Aspects Op Secondary Metabolism in Basidiqmycetes

ASPECTS OP SECONDARY METABOLISM IN BASIDIQMYCETES

BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS

A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF

LENTINUS AND LENTINELLUS

by by.' . WEI-WEI/WANG

B.Sc, National Taiwan University, 1974

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in

THE FACULTY OF GRADUATE STUDIES

(DEPARTMENT OF BOTANY)

We accept 'this thesis as conforming

to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

November, 1977

f7\ Wei-Wei Wang, 1977 In presenting this thesis in partial fulfilment of the requirements for

an advanced degree at the University of British Columbia, I agree that

the Library shall make it freely available for reference and study.

I further agree that permission for extensive copying of this thesis

for scholarly purposes may be granted by the Head of my Department or

by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my

written permission.

Department of Botany

The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5

Date December 6. 1977 ii

ABSTRACT

I. Psilocybe cubensis was cultured successfully in two media. Medium A

was devised by Catalfomo and Tyler and Medium B was a modification of a

medium which has been used for ergot alkaloid production by Claviceps

purpurea. Only when the fungus was kept on Sabouraud agar plates.did it

subsequently produce psilocybin when transferred to liquid media. A

quantitative time-course study of psilocybin production in the two media

was carried out. Maximal production appeared on the fifth day. The

activities of an acid phosphatase, acting on psilocybin, were measured

from mycelia grown in the two media. Enzyme activity from the A culture

was very high and a blue color caused by oxidation of psilocin formed in

five minutes.

The effect of adding L-tryptophan on alkaloid production as well as 14

the fate of tryptophan-C was also investigated. Tryptophan stimulated

significantly psilocybin production in the very beginning in the B medium.

The degradation of tryptophan was different in the two media. It was

converted to kynurenine and anthranilic acid in A medium and to tryptamine

in tryptophan added B medium (B' medium). Radioactive D,L-tryptophan

side chain labeled, gave labeled psilocin and psilocybin.

Potassium deficiency decreased psilocybin production while a potassium

supplement had no effect. The fungus did not produce polyacetylenic

compounds in the medium but ergosterol was detected as a major acetate

derived metabolite when the fungus was kept on MYP agar plates and

transferred subsequently to liquid media. Psilocin has very slight anti•

biotic activity against Candida albicans whereas psilocybin has none. iii

II. Eight species of Lentinus and Lentinellus. were investigated for the

occurrence of a phenol-O-methyltransferase. Only Lentinus lepideus and

Lentinus pbnderbsus showed enzyme activity in both light and dark

conditions. The specificity of the enzyme for a number of substrates was

also examined. Of six compounds tested, methyl p-coumarate, methyl

caffeate and methyl ferulate.served-as substrates. The products of

enzymic activity were identified-by radioautography. iv

TABLE OF CONTENTS

PAGE

ABSTRACT ii

TABLE OF CONTENTS;. iv

LIST OF TABLES vi

LIST OF FIGURES vii

ACKNOWLEDGEMENT ix

I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS

INTRODUCTION 1

REVIEW OF LITERATURE 3

MATERIALS AND METHODS

A. Chemicals 13

B. Medium and culture 15

C. Analytical methods 17

D. Radioautography 20

E. Psilocybin phosphatase activity 21

F. Potassium nutrition and psilocybin production 23

G. Test of UV-mediated antibiotic and phototoxic

activities of medium, psilocin and psilocybin 24

EXPERIMENTAL AND RESULTS

A. Chromatographic separation and identification of

tryptophan metabolites 25

B. pH, Growth and morphological difference of cultures 26

C. Psilocybin production 27

D. Formation of psilocybin and psilocin from tryptophan 28 V

E. Phosphatase activity 39

F. Psilocybin production and potassium nutrition 39

G. Ehrlich-positive compounds in the extracts 42

H. Tryptophan concentration in the medium 42

I. Antibiotic activity of psilocin, psilocybin and

medium filtrate 49

J. Identification of d(-)mannitol 49

DISCUSSION 54

LITERATURE CITED 63

II. A SURVEY OF PHENOL-0-METHYLTRANSFERASE IN SPECIES OF

LENTINUS AND LENTINELLUS

INTRODUCTION 69

MAT ERI AL S A ANDI1ME THOD S

A. Chemicals 73

B. Medium and culture 74

C. Extraction of enzyme 75

D. Enzyme assay 75

E. Identification of reaction products 76

F. Protein concentration 76

RESULTS AND DISCUSSION 77

LITERATURE CITED 81 vi

LIST OF TABLES

page

I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS

1. Some Tryptamine Derivatives Found in Fungi 3

2. Fungi Containing Psilocybin 6

3. Composition of A-medium 15

4. Composition of B-medium 16

5. Fluorescence, Color Reaction and R^ Values of

Some Tryptophan Metabolites 25

6. Distribution of Radioactivity in Tryptophan,

Psilocybin and Psilocin of Mycelium Extract, 14

Administered D,L-Tryptophan, side chain-3-C 28

7. The Growth, pH of Medium and Psilocybin Production

of B-medium, Potassium Deficient and Supplement Media 39

II. A SURVEY OF PHENOL-0-METHYLTRANSFERASE IN SPECIES OF

LENTINUS AND LENTINELLUS

1. Substrates Specificity of Methylating Enzymes from

Lentinus ponderosus and Lentinus lepideus 78 vii

LIST OF FIGURES

page

I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS

1. Locations of some standard tryptophan metabolites on a

two-dimensional cellulose thin layer plate 29

2. pH of A and A' media 30

3. pH of B and B' media 31

4. Growth rate of fungal cells in A and A' media 32

5. Growth rate of fungal cells in B and B' media 33

6. UV spectrum of authentic psilocybin -.34

6!. UV spectrum of psilocybin isolated from fungal cells 35

7. Psilocybin production from A and A' media 36

8. Psilocybin production from B and B' media 37

9. Radioautograph of chromatographed tryptophan

metabolites of a mycelium extract of Psilocybe cubensis,

administered D,L-tryptophan 38

10. Blue color formation when psilocybin incubated

with enzyme extract 40

11. Activities of acid phosphatases of the mycelia

from two different media 41

12. Chromatograms of mycelium extract and medium extract

of different growth period 43

13. Tryptophan concentration of A' and B' media 48

14. Antibiotic activity of psilocin 50

15. IR spectrum of white crystals 51

16. NMR spectrum of acetate derivative of white crystals 53

17. UV spectrum of ergosterol 55 viii

A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF

LENTINUS AND LENTINELLUS

1. Radioautographs of the chromatographed methylated

clnnamate products ix

ACKNOWLEDGEMENT

I wish to express my appreciation to Dr. G. H. N. Towers for his suggestion of this project and his advice, criticism and encouragement.

