in any of the Clauiceps species ,though large amounts can be easily obtained by hydrolysis of other ergot alkaloids. The simplest amide derivative, lysergic acid amide, also known as ergine (its Ha-C-8 epimer is erginine), has been isolated from C. paspali. This compound was also found to be largely responsible for the hallucinogenic activity of ololiuqui, a magic drug prepared by American Indians from the seeds of Zpomoea uiolaceae and Riuea corymbosa (189). However, since it is always accompanied by the corresponding a-hydroxyethylamide, which spontaneously decomposes to acetaldehyde and lysergic acid amide, the natural occurrence of the latter is sometimes questionable. On the other hand, the hydroxylated epimers of lysergic acid amide, 8-hydroxyergine and 8-hydroxyerginine7 were recently isolated from C. paspali (190). Lysergic acid and its amide derivatives easily undergo epimerization at C-8 under a variety of conditions to yield the corresponding isolysergic acid derivatives which are strongly dextrorotatory and pharmacologically inactive (Scheme 36). Another type of facile isomerization recognized in these systems involves double bond migration inside the nonaromatic ring system of the ergolene derivatives. The isomerization of paspalic acid (the major alkaloid in Portuguese C. paspalurn strains and less abundant in C. purpurea) to lysergic acid, which entails a suprafacial [ 1 0 4 hydrogen shift, is readily achieved by means of base treatment. The reverse transformation leading to displacement of the double bond to C-8 requires solvomercuration in methanol (191,192)with subsequent reduction by lithium aluminum hydride-aluminum chloride. The intermediate methyl 10methoxypaspalate was used as a readily available starting material for preparation of several ergoline alkaloids. The susceptibility to isomerization in the ergolene system has been also demonstrated by the easy transformation to the benzindoline system under acidic conditions, which was found to be a substantial problem during syntheses of these alkaloids. Among the simple mono-N-substituted derivatives of lysergic acid amide, ergonovine appears to be the most interesting because of its powerful pharmacological activity. The structure of the alkaloid followed from the finding that alkaline hydrolysis yielded lysergic acid and L-( +)-2aminopropanol. Ergonovine (also known in the literature as ergometrine,

238

R 6 Z A ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

'B3

HO,

H

eC

H

L y s e r' q i c a c i d

H ( + 1- I s o l y s e r g i I;

Paspalic acid

acid

t 1 ymo c 1 a v i ri e

M e t h y l 10-methoxypaspalate

R

& H

H + -

/

N

H

SCHEME 36. Modes of isomerization in the ergolene system.

ergotocin, ergobasine, and ergostetrine) is the main constituent of the water-soluble fraction of ergot alkaloids, but this source appeared to be insufficient to meet demand and led to the elaboration of several total or partial syntheses of lysergic acid and hence of other ergot alkaloids (193195). These and other synthetic approaches to the ergot alkaloids were compiled by Howell in 1980 (196). The first total synthesis of (*)-lysergic acid was reported by Kornfeld et al. (193). This basic fragment derived from ergot alkaloids was synthesized in a 15-stage sequence begining with 3-indolepropionic acid (Scheme 37). The synthetic (*)-lysergic acid was then converted to racemic isolysergic

2.

239

ALKALOIDS FROM MUSHROOMS

resolution via ditolyl-(l )-tartaric amm.salt

d,l-Lysergic acid

d-Lysergic acid hydrazide A

-

1

HO-

d-Isolysergic HNo2 acid hydrazide

y

y

3

H$.I-CHCHzOH

9 d-R-CN3

O=t:

&OH YH2

//h

3

HY-CH CH20H

HO-o=L&

-

\

H

Ergonovinine

SCHEME 37. Kornfeld synthesis of lysergic acid and ergonovine (193).

\

H

Ergonovine

240

R6ZA ANTKOWIAK AND WIESKAW 2. ANTKOWIAK

acid hydrazide. Since its racemic resolution and further conversion to ergonovine was already known (197), Kornfeld's synthesis also completed the synthesis of this ergot alkaloid. In the 1980s several other approaches to the total synthesis of lysergic acid were reported. Some approaches, such as Ramage's synthesis (195) (Scheme 38), involved a central tricyclic ketone intermediate in the form of a 2,3-dihydro-N-benzoyl Uhle ketone derivative. The isomer with the carbonyl in the vicinal4 position (198),which can be derived from the Uhle ketone, was used by Ninomiya et al. (199) in the total synthesis of racemic methyl lysergate along with methyl isolysergate and isofumigaclavine B (roquefortine B) (Scheme 39). This synthesis involved a reductive photocyclization step followed by dihydrofuran ring opening, which led to the formation of the tetracyclic key intermediate of the final carbon-nitrogen skeleton. In Rebek's synthesis (200) the tricyclic ketone was prepared from L-tryptophan and thus already possessed the N-6 atom of the final product located in the defined configuration (Scheme 40). This forced the stereochemistry in further steps of the synthesis and led to the production of optically active products. Because of the possibility of pyrrole double bond migration, all the above syntheses were carried out via 2,3-dihydroindole intermediates which ultimately required a dehydrogenation step. The synthesis elaborated by Oppolzer (201)is exceptional as the indole nucleus was kept intact throughout the entire reaction sequence (Scheme 41). The strategy relied on concomitant formation of rings C and D by an intramolecular [4+2]

aH

q,OOMe H % HCII2H, HCHO

heat, 3h

-

MeOH,reflux

Bz

COOMe

-

+ c-8 epimer

BZ

SCHEME 38. Synthesis of lysergic acid by Ramage et a / . (195).

2.

24 1

ALKALOIDS FROM MUSHROOMS

0

&-

hv,NaBHq

1 . MeNH2

1 .LiA1H4 2 . oso,

BZ

Bz

COOMe

COOMe

1.N a I 0 4

l.PCIJ

2.Cr0,

2 .Et30i3F4 3 . ( PhSeO )*O

H

Bz

SCHEME 39. Synthesis of lysergic acid by Ninomiya

el

al. (199).

cycloaddition followed by a retro-Diels-Alder reaction to release the diene unit and isomerization of the C-8, C-9 double bond to the more stable C-9, C-10 position. In the 1980s Muratake and Natsume (202) in a number of reports presented a new, interesting approach to the synthesis of substituted indole derivatives that focused on their use as intermediates in the preparation of a variety of clavine alkaloids and mycotoxins. The method elaborated by the authors consisted of constructing the specifically substituted benzene portion of the indole nucleus, with 1-methoxycarbonylpyrrole being the starting material. The functionalized 4-alkylindole thus obtained was transformed to a tricyclic indole derivative which appeared to be a common intermediate for the synthesis of several ergot alkaloids, as exemplified by the synthesis of (2)-dihydrosetoclavine (203-205) (Scheme 42). Recently, the application of this method was extended by the authors (206) to the syntheses of a series of marine alkaloids, the hapalindoles. Most of these alkaloids have a tetracyclic framework made up of 3,4-substituted tryptamine and two isoprene units. A representative of the simple lysergic acid amide derivatives is the semisynthetic diethylamide, LSD (lysergic acid diethylamide), which has a questionable reputation. Although LSD is not really a natural product, the discovery of its powerful hallucinogenic properties by Hofmann in the 1940s led to the development of the broad field of psychopharmacology.

242

R6ZA ANTKOWIAK AND WIESEAW Z. ANTKOWIAK

1

Bz 1 .Ac2U 2.A1C13

I

BZ

OgH

+-

3 . Et30BF4

H -

w

Br

BZ

1.MeI 2. HBr

Zn

UEt

BZ

H

H

I

SOC12,MeOH

L u m i 1 a c t o n e It of isolysergic acid I'

P205,MeS03H

H-

Mn02

Methyl isolysergate +

CH2C12

Methyl lysergate

SCHEME 40. Synthesis of methyl lysergate and methyl isolysergateby Rebek et al. (200).

3. Ergopeptide Alkaloids Most typical ergot alkaloids have a lysergyltripeptide structure. One of the first known alkaloids of the ergopeptine type, ergotamine (Fig. 15), which was isolated and investigated by Stoll and Hofmann (207-209), on hydrolysis yielded (+)-lysergic acid, D-proline, L-phenylalanine, pyruvic acid, and 1 equiv of ammonia. The first two components occur in almost all

2.

ALKALOIDS FROM MUSHROOMS

2.Cti. -CH-N02 I.Na;H,MeUH

c

Ts

UHC

,N d i , UMSU

% COOMe

X C O O M e 1.CBr4,PPh3,0MF 2.PBu3,PhH

$I -

243

.,A

\

N Ts

H

MeOOC I .MeOS02F 2. A l/lig 3. KUH,EtUH

-0 H

H

H

SCHEME 41. Synthesis of lysergic acid by Oppolzer et al. (201).

alkaloids of this type, whereas phenylalanine and pyruvic acid are exchangeable for another amino or keto acid, respectively. The keto acid together with ammonia composes the synthon a-hydroxy-a-amino acid, which participates in two peptide linkages and additionally in a cyclic hemiacetal linkage with the carbonyl carbon of proline. In the case of ergopeptam-type ergot alkaloids, the discovery of which was initiated in 1973 by the isolation of ergocristam (210)(Fig. 15), instead of the keto acid a true a-amino acid precursor in ‘theform of an L-valine residue is present in the molecule. The absolute configuration of ergotamine was established by chemical and spectroscopic methods (209) as (5R,8R,2’R,5’S, 1 l’S,12’s) and was confirmed by X-ray analysis of (-)-dihydroergotamine methanosulfonate monohydrate (211). This proved the reversed (L) configuration for the proline residue to that of the D-( +)-amino acid isolated previously (207)as the hydrolysis product of this alkaloid. However, Hofmann et al. (209) indicated that a cyclic dipeptide intermediate, (3S,9S,)-phenylalanylproline lactam, formed during the degradation easily equilibrates under alkaline conditions to the (3S,9R) diastereoisomer in a ratio of 1 to 9, yielding as the final product almost pure (R)-(+)-proline (see Scheme 43). In contrast to ergopeptines, however, the original configuration of proline included in the peptide moiety of the alkaloids of ergopeptam type is indeed D.

244

R 6 Z A ANTKOWIAK A N D WIESJLAW Z. ANTKOWIAK TMSCl

*CHO

Et3N,ZirC12

-

-0TMS

\

1.

-a1 cyJp

0 Y

0-1-0

COOMe

& H

3 . TsCH2NC

2. PCC

COOMe

-x HN'

1.LiA1H4

Z.DnOCOC1

0

SnC12

COOMe

co

n

&

-

'0'

l.Ts0H

-

2.H2,Pd/C

OH *wMe

..

HV(;

3.CH20,H2,Pd/C

('J-Dihydrosetoclavine

SCHEME 42. Synthesis of dihydrosetoclavine by Muratake and Natsume (203-205).

The alkaloids belonging to the ergopeptine and ergopeptam groups already identified as natural products are shown in Table I. New alkaloids, which continue to be discovered, may be ascribed either to those normally produced by the fungi (ergopeptines and ergopeptams) or to those formed by directed biosynthesis by incorporation of different amino acids, often unnatural, in the cyclol system. The group of natural ergot alkaloids has recently been enriched by two new metabolites of Clauiceps purpurea that are methylated derivatives of ergocornine and a-ergokryptine, respectively, at position 0-12', which is considered to be the starting point of the biosynthesis of the cyclol structure (224). As a result of directed biosynthesis using as an example a nutrient medium enriched with D,L-isoleucirie,

TABLE I ERGOPEPTIDE ALKALOIDS Ergopeptines" Amino acid' Phenylalanine Leucine

Ergotamine groupc [CH3COCOOH]

Ergoxine group' [CH3CH2COCOOH]

Ergotoxined group' [(CH,)&H-COCOOH]

Ergotaminef Ergotaminine Ergosine Ergosinine

Ergostine Ergostinine Ergoptine Ergoptinine

Ergovaline Ergovalinine

Ergonine Ergoninine

Ergocristine* Ergocristinine a-Ergokryptine* a-Ergokryptinine P-Ergokryptine P-Ergokryptinine Ergocornhe* Ergocorninine

Isoleucine Valine

Ergopeptams,b with the ergotaxam group' [(CH3)2CH-CH(NH2)COOHl Ergocristam Ergocristinam a-Ergokryptam a-Ergokryptinam P-Ergokryptam Ergocornam Ergocorninam

* Containing L-proline. Containing o-proline. Specific a-amino-a-hydroxy acid precursor. Ergotoxine [mp 174"C, [a]o- 189.7"(212)]was demonstrated by Stoll and Hofmann (213)to be an equimolar mixture of the three alkaloids listed in this group as lysergic acid derivatives (marked with asterisks). ' Specific for the respective horizontal sequences. 'The components of each pair are epimers at C-8.

246

R6ZA ANTKOWIAK AND WIESLAW Z . ANTKOWIAK

Ergotamine

Ergocristam

FIG. 15. Representative ergopeptide alkaloids of the ergopeptine and ergopeptam types.

three new alkaloids have been found (215) in C. purpurea: the 5’ epimers of P-ergokryptine, 6-ergokryptam, and P,P-ergoannam, a lactam alkaloid from a new group of ergopeptam alkaloids containing L-isoleucine as the first and second amino acids. In studies of the numerous ergot alkaloids spectroscopic methods were successfully used; references to some earlier reports are given by Floss

n

(35,95)

(2R,55,115,125)

tlo-

I

d-Lysergyl chloride

(-)-Ergotarnine 5 ’ 5 , l l ‘5,12 ‘5)

(5R,8R,2’RI

plus

(+)-Ergotaminine

(35,9R)

SCHEME 43. Total synthesis of ergotamine by Hofmann and co-workers (209).

2.

ALKALOIDS FROM MUSHROOMS

247

(184). More recently applications of MS (226) and NMR (227-219) for identification and structure determination, respectively, of some natural and synthetic alkaloids have been reported. Extensive studies carried out to elucidate the formation of the ergot alkaloids in nature were reviewed by Floss (284) with further supplements (186,220).A summary of the conclusions resulting mainly from numerous radioactive (I4C, 3H) feeding experiments is presented in Scheme 44. The biogenetic pathway is initiated with (R)-mevalonic acid and L-tryptophan which, when isotopically labeled, were found to be efficiently incorporated (221-223) into the ergoline ring system of clavine and peptide ergot alkaloids. It was demonstrated that L-tryptophan is the immediate alkaloid precursor and that the D isomer, which is also consumed by the culture, enters the pathway after inversion of the configuration. It was also demonstrated that tryptophan condensed directly with an isoprene unit at C-4, although the 4 position is not the most reactive one for electrophilic substitution of the indole ring ( 2 2 4 , and that the N6-methylation took place by enzymatic transfer of the methyl group from methionine prior to or during the formation of ring C. An interesting conclusion followed from experiments carried out by Floss and co-workers (225) which demonstrated that ergolines in fungi and in higher plants are formed from the same precursors. 4. Other Indole Mevalonates

Numerous matabolites other than the ones discussed above have been isolated from ergot alkaloid-producing genera, especially in the 1970s and 1980s. Some, the indole mevalonates, are listed below, as related to ergolines in some way. (Usually, only the most recent references are given, which should enable the reader to follow the studies carried out in connection with a particular alkaloid). One kind of structure of the rapidly growing family of indole diterpene alkaloids is represented by derivatives with a 3-substituted indole nucleus. Aflavinine, along with a few of its natural derivatives (226,227),and nominine (228) were isolated from Aspergillus flauus, the well-known aflatoxin-producing fungus, and A. tubingensis, respectively, whereas emindoles (DA, DB, and SA) were found to be produced by Emericellu species (Eurotiales) (229-231). The structures of these metabolites (Fig. 16) were determined on the basis of spectroscopic and chemical investigations supported by X-ray crystal analyses in some cases. A possible biosynthetic pathway for emindoles was presented by Nozawa et al. (229). Another group of alkaloids of this type is distinguished by the indole nucleus being fused in positions 2 and 3 to the diterpene moiety (Fig. 17). Three alkaloids with such a structure, paspaline, paspalicine, and the

248

RdZA ANTKOWIAK AND WIESKAW Z . ANTKOWIAK

S c h i f f base w i t h p y r i d o x a l phosphate

OH

------L

---L

--.-)

H

H Elymoclavine

-

E n z /s& 0

-

& S,

Lysergyl-CoA

Enz

Enz H 2 N d 0 H R

R

Lysergic acid

0

H R

SCHEME 44. Biosynthesis of ergot alkaloids according to Floss. For ergocornine R' = CHMe2.

=

-

2.

249

ALKALOIDS FROM MUSHROOMS

OH

H Aflavinine

Norninine

/ I

OH

\

H

'

OH'

H E m i n d o l e DB

Ernindole SA

FIG. 16

tremorgen paspalinine, were isolated from Claviceps paspali and another tremorgenic toxin, aflatrem, from Aspergillus Bavus. The structures and absolute configurations of these alkaloids were determined by spectroscopic and X-ray diffraction methods (232-234). The total synthesis of (-)-paspaline was reported by Smith and Mewshaw (235). This group of alkaloids can be completed by paspalitrems A and B (236) isolated from Claviceps paspali, by paxilline found in Penicillium paxilli (237), and by tubingensins A and B present in Aspergillus tubingensis (228). Similar structural features are also demonstrated by penitrems A to F (Fig. 18), which form a small family of tremorgenic mycotoxins produced by the ergot fungus Penicillium crustosum. The structure of these alkaloids was elucidated by Steyn and co-workers (238-241), and an approach to the total synthesis of penitrem D was also presented (242). Tremor-causing properties are also typical of the isoprenylated indole derivatives, which additionally contain a diketopiperazine fragment formed biogenetically as a result of the condensation of tryptophan and another amino acid. Metabolites with such a structure can be represented by fumitremorgins A to C and verruculogen TR-2 (Fig. 19), which were isolated from Aspergillus fumigatus; their synthesis was reported recently (243-248). Other examples are provided by echinulines (249,250), neoechinuline (251-254), brevianamides (255-258), and austamides (259,260) found in A. amstelodami [the echinule family additionally in A. ruber

H

O-Paspaline

Paspalicine

H

0 0-Paspalinine

Paxilline

0

O--

O-Aflatrem

Paspalitrem B

H

H

Tubingensin A

Tubingensin B

FIG. 17

1= O H , R 2 = C 1 ; 2 3 , 2 4 - d - e p o x i d e ) B ( R = R =H; 23,24-a-epoxide) 1 2 A (R

th,R,

C (R,=H,

R,=Cl) D ( R 11= R 2 = H ) 2 -

H%

E (R1=OH,

F (R =H,

Penitrems

FIG. 18

'

R2=H;

23,24-a-epoxide)

R - C 1 ; 23,24-r*-epoxide)

2.

ALKALOIDS FROM MUSHROOMS

Fumitremorgin C

V e r r u c u l o g e n TR-2

Neoechinuline ( X = O ) N e o e c h i n u l i n e 0 (X=Me,H) Cryptoechinuline A (X=CH2)

Echinuline

Austarnide

Lanosulin

AC

Verrucofortine

Roquefortine

FIG. 19

25 1

252

R6ZA ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

(261)],as well as by lanosulin (262)found in Penicillium lanosum, verrucofortine (263) in P. uerrucosum, and roquefortine (264) in P. roqueforti and P. crustosum. The structural features of the epimeric brevianamides A and B resemble those of the mycotoxins marcfortines A to C (265) and paraherquamide (266)found in P. roqueforti and P. paraherquei, respectively (Fig. 20). Amauromine, a dimeric indole alkaloid with hypotensive vasodilator properties, was isolated from Amauroascus species (Gymnoascales, Ascomycotina) (267) and Penicillium verrucosum (263). The structure of this metabolite was determined by chemical and spectroscopic investigations and confirmed by a total synthesis by Takase et al. (268). The symmetrical diketopiperazine made up of two modified tryptophans, each bearing a 1,l-dimethylallyl group at C-3, was synthesized as shown briefly in Scheme 45. The two inverted prenyl groups were introduced simultaneously by a thio-Claisen rearrangement reaction through the sulfonium salt. A close biogenetic relationship to ergoline alkaloids can be easily recognized in the case of cyclopiazonic acid (269-271) and its imine and bissecodehydro derivatives (272,273) (Fig. 21), which were isolated from P. cyclopium and A. versicolor.