I like to thank sincerely to Dr. C. K. Wat for her helpful assist• ance and guidance; to the members of committee in reading the manuscript and making critical comments; and.to the faculty, staff and students of the Department of Botany for being helpful in many ways.

I also acknowledge with gratitude to Mr. Terry Q. F. Ching for his invaluable assistance in the preparation of the manuscript; to Miss Yvonne

Tang for proofreading the final manuscript; and to Mr. H. P. Hsu for his contribution in many ways. X

I, I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS 1

INTRODUCTION

Intoxication, following the ingestion of certain kinds of mushrooms has. been known as early as the tenth century in the Sung Dynasty of China

(69) and an 11th Century tale from Japan is about the eating of "dancing mushrooms" and "laughing mushrooms" (4, 69). Of the psychotropic materials utilized by ancient Nahuatl people of the New World, teonanacatl ("God's mushrooms" or "God's flesh") especially stands out in Mexican history (3).

Since the pre-Columbian era, the Indians of Mexico have made the eating of certain fungi a part of their religious rites (15). The first record of the use of hallucinogenic mushrooms dates from 1502 at the Aztec Coronation festivals of Montezuma II (3, 70). This was recently rediscovered by

R. G. Wasson and U. P. Wasson in 1957 (70). The active hallucinogenic principle was isolated from Psilocybe and therefore named psilocybin

(4-phosphoryl-N,N-dimethyltryptamine). Psilocin is the dephosphorylated derivative. They are the first natural indole derivatives found to possess an OH group in position 4 and psilocybin is the first one known to contain a phosphate group (71).

The chemical synthesis of these two indole alkaloids has been well 14

studied. The biosynthesis has only been studied by feeding C -labeled precursors and from these results a pathway has been proposed. Enzymological

studies are meager. One of the objectives of the present study was to

find better growth conditions for the production of psilocybin so as to

lead to further studies on enzymology of biosynthesis and psychopharmaco-

logy.

Psilocybin has been demonstrated to be derived from tryptophan. An

objective of this study concerned the fate of added tryptophan and of

labeled tryptophan. 2

It was interesting to find out if there is any biological significance in the production of these two indole compounds. A brief examination of cultures for other obvious secondary metabolites, such as polyacetylenes, was also carried out. 3

REVIEW OF LITERATURE

A. The occurrence of tryptamine derivatives in higher fungi

Certain tryptamine derivatives (I) to which psychotomimetic,

properties have been ascribed occur in a number of the Basidiomycetes

(1, 2, 3, 4). Serotonin (II), N-methylserotonin (III), bufotenine (IV)

bufotenine N-oxide (V), N,N-dimethyltryptamine (VI) and 5-methoxy-N,N-

dimethyltryptamine (VII) have been found in either carpophores or

mycelia of species of Amanita (Amanitaceae), Panaeolus, Coprinus

(Coprinaceae), Boletus (I('B6>letaeeaeO) and Sarcodpn (Thelephoraceae)

(Table 1) (5, 6, 7, 8, 9, 10).

Table 1. Some Tryptamine Derivatives Found in Fungi Species Compound Amanita citrinaa. II, Amanita mappa IV Amanita muscaria IV Amanita pantherina IV Amanita porphyria II, Amanita tomentella !V Ranaeolus foenisecii II Panaeolus semiovatus II Panaeolus sphinctrinus II Panaeolus subbalteatus II Coprinus atramentarius I Coprinus comatus I Coprinus micaceous I Boletus erythropus I Sarcodon imbricatum I

Psilocybin (VIII) and psilocin (IX) have been shown to be responsible

for the hallucinogenic effects of mushrooms of the genus Psilocybe

(Strophariaceae). Two analogs of psilocybin, baeocystin (X) and 4

•N-CH,

N-Methylserotonin Bufotenine

(5-Hydroxy-N,N-dimethyltryptamine)

III IV

•N-CH, 0H-

Bufotenine N-oxide N,N-Mmethyltryptamine

V VI 5 0 II _ HO-P-0

5-Methoxy-N,N-dimethyltryptamine Psilocybin (4-Phosphoryl-N-N-dimethyltryptamine)

VII VIII

-N-CH- I 3

Baeocystin Psilocin (4-Phosphoryl|rN^monomethyltryptamine) (4-Hydroxy-N,N-dimethyltryptamine) X IX

Nor-baeocystin H (4-Phosphory1tryptamine) Ergoline ring XI XII 6

nor-baeocystin (XI) have been found in Psilocybe baeocystis (11, 12, 13)

and recently baeocystin was detected in Psilocybe semilanceata (14).

Baeocystin and nor-baeocystin are found to be toxins (74).

B. Chemical synthesis of psilocybin and its physical properties

Hofmann (15) reported the chemical synthesis of psilocybin in 10

stages from O-nitrocresoll'.- This was a commercial synthesis and is

outlined in Scheme I.

Psilocybin and psilocin have been isolated from Psilocybe and

identified critically (15, 16, 17). Psilocybin forms colorless,

monoclinic crystals, soluble in methanol but practically insoluble in

the usual organic solvents. The melting point is 185-195 °C and UV

absorption maxima in methanol are 220, 267, 290 nm (loge 4.6, 3.8, 3.6).

C. Distribution of psilocybin

Psilocybin has been identified in mycelia, spores, carpophores or

s'cierbt:ia

and Psilocybe (Table 2).