0

Brevianamide B

Brevianarnide A

Paraherquarnide

Marcfortine A

FIG. 20

2.

253

ALKALOIDS FROM MUSHROOMS

L-Tryptophan

TiC14-LiA1H4

Amaurornine

SCHEME 45. Total synthesis of amauromine by Takase et al. (268).

p \

\

H

Cyclopiazonic acid

Cyclopiazonic acid imine

Bissecodehydroc y c l o p i a z o n i c acid

FIG. 21

IV. Pyridine Alkaloids A. ORELLANINE

The mushroom Cortinarius orellanus, occurring in several Western European countries, was recognized for the first time by Grzymala (274276) to be responsible for many fatal poisonings in Poland. The toxic properties of this species were attributed by the author, following the experiments on test animals, to a crystalline, colorless substance he isolated and called orellanine (277,278). The responsibility of orellanine for

254

R6ZA ANTKOWIAK AND WIESLAW 2. ANTKOWIAK

the toxicity of the mushroom was confirmed by Antkowiak and Gessner (279) following experiments on mice, as well as very recently by Richard and co-workers (see Ref.280 and also Refs. 2-4 cited therein). The toxin acts slowly over several days (up to 2 weeks) after the consumption of the mushroom, injuring mainly the kidney and to some extent the liver and other organs. It has been also shown that C. orellanus toxin inhibits the DNA-dependent RNA polymerase from rat liver and from Escherichia coli (281,282).Recent studies of C. orellanus intoxication have been described by Rapior et al. (283,284),and previous reports concerning the toxicity of this mushroom were reviewed by Schumacher and Hgiland (285). Besides orellanine in C. orellanus and later [following Steglich’s suggestion (286)l in C. speciosissimus, two other related alkaloids, orellinine and the nontoxic orelline, have been found (279,287) (Fig. 22). Chemotaxonomic studies by Gruber (288) and further investigations by Moser and co-workers (289)and Rapior et al. (290)also revealed the presence of these alkaloids, in different relative amounts, in C. orellanoides, [recently shown to be indistinguishable from C. speciosissimus (292)], C . Juorescens from South America, and many other Cortinarius species. Since numerous authors have issued reports in Europe and even in North America and Japan on intoxications by mushrooms form the genus Cortinarius (285), the list of species containing orellanine may be longer. A successful attempt to prepare a pure culture of C. orellanus was first reported by Rapior and Andary (292), but the orellanine content of the cultured mycelia was found to be lower than that of the carpophore. On the basis of spectroscopic data, chemical properties, and comparision with synthetic compounds with similar structural features, the structures of the orellanines were determined by Antkowiak and Gessner (293,294)in the late 1970s. According to their findings, orellanine, the main toxin of C. orellanus, has the structure 3,3’ ,4,4’-tetrahydroxy-2,2’bipyridyl-1 ,1‘-dioxide, whereas orellinine and the nontoxic orelline are its monode-N-oxide and dide-N-oxide derivatives, respectively. The structural propositions were well documented, mainly on the basis of UV,

Orellanine

Orellinine

FIG.22. Orellanines.

Orelline

2.

255

ALKALOIDS FROM MUSHROOMS

'H NMR, 13CNMR, and particularly the MS fragmentation, the pattern of which was confirmed by high-resolution mass measurements, detection of metastable ions, and a comparision with the mass spectra of partially deutered derivatives (295). The spectroscopic properties of orellanine have been reexamined by other authors (296-301). The correctness of the structural propositions had been questioned (281,302-305) until the mid1980s, when it was confirmed both by synthesis of orellanine (298,299)and X-ray analyses of orellanine trifluoroacetate (300)and the original crystals of orellanine hydrate (306) obtained by Grzymda. These achievements solved the problem of the homogeneity of the natural material called orellanine, which had been questioned most. Orellanine and orelline have been demonstrated to exist in tautomeric forms on the basis of UV and IR spectra [not confirmed, however, by recent I3C-NMR studies (297)] as well as of their chemical behavior toward alkylating agents (Scheme 46). As shown by Gessner (295) and by Dehmlow and Schulz (303, both alkaloids when treated with diazomethane gave three isomeric tetramethyl derivatives, indicating the participation of keto-substituted hydroxylamine and keto-secondary amine forms, respectively, in the tautomeric equilibrium. The synthesis of both orellanine and orelline was elaborated almost simultaneously by two independent teams from Germany and Italy. In both cases the key intermediate step, entailing the coupling reaction to the bipyridyl system, was carried out using the general method elaborated by Tiecco ef al. (308). In the procedure described by Dehmlow and Schulz (298,307)(Scheme 47), 2-chloro-3 ,4-dimethoxypyridine was coupled in the

orellanine

CH2N2

-

M 0e O S M e MeOXN

\

+

Me% :..

0-

OMe

+

/

:I:$Me OhN/

OMe OMe

orelline

CH2N2

-

'8

Me0

Me,N

+

\

\

OMe

Me% :

OMe

+

N/ \

N/

OMe

OMe OMe

SCHEME 46. Tautomeric structures of orellanine and orelline.

\

OMe

256

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK 1.H202/Ac20 I

Me0

NO2

-

1.MeONa

2.PC13

H202/Ac20

2

OMe

*

4

0

\

HEr/AcOH

HO

Me0 -z

____)

'

Orellanine

GNH2

Orelline

OMe N

Br

Me0

5ch.r.

-

c

\

OMe

0H OH

OH

OMe

-

8

-

O1

&

-

, OMe \

OMe

SCHEME 47. Dehmlow and Schulz synthesis of orellanine and orelline (298,307)

presence of a tetrakis(triphenylphosphine)nickel(O) complex in 25% (298) or 52% (307) yield, whereas the use of 2-bromo-3 ,Cdimethoxypyridine by Tiecco et al. (299,301,309)for this purpose gave tetramethoxyorelline in yields up to 87%. An attempt to improve the yield of the reductive dimerization by replacing the chloropyridine derivative with a corresponding bromo derivative in the Dehmlow-Schulz procedure, however, failed

2.

257

ALKALOIDS FROM MUSHROOMS

SCHEME48. Improved synthesis of 2-chloro-3-methoxy-4-nitropyridine N-oxide (310).

(total yield 0.18%) because of the low yield (11%) of the Schiemann reaction (307). Preparation of the 2-chloro-3-methoxy-4-nitropyridine N-oxide intermediate in the synthesis was considerably improved by the methylation of commercially available 2-chloro-3-pyridinol using dimethylsulfate and tetrabutylammonium bromide as the catalyst under phase-transfer conditions (310),instead of diazomethane (Scheme 48). In the synthesis of orellanines elaborated by Tiecco et al. (299,301,309), shown in Scheme 49, the commercially available 3-hydroxypyridine was the starting material. The structures of the synthetic compounds were proved to be identical to those

1HBr

IHBr

HO

-

H O % "

\

OH

7

A

A or uv

or

uv

OH Orelline

Orellinine

SCHEME49. Tiecco et al. synthesis of orellanines (299,301,309).

Orellanine

258

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

of orellanines isolated from natural sources by comparison of chemical and spectroscopic data (298,299,301). The behavior of tetramethylorelline toward the boron tribromidedimethyl sulfide complex is interesting. Demethylation of the methoxy groups occurred selectively in the 3 and 3' positions without affecting those in the 4 and 4' positions of the 2,2'-bipyridyl system (Scheme 50). It was assumed (301) that demethylation at the 3 position is assisted by coordination between the boron atom attached to nitrogen and the oxygen of the methoxy group of the second ring. The third simple method of orelline synthesis and thus potentially of the remaining orellanines, also based on the Tiecco's coupling reaction, has been recently reported by Hasseberg and Gerlach (311)(Scheme 51). The easily accessible 2-bromo-3-hydroxypyridine was converted to the corresponding [2-(trimethylsilyl)ethoxy]methyl (SEM) ether and dimerized to form the bipyridyl derivative. Owing to the chelating effect of the two SEM ether groups, it was possible to form selectively the 4,4'-dilithium derivative. This afforded orelline via direct oxidation with 2-(phenylsulfonyl)-3phenyloxaziridine or bis(trimethylsilyl)peroxide, or via prior reaction with electrophiles followed by oxidation and hydrolysis. It had been observed by Grzymda (277) that orellanine underwent a thermal decomplosition at 270°C with the formation of a yellow product. Later, the problem was investigated by Antkowiak and Gessner, who found that the decomposition product had the orelline structure (Scheme 52). It was also demonstrated (287,294)that orellanine decomposed slowly after reaching 150°C or when irradiated by UV light with formation of a Me0

Me0 BBr3 C2H4C12

or OMS0

OMe

OMe

SCHEME 50. Regioselective demethylation of tetramethylorelline.

2.

259

ALKALOIDS FROM MYSHROOMS

j

HO HCl.aq,MeOH

OSEM

c

(Y:75%)

\

Hog;; h For R=CHO: H202/KOH

(Y :36%)

R

sEM'$OSE '

M

$'OH

R

R = CHO

-

TsOH,MeOH

R = SiMeB ( Y : 6 1 % )

-

HC1. aq

(Y:L5%)

R

R = SiMe3 ( Y : 8 3 % ) R= CHO

( Y : 75%)

SCHEME51. Hasseberg and Gerlach synthesis of orelline (311).

HO OH OH

-1/2

o2

-1/2

OH

o2

* OH

SCHEME52. Decomposition of orellanine. X may denote (1) heating above 1 S O T , (2) UV irradiation, (3) Hz. Pt, or (4) mass fragmentation conditions.

260

~

6 ANTKOWIAK 2 ~ AND WIESJZAWz. ANTKOWIAK

mono-N-oxide, orellinine, first and finally the free base, orelline, by stepwise loss of the two oxygens of the N-oxide functions. The same stepwise process was also observed in the mass fragmentation, as well as during catalytic hydrogenation. The sensitivity of orellanine to elevated temperatures and especially to UV irradiation was later confirmed and investigated by other authors (281,289,307,309,312). To explain the unusual ability of orellanine to lose oxygen either at elevated temperatures or under the influence of UV light, both being contrary to the generally observed stability of N-oxides under these conditions (303),Antkowiak and Gessner (313,314) suggested a mechanism of sigmatropic rearrangement with [ 1,5] oxygen shift from the N-oxide to the hydroxyl group at C-3 of the second ring and the formation of a hydroperoxide (Scheme 53). The elimination of oxygen from the orellanine molecule would then be the result of hydroperoxide decomposition instead of direct N-oxide decay, assuming that both the sigmatropic rearrangement and the hydroperoxide decomposition should be relatively low-energy processes. This hypothesis was substantiated by an experiment in which it was demonstrated that the model compound 2-(2-hydroxyphenyl) pyridine N-oxide, containing the minimum structural features essential for such a rearrangement, underwent deoxidation at 220°C to the corresponding free base (314) (Scheme 54). Recently, the deoxidation process was also observed (315) when the N-oxide model compound as well as its methyl ether were subjected to high pressure (10-20 kbar) treatment at 50°C. In both cases, however, the

Hoh+l

Orellinine

Orelline

SCHEME 53. Mechanism of thermal and photochemical deoxidation of orellanine proposed by Antkowiak and Gessner (313,314).

2.

ALKALOIDS FROM MUSHROOMS

26 1

SCHEME 54. Synthesis of a simple model with an orellanine-like structure (314).

resulting product consisted of an additional component which could be easily separated by TLC. Each of these new components decomposed quickly to hydroxy and methoxy free amine, respectively, when exposed to UV light. This could indicate the initial formation of peroxide intermediates and thus support the suggested mechanism. It was noted that the 2-(2-hydroxyphenyl)pyridine N-oxide and its methyl ether, like orellanine itself and its tetramethyl ethers (295,296,298,314),are more prone to undergo loss not only of the oxygen atom but also of the hydroxyl or methoxy group, respectively, at the begining of MS fragmentation with the formation of an isoxazolinium-type cation. This would confirm the assumption (315) that the deoxidation mechanism also involves the process shown for the model compound in Scheme 55. The coplanarity of the molecules needed for reaching the transition state or even some of the tautomeric forms requires energy to be supplied under the different conditions in which the transformations have been recorded. X-Ray investigations confirmed the more stable nonplanar conformations of the compounds in the ground state, and no indication of a shorter bond between the two aromatic rings and, thus, of conjugation was observed. Even in the case of the hydroxy N-oxide model compound (316)for which the reversible color alteration from colorless to deep green-blue caused by daylight was observed, the molecules appeared to be twisted by 38" from coplanarity with a C-2-C-2' bond length of 1.477 A. The first report on the X-ray data of orellanine by Cohen-Addad et al. (300) ultimately confirmed the structure of this alkaloid. However, since the investigations were carried out on the trifluoroacetic acid salt, no conclusion concerning the conformation of naturally occurring orellanine

262

R = H o r CHJ

R62A ANTKOWIAK AND WIESJLAW 2. ANTKOWIAK

Isoxazolinium c a t ion (100%r.i. 1

SCHEME 55. Initial steps of mass fragmentation of orellanine-like systems.

or of its neat form could be drawn. The almost perpendicular arrangement of the rings found by these authors must have been the result of protonation of the N-oxide oxygen by an external proton causing disruption of possible intramolecular hydrogen bonds. A similar molecular geometry was determined by Kubicki et al. (306) for the Grzymda orellanine hydrate (Fig. 23) almost 30 years after the crystals had been obtained. In this case, however, it appeared that the two rings were coupled by a water molecule. Though this may illustrate the true conformation of orellanine under physi-

FIG. 23. Three-dimensional structure of orellanine hydrate. (From Ref. 306 by courtesy of Plenum Publishing Corporation.)

2.

ALKALOIDS FROM MUSHROOMS

263

ological conditions, it gave no information on the hydrogen bonding and geometry of the neat orellanine. The nonplanar conformation should impart chirality to the molecules; however, no observations of optical activity of orellanine have yet been reported. Special attention has been given to the fluorescent properties of orelline and some related compounds sharing the common feature of having hydroxyl groups in the 3 position of a 2,2'-bipyridyl system. All have a characteristic blue fluorescence when irradiated with UV light, which is used to identify these compounds. The fluorescent properties were interpreted in terms of keto-enol phototautomerism (299,301,317) or proton transfer to form a phenolate anion (318)(Scheme 56). The latter suggestion when applied to the model compound 2-(2-hydroxyphenyl)pyridine N oxide having a green-blue color induced by daylight, as well as to other analogs with rigid conformations (315,319),explained the absence of carbony1 absorption in their IR spectra. The fluorescent properties of orelline were recently utilized by Rapior and co-workers (284,320) to assay orellanine in plasma and renal biopsy specimens, thus permitting precise diagnosis of orellanine poisoning. The biological fluids and the tissue extracts were passed through Amberlite resin, and orellanine was detected by fluorimetry on cellulose chromatograms after TLC and photodecomposition to orelline. Interestingly, this procedure makes it possible to detect orellanine in plasma as late as 10 days after ingestion, and orelline in renal biopsies even 6 months later.

HR o g ! hv

hv 4

f luoresc .

f luoresc.

R R=H,OH o r OMe

SOH/N,O-

l*

hv

SCHEME 56. Alternative interpretationsof the fluorescent properties of the orelline-like chromophore system.

264

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

This proved a particularly slow release of orellanine and its photodecomposition products from blood and renal cells. With all the well-documented observations concerning the chemical symptoms and histopathological changes caused by ingestion of Cortinarius mushrooms containing orellanine (285), little is known about the pathogenesis of the toxin. The results of Heiland’s investigations indicated (321) that the photosynthetic apparatus of the plant Lemna minor was destroyed by orellanine in extracts of C. speciosissirnus in the presence of light. The author, in cooperation with Schumacher (285), also found relationships between the structure and physiological activities of orellanine and two biologically active cationic bipyridines with known herbicidal properties, diquat (N-methylated 2,2’-bipyridyl) and paraquat (4,4’isomer). They also suggested a similar pathogenic mechanism of action for all these compounds. The mammalian toxicity of diquat and paraquat as well as their pathogenic mechanism have been intensively studied, establishing the kidney as the most important target organ of the activity of the toxins, which disrupt redox metabolic processes in the cells. According to the authors orellanine, like other members of bipyridine family with positively charged nitrogen, is supposed to have the ability to inhibit the intracellular metabolic systems dependent on NADPH plus H+, by participation in the electron transport process (Scheme 57). It is interesting, too, that the concomitantly produced superoxide also has cytotoxic properties, either directly or by generating other reactive forms of oxygen such as singlet

HO

SCHEME 57. Intracellular toxic mechanism proposed for orellanine. [From Schumacher and Hfliland (285).]

2.

ALKALOIDS FROM MUSHROOMS

265

oxygen (322).The proposed (322) intracellular toxic mechanism with NADPH plus H f depletion would need a long time to impoverish the cells to the degree of inevitable necrosis, which is in concordance with the delayed toxic effects observed in this type of mushroom poisoning. The assumption that it is the ability of the cationic bipyridines including orellanine to pick up electrons to form radicals, which is responsible for the cytotoxic effects, is also supported by the nontoxicity found for orelline. The conclusion of Schumacher and Hgiland was not accepted by Rifor reasons not mentioned in their paper. Using a chard et al. (305~) method of quantitative structure-activity relationship determination, the above authors tried to evaluate the contribution of hydroxyl substituents to the toxicity of orellanine. The results appeared to be in total contradiction to the experimental data [e.g., calculated LDso -5 g/kg versus an experimentally determined LDS0of -4.9-12.5 mg/kg (276)l and led the authors to the conclusion that the proposal of the exact structure of orellanine may be questioned. It must be mentioned, however, that neither paraquat nor diquat were taken into account in the calculations. Three years later, more substantial arguments were made by Richard et al. (30%) as the result of their studies of the electrochemical behavior of orellanine which was shown to be different from diquat and paraquat. An interesting approach to the pathogenesis of orellanine was also presented by Rapior and co-workers (283,312,323).The authors, inspired by the phototoxicity mechanism of chlorodiazepoxide action (324),assuming that the initial photoisomerization to the corresponding oxaziridine occurred prior to binding with peptides, as well as on the basis of other observations concerning the formation of isoxazolinium cations (314,317), proposed that Cortinarius toxicity is caused by metabolites with the isoxazolinium core derived from the photochemical rearrangement of orellanine (Scheme 58). The isoxazolinium intermediates are supposed to be able to bind covalently with numerous proteins in the body, leading to organ damage. According to Rapior et al. (323),orellanine purified in the dark and administered to laboratory animals showed low toxicity, whereas that extracted in light induced moribundity. These observations indicating the consequence of photoexcitation supported the hypothesis of the proposed phototoxicity mechanism of orellanine. Finally, it may be conceivable that the hydroperoxide intermediates proposed by Antkowiak and Gessner (314) resulting from the sigmatropic rearrangement of orellanine, or their radical decomposition products, play a significant role in the toxicity of this alkaloid. However, putting the ,toxicity problem aside, the ability of a living organism to transform orellanine to orelline has already been sufficiently demonstrated (279,284),and thus the elucidation of this biotransformation mechanism remains to be

266

R62A ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK HO

HO

HO

% '' O 0H ,

% '' OH

/

\

OH

OH

- og HO

- .

intermediates capable of binding with proteins

OH

OH

SCHEME 58. Photodecomposition mechanism for orellanine proposed by Rapior (283,312,323).

solved. It is believed that under physiological conditions the energy required to induce the rearrangement of orellanine via a coplanar transition state originates from neither photochemical nor thermal sources but from the binding to enzyme receptors (325). This idea seems to be supported by the observations of several model compounds undergoing both the photo and thermal decomposition to free bases which showed that the deoxidation temperature was dependent on the extent of molecular planarity when conformational rigidity was achieved by chemical annelation or steric hindrance (315) (Scheme 59). No suggestions concerning the biogenesis of orellanine and its deoxidated metabolites have yet been reported. Studies on the influence of the

___L

(twist conformation) (planar conformation)

SCHEME 59. Possible biotransformation of orellanine to orelline.

OH

2.

ALKALOIDS FROM MUSHROOMS

267

nitrogen source on the growth of C. orellanus (326) and preparation of a pure culture of this mushroom could be considered as a preliminary approach to this problem. Although orellanine has been finally accepted as the true toxic component of several Cortinarius species, the responsibility for the toxicity of these mushrooms has not been completely assigned as yet. During the 1970s and 1980s several observations of the chromatographic resolution of the fluorescent components of Cortinarius species have been reported (281,288,327);usually the effect induced by UV irradiation was supposed to be connected with the toxicity of the compound. In 1970 Testa (328) isolated from C . orellanus four main fractions giving a distinct fluorescence in UV light, naming them grzymaline, benzoines a and b, and cortinarine and suggesting their polypeptide structure. Later, H@iland (327) also obtained more complicated chromatographic patterns for both C. orellanus and C . speciosissimus than expected for those containing only orellanine and its deoxidated derivaties. Kurnsteiner and Moser (281) suggested that both “a peptide and a phenolic structure may be present as part of a larger molecule,” assuming a possible chemical correlation in mushroom tissue between a peptide and orellanine-like molecule. Following these results and the preliminary report of Tebbett and Caddy (302), these authors claimed (304) in 1984 to have isolated three major components of Cortinarius speciosissimus: the toxic cortinarins A and B, and the nontoxic cortinarin C; the structures were determined as being cyclic polypeptides (Scheme 60). The former two have been shown to cause nephrotoxicity in laboratory animals characteristic of Cortinarius mushroom poisoning. According to the authors, cortinarins A and C were found by TLC, HPLC, and reversed-phase HPLC (329)to be present in 60 different species of Cortinarius examined. Only three species, reported to be the most toxic within the genus, were found to contain cortinarin B: C. orellanus, C . orellanoides, and C . speciosissimus. Amino acid sequencing was performed (304) by initial Raney nickel treatment of cortinarin A followed by partial hydrolysis and degradation of the resulting linear peptide employing dansylation of the terminal amino acid. Dansyl amino acids were identified by comparison with standards using two-dimensional TLC on polyamide plates. The amino acid sequence was also determined by the mass spectrometry method (Scheme 61). The sequence reported (330)in 1986 appeared to be a reversal of that shown previously (304). Toxicologic ’studies (304) showed that when cortinarin A was administered tcv mice, which was followed by methanolic extraction of the kidney, the metabolite underwent O-demethylation and S-oxidation to sulfoxide, probably prior to exerting its effect on renal tissue. These observations

268

R6ZA ANTKOWIAK AND WIESLAW Z . ANTKOWIAK

+Amino acids

Cortinarin C C o r t i n a r i n A (R=OCH3)

(R=OCH3)

C o r t i n a r i n B (R=OH)

Ala -T

hr -Gly -Lys -Phe -NH - C H-

I

CO -Val -Or n-

Leu-1 le

+Amino acids

H

SCHEME60. Chemical correlation between cortinarins.

I

I

I

I

526

100

257

687 931 'OLL

1228 1355

I

II

I I

SCHEME 61. Low-resolution mass spectrum af the linear form of cortinarin A after acetylation and permethylation. [From Tebbett and Caddy (304)by courtesy of Birkhauser Verlag, Basel.]

1513

2.

ALKALOIDS FROM MUSHROOMS

269

could imply that the true active toxin is a product of the metabolism in the liver. Later, investigations carried out by Rapior (283)confirmed the presence of cortinarins A and C in more than 40 species of Cortinarius, both toxic ones and those considered to be edible. Rapior was the first to find both orellanines and cortinarins in the same C. orellanus. Apart from the report of Kurnsteiner and Moser (281) on the isolation of both a slow and a fast acting toxin from this mushroom without, however, any structural proposal, the previous extraction studies (287,304) revealed the alternative presence of bipyridine or cyclic peptide groups of alkaloids in the same species. Since the toxicological properties of cortinarins described by Tebbett and Caddy (304)resembled those of orellanine, the presence of a second group of toxins accompanying orellanine in C. orellanus and in the other most toxic Cortinarius species needs to be further confirmed. B. NECATORONES Several species of Lactarius were found to reveal mutagenic properties toward Salmonella in the Ames assay. Among them the ugly milk cap, Lactarius necator (synonyms L . plumbeus, L. turpis),considered by some to be a toadstool and by others to be edible in spite of its acrid flavor, is supposed to have the highest microbial mutagenic activity. The mushroom is widely distributed in Europe, Asia, and North America and can be distinguished by its dark olive brown fruiting bodies which change color to purple violet when exposed to ammonia. Suortti and von Wright (331) isolated an active principle called necatorin from this fungus. The activity of necatorin was comparable with aflatoxin B1in the microbiological mutagenetic test. For this compound the authors suggested the tentative structure 5-hydroxycoumaro[7,8-c]cinnolinebased on spectroscopic data (332) (Fig. 24). Independent studies carried out by Steglich and co-workers at the same time led to the isolation of an alkaloidal pigment, necatorone (333) (Fig.

Q--c&o N=N

-

S u o r t t i ’ s necatorin

Necatorone

FIG. 24

270

R62A ANTKOWIAK AND WIESEAW Z . ANTKOWIAK

24). The red needles of the compound dissolved in DMSO gave a solution with a grass-green color showing a strong green-yellow fluorescence. On gradual addition of aqueous ammonia to this solution a change to a beautiful blue color and next to purple was observed. Systematic analysis of the 13C-NMRspectrum assisted by extensive 'H,'3C-decoupling led to elucidation of the necatorone structure as 5,10-dihydroxy-6-oxodibenzo[de,h][1,6]naphthyridine. Following careful comparison of the properties of Suortti's necatorin with those of necatorone, especially the molecular composition and mass fragmentation, Steglich concluded that both compounds have the same necatorone structure. This has been confirmed by synthesis of necatorone (334) starting from the condensation product of 2-(3,6dimethoxypheny1)ethylamine with 5-methoxy-2-nitrobenzoyl chloride (Scheme 62). Bischler-Napieralski cyclization of the compound followed by successive dehydrogenation, demethylation, and hydrogenation gave an aminophenylisoquinoline derivative which was transformed by an oxidative cyclization to a necatorone identical to the natural product. The last step appeared to occur via a quinone-imine intermediate essential for the electrophilic intramolecular attack on the neighboring phenol ring. In collaboration with Suortti, the synthetic necatorone was found to show strong mutagenic activity in the Ames test (334), in spite of the first negative results (333). Further studies revealed that necatorone exhibits moderate antibiotic activity against Bacillus subtilis, B . brevis, and Acetobacter calcoaceticus (176).

SCHEME 62. Synthesis,of necatorone by Hilger et al. (334).

2.

ALKALOIDS FROM MUSHROOMS

27 1

Additional alkaloids of the necatorone groups, 4,4'-binecatorone and

lO-deoxy-4,4'-binecatorone,were also isolated (63,276,335)from L . necator, whereas in L . atroviridis, a North American species, 10,lO'-dideoxy4,4'-binecatorone was found to be the main alkaloid responsible for the green appearance of this fungus (Fig. 25). The reported synthesis of the dimeric L . necator alkaloids was based on the observation that binecatorone was produced in uitro in 31% yield by the action of horseradish peroxidase on necatorone in the presence of hydrogen peroxide at pH 8. For the synthesis of the remaining dimers, the method of preparation of 10-deoxynecatorone, which had not been found in the toadstools, had to be elaborated. This was achieved by following the necatorone synthesis except that the oxidative cyclization of 1-(2-aminopheny1)-6,7-dihydroxyisoquinoline had to be carried out by means of potassium peroxydisulfate in 1% aqueous potassium hydroxide instead of oxygen in alkaline solution (Scheme 63). In the coupling reaction induced by horseradish peroxidase the dideoxy and unsymmetrical dimers were obtained from either 10deoxynecatorone or an equimolar mixture of necatorone and its deoxy derivative in 53 and 20% yield, respectively. As a step toward a total chemical synthesis of these dimeric alkaloids, a simple and efficient method for the preparation of monomers functionalized at 4 position has been elaborated (Scheme 64). This procedure consisted of heating a corresponding nitrophenyldihydroisoquinoline derivative in concentrated sulfuric acid up to 200°C. The biogenesis of necatorone and related Lactarius alkaloids is not yet fully understood. However, in a preliminary feeding experiment, a high incorporation of tyrosine-3-13Cinto necatorone was found when the precursor was administered to a young specimen of Lactarius necator. These

4,4'-Binecatorone

10-0eoxy-4,4'-binecatorone

FIG. 25

10,10'-Dideoxy-4,4 -binecatorone

-

272

Hzq- H:q

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK prse-radish

HO

K2S208

H2N

‘

eroxidase

/

1%KOH

/

‘

R

R = H o r OH

2

monomers R=H

/

H202

R

\

R

d i m e r s : R = O H , OH

o r OH

R=H,

OH

R=H,

H

SCHEME 63. Synthesis of dirneric Lactarius necator alkaloids.

observations strongly supported the hypothesis that the alkaloids can be derived biosynthetically from 3-(3,4-dihydroxyphenyl)alanine (dopa) and anthranilic acid (176) (Scheme 65). C. ILLUDININE Omphalotus illudens (until recently classified as Clitocybe illudens), known as jack-0-lantern, is a source of several toxins which can be derived from the same basic “protoilludane” skeleton and probably have the same biogenetic origin. The most toxic component was found to be the nitrogen-free antitumor sesquiterpene known as illudin S (accompanied by illudin M), also isolated from Lampteromyces japonicus (Tricholomaceae) and 0.oliuascens (336),for which the structure and absolute configuration have recently been proved by X-ray analysis (337).The extreme toxicity of illudins which is compared with that of ricin, one of the most potent toxins known, is connected with their cytotoxic activity toward human leukemia cells and their primary effect on DNA synthesis. The only nitrogen-containing secondary metabolite of 0. illudens is illudinine (mp 228-229”C, dec.), the structure of which was established by 0

R = H o r OH

SCHEME 64. Synthesis of 4-functionalized necatorones.

OH

2.

ALKALOIDS FROM MUSHROOMS

273

SCHEME 65. Biogenesis of Lactarius alkaloids. The position of the isotope is marked by a dot.

Nair et af. on the basis of spectroscopic and chemical evidence (338) (Scheme 66). On pyridine hydrochloride cleavage of the methoxyl group illudinine underwent simultaneous decarboxylation to yield an isoquinoline system with a gem-dimethyl cyclopentane fused ring. The same product was also obtained by treatment of illudalic acid, another metabolite of 0. illudens, with ammonia and subsequent decarboxylation. On the other hand, illudin M when kept for several hours at room temperature in a very diluted strong acid gave the cyclopropane ring-opened triol. Both transformations clearly illustrate the ease of chemical interconversion among members of this metabolite family. It has been suggested (338,340)that illudinine may originate biogenetically from farnesyl pyrophosphate via a “protoilludane” cation and illudalic acid (Scheme 67). Interestingly, illudinine was found as a new compound in a mutated strain of the fungus originally selected for enhanced production’ of illudalic acid. The fact that illudalic acid could no longer be isolated from the culture which produced illudinine, as well as the finding

R~ZA ANTKOWIAK AND WIESKAW z. ANTKOWIAK

274

PyH.HC1

-

&-

1.NH3 2. -co2

COOH

H

HO

Illudinine

P

0

0

Illudalic acid

Illudin M,X=H (Illudin S,X=OH)

SCHEME 66. Chemical correlations between active principles of jack-o’-lantern.

F a r n e s y l OPP

I

I &COO,

H Mevalonic acid

I I

Protoilludane cation

Hall f!

I

x -*

SOH Illudol (naturally occuring)

I

---1 I I

i Illudalic acid

----

SCHEME 67. Hypothetical biogenesis of illudinine.

Illudinine

2.

275

ALKALOIDS FROM MUSHROOMS

that, like other members of this family of metabolites, illudinine incorporated mevalonic acid-2-14C, seem to support strongly the suggested biogenetic pathway.

V. Hydrazine Alkaloids Since.the middle of the 1900s, the occurrence in nature of numerous toxins and antibiotics containing a nitrogen-nitrogen bond has been reported. In the late 1970s the structures and physiological implications of these metabolites were reviewed by LaRue (341).

A. GYROMITRINE The tumorigenic metabolite gyromitrine (342) was isolated from Gyromitra esculenta, the false morel also known as the brain mushroom or lorchel, by List and Luft (343). The mushroom looks somewhat like a true morel (Morchella esculenta) and is treated as an edible and for that reason also collected for commercial purposes in Europe and North America. Consumption of false morel or even inhalation of the fumes from cooking mushrooms appeared to be responsible for serious illness or even death in some cases. The harmful or lethal level of hydrazine toxins ingested by a person depends on the amount consumed and the mode of preparation of the mushroom dish. This is reasonable since gyromitrine, its decomposition product monomethylhdrazine(MMH), and related derivatives are soluble in water and volatile with steam. The structure of gyromitrine was found to be acetaldehyde N-methyl-Nformylhydrazone on the basis of spectral analysis and chemical transformations. On acidic hydrolysis gyromitrine was converted to monomethylhydrazine (343),also a natural constituent of G. esculenta (344,345), which is the highly toxic substance used as rocket fuel to help send Apollo

0

fH3

+

H2N-N ‘H

EtOQ

H

-

H2N-N

CH3

/

\

/CH3

CH3CH0

-.-

CHO

CH,CH=N-N

\

CHO

Gyromitrine

SCHEME 68.’ Synthesis of gyromitrine.

276

R 6 Z A ANTKOWIAK A N D WIESJLAW 2. ANTKOWIAK /CH3

R=N-N

\

CHO

FIG. 26. Homologs of gyromitrine.

astronauts to the moon (3). Physiopathologically, MMH appeared to attack the nervous system and caused liver and intestinal tumors in animals. The correctness of the gyromitrine structural assignment was proved by its synthesis (343)from methylhydrazine and ethyl formate followed by condensation of the resulting N-methyl-N-formylhydrazine with acetaldehyde (Scheme 68). Apart from gyromitrine and MMH, other toxic hydrazine derivatives were identified in G. esculenta, namely, the N-methyl-N-formylhydrazine isolated by Stijve (346)and three higher homologs of gyromitrine found by Pyssalo (347):2-pentylidine-, 2-(3-methyl)butylidene-, and 2-hexylidene1-methyl-1-formylhydrazine(Fig. 26). The concentration of the 3methylbutylidene derivative in the mushroom was found to be even higher (3.3%) than that of gyromitrine (0.9%). The presence of such hydrazine derivatives in several other species of the subdivision Ascomycotina was demonstrated by Andary et al. (348). B. AGARITINE Agaritine, a “harmless” hydrazine derivative, was first isolated by Levenberg (349,350)from Agaricus bisporus, the champignon, the mushroom commercially cultivated in the Western hemisphere. This metabolite was identified as N 2 [ y - ~+)glutamyl]-N1-(4-hydroxymethylpheny1)hydra( zine. Later was also detected in several other Agaricus species, including A . augustus, A . silvicola, A . arvensis, and A . campester. However, many edible and commercially available mushrooms examined, such as shiitake (Lentinus edodus), the padi straw mushroom (Volvariella volvacea, Pluteaceae, Agaricales), the inky cap (Coprinus comatus), and the oyster mushroom (Pleurotus ostreatus, Polyporaceae), were found to be devoid of agaritine (351). Although it is believed that agaritine precursors or breakdown products rather than agaritine itself can be harmful, LaRue (341)mentions a report by Toth et al. (352)concerning the death of Swiss mice caused by agaritine injection. Cleavage of agaritine with hot, dilute hydrochloric acid or by the action

2.

277

ALKALOIDS FROM MUSHROOMS

H+

___)

or

+

4-Hydroxymethyl-

enzyme Agaritine

SCHEME69. Chemical degradation of agaritine.

of the enzyme y-glutamyltransferase, also present in the mushroom tissues, resulted in the liberation of L-glutamic acid and 4-hydroxymethylphenylhydrazine (349) (Scheme 69). The latter compound can be oxidized by a laccaselike enzyme to the murine carcinogen 4-hydroxymethylbenzenediazonium ion, which was reported to exist in the mushroom, though in a small concentration (351,353,354). The 4-hydroxymethylphenylhydrazine system occurring in agaritine appeared to be very unstable, easily undergoing elimination of water across the benzene ring (Scheme 70). Mainly for this reason, the yield of the first synthesis of agaritine by Kelly et al. (353,based on the condensation of the y-azide of N-carbobenzoxy-L-glutamicacid with 4-hydroxymethylphenylhydrazine, was only 1%. Later, Wallcave et al. (356) reported an improved synthesis in which the mixed anhydride derived from 1-benzylN-benzyloxycarbonyl-L-glutamateand ethylchloroformate was condensed with 4-carboxyphenylhydrazine to yield the y-hydrazide of the protected glutamic acid derivative (Scheme 71). Reduction of the aromatic carboxylic group of the intermediate with diborane folowed by hydrogenolysis of the protecting groups afforded agaritine in a yield 25-fold higher than previously obtained. The procedure was somewhat modified by Stijve (351).

The proposed biosynthesis (341) (Scheme 72) of agaritine by A. bisporus

R - H or O=C

SCHEME70. Mechanism of water elimination in agaritine-like systems.

278

R6ZA ANTKOWIAK AND WIESKAW Z. ANTKOWIAK H

SCHEME71. Synthesis of agaritine.

was based on 'observations gathered by Schutte et al. (357) in feeding experiments with isotopically labeled compounds. Since the incorporation of shikimic acid-U-I4C, glutamic acid-2-I4C, and p-arninobenzoi~-3,5-~H acid into agaritine was demonstrated, it was assumed that p-aminobenzoic acid is a precursor in biosynthesis of this alkaloid. The biosynthetic formation of p-aminobenzoic acid from shikimic acid via chorismic acid was investigated by Gibson and co-workers (358-360). The isolation from A. bisporus of two of the postulated intermediates, namely, p-hydrazinoben-

Glutamine-

Shikimic acid

Chorismic acid

Glutamic acid

p-Aminobenzoic acid

SCHEME72. Biosynthesis of agaritine proposed by LaRue (341).

2.

ALKALOIDS FROM MUSHROOMS

279

zoic acid and N2-(y-~-glutamyl)-4-carboxyphenylhydrazine, was reported by Chauhan et al. (361,362); this supported the biogenetic proposal. However, the presence of these metabolites in A. bisporus was not confirmed in the chromatographic investigations by Stijve (351). C. AGARICONE Agaricone, a yellow pigment with antibiotic activity, was recently isolated in the laboratories of Steglich and Anke (363) from the slightly poisonous Agaricus xanthoderma, and its structure was also elucidated. The intense chrome-yellow coloring of the toadstool changes to orangeyellow when the fruiting bodies are bruised, indicating a substance sensitive to air oxidation. This was confirmed by the synthesis of leucoagaricone, which appeared to be easily oxidized to agaricone when exposed to atmospheric oxygen or treated with sodium periodate (Scheme 73). U

1.MeOH

Butyrolactim methyl ester

U

Leucoagaricone

Agaricone

SCHEME 73. Chemical synthesis of agaricone.

D. XANTHODERMIN Xanthodermin, another chromogen isolated from A . xanthoderma, was identified as being y-glutamyl-N'-(4-hydroxyphenyl)hydrazide (363).The structure of this metabolite as indicated by its spectral and chemical properties was confirmed by synthesis (Scheme 74). In the elaborated procedure N-(4-benzyloxyphenyl)hydrazine was converted to the corresponding hydrazide using an acyl-transfer agent derived according to Steglich's method (364).To prepare this reagent the y-carboxyl group of a-benzyl N-(benzyloxycarbony1)-L-glutamate was activated by esterification with a thiophene-1,l-dioxide derivative. After removal of the protecting groups by hydrogenolysis the product of transacylation afforded the oxidation-sensitive xanthodermin in quantitative yield. The anion of the acylazo compound is responsible for the color reaction; presumably this is due to the possible occurence of quinonoid chromophore formation. Both leucoagaricone and xanthodermin participate in the yellow discoloration of the A. xanthoderma fruiting body and in its color reaction with sodium hydroxide.