Table 2. Fungi Containing Psilocybin Species Reference Conocybe cyanopus 9, 12 Conocybe smithii 9, 12 Panaeolus africanus 4 Panaeolus ater 4 Panaeolus cambodginiensis 4, 18 Panaeolus campanulatus 4 Panaeolus castaneifolius 4 Panaeolus cyanescens 4 Panaeolus fimicola 4 Panaeolus foenisccii 4, 20 7

Panaeolus retirugis 4 Panaeolus sphinctrinus 4, 9, 12 Panaeolus subbalteatus 4, 9, 18 Panaeolus tropicalis 4 Psilocybe aztecorum 9, 12 Psilocybe baeocystis 9, 12 Psilocybe bonetii 18 Psilocybe caerulescens 12 Psilocybe eaerulipes 4, 12 Psilocybe collybioides 3 Psilocybe cubensis 1, 9, 12 Psilocybe cyanesis 9, 12 Psilocybe fimetaria 9, 12 Psilocybe mexicana 9, 12, 20 Psilocybe mixanesis 9, 12, 21

Psilocybe pelliculosa 9, 12 Psilocybe quebecensis 22 Psilocybe semilanceata 9, 12

Psilocybe semperviva 9, 12, 21 Psilocybe stricitipes 25 Psilocybe stuntzii 18 Psilocybe subaeruginosa 3, 24 Psilocybe wassonii 9, 12 Psilocybe zapotecorum 9, 21

Other species of Psilocybe have been reported to be hallucinogenic

; and/or Coxi" , bul whether ;'• v" • -v:- . ..• ^ .jcy^iu .-- • -/ and/or toxic, but whether or not they contain psilocybin is not known eg,. Psilocybe r> o: issir\, Psilocv.Le agge- id.-r.-4 , IVixocvbe p.-_-

•ea g>0Psi1 ocybggacu 11ssima-, Psilcycybe\ agg.er.i.cola,^Psilocybe-,bolivari,

PsixocyBe b6 o^sbligehii^aPsilb'cybe callosa, Psilocybe candidipes,

Psilocybe cordispora, Psilocybe fagicola, Psilocybe fasciata, Psilocybe isauri, Psilocybe kumaenorum, Psilocybe macrocystis, Psilocybe silvatica, Psilocybe subcaerulipes, Psilocybe yungensis (3)i and

Psilocybe argentipes (26). 8

OCH_C,H_ I 2 6 5

0=P-0CHoC,H Cl

Scheme I Nutritional studies

Heim and Cailleux succeeded in growing several mushrooms belonging

to the family of Strophariaceae and even obtained fruiting bodies, on natural substrates (15). Submerged cultures of Psilocybe was first

attempted by Catalfomo and Tyler (1). They developed conditions for the

production of mycelialrpellets yielding psilocybin in shaken culture of

Psilocybe cubensis. However, Psilocybe cyanescens and Psilocybe

pelliculosa failed to produce psilocybin under these conditions. Of

various species of the family Strophariaceae studied by Leung (12), only

Psilocybe baeocystis was successfully grown on a modified nutrient

solution of Catalfomo and Tyler. The fungus was found to produce

psilocybin and baeocystin under these conditions. The nutrient solution

used for producing ergot alkaloids of Claviceps species has been tried

with Psilocybe but without success. The influence of phosphate on the

distribution pattern of nitrogen between soluble and insoluble cellular

components was studied on Psilocybe cubensis, Psilocybe cyanescens and

Panaeolus campanulatus (27). Kitamoto et al. (28) showed that Psilocybe

panaepllf-ormisjformed fruiting bodies within two weeks on a liquid medium

consisting of glucose, amino acids, mineral salts and various growth

factors. Ammonium and nitrate nitrogen were utilized for mycelial

growth but not for fruiting. Casamino acids were a good nitrogen source

for fruiting.

Biogen;esiso:5fp psMocy.bin

The structural similarity between psilocybin and tryptophan suggests

that it is derived from that amino acid. Brack et al. (23) demonstrated

that psilocybin isolated from surface-cultures mycelium of Psilocybe

semperviva was derived from labeled tryptophan added to the medium. 10

14 Agurell and Nilsson (2, 29, 30) using C -labeled substrates demonstrated a biosynthetic sequence from tryptophan to psilocybin via decarboxylation,

N-methylations, hydroxylation and phosphorylation (Scheme II). Their results indicated however that this is not the only pathway to psilocybin

and that a grid of pathways probably exists in the fungus.

H H

Scheme II

F. Blueing phenomenon in psilocybin-containing mushrooms

Psilocybin-containing mushrooms are well known for the blueing

which results from damage, Although the blueing reaction has not been

studied in hallucinogenic mushrooms per se, a number of other studies 11 shed light on this phenomenon (4). Blaschko and Levine (31, 32) found that an enzyme from the gill plates of the mussel (Mytilus edulis) or ceruloplasmin, a copper containing oxidase, from pig plasma, converted psilocin to a blue product. Horita and Weber (33) found that psilocybin was readily dephosphorylated by various mammalian alkaline phosphatases and that the liberated psilocin was oxidized to blue metabolites.

Using a preparation of cytochrome oxidase, they (34) found psilocin was oxidized to a dark blue product and the synthetic hallucinogen 4-hydroxy-

N,N-diethyltryptamine to a bluish-green metabolite. 4-Hydroxy-tryptamine gave a blackish-brown pigment. Levine (35) found that the oxidative formation of a blue color could also be elicited without enzyme in the presence of ferric ion. EDTA blocked this reaction. With psilocybin, neither formation of blue color nor uptake of 0^ occurred during incu• bation with ceruloplasmin or ferric ion.

para-Diphenol oxidase (laccase), a copper containing enzyme from the fungus Polyporusv versicolor, is capable of oxidizing psilocin to a blue product (36). The blue product is hydrophilic and has a;;sp'ectrum withtabsorption at 610 nm and 395 nm. The blue product was readily reduced to a colorless product by ascorbic acid and is thus probably quinonoid in nature. It appears that a free OH group is essential for oxidation because psilocybin is not oxidized under identical conditions.

Bocks;(37) also found a laccase type of enzyme in homogenates of the mycelium of Psilocybe cubensis which is capable of catalyzing the

oxidation of 2,6-dimethoxyphenol and of psilocin. The mechanism of

the catalytic action of laccase on 2,6-dimethoxyphenol could be written as follows (38). 79); " 12

Adventitious browning occurs in the flesh of certain ripe fruits

such as apples and pears following injury and this rapid browning

results from the oxidation of endogenous phenols by enzymes having

laccase and/or phenolase activity. The blueing reaction in Psilocybe

is most likely a similar type of reaction mediated by these types of

oxidizing enzymes (4).

G. Other chemical constituents of Psilocybe

Very little chemical work has been carried out on members of this

genus. Brack e_t al. (73) reported the production of certain ergot

alkaloids in Psilocybe semperviva. Polyacetylenic compounds, such as

s 2 the triynol, CH3-C C-C C-C=C-CH20H (2,4,6-octatriynol) and triynoic

acid CH3-C=C-C=C-C=C-COOH (2,4,6-octatriynoic acid) were found in the

culture fluids of Psilocybe merdaria (39). The polyacetylene,

HC=C-C=C-C=C-C=C-COOH (4,6,8-nonatriyn-2-enoic acid) was reported from

Psilocybe sarcocephala (40). These fungal acetylenes often show anti•

biotic activity. 13

MATERIALS AND METHODS

Chemicals: The source of the chemicals used was as follows:

Chemical Source

MgS04. 7H20 Fisher Scientific Co.

(NH4)6MS7024. 4H20 Fisher

ZnSO,. 7H.0 B. D. H. Laboratory 4 2

MnCl2. 4H20 B. D. H.

FeSO.. 7H.0 Allied Chemical Ltd. 4 2

CuS04. 5H20 Fisher

NaN0 Mallinckrodt Inc. 2,

KH2P04 Fisher

NaHoP0. Fisher 2 4 KCI Fisher

NaOH Mallinckrodt

NH. OH Allied 4 Allied H2S°4 HC1 Allied

NaCI Amachem

NaCN Mallinckrodt

(NH4)2C03 J. T. Baker Chemical Co

Acetic acid, glacial Allied

Isopropanol Amachem

Methanol Fisher

n-Butanol Fisher

Thiamine hydrochloride J. T. Baker

D(-)Glucose Mallinckrodt

D(-)Mannitol ICN

Sucrose Fisher 14 Yeast extract Difco Laboratory

Sabouraud dextrose agar Difco

Malt extract Difco

Soytone Difco

Glycine Fisher

L-Tryptophan Sigma

p-Dimethylaminobenzaldehyde Matheson Coleman and Bell Co.

Tryptamine hydrochloride Sigma

N-Methyltryptamine Sigma

N, N-5-Dimethyltryptamine Gifts from the Dept. of National'- Psilocin •Health and Welfare, Health Protection Branch, Ottawa Psilocybin

5-Hydroxytryptophan Sigma

5 -Hydroxy t ryp t amine (oxalate in ) (serotonin) Sigma

Kynurenine Sigma

Kynurenic acid Sigma

Anthranilic acid Calbiochem

3-Hydroxyanthranilic acid Mann Research Laboratory

3-Methy1indole(skatole) Sigma

r3=Ethanol indole(tryptophol) Sigma iiua Urea Fisher

Allantoic acid ICN

Allantoina Eastman OrganicCChemicals

N-Acetyl-D, L-tryptophan Sigma 14 D, L-Tryptophan(side chain-3-C ) specific activity 3.76 mCi/m mole ICN (2.71 mg 50 uCi) Aquasol New England Nuclear

Dowex 50 WX-8 J. T. Baker 15

Phenolphthalein . diphosphate (PDP) Sigma

l-Nitroso-2-naphthol BDH

Ethylene dichloride Matheson Coleman and Bell Manufact• uring Chemists

Ammonium succinate was prepared by adding 40 ml NH^OH to 11.8 gm

succinic acid.. To this solution, 95% ethanol was added until

white crystals precipitated. The crystals were collected and

air dried.

Medium and culture

Medium:

Two kinds of liquid media were employed in this work. A-medium

was devised by Catalfomo and Tyler (1) (Table 3). B-medium was a

modified version of Stoll's medium for ergot alkaloid production from

Claviceps purpurea (41) (Table 4).

Table 3. Composition"offA^medium-s Ammonium succinate 1.0 gm Glycine 9.0 gm D(-)Glucose 10.0 gm Yeast extract 0.5 gm

KH2P04 0.1 gm Thiamine hydrochloride 3.0 mg

MgS04. 7H20 0.5 gm

(NH4)6Mo7024. 4H20 0.05 mg ZnSO.. 7H„0 0.3 mg 4 2

MnCl2. 4H20 0.35 mg FeSO.. 7H£0 2.5 mg 4 2 CuSO,. 5H-0 0.5 mg 4 2 Distilled water 1 liter The pH was adjusted^fco::-555vwith"HGl. 16

Table 4. Composition;.of B-medium-- D(-)Mannitol 50.0 gm Sucrose 50.0 gm Succinic:..acid" 5.4 gm Yeast extract 1.0 gm

KH2P04 1.0 gm

FeS04. 7H20 O.Olgm

MgS04. 7H20 0.3 gm

ZnSO^. 7H20 4.0 mg Distilled water X>1 liter The pH was adjusted to 5.4 with NH^OH.

One hundred ml of each medium in a 500 ml Erlenmeyer flask with a cotton plug was sterilized at 15 lb, 121 °C for 15 minutes.

L-Tryptophan containing media, A'-medium and B'-medium, were prepared by adding aseptically 10 ml L-tryptophan solution (2.5 mg/ml

A-medium or B-medium) to a flask containing 90 ml sterilized A-medium or B-medium.

Culture:

Psilocybe cubensis was obtained from Dr. S. H. Pollock, Dept. of

Pharmacology, University of Texas, USA. It was reported to produce both psilocybin and psilocin. The fungus was maintained on slants of malt extract-yeast extract-soytone (MYP, 7 gm—0.5 gm—1 gm) agar.

After growth, the cultures were covered with sterile mineral oil and

stored in closed screw-cap tubes at 4 C.

Mycelium from a slant was transferred to a Sabouraud agar plate

(neopeptone-dextrose-agar, 10 gm--~40 gm —15 gm) and incubated at room

temperature for approximately two weeks. After growth, this plate

culture served as a "working plate". Mycelial tissues from the working

plate were transferred to a sterilized Waring Blendor and homogenized 17

for 30 seconds with sterile distilled water (50 ml for half a plate).

Ten ml of the resulting suspension was pipetted under sterile conditions

into a 500 ml Erlenmeyer flask, containing 100 ml A-medium: or B-medium.

The culture was incubated for 5 days at 23+1 °C on a reciprocal shaker

(80 rpm) and served as "liquid inoculum". The mycelial pellets of

this liquid inoculum were aseptically transferred from flask to a

sterile Waring Blendor and homogenized for 30 seconds. Five ml aliquots

were added to 500 ml Erlenmeyer flasks containing 100 ml of A-medium

or B-medium.