280

R 6 Z A ANTKOWIAK A N D WIESJLAW 2. ANTKOWIAK 02

phY?ph Ox0

HO P -h HhKQPh

0

0 PYH

O2 Ph php&"'Ph HN+Ph

-co, L

0

H

o KJ[Fe(CN)6] D

NaOH Xanthoderrnin

y e l l o w pigment

SCHEME 74. Chemical synthesis of xanthodermin.

E. PSALLIOTIN Psalliotin, an antibiotically active, true constituent of A. xanthoderma with a 4-diazo-2,5-cyclohexadien-l-one structure (363),can be detected in methanolic extracts of the fungus by azo coupling with resorcinol or @naphthol. This metabolite is responsible for the formation of some artifacts, such as 4-hydroxybenzenediazosulfonate or 4,4'-dihydroxyazobenzene, also found in the mushroom by different work-up procedures. Earlier searches for constituents of A. xanthoderma with antibiotic activity led to the isolation, without structural determination, of two compounds called psalliotin and agaricin by Atkinson (365,366)and Dornberger (367), respectively. In recent studies (363) these two compounds have been and 4-hydroxybenzeneidentified as 4-diazo-2,5-cyclohexadien-l-one diazosulfonate, respectively (Scheme 75).

F. LYOPHYLLIN Lyophyllin was isolated from Lyophyllum connatum (Tricholomataceae) by Fugmann and Steglich (368) along with two other carbamide derivatives: N'-hydroxy-N,N-dimethylureaand an amino acid, connatin.

2.

6

28 1

ALKALOIDS FROM MUSHROOMS

OH

N+

II

-NIAgaric in

Psalliotin

SCHEME 75. Formation of agaricin.

On the basis of spectral data and hydrogenolysis to dimethylurea, the structure of lyophyllin was established as N,N-dimethyl-methylazoxycarboxamide, and it was confirmed by synthesis (Scheme 76).

8

(CH3IZN-C-NHOH

FI

(CH3)ZN-C-NH-NHCH3

N’-Hydroxy-N,Ndimethylurea

H202

-

F I f0 (CH3)2N-C-N=N

yy

‘CH3 Lyophyllin

SCHEME 76. Synthesis of lyophyllin.

VI. Nucleoside-Type Alkaloids In various fungi, including those of Basidiomycotina, numerous nucleoside-type metabolites and related free bases have been found (369). Apart from the typical components that are structural units of nucleic acid chains, like adenosine from Amanita muscaria (9), some of the nucleoside analogs apparently deserve special interest because of their biological and pharmaceutical activities, which are usually induced by a slight difference in structural features. In addition, the role played by such nucleoside antibiotics in protein and nucleic acid biosynthesis might be of great importance. A. CLITIDINE Clitidine was isolated among other nitrogen-containing principles from Clitocybe acromelalga, the toadstool long known in Japan as the cause of markedly increased hyperemia and hyperesthesia in fingers and toes. Pure

282

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

crystals of clitidine were obtained in a 0.019% yield from frozen fruiting bodies through the sequential extraction with water, precipitation with acetone, dialysis, and chromatography on Sephadex G- 10 followed by recrystallization of the crude product from water (370). It was found to be biologically active, and its toxicity (LD50) was determined as about 50 mg/kg by intraperitoneal injection in mice. The structure of clitidine was found to be 3-carboxy-4-imino-1-@+-ribofuranosy1)-1,4-dihydropyridine by 'H-NMR, I3C-NMR, and mass spectral analysis, along with chemical degradation studies. When heated in a sealed tube with concentrated aqueous HC1, clitidine afforded 4-aminonicotinic acid hydrochloride in quantitative yield. The suggested structure based on spectroscopic investigations was confirmed by a synthesis (371) which consisted of condensation of methyl 4-aminonicotinate with 3,5-di-O-benzoyl-~-~bofuranosyl chloride and subsequent hydrolysis (Scheme 77). Alkylation at the ring nitrogen and not at the exocyclic nitrogen bonded to C-4 was verified by the synthesis of nicotinamide riboside as shown in Scheme 77.

Et3N-H20-MeOH

83%

89%

r

Cl

26%

HO OH SCHEME 77. Synthesis of clitidine.

2.

283

ALKALOIDS FROM MUSHROOMS

B. CORDYCEPIN Cordycepin, a metabolic product revealing antitumor activity (372), was isolated from cultures of Cordyceps militaris (Clavicipitales).Acid hydrolysis of cordycepin yielded adenine and a "branched-chain'' sugar called cordycepose (373); the structure of the latter was subsequently verified to be 3-deoxyribose by comparing cordycepin with 3'-deoxyadenosine, already known as an active metabolite from Aspergillus niduluns. The improved synthesis of 3'-deoxyadenosine reported by Walton et al. (374) was based on the coupling of chloromercuri-6-benzamidopurine with the 2,5di-O-benzoyl-3-deoxy-~-ribofuranosyl bromide obtained by stereospecific hydrogenation over Raney nickel catalyst of 2,3-anhydro-P-~-ribofuranoside followed by benzoylation and conversion to the corresponding bromide (Scheme 78). Studies on the biogenesis of cordycepin with labeled precursors demonstrated (375,376) that glucose-l-I4C and gl~cose-6-'~C, as well as adenine8-14C, adenosine-U-I4C, and formate-I4C, were incorporated into cordycepin, whereas ribose-l-I4Cwas essentially unutilized. Determining the radioactivity distribution led to the conclusion that cordycepin is generated from adenosine, with retention of the glucoside bond intact. HoQoMe

l.H~,R-Ni EtOH, 80"

/

Her, AcOH

BzO

l.xylene,refl.

0

'2. PhCOCl

0

0 Bz

HgCl

OH Cordycepin

SCHEME 78. Synthesis of cordycepin.

C. CLITOCINE Clitocine, the nucleoside with strong insecticidal activity against the pink bollworm, was isolated (377)from Clitocybe inuersa, a medium sized,

284

R6ZA ANTKOWIAK AND WIESLAW 2. ANTKOWIAK

buff-colored, poor quality edible mushroom that can be found close to conifers in western North America. The crude clitocine, obtained after the dilution of the methanol extract of the mushroom with water and taken by partition into ethyl acetate, was contaminated with adenosine. Chemically, both nucleosides appeared to be closely related, though clitocine revealed biological activity whereas adenosine was inactive. Thorough spectral analysis and demonstration of the strong similarities of the spectral properties of clitocine and those of adenosine indicated that clitoIt is interesting to cine is 6-amino-5-nitro-4-ribofuranosyliminopyrimidine. note that clitocine was the first nitro-containing nucleoside isolated from a biological material. It can be assumed that in nature clitocine is the product of biosynthetic oxidation of adenosine removing the C-8 carbon followed by the oxidation of the N-7 nitrogen to a nitro group (Scheme 79). The structural similarity of clitocine and adenosine may imply that clitocine has similar biological properties and could act as an adenosine analog. In fact, the biochemical studies carried out by Moss et al. (378) demonstrated that clitocine is a substrate and inhibitor of adenosine kinase, with Ki of 3 X M , and furthermore that it has a potent inhibitory effect on some leukemia cell lines, with an IDso of 3 x lO-'M. The first total synthesis of clitocine accomplished by Moss et al. (378) consisted of glycosylation of 4,6-diamino-5-nitropyrimidinewith 1-0acetyl-2,3,5-tri-O-benzoyl-~-ribofuranose, which afforded the protected nucleoside exclusively as the p anomer (Scheme 80). Subsequent deprotection gave clitocine with less than 1% of the a anomer. The procedure reported next by Kamikawa et al. (379) was based on the condensation of 2,3,5-tribenzoylribofuranosylaminewith 4,6-dichloro-5nitropyrimidine followed by amination (Scheme 81). Because of the low yield of the condensation step, the procedure was improved by treatment of the protected ribofuranosylamine in the form of the toluenesulfonic acid

biosynthetic oxidation (proposal)

c -

HO OH Clitocine

Adenosine

SCHEME 79. Assumed bioformation of clitocine.

2.

285

ALKALOIDS FROM MUSHROOMS

02N$

+ BzO OBz

Ho4 HO OH

Clitocine

SCHEME80. Synthesis of clitocine by Moss et a/. (378).

salt and 4-chloro-5-nitro-6-aminopyrimidine with triethylamine. In both cases the synthesis gave mixtures of a and /3 anomers in the ratio of 7 : 1 and 1 : 2.8, respectively. The interesting biological activity of clitocine prompted several research groups to prepare some of its analogs. The synthesis of carbocyclic clitocine, 4-(2,3-dihydroxy-4-hydroxymethylcyclopentenyl)-5-nitro-6-aminopyrimidine, was reported by Palmer et al. (380) and is depicted in Scheme 82. The improved synthesis of carbocyclic clitocine and its interesting biological activity were announced by Baxter et al. (381)in the symposium papers of the Ninth International Round Table on Nucleosides, Nucleotides, and Their Biological Applications held in Uppsala in 1990. In the

B z 0 w H 2 I

I

BzO OBz

Et3Nl +, OMF

SCHEME81. Synthesis of clitocine by Kamikawa et a/. (379).

["N

C1

286

R 6 2 A ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

MCPBA AcOH

HO

TsOH

H6

HO

R=SiPh2tEu cat.

CI

-

1.NH3 2. TEAF 3.H+

HO OH 3-Deazaclitocine

Carbocyclic clitocine

SCHEME 82. Synthesis of carbocyclic clitocine by Palmer et al. (380).

same collection of papers Franchetti et al. (382) reported on the synthesis of 3-deaza analog of clitocine, 2-amino~3-nitro-4-(p-~-ribofuranosylamino)pyridine, and its cytotoxic properties. AND METHYLATED DERIVATIVES D. NEBULARINE

Nebularine has long been known as a metabolite of Clitocybe nebularis (383), revealing tuberculostatic and antimitotic activity (383-385). Few syntheses of this compound having a 9-(~-ribosyl)purinestructure have been reported, namely, the Brown (386) approach, starting from a chloromercuri-6-purineand 2,3,5-tri-O-acetyl-~-ribofuranosyl chloride, the Fox (387)procedure, based on the transformation of inosine to nebularine via a desulfurization by Raney nickel of the thioinosine intermediate, and the Iwamura-Hashizume (384,388) method, in which nebularine and its N-7 isomer were synthesized simultaneously by the fusion of purine and tetra0-acetyl-D-ribofuranose using bis(pnitropheny1)hydrogen phosphate as the catalyst. Recently, a search for natural antiviral agents led to the isolation (389,390),along with 6-methylpurine, of two nucleosides, 6-methylnebularine and 6-hydroxymethylnebularine,from mycelial cultures of Collybia rnaculata. All the alkaloids revealed antifungal, cytotoxic, and antiviral activities; however, no antibacterial activities were observed. The structures of the metabolites were established on the basis of 'H- and 13C-NMR data (Fig. 27). The data appeared to correlate exactly with the values

2.

6-Methylpurine

ALKALOIDS FROM MUSHROOMS

Nebularine

6-Methylnebularine

287

6-Hydroxymethylnebularine

FIG. 27

reported for nebularine (392,392) apart from the substituted C-6 carbon. Previously, the compounds had been obtained separately by synthesis (393-399, but they had not been identified as natural products until their isolation from fungus.

E. ERITADENINE Eritadenine (396), a hypocholesterolemic substance which was given two different trivial names when discovered, lentinacin and lentysine, was isolated simultaneously and independently in two laboratories in Osaka. It was isolated from Lentinus edodes, an edible mushroom extremely popular in the Orient and known as shiitake (from the Japanese shii meaning oak and take fungus) or hoang me. Besides Agaricus bisporus (champignon), cultivated in the West, shiitake is the second most important mushroom in world commerce, and its cultivation on wood is a major industry in a number of Asian countries, especially China and Japan. In Japan alone over 2 x lo6 m3 of wood is converted to over U.S. $1 billion worth of mushroom product (397) annually. For centuries, the shiitake has been believed to be not only extremely tasty but also to have broad therapeutic properties (3). This opinion was confirmed by the isolation of physiologically active principles, namely, a high molecular weight polysaccharide composed mainly of pentoses which selectively inhibited the multiplication of myxovirus, the cause of influenza infection (398),and eritadenine, which was found to be effective in lowering blood cholesterol levels by Chibata et al. (lentinacin) (399)and Kamiya et al. (lentysine) (400). The structure and absolute configuration of eritadenine as being 2(R),3(R)-dihydroxy-4-(9-adenyl)butyric acid were established (399,400) by spectral analysis (including the identity of the UV pattern with that of 9-substituted adenine), by cleavage of the molecule with aqueous HCl resulting in the liberation of glycine and (+)-4-amino-2,3-erythro-

288

R 6 Z A ANTKOWIAK A N D WIESKAW Z. ANTKOWIAK 1.

QJjK 0

OQ 0

0

x

OV'

*

Ox0

1 .H7, R - N i

NaOH.aq

-

oQ HO OH Eritadenine

SCHEME 83. Synthesis of eritadenine by Kamiya et al. (400).

dihydroxybutyric acid, and finally by total syntheses. Few synthetic approaches have been reported independently. Structural studies presented by Chibata and by Kamiya included similar total syntheses of eritadenine, each consisting of the condensation of 4-amino-6-chloro-5-nitropyridine with 4-amino-4-deoxy-2,3-O-isopropylidenem-erythromic acid, followed by catalytic reduction of the nitro group and imidazole ring closure by formic acid or its amide. This can be exemplified by Kamiya's procedure (400)shown in Scheme 83. Soon improved syntheses were reported from both laboratories by Kawazu et al. (401) and Kamiya et al. (402),each starting from adenine and methyl 2,3-O-isopropylidene-5-O-tosyl-~-~-ribofuranos~de or Derythronolactone acetonide, respectively (Schemes 84 and 85). Kamiya

reversed nucleoside I'

Eritadenine

SCHEME84. Improved synthesis of eritadenine by Kawazu et a / . (401).

2.

Nap3 DMF. ref 1.

289

ALKALOIDS FROM MUSHROOMS

-

lO%AcOH,refl (catal. red. )

major

3- i s o m e r

Eritadenine

SCHEME 85. Improved synthesis of eritadenine by Kamiya et al. (402).

(402) found that although the condensation of adenine with D-erythronolactone led to a mixture of the 9 isomer and a small amount of the 3 isomer, the use of adenine 1-oxide gave eritadenine as the only product. Direct alkylation of adenine, and other substituted purines that could be converted to adenine, with 2,3-O-protected D-erythronolactone in the presence of alkali was also investigated by Okumura et al. (396), who found this reaction to provide a simple route to the preparation of eritadenine . The syntheses of eritadenine stereoisomers, D-threo and L-threo, were reported by Hashimoto et al. (403). It was found that 2,3-0isopropylidene-D-erythronolactonewhen treated with methoxide anion underwent transformation with epimerization to methyl 2,3-0isopropylidene-D-threonate. Following that interesting observation, eritadenine acetonide methyl ester was treated with sodium methoxide, and the epimerized product in a stereoisomeric ratio of 1 : 6 was obtained. Consecutive alkaline and acidic hydrolysis yielded D-threo-eritadenine. The enantiomeric L-threo-eritadenine was obtained by the total synthesis outlined in Scheme 86.

VII. a-Amino Acids Only some of the amino acids found in mushrooms have been selected for discussion here to show their unusual structures and physiological activities. Ibotenic acid and glutamyl hydrazides which also belong to this group of nitrogen-containing metabolites are discussed separately. A more comprehensive review of the secondary amino acids of mushrooms was published by Chilton in the early 1980s (404).

290

R 6 Z A ANTKOWIAK AND WIESEAW Z. ANTKOWIAK

0-t hreo-

Methyl O-erythroeritadeninate acetonide

HBr,EtOH,

Eritadenine

O C O H

1. TsCl 2 . NaN3

~

3.H2,Pd

BzO b B z L-Threonolactone dibenzoate

HO

8H

L-threoEritadenine

SCHEME 86. Syntheses of D- and L-threo-eritadenine stereoisomers.

A. ACTIVEPRINCIPLES OF Clitocybe acromelalga The poisonous mushroom Clitocybe acromelalga which can only be found in Japan where it is known as dokusasako, causes a violent pain and a red coloration of the fingers and toes similar to acromelalgia disease for 2-4 weeks after ingestion. Several alkaloids were found in this toadstool by Japanese scientists from the team of Matsumoto (405). Fractionation by successive chromatography of the aqueous extract on charcoal, Amberlite, and cellulose, guided by the lethal effect on mice, led to the isolation of the following principles: the mammalian toxins acromelic acids A and B, the potentially bioactive pyridine-2,3-dicarboxylicacid, the nontoxic amino acid betaine clithioneine, and the toxic nucleoside clitidine (Fig. 28). The latter is discussed separately. 1. Acromelic Acids

The isomeric acromelic acids A and B display a powerful neuroexcitatory action and are structurally related to the anthelmintic kainic acid (406,407) and domoic acid (408),the known principles from the marine red algae Digenea simplex and Chondria armata, respectively. Both of the latter amino acids, the structures of which were determined by chemical and X-ray studies, attracted considerable interest as well because of their

2.

29 1

ALKALOIDS FROM MUSHROOMS

A c r o m e l i c acid A

A c r o m e l i c acid B

POzH C02H

H, NH3 +

Domoic acid

Kainic acid

4-Aminopyridine-2,3-dicarboxylic a c i d

(I 'Z, 3 ' E , 5 ' R )

tj

co:

&02H

- O r HO c / SH C s 1H~ ? k k 3

OH OH Cli t i d i n e

Clithioneine

FIG. 28

potent neurotransmitting activities in the central nervous system; domoic acid was also studied for its insecticidal properties (409). The structures and configuration of acromelic acids A and B have been elucidated both by thorough spectral analyses, especially 'H-NMR, I3CNMR, and UV spectra (410),and on the basis of biogenetic considerations (411). The absolute configuration of both kainoids was determined by means of comparing the CD spectra and J values with those of kainic acid and domoic acid. The configuration turned out to be L , and that of all three chiral centers of the pyrrolidine ring to be (S).As a result, the relative configuration around these centers appeared to be 2,3-trans and 3,4-cis. The main conformations of acromelic acids A and B shown in Fig. 29 were deduced from J values and inspection of models. The anomalous downfield shift of the pyrrolidine H-4 peak (3.82 and 4.47 ppm, respectively) was interpreted as the result of the anisotropic effect of the carbonyl or carboxyl group, respectively, closely orientated to that proton.

292

R6ZA ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

H

O

HO2C / H

2

c

H % COzH

j 3 q. 8 2 p p m

-

$L.L7 ppm

3

HO2C

' NH

CO2H

\

NH

0

C02H Acromelic acid A

Acromelic acid B

FIG. 29. Main conformations of acromelic acids A and B

The results of structural and stereochemical investigations were confirmed by a synthesis (411,412) starting from L-a-kainic acid (Scheme 87); linked with the previously elaborated syntheses of kainic acid (413-413, this can be considered to be a total synthesis of acromelic acids A and B. The optically pure L-a-kainic acid, commercially available, was first transformed to the corresponding aldehyde, the key intermediate in the preparation of both target compounds, by successive protection of the functional groups and subsequent allylic oxidation via an epoxide intermediate. The problem of pyridine ring construction was solved in both cases separately (Scheme 88) by cyclization in the presence of ammonia of an unsaturated 1,5-dicarbonyl system. This was prepared by the use of the Pummerer rearrangement in the final stage, which was carried out under mild and neutral conditions. Finally, acromelic acids A and B were obtained by

1.MCPBA 2.130.C

H

-Po,,. Y COCF3

4.s-C02Me 180'

c --

r

COzMe

COCF3

-

4.