To investigate the effect of tryptophan, the liquid inoculum was

homogenized as above and a five ml portion added to A'-medium or B'-

medium.

All cultures were incubated at 23+1°C on a reciprocal shaker

(80)rpm) and illuminated for 8 hours a day. Cultureswwerehharvested

on alternate days five to fifteen days after inoculation.

C. Analytical methods

1. Preparation of samples for chromatography:

Six replicate cultures were sampled each time. Each culture

was filtered through four-layered cheesecloth and the mycelial

pellets washed thoroughly with distilled water and freeze dried

(VIRTIS) for two days. The weight of the freeze-dried pellets was

recorded. Using a Waring Blendor, freeze-dried pellets were

homogenized with distilled methanol and then decanted into a 500 ml

Erlenmeyer flask. The blender was rinsed with methanol three times

and the washings combined with the homogenate and the flask covered

with a paraffin membrane. The flask was agitated on the rotary 18

shaker (80 rpm) overnight. The suspension was filtered with suction

through a Buchner funnel and the residue washed with methanol three

times, the washings being added to the filtrate. The methanolic

extract was transferred to a round bottom flask and evaporated to

dryness in vacuo at 40 °C and the residue redissolved in 1-2 ml methanol.

A 1 x 5 cm column of ion exchanger (Dowex 50 WX-8(H^')) was washed

in sequence with 4 ml 1 N NaOH, 8 ml distilled water, 4 ml 1 N HC1,

8 ml distilled water and 5 ml 80% methanol. The concentrated

methanolic extract was added to the top of the column and washed in

with 5 ml 80% methanol. The effluent was discarded since it did not

show psilocybin-type ultraviolet absorption. The column was then

eluted with 20 ml of 5% concentrated NH.OH in 80% methanol. The 4

solvent was evaporated and the residue dissolved in 0.6 ml distilled

methanol. Extracts prepared in.this manner were stored in a refrig•

erator until used in chromatographic and UV spectral analyses.

The filtered medium of A and-B media, after determination of the

pH was extracted with ethyl ether. The ethereal layer was tested

for antibiotic activity after concentration to 5 ml (see below) and

the aqueous phase was evaporated to dryness in a rotatory evaporator

at 40 °C under reduced pressure. The residue was extracted with 2-5

ml portion of distilled methanol and transferred to a small vial.

After standing for a while, the solution was refiltered through

cotton and evaporated to dryness. Using 1 ml distilled methanol, the

residue was redissolved and passed on to the cation exchanger as

above and the final concentrated extract (0.6 ml) was stored until

ready to use. 19

A 10 ml aliquot of the filtered medium (A'-medium or B'-medium), after determination of the pH, was used for determination of trypto• phan concentration. The remainder of.the medium was evaporated and treated as above for medium A or B, i.e. subjected to ion exchange, fractionation.etc.

Location and identification of.Ehrlich-positive compounds on chroma• tograms

Two directional thin layer chromatography (2-D TLC) was employed. 2

A 30 ul aliquot of extract was spotted on a 10 x 10 cm cellulose plate (Eastman Chromatogram 13254 cellulose) and the plate developed in two directions using n-Butanol-Acetic acid-Water (BAW, 4:1:1) followed by Isopropanol-Ammonium hydroxide-Water (IAW, 8:1:1). After drying, the plate was examined for fluorescence withwaishort".wave- ; length UV lamp before spraying with Ehrlich reagent (1 gm p-dimethyl- aminobenzaldehyde dissolved in a mixture of 75 ml distilled methanol and 25 ml cone. HCI).

Isolation and purification of psilocybin

Descending paper chromatography was employed. An aliquot of a suitable extract (200 ul) was streaked on Whatman #1 chromatography paper and an aliquot of 50 ul was spotted with authentic psilocybin solution.^. In addition, authentic psilocybin was spotted alone. The paper was developed in the BAW system overnight. The developed chromatogram was air dried and examined for fluorescent compounds with UV light. The band corresponding to reference psilocybin was eluted with 80% methanol and the eluate concentrated under reduced pressure. It was streaked on paper again and developed with solvent

IAW overnight. The band migrating at the same rate as reference 20

psilocybin was eluted as above and the eluate evaporated to dryness

and the residue redissolved in 1 ml methanol. The ultraviolet absorp•

tion spectrum of the solution was obtained (UNICAM sp 800) and the

optical density at 267 nm was recorded (UNICAM sp 500 PYE Series 2).

The concentration of psilocybin could then be calculated from a

standard curve of psilocybin in which optical density was plotted

against concentration. This was linear over the range from 0 to

50 ug/ml.

4. Determination of tryptophan concentration in A' and B' media

Tryptophan- was assayed in the aqueous medium filtrate by the

Spiess and Chambers (42) method. Eight ml of 23.8 N l^SO^ and 1 ml

of 2 N H^SO^ containing 30 mg of p-dimethylaminobenzaldehyde were

o

mixed and.cooled to 25 C. To this solution was added 1 ml of medium

filtrate. The mixture after shaking and cooling to 25 C was kept in

the dark for one hour after which 0.1 ml of 0.04% NaM^ solution was

added. The mixture was shaken and the color allowed to developififor

30 minutes at room temperature in the dark. The optical density at

600 nm was read and converted to the concentration of tryptophan from

standard curves.

D. Radioautography 14

The fate of C -labeled tryptophan in the fungal cells was investi-

gagated by radioautography of the chromatographically separated compounds

in the fungal extracts. 14

D,L-Tryptophan, side chain-3-C , 5uCi/250 mg/100 ml B-medium was

added aseptically to a 3-day-old culture. Three flasks were used.

After 48 hours, the pellets were collected and the medium filtrate 21

processed as above. The extracts were spotted on paper together with

authentic tryptophan metabolites and developed in two directions, BAW

followed by IAW. After drying and examination under UV light, the

chromatogram was exposed to Kodak X-ray film (Blue brand) for two weeks.

The film was developed in Kodak X-ray developer for 4-5 minutes,

washed with dilute acetic acid and transferred to a fixative solution

for about 10 minutes. After washing in running tap water for 20-30

minutes it was dried and examined. The dark spots on the radioautograph

represented the radioactive substances on the chromatogram.

Radioactive spots were eluted with 80% methanol and the eluates

evaporated to dryness under reduced pressure at 40 °C. The residue was

redissolved in 0.7 ml methanol and pipetted into a scintillation vial.

A 9.3 ml aliquot of scintillator (Aquasol) was added and the mixture

counted in a Searle Isocap/300.

E. Psilocybin phosphatase activity

Method 1:

Enzyme extract: All extraction steps were carried out in a cold

room. Eight-day-old cultures from A ordB medium were collected and

the pellets thoroughly washed with distilled water after determination

of the fresh weights, the pellets were homogenized in a chilled Waring

Blendor for about 30 seconds with glycine-NaOH buffer (pH 9.0, contain•

ing 2 mM mercaptoethanol) and polyclar AT (0.1% of fresh weight) was

added. The slurry was filtered through 4 layers of cheesecloth and the

suspension centrifuged at 7,000 g (SORVALL RC 2-B) for 15 minutes. The

supernatant was used as crude enzyme extract.

Enzyme assay: Psilocybin aqueous solution (1.76 mM) was used as 22 substrate. An aliquot of 0.5 ml crude enzyme extract and 0.4 ml psilocybin solution were incubated at 30 °C for 30-90 minutes and 0.5 ml of the reagent (2';gm p-dimethylaminobenzaldehyde dissolved in the mixture of 65 ml cone. U^SO^ and 35 ml H^O containing 0.1 ml 3% FeCl^) was ,]) added to the test.tube after the appropriate time (29). The mixture was stirred and irradiated for 15 minutes under an UV lamp. The optical density of the solution which turned purple on irradiation, was deter• mined at 570 nm.

A blue product (max. absorption at 630 nm) was always produced during the incubation, however, and this color interferred with the purple color developed by psilocybin. The concentration of psilocybin could not be measured and this method was therefore abandoned.

Method 2:

Enzyme extract: All the extraction procedures were similar to those described in.Method.1 except that a succinate-NaOH buffer

(pH 5.0, containing 2 mM mercaptoethanol and 0.001 M NaCN) replaced the glycine-NaOH buffer.

Enzyme assay: Three substrates were employed in this assay.

Phenolphthalein diphosphate (PDP, 0.1 M, 0.1 ml) was added to 1 ml of enzyme extract and incubated at 34 *C. After an appropriate incubation period, 3 ml of 2 M (NH^^CO^ was added and the absorbance of the red color which developed was measured at 540 nm. Psilocybin (1.76 mM,

0.4 ml) was added to 1 ml of enzyme extract and the reaction mixture was incubated at 34°C. After an appropriate incubation period, 5 ml of n-butanol was added and the psilocin liberated was extracted and its concentration determined. Psilocin aqueous solution (2.46 mM,

0.4 ml, prepared freshly) was added to 1 ml of enzyme extract and the 23

mixture was incubated at 34 °C. After an appropriate incubation.period,

5 ml of n-butanol was added and the residual psilocin was extracted and

its concentration determined.

Colorimetric analysis for psilocin: To the reaction mixture (enzyme

extract, substrate and 5 ml of n-butanol), 0.5 ml borate buffer (KCl-

H3B03, pH 9.0) and 1 gm NaCl were added (33, 43, 44). The test tube

was shaken and then.the supernatant was pipetted into a centrifuge tube.

The organic phase was washed by shaking with 2 ml borate buffer and the

butanol phase was transferred to another centrifuge tube containing

5 ml heptane and 0.4 ml 0.1 N HCI. The mixture was shaken. The super•

natant was discarded. To the acidic phase, 1 ml of l-nitroso-2-naphthol

reagent (0.1% l-nitroso-2-naphthol in 95% ethyl alcohol) and 1 ml of

nitrous acid reagent (0.2 ml of 2.5% NaN02 in 5 ml of 2 N l^SO^, pre•

pared freshly) were added. The mixture was stirred and incubated in a

water bath at 55 C for 5 minutes. Ten ml of ethylene dichloride was

added and the tube was centrifuged at a low speed for 5 minutes and the

absorbance at 430 nm of the supernatant (brownish orange color) was

recorded. The psilocin concentration was calculated from a standard

curve. This was linear over the range from 0 to 0.5 mg/ml.

Protein concentration was determined by the method of Lowry ejt al.

(45). Enzyme activity was expressed as mg psilocin released/mg protein.

F. Potassium nutrition and psilocybin production

Two treatments were studied, potassium deficiency and high levels of

potassium. Replacement cultures were used.

K+-deficiency: Instead of 1 gm of KJ^PO^ as used in B-medium, 1 gm

NaH0PO/ and 20 mg KHoP0/i were used. After growing for 5 days, the pellets 24

were harvested and the concentration of. psilocybin determined.

K+-?supplement: In addition to 1 gm KR^PO^ as used in B-medium, 2 gm

of KCI was added. After growing for 5 days, the pellets were collected

and the concentration of psilocybin determined. A culture growing in

B-medium served as control.

G. Test of UV-mediated antibiotic and phototoxic activities of medium,

psilocybin and psilocin

A Candida albicans assay was employed (46) . Candida albicans was

streaked on Sabouraud's agar plate with sterile cotton swabs in one

direction and cross-wise in other direction. Several small filter paper

discs were sterilized. About 200ul of an ethereal extract of medium was

dropped on to the paper disc and allowed to dry. Discs were placed

uniformly around the agar plate so that zones of inhibition would not

overlap. A control consisting of solvent alone and a sample of

8-methoxy-psoralen, a known phototoxic compound was also included in

the test. Test plates were incubated at room temperature for at least

12 hours under an UV lamp. Addtiplicate plate was incubated in the dark

at room temperature. Extracts which caused a greater area of clearing

after light incubation than after dark were termed phototoxic. Those

extracts which caused the killing of Candida in both light and dark were

termed antibiotic. Authentic crystals of psilocybin and psilocin were

tested as above. 25

EXPERIMENTAL AND RESULTS

Chromatographic separation and identification of tryptophan metabolites

The color reactions, Rjl values and fluorescence colors in UVor, of f 254

some tryptophan metabolites are listed in Table 5. Figure 1 is a map

showing the location of these compounds on a two-dimensional chromatogram

using the BAW and IAW solvent systems.