,-COzH

QC02H H

SCHEME 87. Total synthesis of racemic kainic acid via an intramolecular ene cyclization reaction by Oppolzer and Andres (413).

2.

ALKALOIDS FROM MUSHROOMS

293

CH,COCH,PO(OEt), c

OIBAH

Intermediate aldehyde

1.Se0, 2. CHZNZ

3. Bu4NF 4 . POC-DMF

-

KPBA

5 . CH2N2

Boc

Acrornelic a c i d A

SCHEME 88. Synthesis of acromelic acids A and B from a mutual intermediate aldehyde.

oxidation of the a-methyl group followed by subsequent oxidation at room temperature to 2-pyridone via N - oxide rearrangement induced by mild trifluoroacetic anhydride-DMF conditions (416). The biosynthesis of kainic acid and domoic acid was assumed to follow the path of condensation of an isoprene unit with glutamic acid and subsequent cyclization to form a pyrrolidine ring (Scheme 89). Consequently, it was proposed that the pyrrolidine moiety of acromelic acids A and B

294

R6ZA ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

Isoprene

unit

Glutamic acid

Kainic acid or Oomaic acid

SCHEME 89. Assumed biogenesis of kainic and dornoic acids.

arose from glutamic acid. It was suggested that the aromatic portion originated from L-dopa since fission of the catechol ring and subsequent recyclization to a pyrone ring had been established biosynthetically in the case of stizolobic and stizolobinic acids (61). Accordingly, extradiol cleavage (Scheme 90, path a) and intradiol cleavage (path b) (417)followed by cyclization with ammonia would produce pyridone carboxylic acids, which would then condense with glutamic acid along with deamination and decarboxylation to afford acromelic acids A and B, respectively (411) (Scheme 90). 2. 4-Aminopyridine-2,3-dicarboxylicacid acid, was recently A new metabolite, 4-aminopyridine-2,3-dicarboxylic isolated from C. acromelalga in a yield of 0.000056% based on the weight of frozen fruiting bodies (405). A lethal effect of this compound on mice has not been observed; however, bioactivity is still expected because of the similarity of its structure to the physiologically active pyridine-2,3dicarboxylic acid. The structure of this compound was deduced from 'H-NMR, I3C-NMR, and mass spectra, and the position of the substituents was assumed from comparison with clitidine, which contains a 4aminopyridine-3-carboxylicacid moiety. The structural proposal was confirmed by synthesis from 2,3-dimethyl-4-nitropyridine 1-oxide (Scheme 91); when this compound was subjected to successive reduction and oxidation it yielded a product identical to the one occurring naturally. 3. Clithioneine Clithioneine was obtained in a yield of 0.00023% from fresh fruiting bodies of C. acromelalga (418,419).Its structure was revealed by spectral analyses and chemical degradation studies and turned out to be composed of residues of two amino acids, threonine and hercynine, bonded together by a sulfide bridge. The molecular formula was deduced as being C I~H~~N from ~ O the~field S desorption (FD) mass spectrum. Interestingly, the electron-impact (EI) mass spectrum of the compound revealed no trace of the M+ ion peak, presumably owing to a smooth double McLafferty

2.

E&

295

ALKALOIDS FROM MUSHROOMS

/

HOzC

NH2

HozcG N

O

HO~C*NH~

t

Acromelic acid A

Acromelic acid B

SCHEME 90. Proposed biogenesis of acromelic acids A and B.

296

I;&

R6ZA ANTKOWIAK AND WIESEAW 2. ANTKOWIAK

Fe

-

1.Ac20 2.KMn04

&H3 CH3

AcOH

4

-

&COOH N'

3.H'

COOH

0 SCHEME 91. Synthesis of 4-amino-2,3-dicarboxylicacid.

rearrangement leading to a fragmentation pattern completely identical to that of ergothioneine (Scheme 92). The structure deduced from proton and carbon NMR, UV spectra, and comparison with the known S-(P-amino-P-carboxyethy1)ergothioneine was further confirmed by chemical degradation in which treatment with Raney nickel gave L-threonine, L-a-amino-n-butyric acid, and L-hercynine (Scheme 93). The L configuration of the amino acids was established by CD spectral comparisons with authentic samples. The essential fragments of the clithioneine molecule, ergothioneine and its biogenetic precursor hercynine, were found to be produced by several mycobacteria (but not by bacteria) as well as by micro- and macrofungi (420,421), for example, Claviceps purpurea, Neurospora crassa (Sor-

m/z 229

m/z 184

S

S I Me m/z 140

H m/z 126

m/z 58

SCHEME 92. Mass fragmentation(EI) of clithioneine and ergothioneine.

2.

297

ALKALOIDS FROM MUSHROOMS

H COj NMe3 H3N H

HO OH

S-(p-Amino-p-carboxyethy1)-

ergothioneine

SAM

Clithioneine

L-Threonine

L-Hercynine

SCHEME 93. Chemical degradation of clithioneine.

dariales, Ascomycotina), Coprinus atramentarius, and C . comatus, whereas hercynine alone was found in Amanita muscaria. Studies by Melville and colleagues of the biosynthesis pathway of ergothioneine in Neurospora crassa demonstrated that histidine undergoes successive triple transmethylation from S-adenosylmethionine (SAM) to hercynine (422-424) (Scheme 94). Subsequent thiolation of hercynine by cysteine to ergothioneine occurs via a sulfoxide intermediate (425).

L-Histidine

Hercynine

r

H CO?

5-Methylcysteinase

-CH~COCOO-+NH~ L

H

1

NMe3

SCHEME 94. Biosynthetic pathway of ergothioneine.

H Ergothioneine

3

298

R6ZA ANTKOWIAK AND WIESKAW 2. ANTKOWIAK

B. COPRINE Coprine, a constituent of Coprinus atrarnentarius, the common inky cap, was isolated and characterized almost simultaneously by Hatfield and Schaumberg (426) in the United States, and Lindberg et al. (427) in Sweden. This mushroom is frequently collected as an esculent and is nontoxic, provided that is not consumed with ethanol. When ingested with alcohol, the mushroom slows down the rate of ethanol metabolism and induces an elevated level of acetaldehyde in the blood, causing symptoms similar to those observed after administration of the drug disulfiram [Antabuse, bis(diethylthiocarbamyl)disulfide, Et2N--CS-SS--CS-NEt2], which is used in the treatment of chronic alcoholism. The active constituent of the mushroom responsible for the Antabuselike hyperacetaldehydemia was found to have the structure N5-( lhydroxycyclopropy1)-L-glutamineand was called coprine. The occurrence of this metabolite in other common edible inky caps is still questionable, but at least two collected in North America, the glistening inky cap (Coprinus rnicaceus) and the tastiest shaggy cap or shaggy mane (Coprinus cornatus) were found to be devoid of coprine (3). The elucidation of the structure of coprine was based on spectral analysis and chemical degradations (Scheme 95). Acidic hydrolysis (426,427)of coprine afforded L-glutamic acid and the 1-hydroxycyclopropylammonium ion, which rearranged to propionic acid after a brief treatment with

CO2H

ii/ HF + HO-

0

+ SCHEME 95. Chemical degradation of coprine.

2.

299

ALKALOIDS FROM MUSHROOMS

alkali. Alkaline hydrolysis (427) yielded L-pyroglutamic acid and propionamide, whereas catalytic hydrogenation gave N5-isopropyl-~-glutamine together with minor amounts of acetone and L-glutamine. The synthesis of coprine by Lindberg et al. (427) (Scheme 96) was carried out by acylation of the stable salt intermediate (427,428), 1hydroxycyclopropylammonium chloride, with N-phthaloyl-L-glutamic anhydride in the presence of triethylamine. This step was followed by removal of the blocking group with hydrazine. Though the symptoms of the biological action of coprine and disulfiram are similar, it was demonstrated that the mechanisms of action are different. Contrary to cyclopropanone hydrate, coprine inhibits mouse liver aldehyde dehydrogenase only in uiuo but not in uitro. Based on this observation Wiseman and Abeles (429) assumed that coprine itself is inactive in uiuo but is activated by hydrolysis to give initially cyclopropanone hemiaminal and ultimately cyclopropanone hydrate. After enzymatic dehydration to cyclopropanone, this compound forms a kinetically stable thiohemiketal with the enzyme active-site thiols, leading to inactivation of aldehyde dehydrogenase in the enzyme-catalyzed oxidation of acetaldehyde to acetic acid (Scheme 97).

INaoH BNH2

OR Coprine

SCHEME 96. Synthesis of coprine by Lindberg et al. (427).

c. ANILINEDERIVATIVES FROM Agaricus SPECIES Apart from the phenylhydrazine glutamates discussed earlier, the common Agaricus species were also found to produce various structurally and

300

R6ZA ANTKOWIAK AND WIESKAW Z . ANTKOWIAK

$

RCH

E-SH

TH

E-SH

NAD

E-S-CHR

. --

F1

E-SH

E-SCR

+ RCO?

NADH

E-SH

E-S

E-SH ___c

X

OH

SCHEME97. Mechanism of biooxidation catalyzed by aldehyde dehydrogenase and of enzyme inhibition by coprine.

biosynthetically related aniline derivatives. In particular, these metabolites were found in the sporulating gill tissue of A. bisporus. 1. y-~-Glutaminyl-4-hydroxybenzene

y-~-Glutaminyl-4-hydroxybenzene (GHB) was isolated from Agaricus bisporus by Weaver et al. (430).Elucidation of its structure resulted from chemical and spectral studies and was verified by a synthesis consisting of coupling of p-benzyloxyaniline with N-carbobenzoxy-a-benzyl-Lglutamic acid in the presence of dicyclohexylcarbodiimine followed by hydrogenolysis over palladium on carbon (Scheme 98).

GHB

SCHEME98. Synthesis of GHB.

2. Agaridoxin

Agaridoxin, a strongly autoxidizable substance, was isolated from Agaricus campestris by Szent-Gyorgyi et al. (431) and from A. bisporus (along with L-dopa) by Tsuji et al. (432).The structure of this metabolite was established (431) to be y-~-glutaminyl-3,4-dihydroxybenzene

2.

301

ALKALOIDS FROM MUSHROOMS

(GDHB), largely by the use of NMR, MS, UV, and polarography. It was then synthesized (431), starting with the reaction between 3,4(isopropy1idenedioxy)aniline and N-phthaloylglutamic anhydride, followed by successive removal of the protecting groups by hydrazine and boron trichloride (Scheme 99). 1.

0

GDHB

GBQ

SCHEME99. Synthesis of agaridoxin and GBQ.

3. y-~-Glutaminyl-3 ,4-benzoquinone

y-~-Glutaminyl-3,4-benzoquinone (GBQ), a sulfhydryl enzyme inhibitor, was identified as a constituent ofA. bisporus (433)and was obtained in uitro by enzymatic conversion (430) of GHB accompanying GBQ in this mushroom. This red colored quinone, for which a h,,,490 nm was claimed by Weaver et al. (433): was found to be extremely unstable and yielded glutamic acid on acid hydrolysis; however, the corresponding aminoquinone moiety was not identified by the authors. Tiffany et al. (434) obtained GBQ by oxidation of y-~-glutaminyl-3,4dihydroxybenzene with sodium metaperiodate; they found that the quinone had an absorption maximum at 440 nm rather than at 490 nm and demonstrated notably less sulfhydryl reactivity than for the quinoid metabolite absorbing at 490 nm. These and other findings support the conclusion that GBQ is not the correct structure for the 490 quinone ( 4 3 4 , as had been postulated previously (433),and that the latter probably occurred as a contaminant of GBQ investigated by Weaver. 4. 490 Quinone 490 Quinone, a real potent inhibitor of numerous enzymes containing sulfhydryl groups at their active sites, which also shows antibacterial and antitumor activities (434-436), was finally identified as 2-hydroxy-4-imino2,5-cyclohexadienone by Mize et al. (437). Elucidation of the structure of the iminoquinone, which was hampered by its instability at concentrations above M , was based on a combination of biosynthetic considerations, cyclic voltammetry experiments , and chemical studies. A preparation of the 490 quinone identical to the natural product was obtained by air oxidation of 4-aminocatechol in an aqueous solution at pH 7.8 (Scheme

302

R62A ANTKOWIAK AND WIESJLAW Z . ANTKOWIAK

: I ~ N H z

o2 pH

(7.8 air)I

, "0 O D N/ H

-

490-Quinone

SCHEME100. Chemical correlation of 490 quinone.

100). Further confirmation of the iminoquinone structure was provided by conversion of the natural quinone to 3,4-diacetoxyacetanilidevia reduction with sodium borohydride and subsequent acetylation. All the above-mentioned glutamine-derived metabolites were found to be intermediates in the biosynthesis of the 490 quinone (438),which entails oxidation catalyzed by tyrosinase purified from extracts of A. bisporus (433). It was observed ( 4 3 3 , however, that enzymatic oxidation of the catechol GDHB carried out at pH 6.5 produced GBQ, whereas the latter could be converted to the 490 quinone when the pH was raised to 7.8, with or without tyrosinase being present (Scheme 101). This indicated that the intramolecular transformation which provided 490 quinone in the last step was nonenzymatic. The studies of the origin of GHB in A. bisporus demonstrated the involvement of the shikimate-chorismate pathway (Scheme 102). Labeling experiments showed an efficient incorporation of 3H- and 14C-labeled shikimic acid (439,440) and 14C-labeledchorismic acid (441)into the 4hydroxyaniline moiety of GHB. It was also demonstrated that in the biochemical shikimate-4-hydroxyaniline conversion in the mushroom, amination occurred at the 4 position of one of the carboxylic acid intermediates [initially assumed to be shikimic acid (439)l. Additionally, the paminobenzoic acid, which proved to be (441) the precursor of 4hydroxyaniline, underwent a decarboxylative hydroxylation catalyzed by a FAD-dependent monooxygenase 4-aminobenzoate hydroxylase in the presence of NAD(P)H and 02.This enzyme from A. bisporus was recently purified to homogeneity by Tsuji et al. (442).

GBQ

490-Quinone

SCHEME101. pH dependence of 490 quinone formation.

2.

CH2 '%OOH

.9,6 OH

O H u H 0 X & O H

0

'OH

S h ik i m i c

acid

303

ALKALOIDS FROM MUSHROOMS

0

+

HDNH GHB

0

Chorismic acid

PABA

SCHEME 102. Probable pathway in the biosynthesis of GHB.

D. ACETYLENIC AMINOACIDS Several amino acids containing a triple bond in their structures were found in the Basidiomycotina (Fig. 30). Most revealed antibacterial or antimetabolic activity. 2(S)-Amino-5-hexynoic acid, an antibacterial metabolite that strongly inhibits the growth of Bacillus subtilis B-50, was isolated from Cortinarius claricolor (443). L-2-Amino-4-pentynoic acid, with hepatotoxic properties, was isolated along with the physiologically inactive ~-2-amino-4,5-hexadienoic acid from Amanita abrupta (444), A . solitaria, and A . pseudoporphyria. Examples of fungal metabolites revealing such structural features were mentioned by Aoyagi and Sugahara (443), as previously discussed by Chilton (404). These are 2-amino-4hexynoic acid, found in Tricholomopsis rutilans (Tricholomataceae) and Amanita pseudoporphyria; threo- and erythro-2-(S)-amino-3-hydroxy-4hexynoic acids from Tricholomopsis rutilans; 2-amino-6-hydroxy-4hexynoic acid from Amanita onusta; and 2-amino-4-heptene-6-ynoic acid from A . pseudoporphyria.

threo

FIG. 30. Acetylenic amino acids from mushrooms.

erythro

304

R 6 Z A ANTKOWIAK A N D WIESJLAW Z. ANTKOWIAK

5% H 50

HoQ 0 H NH2

SCHEME 103. Chemical degradation of connatin.

E. CONNATIN Connatin, the main chromogen of Lyophyllum connatum (Tricholomataceae) responsible for the color reaction with FeC13, was isolated from this mushroom by Fugmann and Steglich (368). The structure of this metabolite was determined by spectral analysis and chemical transformations to be N*-hydroxy-N", Nm-dimethyl-2-(S)-citrulline [N5-(dimethylaminocarbonyl)-N5-hydroxy-~-ornithine] (Scheme 103). When treated with trifluoroacetic anhydride connatin gave 4-substituted 2-trifluoromethyl-5(2H)-oxazolone,indicating that the cu-NHz group was initially unsubstituted. When hydrolyzed by dilute sulfuric acid or hydrochloric acid connatin was converted to (S)-3-amino-l-hydroxy-2piperidone and (S)-N'-hydroxyornithine hydrochloride, respectively. F. P-NITROAMINOALANINE P-Nitroaminoalanine and its decarboxylation product N-nitroethylenediamine (Fig. 3 1) were identified in Agaricus siluaticus (445).The isomeric compound alanosine and related N-nitroglycine were also found as natu-

p-Nitroaminoalanine

N-Nitroethylenediamine

Alanosine

FIG. 31

N-Nitroglycine

2.

305

ALKALOIDS FROM MUSHROOMS

rally occurring amino acids but in filamentous bacteria, Streptomyces spp. (Actinomycetes).

MIL Alkaloids of Miscellaneous Structures A. LEPISTINE Lepistine was found in Clitocybefasciculata (446),previously known as Lepista caespitosa, the agaric commonly growing in the northern part of New Zealand. The structure of lepistine was determined on the basis of X-ray investigations of the alkaloid hydrobromide salt and on NMR considerations (Fig. 32).

@YCOCH3 Lepistine

FIG. 32

B. PISTILLARIN Pistillarin, a bitter metabolite responsible for the green color reaction of fruiting bodies with FeC13, was isolated from Clavariadelphus pistillaris and several Ramaria species (Gomphaceae, Aphyllophorales) (447). The structure was determined as being N' ,N8-bis(3,4-dihydroxybenzoyl)spermidine on the basis of spectral data. This was confirmed by synthesis from N4-benzylspermidine and veratryl chloride followed by removal of the protecting groups by successive hydrogenolysis and boron tribromide treatment (Scheme 104). 1.

O*C/Cl

8.. 'dmNp;

2 . ti2,Pd/C OH

3.88r3

H

D

H'

Pistillarin

SCHEME104. Synthesis of pistillann.

0

306

R 6 Z A ANTKOWIAK A N D WIESKAW Z. ANTKOWIAK

C. CHALCIPORONES Chalciporone was found (448) to be the main pungent principle of Chulciporus piperutus, a small mushroom distinguished from other boletes by its peppery taste and colorful lemon-yellow stem bases. Chalciporone, having high antimicrobial activity, is accompanied in the fungus by an equally pungent norchalciporyl propionate and the nonpungent isochalciporone and dehydroisochalciporone (Scheme 105). Chalciporone ( [ a ] ~ -452") was found to be susceptible to spontaneous isomerization to isochalciporone with total loss of optical activity and pungency when kept in a chloroform solution. The azepine structures of these metabolites were determined by intensive NMR studies and supported by other spectroscopic methods and chemical transformations.

+

isornerization

\ /

Chalciporone

Norchalciporyl propionate

-e -

Isochalciporone

Dehydroisochalciporone

SCHEME 105. Structural relationships between chalciporones.

D. RETICULINE Reticuline (Fig. 33), well known for about half a century now as an alkaloid of plant but not mushroom origin and as a biosynthetic precursor to several alkaloids of great importance, was recently claimed (449) to be

2.

ALKALOIDS FROM MUSHROOMS

10‘

307

(+)-Reticuline

Me0

(depicted inversely)

isolated from the parasitic fungus Laurobasidiurn lauri (Exobasidiales, Hymenomycetes). Alcoholic extracts of this fungus are used in the traditional folk medicine of the Madeira Islands, and its effectiveness as a sedative and uterine relaxant has been ascribed to the physiological properties of reticuline. The formula presented in the paper, and the physical properties of the isolated metabolite described by the authors are almost identical to those of reticuline reported in other papers (450452) but not in every respect.

IX. Addendum A. CYTOCHALASANS The cytochalasans comprise a large group of biologically active metabolites from molds characterized by a skeleton which includes a highly hydrogenated isoindolone unit fused to a macrocyclic ring of varying size. This ring, composed of 11, 13, or 14 atoms (the number is indicated in a systematic name in square brackets), can either be a lactone, a carbonate, or a carbocycle. As far as we know all the cytochalasans discovered up to now are metabolites of lower fungi, with the exception of cytochalasin D which was also found to be produced by Coriolus vernicipes, a species in the Basidiomycotina. The trivial names of cytochalasans, namely, cytochalasins, aspochalasins, zygosporins, chaetoglobosins, and so on, have been formed according to the origin or biological activity of the alkaloids. It appears, however, that designations formed in this way can often be misleading, as, for example, in the case of cytochalasins K, N, 0, P, Q,

308

R6ZA ANTKOWIAK AND WIESJLAW Z. ANTKOWIAK

and R, each of which is represented by two different structures. A systematic nomenclature is described in Refs. 453-455. The following tabulation gives the structures and fungal sources for the cytochalasans along with notes and references relating to studies of the alkaloids.

24-0xa[ 14]cytochalasans

I . Cytochalasin A (dehydrophomin) X

=

0

Helminthosporium dematioideum (Hyphomycetes, Deuterom ycotina)

Phoma species (Coelomycetes, Deuteromy cotina) HO

Ascochyta heteromorpha (Coelomycetes) 2. Cytochalasin B (phomin) X = H, a-OH

Isolation, structure, mp 182-185"C, MS, IR, IH NMR, chemical transformations (456,457);derived from cytochalasin B labeled by I4C or 3H (458) Isolation, structure, mp 185-187"C, [a]D +92" (EtOH), UV, IR, 'H NMR, chemical transformations, biological activity (459);partial synthesis (460);biosynthesis (461 ) Isolation, identification (462)

Helminthosporium dematioideum

Isolation, structure, mp 218-221°C, IR, MS, 'H NMR, chemical transformations (456,457);biosynthesis

Phoma species

Structure, [a]D +83" (MeOH), UV, IR, MS, 'H NMR, chemical transformations, biological activity (459);X-ray structure (463);biosynthesis, I3C and 'H NMR (461,464,465) Isolation, identification (462);total synthesis (466,467)

(458)

Ascochyta heteromorpha

2.

ALKALOIDS FROM MUSHROOMS

309

3. Cytochalasin F X = H, &-OH (P,P-6,7-epoxide, 6-a-methyl derivative of cytochalasin B)

Helrninthosporiurn dernatioideurn

Isolation, structure, 'H NMR (468,469);total synthesis (467)

4. Cytochalasin L X = H, P-OAC, Y 21.22-unsaturated

Chalara microspora (Hyphomycetes)

Isolation, structure, amorphous, [a]D - 165" (EtOH), UV, IR, 'H and I3C NMR (470)

Chalara microspora

Isolation, structure, mp 161-162"C, [(Y]D + 18.7" (EtOH), UV, IR, 'H and "C NMR(470); X-ray structure (471)

Rosellinia necatrix (Sphaeriales, Aspergillus Ascomycotina) clavatus

Isolation, structure, 'H NMR (468,469) X-Ray structure, mp

=

0,

5. Cytochalasin M X = 0, Y = H, P-OH, 21,22saturated

21,23-Dioxa[ 13]cytochalasans 6. Cytochalasin E

&o

(Hyphomycetes)

NH

d EH

206-208"C, [(Y]D -25.6" (MeOH), 'H NMR, biological activity (472); chemical transformations, 'H and "C NMR(473)

Ph

7. Cytochalasin K

Aspergillus clavatus

HO

Ph

Isolation, structure, mp 246-248"C, UV, IR, 'H and I3C NMR, chemical transformations (473)

310 ~~

R ~ Z AANTKOWIAK AND WIESEAW

z.

ANTKOWIAK

~

[13]Cytochalasans

8. Protophomin

Phoma species

Isolation, structure, mp 252-256"C, [(Y]D -112" (CHC13),IR, MS, 'H NMR(474); biosynthesis (461)

9. Chaetoglobosin B X = H, p-OH, Y = 0, 21,22unsaturated, R = 3-indolyl

Chaetomium globosum (Sordariales, Ascomycotina)

Isolation, structure, mp 186-187"C, [(Y]D -176" (MeOH), UV, IR, 'H NMR(475); bioproduction, cytotoxicity (476,477); IH NMR, structure (478), I3C NMR(479)

10. 19-0-AcetylchaetoglobosinB X = H, p-OAc, Y = 0, 21,22-unsaturated, R = 3indolyl

Chaetomium

Isolation, structure, mp 154-157"C, [(YID -148' (CHC13), UV, IR, MS, 'H and I3C NMR (480)

11. Chaetoglobosin E X = 0, Y = H, p-OH, 21,22saturated, R = 3-indolyl

Chaetomium globosum

Isolation, cytotoxicity (476); structure, mp 279-280°C, [ a ]+~158" (MeOH), UV, IR, 'H NMR (481); bioproduction, cytotoxicity (477); 'H NMR, conformations, chemical correlations (482); I3C NMR(479)

12. Chaetoglobosin G X = Y = 0, 21,22-saturated, R = 3-indolyl

Chaetomium globosum

Isolation, structure, mp 251-253"C, [(Y]D +89" (MeOH), chemical correlations, UV, IR, MS (483); bioproduction,

Ph

globosum

~

2.

ALKALOIDS FROM MUSHROOMS

311 cytotoxicity (477); 'H NMR, conformations, chemical correlations (482);I3C NMR(479)

13. Proxiphomin

Phoma species

Isolation, structure, amorphous, [ a ]-140" ~ (CHCI3), UV, IR, MS, 'H NMR(474); biosynthesis (461); total synthesis (484)

Chaetomium globosum

Isolation, structure, mp 149-151°C, UV, IR, MS, chemical correlations (483); bioproduction, [aID +41" (MeOH), cytotoxicity, (477); 'H NMR, conformations, chemical correlations (482);I3C NMR(479)

Phoma species

Isolation, structure, amorphous, [a]D -50.8" (CHCI3), UV, IR, MS, 'H NMR, chemical transformations, biological activity (485) Isolation, identification 'H NMR (486)

Ph

14. Chaetoglobosin J R = 3-indolyl

f H

15. Deoxaphomin 2 1,22-unsaturated

HO

16. Ascochalasin 21,22-saturated

Ascochyta heteromorpha

Ascochyta heteromorpha

Isolation, structure, amorphous, UV, IR, MS, 'H and I3C NMR (486)

312

R6ZA ANTKOWIAK AND WIESKAW Z. ANTKOWIAK

17. Chaetoglobosin D Rl = R2 = R3 =H, R = 3indolyl

Chaetomium globosum

Isolation, cytotoxicity (476); structure, mp 216"C, [(Y]D -269" (MeOH), UV, IR, 'H NMR (481); bioproduction, cytotoxicity (477); structure, 'H NMR (478); "C NMR (479)

Diplodia macrospora (Coelomycetes)

Isolation, structure, IR, MS,'H NMR (487)

Chaetomium globosum

Isolation, structure, mp 239-24I0C, [a]D -176" (CHC13), UV, IR, MS, 'H and I3C NMR (480)

20. Chaetoglobosin A RI = R2 = R3 = H, R = 3indolyl

Chaetomium globosum

Isolation, structure, mp 168-170"C, [a]D -270" (MeOH), MS, UV, IR, 'H NMR(475,478); biological activity (476); bioproduction, cytotoxicity (477); identification by TLC (488); structure, mp 1.51-153"C, [ a ] D -372" (CHCI3), UV, MS, IR, 'H and I3C NMR (480);X-ray analysis (489,490); biosynthetic studies, 13C NMR (479,491)

21. 19-0-AcetylchaetoglobosinA RI = Ac, R2 = R3 = H, R = 3-indolyl

Chaetomium globosum

Isolation, structure, mp ~ 223-225"C, [ a ] -304" (CHC13), UV, IR, MS, 'H and I3C NMR (480);biosynthetic studies, I3C NMR (479,491)

22. Cytochalasin K RI = Ac, R2 = R3 = H, R = phenyl

Chalara microspora

Isolation, structure, amorphous, [(Y]D -177" (EtOH), UV, IR, 'H and I3C NMR (470)

HO

18. Chaetoglobosin L R2 = R3 = Me, RI R = 3-indolyl

=

H,

19. 19-0-AcetylchaetoglobosinD

Rz = R3 = H, Rl = Ac, R = 3-indolyl

2.

313

ALKALOIDS FROM MUSHROOMS

23. Chaetoglobosin K R I = H, R2 = R3 = Me R = 3-indolyl

Diplodia macrospora

Isolation, structure, mp 264-266"C, UV, IR, MS, 'H and I3C NMR, biological activity (492); X-ray analysis (493); isolation, mp 235-24OoC, IH NMR (487)

24. Chaetoglobosin C X = 0, R = 3-indolyl

Chaetomium globosum

Penicillium aurantiovirens

25. Chaetoglobosin F X = H, P-OH, R = 3-indolyl

Chaeromium globosum

Isolation, mp 259-261°C (475); cytotoxicity (476); structure, mp 260-263"C, [(YID -30" (MeOH), MS, UV, IR, IH NMR, chemical correlations (481); bioproduction, cytotoxicity (477); isolation, UV, IR, I3C NMR (480); 'H NMR, conformations, chemical correlations (478,482); biosynthetic studies, incorporation of [1-'3C]-, [2-'3CI-, and [ 1,2J3C2]acetate, [ I3C-merhyl]-~methionine, and D L - [ ~ I3C] and DL[2-I3C]tryptophan, I3C NMR (479) Isolation, mp 257-259"C, IR, 'H NMR, X-ray structure (494)

Isolation, mp 177-178"C, cytotoxicity (476); structure, mp 177178"C, [OI]D -69" (CHC13), MS, UV, IR, 'H NMR, chemical correlations (481); bioproduction, cytotoxicity (477); 'H NMR, conformations, chemical correlations (482); I3C NMR (479)

314

R6ZA ANTKOWIAK AND WIESKAW Z. ANTKOWIAK

[ll]Cytochdasans 26. Cytochalasin C

Metarhiziurn anisopliae

HO Hypoxylon terricola (Sphaeriales, Ascomycotina)

Isolation, structure, 'H NMR (495);From cytochalasin D, mp 261.5-267"C, [(Y]D - 14.7" (dioxane) (496); structure-activit y relationship (497) Isolation, 'H and I3C NMR, chemical correlations (498)

27. Cytochalasin N (5,Gepoxide of cytochalasinC)

Hypoxylon terricola

Isolation, structure, mp 272"C, [(Y]D -4" (MeOH), IR, 'H and 13C NMR, chemical correlations (498)

28. Cytochalasin 0 (6-hydroxy-5,6dihydrocytochalasin C)

Hypoxylon terricola

Isolation, structure, mp 258-265"C, [(Y]D -39.3" (MeOH), IR, 'H and I3C NMR, chemical correlations (498)

29. Cytochalasin P (6-epimer of cytochalasin 0)

Hypoxylon terricola

Isolation, structure, mp 169-173"C, [(Y]D -35.8" (MeOH), IR, 'H, I3C NMR, chemical correlations (498)

30. Cytochalasin Q (6,7-epoxide, isomeric with cytochalasin N)

Hypoxylon terricola

Isolation, structure, mp 145-147"C, [a]D -94.5" (CHCI3), IR, 'H, I3C NMR, chemical correlations (498)

31. Cytochalasin R (13,14-epoxide of cytochalasin Q, ignoring steric features)

Hypoxylon terricola

Isolation, structure, mp 159-167"C, [(Y]D -73.1" (MeOH), IR, 'H, I3C NMR, chemical correlations (498)

32. Cytochalasin D (zygosporin A)

Zygosporium masonii (Hyphomycetes)

Isolation (499);X-ray structure (500,501); structure, mp 268270"C, [a]D -7.5" (dioxane), IR, 'H NMR, chemical transformations, cytotoxicity (502,503); chemical correlations (504); structure-

HO

2.

315

ALKALOIDS FROM MUSHROOMS

Metarrhizium anisopliae Engleromyces goetzii (Sphaeriales) Coriolus vernicipes (Polyporaceae) Hypoxylon terricola

33. Zygosporin D (deacetylcytochalasin D)

Zygosporium masoni

activity relationship (496,497); biosynthesis (458,464,465,505,506) 'H NMR, chemical transformations (495) Biological activity (507) Bioproduction (508) Isolation, 'H and I3C NMR (498);partial synthesis (509); synthesis of analogs (510) Isolation, structure, mp 180-190°C, ID -14.9" (dioxane), IR, 'H NMR, chemical correlations, cytotoxicity (503); from Zygosporin A, mp 187-190°C (502); structure-activity relationship (496); X-ray structure of 7-0acetyl derivative (500,501)

34. Engleromycin (19,20epoxide of zygosporin D)

Engleromyces goetzei (Sphaeriales)

Isolation, structure, mp 226-228"C, [a]D +64" (EtOH); 'H and I3C NMR(511)

35. Zygosporin E (18-dehydroxycytochalasinD)

Zygosporium masoni

Isolation, structure, mp 218-223 S"C, [a]D +6.2", IR, 'H NMR, chemical correlations, cytotoxicity (503); structure-activity relationship (496)

36. Zygosporin F (7-acetate of cytochalasin D)

Zygosporium masoni

Isolation, structure, mp 126-129"C, [ a ]-12", ~ IR, 'H NMR, chemical correlations, cytotoxicity (503); structure-activit y relationship (496);from cytochalasin D (502,504)

316

R6ZA ANTKOWIAK AND WIESEAW Z . ANTKOWIAK

37. Zygosporin G

-

Zygosporium masoni

Isolation, structure, mp 115-12s0c, [&ID -82'9 IR, 'H NMR, chemical correlations, cytotoxicity (503); from cytochalasin D (504);structureactivity relationship (496);synthesis and X-ray structure of analogs (454,510)

Nigrosabulum species (Pseudeurotiaceae, Eurotiales) Pseudeurotium zonatum (Pseudeurotiaceae)

Isolation, mp 255-257"C, [(Y]D -99" (MeOH), 'H NMR, X-ray structure (512) Isolation, identification, UV, IR, 'H and I3C NMR (480);total synthesis (513)

Phomopsis paspalli

Isolation, mp 268-269"C, [ a ] +63" ~ (MeOH) (514); mp 258-263"C, [(Y]D -9.0" (CHCI3), +91.2" (MeOH), IR, 'H NMR, toxicity (515); X-ray structure (516,517);'H, I3C NMR(518); I3C NMR spectrum (519); 'H and I3C NMR of derivatives (520);EI and CI mass spectra of cytochalasin H and analogs (521); Isolation, mp 250251"C, [ a ] D +32.5" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (522,523). Total synthesis (524,525) Insecticidal activity (526)

Ph

38. Cytochalasin G R = 3-indolyl

0

39. Cytochalasin H (paspaline P1, or kodocytochalasin I), R = H

HO

R

Aspergillus f l a w s

2.

317

ALKALOIDS FROM MUSHROOMS

40. Cytochalasin J (deacetylcytochalasin H) R=H

Phomopsis paspali

41. Epoxycytochalasin H (P,P-6,7-epoxide, 6-a-methyl derivative of cytochalasin H) R=H

Phornopsis species

Isolation, mp 128-13OoC, [a]D -84.68" (CHC13), 13CNMR, toxicity (527); mp 122-123"C, [ a ]+26" ~ (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (522,523)

42. Epoxycytochalasin J (P,p-6,7-epoxide, 6-a-methyl derivative of cytochalasin J), R=H

Phomopsis species

Isolation, mp 192-194"C, [a]D -49. I" (CHCI3), I3C NMR, toxicity (527); mp 125-126"C, [a]~ +69.8" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (522,523)

43. Pyrichalasin H R = OCH3

Pyricularia grisea (Hyphomycetes)

Isolation, structure, mp 207-209"C, [a]D -18.4" (CHC13), +59" (MeOH), UV, IR, MS, CD, 'H and I3C NMR, 2D homonuclear and heteronuclear COSY spectra (528); phytotoxic activity (529)

Isolation, mp 151-165"C, [ a ] +45" ~ (MeOH) (514); mp 135-138"C, IR, MS, 'H NMR (515);mp 158-160"C, [U]D +32.8" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (522,523); structure, mp 274276"C, [ a ] D +47.8" (MeOH), 'H and I3C NMR, biological activity (519,520)

~

~

~

~~

~~

318

R6ZA ANTKOWIAK AND WIESJZAW 2. ANTKOWIAK

44. Cytochalasin N

Phomopsis species

Isolation, structure, mp 253-254"C, [(Y]D +85.4" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (522,523)

45. Cytochalasin 0 (deacetylcytochalasin N)

Phomopsis species

Isolation, structure, mp 170-172"C, [@I.D +59.7" (MeOH), UV, IR, 'H and "C NMR, chemical correlations (522,523)

46. Cytochalasin P R, = & = Me, R2 = OH, R3 = H , Rs = AC

Phomopsis species

Isolation, structure, mp 117-1 18"C, [(Y]D -116.3" (MeOH), UV, IR, 'H and 13CNMR, chemical correlations (522,523)

47. Cytochalasin Q Rl = R4 = Me, R2 = OH, R3 = RS = H

Phomopsis species

Isolation, structure, mp 158-159"C, [(Y]D -47.8" (MeOH), UV, IR, 'H and 13CNMR, chemical correlations (523)

48. Cytochalasin R RI = OH, R2 = R4 = Me, R3 = RJ = H (6-phydroxycytochalasin J)

Phomopsis species

Isolation, structure, mp 106-107"c, [(Y]D -46.0" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (523)

HO

2. ALKALOIDS FROM

319

MUSHROOMS

49. Cytochalasin S Rl = R3 = Me, & = OH, R2 = RS = H (6-(Yhydroxycytochalasin J)

Phomopsis species

Isolation, structure, mp 149-15loC, [(Y]D -62.9" (MeOH), UV, IR, 'H and I3C NMR, chemical correlations (523)

50. Aspochalasin C X = H, (Y-OH,Y = P-OH, H

Aspergillus microcysticus (Hyphomycetes)

Isolation, structure, amorphous, [a]D -86" (CHCI3), UV, IR, MS, 'H and I3C NMR, chemical transformations (530); X-ray structure of 17,18-di-O-acetyl derivative (531); totaf synthesis of 1 3 2 isomer (isoaspochalasin C) (455)

51. Aspochalasin A X = 0, Y = 0, 19,20dihydro

Aspergillus microcysticus

Isolation, structure, amorphous, [a]D -20" (CHCI3), UV, IR, MS, 'H and I3C NMR, chemical transformations (530)

52. Aspochalasin B X = H, (Y-OH,Y = 0

Aspergillus microcysticus

Isolation, structure, amorphous, [a]D - 118°C (CHC13), UV, IR, MS, 'H NMR, chemical transformations (530); total synthesis (532)

53. Aspochalasin D X = Y = H , a-OH

Aspergillus microcysticus

Isolation, structure, mp 148"C, [(Y]D -81" (EtOH), UV, IR, MS, 'H NMR, chemical transformations (530); synthesis of a degradation product (533)

320

R6ZA ANTKOWIAK AND WIESKAW Z. ANTKOWIAK

B.