Table 5. Fluorescence and Color Reactions of Some Tryptophan Metabolites Fluorescence Color reactions color in UV 254 Compounds Ehrlich Ninhydrin 1. L-Tryptophan dark blue purple purple 2. 5-OH-Tryptophan black violet purple 3. Tryptamine dark' blue purple violet 4. 5-OH-Tryptamine dark blue violet brownish (Serotonin) green dark blue purple brownish 5. N-Methyltryptamine purple yellowish brownish brownish 6. N,N-Dimethyltryptamine blue purple purple dark blue pink 7. Psilocybin dark blue blue 8. Psilocin azure orange purple 9. Kynurenine yellowish 10. Kynurenic acid blue 11. 3-Methylindole dark blue violet 12. Indole-3-ethanol dark blue violet 13. N-Acetyltryptophan dark blue purple - 14. Allantoic acid bright yellow 15. Allantoin yellow light blue bright . 16. Anthranilic acid yellow light blue yellowish - 17. 3-0H-Anthranilic acid orange bright yellow - 18. Urea 19. Indole-3-acetic acid grey brown 26

Table 5 (continued). Rf Values of Some Tryptophan Metabolites

Cellulose plate Whatman #1 paper

# Compounds BAW LAW BAW IAW

1. L-Tryptophan 0.44 0.24 0.39 0.26 2. 5-OH-Tryptophan 0.18 0.15 0.12 0.16 3. Tryptamine 0.68 0.75 0.61 0.73 4. 5-OH-Tryptamine 0.39 0.57 0.34 0.49 5. N-Methyltryptamine 0.78 0.86 0.65 0.81 6. N,N-Dimethyltryptamine 0.78 0.95 0.68 0.86 7. Psilocybin 0.38 0.02 0.31 0.06 8. Psilocin 0.72 0.90 0.62 0.82 9. Kynurenine 0.37 0.26 0.26 0.25 10. Kynurenic acid 0.53 0.55 0.55 0.40 11. 3-Methylindole 0.96 0.98 0.92 0.88 12. Indole-3-ethanol 0.93 0.92 0.87 0.85 13? «NzrAeetyit ryp t ophan- 0.90 0.60 0.87 0.60 14. Allantoic acid o.nio 0.02 0.16 0.15 15. Allantoin 0.26 0.07 0.18 0.13 16. Anthranilic acid 0.91 0.46 0.85 0.39 17. 3-OH-Anthranilic acid 0.80 0.33 0.76 0.27 18. Urea 0.4'6 0.44 0.45 0.50 19. Indole-3-acetic acid 0.86 0.51 0.86 0.39

B. pH, Growth and morphological difference of cultures

Cultures of the organism in A, A', B and B' media were harvested on

alternate days, five to fifteen days after inoculation. The pH of the

medium and the dry weight of the mycelium were determined at the

termination of this experiment. The results are summarized in Figures

2, 3, 4 and 5.

These data revealed that the pH of B and B' media remained acid in

the region of 5.35-5.55 through the whole growth period. The pH of the

A and A' media decreased gradually from the fifth day to the ninth day. After the ninth day, the pH of the A'-medium increased gradually to

7.35 on the fifteenth day. The pH of the A-medium, after the ninth day, increased rapidly until a plateau was reached near pH 7.4.

The dry weight of mycelia from A and A' media increased linearly

from the fifth day to the nineth day and continued to increase to a maximum on the eleventh day. There was then a rapid decrease in the dry weight and a final levelling off. The growth rate was poor in either

B or B' medium in comparison with A and A' media. The dry weight of mycelia from B-medium gradually increased until the eleventh day and

then leveled off. The dry weight of mycelia from B-medium increased

slowly in the beginning and then rapidly increased until a plateau was

reached.

The mycelial pellets growing in A or A' medium were round, smooth

and bigger compared with those in B or B' medium. Pellets in B or B1

medium were fuzzy. The pellets of earlier stages from A-medium were

bluish green in color and gradually turned dark grey. Pellets from A'-

medium were also bluish green color at an earlier stage, but they turned

brown to dark brown after eleven days. The color of A'-medium filtrate

at a later stage was dark brown while A-medium filtrate remained yellowish.

The pellets of earlier stages from B and B' media were silvery grey.

At a later stage, pellets from B-medium were still silvery grey while

those from B'-medium were dark brown. The B-medium filtrate gave a

dark brown color at a later stage and B-medium filtrate remained yellowish.

Psilocybin production

Psilocybin was identified by chromatography, color reactions and its

UV spectrum. The spectra of authentic psilocybin and that isolated from 28

fungal cells are shown in Figures 6 and 6'. Psilocybin was produced

on both A and B media. Media to which tryptophan had been added gave

better yields in the earlier periods but decreased later. Psilocybin

was never detected in the medium. The data are shown in Figures 7 and 8.

It can be seen that the fungus produced psilocybin very early and that

maximum production was on the fifth day.

D. Formation of psilocybin and psilocin from tryptophan

14

D,L-Tryptophan, side chain-3-C , was administered to 3-day-old

cultures for 48 hours. After treatment, extracts of the pellets and

the medium were chromatographed two-dimensionally and the chromatograms

radioautographed. In extracts of the medium, no radioactive metabolites

were identifiedd on chromatograms, the bulk of the radioactivity being in

unchanged tryptophan. In extracts of pellets, psilocybin and psilocin

were labeled although most of the radioactivity was still in unchanged

tryptophan. Psilocin was only faintly discernible on radioautograph.

The distribution of radioactivity in tryptophan, psilocin and psilocybin

is shown in Table 6. A photograph of the radioautograph of the mycelium

extract is present in Figure 9.