SELECTED OTHER

ALKALOIDS FROM LOWER FUNGI

The following lists structures, sources, and references pertaining to studies of other alkaloids isolated from lower fungi, compiled from recent reports in the literature. 1. Tenuazonic acid

Alternaria species (Hyphomycetes) Pyricularia oryzae (Hyphomycetes)

Phoma sorghina (Coelomycetes)

Isolation, identification, phytotoxicity (534) Isolation, identification (535,536);synthesis and phytotoxicity of analogs (537) Isolation and characterization of magnesium and calcium complexes (538); 'H and 13CNMR, structural investigations, structurebioactivity relationship (539)

2. FR900483

Nectria lucida (Hypocreales, Ascomycotina)

Isolation, structure, [aID +22" (H,O), 'H and I3C NMR, synthesis (540)

3. Swainsonine

Rhizoctonia leguminicola (Agonomycetes)

Isolation, structure, mp 144145"C, [OI]D -87.2" (MeOH), IR, MS, 'H and I3C NMR (541,542);chiral synthesis from D-glucose (543);biological activity (544)

4. S(R)-Fuligorubin A

Fuligo septica (Myxomycetes)

Isolation, structure, mp >150"C, UV, CD, IR, MS, 'Hand I3C NMR (545,546)

2. 5 . Physarochrome A

ALKALOIDS FROM MUSHROOMS

32 1

Physarum polycephalum (Myxomycetes)

Isolation, structure, amorphous, [aID +7.2” (MeOH), UV, IR, MS, ‘H and I3C NMR (546,547)

6. Piperine

Ulocladium species (H yphomycetes)

Isolation, identification, UV, IR, MS, ‘H NMR (548)

7. Phomopsin A

Phomopsis leptostromiformis (Coelomycetes)

Isolation, structure, mp 205°C (dec.), UV, IR, ‘H and 13C NMR (549,550)

8. Rhizonin A

Rhizopus microsporus (Mucorales, Zygomycetes)

Isolation, structure, conformational analysis in solution and crystal, IH and I3C NMR, X-ray

COCH,

I H O P : %H2N”

(551,552)

322

R62A ANTKOWIAK AND WIESEAW Z. ANTKOWIAK ~

~

9. AK-toxin I R = CH3 AK-toxin I1 R = H

~~

Alternaria alternata (Hyphomycetes)

Isolation, structure determined by chemical, spectral, and X-ray crystallography studies (553,554);total synthesis AK-toxin I1 (555)

Acremonium coenoohialum (Hyphomycetes)

Isolation, semisynthesis, 'H and "C NMR studies of derivatives (556); total synthesis (557)

Fusarium equiseti (Hyphomycetes)

Isolation, structure, UV, CD, IR, MS, 'H and I3C NMR, phenylboronic ester derivative in structure assignment (558); total synthesis (559)

Ascochyta chrysanthemi (Coelomycetes)

Isolation, mp 193"C, [aID -37.2" (PyH), UV, IR, 'H and 13CNMR, X-ray structure (560)

Cylindrocladium ilicicola (Hyphomycetes)

Biosynthetic study with I3Clabeled acetates, "Nlabeled phenylalanine, and I4C-labeledphenylalanine

,,a

R r

10. Loline (festucine)

&CH3

11. Equisetin

HO,

*-Me

12. Chrysanthone O M -e

0

13. Ilicicolin

OH

(561)

H

2. ALKALOIDS FROM MUSHROOMS

323

14. Gliovictin

Asteromyces cruciatus

Isolation, identification [a],, -62" (CHC13), 13C NMR

15. Bohemamine

Aztinosporangium species (Actinomycetes) (Fungi Imperfecti)

Isolation, structure, mp 199200°C (dec.), UV, IR, MS, ' H and I3C NMR, X-ray (563)

16. Olivoretin A R = CH3 Olivoretin D R = H

Streptoverticillium olivoreticuli (Actinomycetes)

Isolation, structure, mp 251253"C, [ a ] -314.9" ~ (CHC13)for A, mp 228229"C, [ a ]-141.5" ~ (MeOH) for D; UV, IR, MS, CD, ' H and "C NMR, X-ray study of olivoretin D (564)

17. Meleagrin R=R'=H

Penicillium rneleagrinum

Isolation, structure, mp 250°C (dec.), UV, IR, MS, CD, 'H NMR, X-ray analysis of 9-0-pbromobenzoyl derivative

OyNH

OR

(565,566)

L I

Me0 R

18. Peramine

Acremonium lolli (Hyphomycetes)

Isolation, HBr salt: mp 242243"C, UV, MS, ' H and 13C NMR, biological activity (567)

324

R 6 Z A ANTKOWIAK A N D WIESJZAW 2. ANTKOWIAK

Acknowledgments

The authors express their deep gratitude especially to Professors W. Boczon, A. Brossi, W. Gessner, K. H@iland, S. Rapior, W. Steglich, and T. Stijve for kind compliance with literature requests, to Mr. M. Court and Mrs. E. Krygier-Court for watching over our English, to Professor A. Bujakiewicz for introducing us to the world taxonomy of fungi, and to Mrs. K. Sternal, Miss L. Sadek, and Mr. J. Bartoszewicz for technical assistance. We also thank the Institute of Organic Chemistry, Polish Academy of Sciences, for financial support (Grant No. CPBP-01.13.2.19). REFERENCES 1. D. L. Hawksworth, B. C. Sutton, and G. C. Ainsworth, “Ainsworth and Bisby’s

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339

525. R. Sauter, E. J. Thomas, and J. P. Watts, J. Chem. Soc., Perkin Trans. 1 , 519 (1989). 526. P. F. Dowd, R. J. Cole, and R. F. Vesonder, U.S. Patent Appl. U.S. 201,143, November 15, 1988; Chem. Abstr. 111,73094d (1989). 527. R. J. Cole, D. M. Wilson, J. L. Harper, R. H. Cox, T. W. Cochran, H. G. Cutler, and D. K. Bell, J. Agric. Food Chem. 30, 301 (1982). 528. M. Nukina, Agric. Biol. Chem. 51,2625 (1987). 529. M. Nukina, Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 29, 705 (1987); Chem. Abstr. 108, 91432e (1988). 530. K. Neupert-Laves and M. Dobler, Helu. Chim. Acta 65, 1426 (1982). 531. W. Keller-Schierlein and E. Kupfer, Helu. Chim. Acta 62, 1501 (1979). 532. B. M. Trost, M. Ohmori, S. A. Boyd, H. Okawara, and S. J. Brickner, J. A m . Chem. SOC.111,8281 (1989). 533. E. Kupfer and W. Keller-Schierlein, Helu. Chim. Acta 62, 1525 (1979). 534. A. C. Stierle, J. H. Cardellina 11, and G. A. Strobel, J . N a t . Prod. 52,42 (1989). 535. S. Iwasaki, H. Muro, S. Nozoe, S. Okuda, and Z. Sato, Tetrahedron Lett., 13 (1972). 536. N. Umetsu, J. Kaji, and K. Tamari, Agric. Biol. Chem. 36,859 (1972). 537. M. H. Lebrun, L. Nicolas, M. Boutar, F. Gaudemer, S. Ranomenjanahary, and A. Gaudemer, Phytochemistry 27,77 (1988). 538. P. S. Steyn and C. J. Rabie, Phytochemistry 15, 1977 (1976). 539. M. J. Nolte, P. S. Steyn, and P. L. Wessels, J . Chem. SOC.,Perkin I, 1057 (1980). 540. H. Kayakiri, S. Takase, H. Setoi, I. Uchida, H. Terano, and M. Hashimoto, Tetrahedron Lett. 29, 1725 (1988). 541. M. J. Schneider, F. S. Ungemach, H. P. Broquist, and T. M. Harris, Tetrahedron 39,29 (1983). 542. F. P. Guengerich, S. J. DiMari, and H. P. Broquist, J. A m . Chem. Soc. 95,2055 (1973). 543. M. H. Ali, L. Hough, and A. C. Richardson, J. Chem. Soc., Chem. Commun., 447 (1984). 544. P. R. Dorling, S. M. Colegate, and C. R. Huxtable, Toxicon 3, (Suppl.) 93 (1983). 545. V. I. Casser, B. Steffan, and W. Steglich, Angew. Chem. 99,597 (1987). 546. W. Steglich, Pure Appl. Chem. 61,281 (1989). 547. B. Steffan, M. Praemassing, and W. Steglich, Tetrahedron Lett. 28,3667 (1987). 548. J. S. Dahiya, D. L. Woods, and J. P. Tewari, Phytochemistry 27,2366 (1988). 549. C. C. J. Culvenor, J. A. Edgar, M. F. Mackay, C. P. Gorst-Allman, W. F. 0. Marasas, P. S. Steyn, R. Vleggaar, and P. L. Wessels, Tetrahedron 45,2351 (1989). 550. C. C. J. Culvenor, P. A. Cockrum, J. A. Edgar, J. L. Frahn, C. P. Gorst-Allman, A. J. Jones, W. F. 0. Marasas, K. E. Murray, L. W. Smith, P. S. Steyn, R. Vleggaar, and P. L. Wessels, J. Chem. SOC.,Chem. Commun., 1259 (1983). 551. M. Potgieter, P. S. Steyn, F. R. van Heerden, P. H. van Rooyen, and P. L. Wessels, Tetrahedron 45,2337 (1989). 552. P. S. Steyn, A. A. Tuinman, F. R. van Heerden, P. H. van Rooyen, P. L. Wessels, and C. J. Rabie, J. Chem. Soc., Chem. Commun., 47 (1983). 553. T. Nakashima, T. Ueno, and H. Fukami, Tetrahedron Lett. 23,4469 (1982). 554. T. Nakashima, T. Ueno, H. Fukami, T. Taga, H. Masuda, K. Osaki, H. Otani, K. Kohmoto, and S. Nishimura, Agric. Biol. Chem. 49,807 (1985). 555. K. Ando, T. Yamada, Y. Takaishi, and M. Shibuya, Heterocycles 29, 1023 (1989). 556. R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell, J. Nut. Prod. 52,810 (1989). 557. J. J. Tufariello, H. Meckler, and K. Winzenberg, J. Org. Chem. 51,3556 (1986). 558. N. J. Phillips, J. T. Goodwin, A. Fraiman, R. J. Cole, and D. G. Lynn, J . A m . Chem. Soc. 111,8223 (1989). 559. E. Turos, J. E. Audia, and S. J. Danishefsky, J. A m . Chem. Soc. 111, 8231 (1989).

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560. A. Albinati, A. Arnone, G. Assante, S. V. Meille, and G. Nasini, Phytochemistry 28, 923 (1989). 561. M. Tanabe and S. Urano, Tetrahedron 39,3569 (1983). 562. J. Shin and W. Fenical, Phytochemistry 26,3347 (1987). 563. T. W. Doyle, D. E. Nettleton, D. M. Balitz, J. E. Moseley, and R. E. Grulich, J. Org. Chem. 45, 1324 (1980). 564. S. Sakai, N. Aimi, K. Yamaguchi, Y. Hitotsuyanagi, C. Watanabe, K. Yokose, Y. Koyama, K. Shudo, and A. Itai, Chem. Pharm. Bull. 32,354 (1984). 565. K. Kawai, K. Nozawa, S. Nakajima, and Y. Iitaka, Chem. Pharm. Bull. 32,94 (1984). 566. K. Nozawa and S. Nakajima, J. Nat. Prod. 42,374 (1979). 567. D. D. Rowan and B. A. Tapper, J . Nut. Prod. 52, 193 (1989).

CUMULATIVE INDEX OF TITLES Aconitum alkaloids, 4,275 (1954), 34,95 (1988) diterpenoid, 7,473 (1960) Ci9 diterpenes, 12,2 (1970) Czoditerpenes, 12, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine, 21, 1 (1983) Actinomycetes, isoquinolinequinones, 21,55 (1983) N-Acyliminium ions as intermediates in alkaloid synthesis, 32,271 (1988) Ajamaline-Sarpagine alkaloids, 8, 789 (1965), 11,41 (1968) Alkaloid structures forensic chemistry of, 32, 1 (1988) spectral methods, study, 24,287 (1985) unknown structure minor alkaloids, 5,301 (1955), 7,509 (1960) unclassified alkaloids, 10,545 (1%7), 12,455 (1970), 13,397 (1971), 14,507 (1973), 15,263 (1975), 16,511 (1977) Alkaloids histochemistry of, 39, 165 (1990) Alkaloids in Cannabis satiua L., 34,77 (1988) the plant, 1, 15 (1950), 6, l(1960) Alkaloids from Ants and insects, 31, 193 (1987) Aspergillus, 29, 185 (1986) Puuridiantha species, 30,223 (1987) Tabernaemontana, 27, 1 (1986) Alstonia alkaloids, 8, 159 (1965), 12,207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2,331 (1952), 6,289 (1960), 11,307 (1968), 15,83 (1975), 30, 251 (1987) Amphibian alkaloids, 21, 139 (1983) Analgesics, 5, 1 (1955) Anesthetics, local, 5,211 (1955) Anthranilic acid, related to quinoline alkaloids, 17, 105 (1979), 32,341 (1988) Antimalarials, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985), 37, 1, 205 (1990) Taxus alkaloids, 25,6 (1985) Sesbania alkaloids, 25, 18 (1985) Pyrrolizidine alkaloids, 25,21 (1985) Acronycine, 25,38 (1985) Emetine, 25,48 (1985) Cephalotaxus alkaloids, 25,57 (1985) Colchicine, 25,69 (1985) Camptothecine, 25,73 (1985)

341

342

CUMULATIVE INDEX OF TITLES

Ellipticine, 25, 89 (1985) Maytansinoids, 25, 142 (1985) Phenanthroindolizidines,25, 156 (1985) Bisisoquinolines, 25, 163 (1985) Benzophenanthridines, 25, 178 (1985) Protoberberines, 25, 188 (1985) Amaryllidacea alkaloids, 25, 198 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, 1 (1967), 24, 153 (1985) Aristolochia alkaloids, 31,29 (1987) Aristotelia alkaloids, 24, 113 (1985) Aspidosperma alkaloids, 8,336 (1965), 11,205 (1968), 17, 199 (1979) Azafluoranthene alkaloids, 23,301 (1984) Bases simple, 8, 1 (1965) simple indole, 10, 491 (1967) Benzodiazepine alkaloids, 39,63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10,402 (1967) Betalains, 39, 1 (1990) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 439 (1960), 9, 133 (1967), 13, 303 (1971), 30, 1 (1987) occurrence, 16,249 (1977) structure, 16,249 (1977) pharmacology, 16,249 (1977) synthesis, 16, 319 (1977) Bisindole alkaloids, 20, 1 (1981), 37, 1 (1990) isolation, structure elucidation, and biosynthesis of, 37, 1 (1990) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37,205 (1990) therapeutic use of, 37,229 (1990) Bums alkaloids, steroids, 9,305 (1967), 14, 1 (1973) Cactus alkaloids, 4,23 (1954) Calabar bean alkaloids, 2,438 (1952), 8,27 (1965), W, 213 (1971), 36,225 (1989) Calabash curare alkaloids, 8,515 (1%5), 11, 189 (1968) Calycanthaceae alkaloids, 8,581 (1965) Camptothecin, 21, 101 (1983) Cancentrine alkaloids, 14,407 (1973) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum species, pungent principle of, 23,227 (1984) Carbazole alkaloids, 13, 273 (1971), 26, 1 (1985) Carboline alkaloids, 8,47 (1965), 26, 1 (1985) P-Carboline congeners and ipecac alkaloids, 22, 1 (1983) Cardioactive alkaloids, 5,79 (1955) Celestraceae alkaloids, 16,215 (1977) Cephalotaxus alkaloids, 23, 157 (1984) Chemotaxonomy of papaveraceae and fumariaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids, 32,241 (1988)

CUMULATIVE INDEX OF TITLES

343

Chromone alkaloids, 31,67 (1987) Cinchona alkaloids, 14, 181 (1973), 34,331 (1988) chemistry, 3, 1 (1953) Colchicine, 2, 261 (1952), 6, 247 (1960), 11,407 (1968), 23, 1 (1984) Configuration and conformation, elucidation by X-ray diffraction, 22,5 1 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4,249 (1954), 10,463 (1967), 29,287 (1986) Curare-like effects, 5,259 (1955) Cyclic tautomers of tryptamines and tryptophans, chemistry and reactions, 34, 1 (1988) Cyclopeptide alkaloids, 15, 165 (1975) Daphniphyllum alkaloids, 15,41 (1975), 29,265 (1986) Delphinium alkaloid, 4,275 (1954) diterpenoid, 7,473 (1960) Clo-diterpenes, 12, 2 (1970) Czo-diterpenes, 12, 136 (1970) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8, 336 (1965) C 19-Diterpenealkaloids Aconitum, 12,2 (1970) Delphinium, 12, 2 (1970) Garrya, 1 2 , 2 (1970) structure, 17, l(1970) synthesis, 17, l(1979) Czo-Diterpenealkaloids Aconitum, 12, 136 (1970) chemistry, 18,99 (1981) Delphinium, 12, 136 (1970) Garrya, 12, 136 (1970) Distribution of alkaloids in traditional Chinese medicinal plants, 32,241 (1988) Diterpenoid alkaloids Aconitum, 7,473 (1960), 12,2 (1970) Delphinium, 7,473 (1960), 12, 2 (1970) Garrya, 7,473 (1960), 12,2 (1960) general introduction, 12, xv (1970) C19-diterpenes, 12, 2 (1970) Cz0-diterpenes, 12, 136 (1970) Eburnamine-Vincamine alkaloids, 8,250 (1965), 11, 125 (1968), 20,297 (1981) Elaeocarpus alkaloids, 14,325 (1973) Ellipticine alkaloids and related compounds synthesis and antitumor activity of, 39,239 (1990) Elucidation, by X-ray diffraction structural formula, 22,51 (1983) configuration, 22,51 (1983) conformation, 22,51 (1983) Enamide cyclizations, application in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in uitro, 18, 323 (1981) Ephedra bases, 3,339 (1953), 35,77 (1989) Ergot alkaloids, 8,726 (1965), 15, 1 (1975), 38, 1 (1990)

344

CUMULATIVE INDEX OF TITLES

Erythrina alkaloids, 2,499(1952), 7,201(1960),9,483 (1967),18,1 (1981) Erythrophleum alkaloids, 4,265 (1954), 10,287 (1967) Eupomatia alkaloids, 24, 1 (1985)

Forensic chemistry, alkaloids, 12, 514 (1970) by chromatographic methods, 32,1 (1988) Galbulimima alkaloids, 9,529 (1%7), 13,227 (1971) Gardneria alkaloids, 36,1 (1989) Garrya alkaloids diterpenoid, 7,473 (1960) C19 V-diterpenes, l2,2(1970) Czo-diterpenes, 12, 136 (1970) Geissospermum alkaloids, 8,679(1965), 33,84(1988) Gelsem‘um alkaloids, 8,93 (1965),33,83 (1988) Glycosides, monoterpene alkaloids, 17,545 (1979) Guatteria alkaloids, 35,1 (1989) Haplophyton cimicidum alkaloids, 8,673(1965) Hasubanan alkaloids, 16,393 (1977), 33,307(1988) Holarrhena group, steroid alkaloids, 7,319 (1960) Hunreria alkaloids, 8,250(1965) Iboga alkaloids, 8,203(1965),11,79(1968) Imidazole alkaloids, 3,201 (1953), 22,281 (1983) Indole alkaloids, 2,369 (1952), 7,1 (1960),26, 1 (1985) distribution in plants, 11,1 (1968) simple, including P-carbolines and P-carbazoles, 26, 1 (1985) Indole bases, simple, 10,491 (1967) Indolizidine, simple and quinolizidine alkaloids, 28, 183 (1986) 2.2’-Indolylquinuclidinealkaloids, chemistry, 8,238(1965),11,73(1968) Ipecac alkaloids, 3,363 (1953),7,419 (1960), 13,189 (1971),22, 1 (1983) P-Carboline alkaloids, 22,1 (1983) Isolation of alkaloids, 1,1 (1950) Isoquinoline alkaloids, 7,423 (1960) biosynthesis, 4,1 (1954) I3C-NMR spectra, 18,217(1981) simple isoquinoline alkaloids, 4,7(1954),21,255 (1983) Isoquinolinequinones, from actinomycetes and sponges, 21,55 (1983)

Khat alkaloids, 39,139 (1990) Kopsia alkaloids, 8, 336 (1965) Lead tetraacetate oxidation, 36,69(1989) Local anesthetics, alkaloids, 5,211(1955) Localization of alkaloids in the plant, 1,15 (1950),6,1 (1960) Lupine alkaloids, 3,199 (1953), 7,253(1960), 9,175 (1967),31,116 (1987) Lycopodium alkaloids, 5,265(1955),7,505 (1960), 10,306(1967),14,347(1973),26,241

(1985)

Lythracae alkaloids, 18,263(1981), 35,155 (1989)

CUMULATIVE INDEX OF TITLES

345

Mammalian alkaloids, 21, 329 (1983) Marine alkaloids, 24,25 (1985) Maytansinoids, 23,71 (1984) Melanins, chemistry of, 36,253 (1989) Melodinus alkaloids, 11,205 (1968) Mesembrine alkaloids, 9,467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in uitro enzymatic transformation of alkaloids, 18,323 (1981) Mitragyna alkaloids, 8, 59 (1965), 10, 521 (1967), 14, 123 (1973) Monoterpene alkaloids, 16,431 (1977) glycosides, 17, 545 (1979) Morphine alkaloids, 2, 1 (part 1, 1952),2, 161 (part 2, 1952),6,219 (1960), 13, 1 (1971) Mushrooms, alkaloids from, 40, 189 (1991) Mydriatic alkaloids, 5,243 (1955) a-Naphthaphenanthridine alkaloids, 4,253 (1954), 10,485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986) Narcotics, 5, 1 (1955) I3C-NMR spectra of isoquinoline alkaloids, 18,217 (1981) Nuphar alkaloids, 9,441 (1967), 16, 181 (1977), 35,215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11,205 (1968) Ourouparia alkaloids, 8, 59 (1965), 10, 521 (1967) Oxaporphine alkaloids, 14,225 (1973) Oxazole alkaloids, 35,259 (1989) Oxindole alkaloids, 14,83 (1973)

Papaveraceae alkaloids, 10,467 (1967), 12,333 (1970), 17,385 (1979) pharmacology, 15,207 (1975) toxicology, 15,207 (1975) Pavine and isopavine alkaloids, 31,317 (1987) Penraceras alkaloids, 8,250 (1965) Peptide alkaloids, 26,299 (1985) Phenanthrene alkaloids, 39,99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) P-Phenethylamines, 3,313 (1953), 35,77 (1989) Phenethylisoquinoline alkaloids, 14, 265 (1973), 36, 171 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7,433 (1960), 9, 117 (1967), 24, 253 (1985) Picralima alkaloids, 14, 157 (1973) Picralima nitida alkaloids, 8, 119 (1965), 10,501 (1967) Piperidine alkaloids, 26,89 (1985) Plant Biotechnology, for production of alkaloids, 40, 1 (1991) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8,336 (1965), 11,205 (1968) Polyamine alkaloids, putrescine, spermidine, spermine, 22,85 (1983) Pressor alkaloids, 5,229 (1955) Proroberberine alkaloids, 4,77 (1954), 9,41 (1967), 28,95 (1986), 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1988) Pseudocinchona alkaloids, 8,694 (1965) Purine Alkaloids, 38,225 (1990)

346

CUMULATIVE INDEX OF TITLES

Putrescine and related polyamine alkaloids, 22,85 (1983) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11,459 (1968), 26,89 (1985) Pyrrolidine alkaloids, 1,91 (1950), 6, 31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960), 12, 246 (1970), 26, 327 (1985) Quinazolidine alkaloids, see Indolizidine Alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29,99 (1986) Quinazolinocarbolines, 8 , 5 5 (1965), 21,29 (1983) Quinoline alkaloids other than Cinchona, 3,65 (1953), 7,229 (1960) related to anthranilic acid, 17, 105 (1979), 32,341 (1988) Rauwo&a alkaloids, 8,287 (1965) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8,287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9,427 (1967) Sceleriuim alkaloids, 19, 1 (1981) Senecio alkaloids, see Pyrrolizidine alkaloids Secoisoquinoline alkaloids, 33,23 1 (1988) Securinega alkaloids, 14,425 (1973) Sinomenine, 2,219 (1952) Solanum alkaloids chemistry, 3,247 (1953) steroids, 7,343 (1960), 10, 1 (1967), 19,81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermine and related polyamine alkaloids, 22, 85 (1983) Spirobenzylisoquinoline alkaloids, 13, 165 (1971), 38, 157 (1990) Sponges, isoquinolinequinones, 21, 55 (1983) Sremona alkaloids, 9,545 (1967) Steroid alkaloids Apocynaceae, 9,305 (1967), 32,79 (1988) Buxus group, 9,305 (1967), 14, 1 (1973), 32,79 (1988) Holarrhena group, 7,319 (1960) Salamandra group, 9,427 (1967) Solanum group, 7,343 (1960), 10, 1 (1967), 19,81 (1981) Verarrum group, 7,363 (1960), 10, 193 (1967), 14, (1973) Stimulants respiratory, 5, 109 (1955) uterine, 5, 163 (1955) Structural formula, elucidation by X-ray diffraction, 22,51 (1983) Strychnos alkaloids, 1,375 (part 1, 1950),2,513 (part 2, 1952), 6, 179 (1%0), 8,515, 592 (1965), 11, 189 (1%8), 34,211 (1988), 36, 1 (1989) Sulfur-containing alkaloids, 26,53 (1985) Taxus alkaloids, 10,597 (1%7), 39, 195 (1990) Toxicology, Papaveraceae alkaloids, 15,207 (1975)

CUMULATIVE INDEX OF TITLES

347

Transformation of alkaloids, enzymatic, microbial and in uitro, 18,323 (1981) Tropane alkaloids, 1,271 (1950), 6, 145 (1960), 9,269 (1967), 13,351 (1971), 16,83 (1977), 33, (1988) Tropoloisoquinoline alkaloids, 23,301 (1984) Tropolonic Colchicum alkaloids, 23, 1 (1984) Tylophora alkaloids, 9,517 (1967) Uterine stimulants, 5, 163 (1955) Verarrum alkaloids chemistry, 3,247 (1952) steroids, 7,363 (1960), 10, 193 (1967), 14, 1 (1973) Vinblastine, 37, 133 (1990) Vinblastine-Type Alkaloids, 37,77 (1990) “Vinca” alkaloids, 8, 272 (1965), 11,99 (1968), 37, 1 (1990) Voacanga alkaloids, 8,203 (1965), 11,79 (1968)

X-Ray diffraction, elucidation of structural formula, configuration, and conformation, 22, 51 (1983) Yohimbe alkaloids, 8,694 (1965), 11, 145 (1968), 27, 131 (1986), see also Coryantheine

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A Acetylenic amino acids, in mushrooms, 303 17-O-Acetylnorajmaline, 146 Acetylnorrauglucine, 147 Acetylrauglucine, 147 Acetyltropine, 69 Aflatoxin, 234 Aflatrem, 250 Aflavinine, 247,249 Agaricone, 279 Agaridoxin, 300 Agaritine, 276 biosynthesis of, 278 synthesis of, 278 Agroclavine, 236 Ajmalicine, 110, 114, 117 Ak-toxins, 322 Aladeoxoviroidin, 219 Alaviroidin, 219 Alkaloids from, Lactarius necator, 272 Alkaloids, production by genetically engineered microorganisms, 6 production by plant cell cultures, 8 production by plants, 8 production of, 2 production prices, 4 types of, 2 precursor bioconversion, 7 Allomuscarine, 195 synthesis of, 197 Amanin amide, 218 Amanin, 218 Amanita, physiological principles, 194 Amanitin, 217, 218 Amanullin, 218 Amanullinic acid, 218 Amatoxins, 218 Amauronine, 253

Amino acids, in mushrooms, 289 4-Aminopyridine-2,3-dicarboxylicacid, 295,296 Anabasine, 45 Anatabine, 45 Anatmanide, 225 3',4'-Anhydrovinblastine, 139 Antiphallotoxin, 225 Apparicine, 9 Apparicine, 151 Arcyriarubin B, 230 Aromelic acids, 290,291 synthesis of, 293, 294 Aromoline, 76 Ascochalasin, 3 11 Aspergillic acids, 227 Aspochalasin, 319 Atropine, 52 Austamides, 249,251

B Baeocystine, 229 Benzylisoquiniolines, biosynthesis of, 79 Berbamine, 76 Berbamunine, 76 Berberine, from Berberis species, 94 from Coptis species, 95 occurrence in plant tissues, 98 patents on bioproduction, 96 Betalamic acid, 209,215 Betalanine alkaloids, 208 Betanidine, 209 Betaxanthins, 209 4,4'-Binecatorone, 271 Biosynthesis of, ajmalicine, 19J berberine, 19,72,94 indole alkaloids, 19 349

350

INDEX

ipecuhana alkaloids, 72 scopolamine, 19 Bisbenzylisoquinoline alkaloids, production by cell cultures, 75 patents, 75 Bissecodehydrocyclopiazonic acid, 253 Bohemamine, 323 Brevianamides, 249,252 Bufotenine N-oxide, 227 Bufotenine, 226 Bufothionine, 227 C

Caffeine, 154,155 Camptothecine, 153 Carbolin-1-propionic acid, 231 p-Carbolines, 231 p-Carbolines, 231 p-Hydroxyhyoscyamine epoxidase, 71 Cardycepin, 283 Catharanthine, 114,139 Cathenamine, 151 Cephaeline, 73 Cephalotaxine, 75 Cephalotaxus alkaloids, 75 production by cell cultures, 75 Chaconine, 157 Chaetoglobosin, 310 Chaliciporones, 305 Chanolclavine I, 236 Cheilanthifoline, 79 Chelerythrine, 88 Chelilutine, 88 Iq 2 Chelirubine, 88, 12 Chrysanthone, 322 Cinchona alkaloids, 104 plant biotechnology, 105 cell cultures, 105 extraction of, 107 bioconversions, 108 patents, 105 Cinchoninone, 108 Clavine alkaloids, 236 Clithioneine, 291 Clitidine, 281 Clitocine, 285,286 Codeine, 77,81 Codeinone, 84 Colchicine, 74

Conessine, 161 Connatin, 280 Connatin, 304 Coprine, 298 Cortinarins, 268 Cryptopine, 83,86 Culture types, 18 Cuscohygrine, 68 Cyclopiazonic acid, 253 Cyclopiazonic acid imine, 253 Cyclostizolobic acid, 213 Cytochalasanes, 307-310 Cytochalasins, 312-318 D Dehydrobufotenine, 227 Dehydrocommersonine, 159 Deoxophomin, 311 Deoxoviroidin, 219 Deoxoviroisin, 219

lO-Deoxy-4,4’-binecatorone, 271 10,10‘-Dideoxy-4,4’-binecatorone, 271 Dihydrochelerythrine, 88 Dihydrochelirubine, 88 Dihydromarcapine, 88 Dihydrosanguinarine, 87 Dihydrosetoclavine, 244 Domoic acid, 291

E Echinulines, 249,251 Elegagnine, 233,235 Ellipticine, 150 Elymoclavine, 236 Emetine, 73 Engleromycin, 315 Epchrosine, 151 Epiallomuscarine, 195 Epimuscarine, 195 synthesis of, 197 Epivincamine, 150 Equisetin, 322 Ergine, 237 Ergoannam, 246 Ergobasine, 238 Ergocornam, 245 Ergocorninam, 245

35 1

INDEX

Ergocornine, 245 Ergocorninine, 245 Ergocristam, 243,245 Ergocristam, 245,246 Ergocristine, 245 Ergocristinine, 245 Ergocryptam, 245 Ergocryptianm, 245 Ergocryptines, 245,246 Ergoline, 236 Ergometrine, 237 Ergonine, 245 Ergoninine, 245 Ergonovine, 237 Ergonovine, 239 Ergonovinine, 239 Ergopeptide alkaloids, 242 Ergopeptine, 242 Ergopeptide alkaloids, tabulation of, 245 Ergophine, 245 Ergophinine, 245 Ergosine, 245 Ergosinine, 245 Ergostetrine, 238 Ergostine, 245 Ergostinine, 245 Ergot alkaloids, 233 Ergotamine, 242,243,246 Ergotaminine, 245 Ergothioneine, 295,2% Ergotocin, 238 Ergovaline, 245 Ergovalinine, 245 Eritadenine, 287, 288 Escholtzia species, alkaloid production, 93 Eudistomine S, 232

F Fuligorubin A, 320 Fumitremorgins, 249, 251

G Genetic engineering, 142 Genetic modification, 19 Gliovictin, 323 -~-Glutaminyl-3,4-benzoquinone, 301 ~-Glutaminyl-4-hydroxybenzene, 300

Grzymaline, 267 Gyromitrine, 275

H Harman, 232,235 Harringtonine, 75 Hercynine, 195 L-Hercynine, 297 Homodeoxyhamngtonine, 75 3-Hydroxyvoafrine A and B, 152 4-Hydroxymethylphenylhydrazine,277 5-Hydroxytryptophan, 227 6-Hydroxymethylnebularine,287 6P-Hydroyhyoscyamine, 64 &Hydroxyergine, 237 %Hydroxyerginine, 237 Hyoscyamine-P-hydroxylase, 71 1-Hyoscyamine, 64 Hydrazine alkaloids, 275 Hydroxyinfractin, 232, 233 1

Ibotenic acid, 195,203 biosynthesis of, 208 photoreaction of, 205 synthesis of, 207 Ilicicolin, 322 Illudalic acid, 274 Illudin, 274 Illudinine, 272, 274 Illudol, 274 Indicaxanthin, 212 Indigo, 229 Indirubin, 230 Indole alkaloids biotechnology of, 109 patents on bioproduction, 111 high producing cell lines, 110 culture medium, 113 precursor feeding, 122 bioregulators, 123 large-scale culturing, 129 economics, 137 production of dimeric alkaloids, 138 bioconversions, 138 plant biotechnology, 141 Infractin, 233

352

INDEX

Infractopicrin, 231 Isobetanidine, 210 Isofumigaclavine, 236 Isolysergic acid, 237 Isoquinoline alkaloids, 72 Isoreserpiline, 151 Isothebaine, 83

J Jatrorrhizine, 76,95 K

Kainic acid, 291,292

L Lanosulin, 251 Lentinacin, 287 Lentysine, 287 Lepistine, 305 Leucoagaricone, 279 Loline, 322 LSD, 241 Lyophyllin, 230 Lysergic acid amide, 237 Lysergic acid, synthesis of, 239 Lysergol, 236

M Macarpine, 38 Macleaya species, Magnoflorine, 83,86, 101 Marcofortines, 252 Melanin, 215 Meleagrin, 323

N-Methyl-N-formylhydrazine,275 Methyl-10-methoxy-paspalate, 238 6-Methylnebularine, 287 10-Methoxyellipticine, 150 Miraxanthins, 212 Monogagamine, 153 Morphine, biosynthesis of,79 Muscaflavin, 210,215 Muscapurpurin, 214 Muscapurpurinic acid, 215 Muscaridine, 195 Muscarines, 194 synthesis of, 197

Muscarufin, 209 Muscaurins, 210,212 Muscazone, 195 Muscimol, 195, 203

N Narceine, 77 Nebularine, 286 Necatorin, 269 Necatorone, 269 synthesis of, 270 Necatorones, 269 Neobetanidine, 209 Neoechinulines, 249, 251 Nicotine, by root cultures, 50 in large-scale suspension cultures, 48 production in tissue cultures, 44 Nominine, 247,249 Nonmorphinan alkaloids, 83 Norbaeocystine, 229 2-Norberbamunine, 76 P-Nitroaminoalanine, 304 biosynthesis of, 7,72 N(8)-Norphysostigmine, 154 precursor bioconversion, 7 synthesis of, 293,294 Norlluorocurarine, 151 Nornicotine, 45 Norsanguinarine, 88 Noscapine, 83 Noscopine, 77 I

0 Olivoretins, 323 Opium alkaloids, biotechnology of, 78 patents on biotechnology methods, 78 bioconversions of, 84 Optisine, 95 Orellanine, 253 Orellanine, 254 demethylation of, 258 fluorescence of,263 mass fragmentation of, 262 model compounds, 261 photodecomposition of, 266 synthesis of, 256,257,259 thermal decomposition of, 259,260

353

INDEX

toxicity of, 264 X-ray analysis of, 262 Orientalidine, 83 Oxysanguinarine, 87

P Palmatine, 95 Pantherine, 203 Papaver species, alkaloid production, 93 Papaverine, 77 Paraherquamide, 252 Paspalic acid, 238 Paspaline, 250 Paspalinine, 250 Paspalitrem B, 250 Paxilline, 250 Penitrems, 250 Penniclavine, 236 Peptide alkaloids, 216 Peramine, 323 Pericine, 9 Phallacidin, 219 Phallacin, 219 Phallisacin, 219 Phallisin, 218 Phalloidin, 217, 219 Phalloin, 219-221 Phallolysin, 224 Phallotoxins, 218, 220 Phomopsin A, 321 Physarochrome A, 321 Physostigmine, 154 Picralina nitida, alkaloids of, 9 Piperine, 321 Pistillamine, 305 Plant biotechnology, cell density, importance of, 13 cell populations, selection of, 11 cellular transport, 15 culture conditions, 12 elicitation in alkaloid formation, 16 feeding of precursors, 16 gas composition, 14 immobilization of plant cells, 17 light, importance of, 13 medium composition, 12 pH, influence of, 12 premeabilization of cells, 17

screening, 11 storage compartments, 14 temperature, importance of, 13 Plant cell cultures, new compounds from, 9 Pleiocarpamine , 15 1 Proamanullin, 218 Process design of, 32,40 Production of alkaloids bioreactors, 34 cost estimation, 25 design stratergy for production of, 22 economic feasibility, 22 forced release of alkaloids, 36 large-scale production of, 20 market volume of alkaloids, 24 process parameters, 40 recovery from biomass, 35 recovery from medium, 37 Prophalloin, 219,221,223 Protophomin, 310 Protopine, 79 Proxiphomin, 3 11 Psalliotin, 230 Psilocin, 223 Psilocybin, 228 Pulcherriminic acids, 227 Pyrichalasin, 3 17 Pyridine alkaloids, 253

Quinidine, 104 Quinidinone, 108 Quinine, 104 Quinone, 301

Q

R Raucaricine, 145 Rauglucine, 147 RauvolJa alkaloids, 142 plant biotechnology, 144 cell cultures, 144, 147, 148 patents on biotechnology of, 147 enzyme production, 149 (R)-Reticuline, (S)-Reticuline, 79 Rhizonin A, 321 Roquefortine, 25 1 Rugulovasine A, 236

354

INDEX

S

Sanguinarine, 79,85 antimicrobial properties, 85 elicitation, 87 growth conditions, 86 patents for bioproduction, 86 Scopolamine, 52,64 Scoulerine, 79 Secologanin, 7 Serotonin, 225 Serpentine, 110, 114, 117 Silybin, 225 Solamargine, 157, 159 Solanidine, 157 Solanine, 157 Solasodine, 157, 159 Solasonine, 157, 159 Stizolobic acid, 210,212,215 Stizolobinic acid, 215 STOX (S)-tetrahydroberberine oxidase, 7,72 Strictosidine lactam, 150 Strictosidine synthase, 6 , 7 Stylopine, 79,83 T Tabersonine, 151 Tenuazonic acid, 320 Tetrahydroalstonine, 151 Tetrahydroharman-3-carboxylic acid, 23 1 from Thalictrum species, 100 Thalifendine, 101 Thebaine, 77,81 Theobromine, 154, 155

Tomatidine, 161 Trichomolic acid, 204 Tropane alkaloids, 68 large-scale cultures, 68 precursor feeding, 69 plant biotechnology, 71 Tropine, 69 Tryptathionine thioether, 223 Tryptophan decarboxylase, 6 Tryptophan, 227 Tubigensin A and B, 250

V Vermcofortin, 25 1 Vermculogen TR-2,249,251 Vinblastine, 109, 137, 139 Vinca alkaloids, 149 Vincamine, 149 Vincrictine, 109, 137 Vindoline, 139 Viroidin, 219, 222 Viroisin, 219 Virotoxins, toxicity of, 221 Voafrine A and B, 152 Vomilenine, 145 Vulgaxanthins, 212

X Xanthodermin, 279,280 Z

Zygosporins, 3 14-3 16