Table 6. Distribution of Radioactivity in Tryptophan, Psilocin and Psilocybin of Mycelium Extract,1,4 Administered D,L-Tryptophan, side chain-3—C

Percentage Distribution of Radioactivity

Tryptophan 0.65

Psilocin 0.004

Psilocybin 0.062 29

+

0.9 + + 12 16 + 13 19 + 17 + •5 + 6 0.7 + + 8 3

+ 10..S 4- 10 + 1 18 + + + 9 4 7 0.3 +

15 + 2

0.1 + 14

e

°-l 0.3 0.5 0.7 0.9

Figure 1. Location of some standard tryptophan metabolites on a two-dimensional cellulose thin-layer plate. Numbering of spots corresponds to that in Table 5 30

l_7y 1 1 1—: 1 1 ,_

0 5 7 9 11 13 15

days

Figure 2. pH of A and A' media 31

Figure 3. pH of B and B' media 32

Figure 4. Growth, rate of fungal cells in A and A' media 33

Figure 5. Growth"rate of fungal cells in B and B' media wavelength millimicrons Figure 6. UV spectrum of authentic psilocybin 200 225 250 275 300 325 3* wavelenj^

Figure 6'. UV spectrum of psilocybin isolated from fungal cell 36

Figure >7-. Psilocybin production from A and A' media 37

A B-Medium

•

L-// 1 1 I \ \ h 0 5 7 9. . 11 13 .15 days

Figure 8'. Psilocybin production from B and B' media 38

Trp - Tryptophan Pb - Psilocybin Pc - Psilocin 39

Phosphatase activity

Incubation of psilocybin or psilocin with a crude enzyme extract resulted in the formation of a.blue color usually after 5-15 minutes incubation and gave a linear rated over 180-minute-period (Figure 10).

The addition of NaCN was effective in delaying the blue color formation, but did not influence the phosphatase activity'.

The dephosphorylation of PDP by a crude enzyme extract proceeded at a rapid rate as shown the liberation of phenolphthalein (30-120 seconds for enzyme extract A and 5-25 minutes for enzyme extract B). The dephos• phorylation of psilocybin was rapid for both enzyme extracts. The blueing phenomena occurred in 3-5 minutes incubation in enzyme extract

A and after 15 minutes in enzyme B. The liberation of psilocin reached a maximum just before the reaction mixture turned blue (Figure 11).

When the psilocin was used as substrate, its concentration remained unchanged before it turned blue.

Partial purification of enzyme was attempted. The fraction which precipitated with ammonium sulfate^ at a concentration of 40-70% showed most activity.

Psilocybin production.and potassium nutrition

The pH, growth and psilocybin content of 5-day-old cultures of K+- deficient, K+-supplement and control conditions were compared. The data are indicated in Table 7.

Table 7. The Growth, pH of Medium and Psilocybin Production of B-medium, Potassium Deficient and Supplement Media B-medium K+-deficient K+-supplement pH £.533 E 5.3 5.45 Growth 70.0 78.1 70.0 mg/100.ml medium Psilocybin 3.51 1.92 3.51 mg/g dry wt 0. D. at 630 nm

- Figure ,10. Blue color formation when.psilocybin incubated with "enzyme extract 41

O A-Medium

A B-Medium

i i i i 0 5 10 15 20 . minutes

Figure H-. Activities of acid phosphatases of the mycelia from two different media 42

G. Ehrlich-positive compounds in extracts

Diagrams of chromatograms visualized with Ehrlich.reagent are shown

in Figure 12.

Spot Y, near the origin gave a yellow color after spraying with

Ehrlich reagent and a purple color with Ninhydrin reagent. On chroma•

tograms of extracts of mycelium of the thirteenth day and the fifteenth

day of growth on B'-medium, there was a spot Z close to psilocin which

gave a purple color with either Ehrlich or Ninhydrin reagent. Attempts

were made to identify these two compounds. After streaking and eluting

in two solvent systems, the R^ values of spot Y were 0>.ll and 0.19 in

BAW and IAW respectively. It was identified as glycine on the basis of

its color reactions and co-chromatography with this amino acid. The

R^ values of spot Z were 0.61 and 0.73 in BAW and IAW respectively. A

methanolic solution of this compound gave an UV spectrum of an indolyl

derivative with maximum at 290, 281, 270 and 220 nm. The R^ values and

color reactions with Ehrlich and Ninhydrin reagents were the same as

those of tryptamine and it was identified as such.

H. Tryptophan concentration in the medium

The tryptophan concentrations of the filtrates of medium A' and B'

were determined. The data are indicated in Figure 13. Tryptophan was

utilized by fungal cells more in A'-medium than in B-medium. From the

beginning to. the fifth day, there was a great decrease in tryptophan in

both media. After five days, tryptophan levels in B'-medium did not

changed too much, while in A-medium, a slow decrease was followed by

a sudden drop and a levelling off. Figure 12. Chromatograms of mycelium extract and medium extract of different growth period 44

mycelium extract 0

psilocybin psilocin tryptophan kynurenine anthranilic acid unknown yellow spot unknown spot, no color reaction with Ehrlich reagent, showing light blue fluorescence 45

z unknown purple spot 46

48

L-// 1 1 1 1 1 f- 0 5 7 9 11 13 15 days

Figure 13.. Tryptophan concentration of A' and B' media 49

I. Antibiotic activity of psilocin, psilocybin and medium filtrate

Neither A nor B medium filtrate showed antibiotic activity. The

characteristic polyacetylenic UV spectra in which normally two sets of

absorption bands are observed were not obtained.

Authentic psilocybin crystals did not show any antibiotic activity

towards Candida albicans while psilocin showed only very slight activity.

With psilocin, there was a clear zone in light and in dark. The control,

8-methoxypsoralen gave a clear zone in UV light but not in the dark.

The agar plate indicating the activity is diagrammed in Figure 14.

J. Identification of D(-)mannitol

Mycelial extracts from B-medium usually gave a deposit of white

crystals after evaporation of the methanol. The crystals melted at

164.5 °C and the IR spectrum (UNICAM 200G) proved to be that of a sugar

alcohol (Figure 15). The acetate derivative was prepared with anhydrous

pyridine and acetic anhydride (47) and the NMR spectrum was taken (EM-390

90 MHz NMR Spectrometer)(Figure 16). The IR and NMR spectra were found

to be identical to those of D(-)mannitol. 50

Figure 14, Antibiotic activity of psilocin (shaded area covered with Candida albicans) Figure 15. IR spectrum of white crystals

Figure 16. NMR spectrum of acetate derivative of white crystals 54

DISCUSSION

It was noticed that only when the fungus was maintained on Sabouraud agar plates^ (neopeptone-dextrose, 10 gm-r-40 gm). would, it produce psilocybin on subsequent, transfer, to . liquid, medium. ... If ..it was kept on malt extract-yeast- soytone agar plate (MYP, 7 gm—0.5 gm—1 gm) , it did not produce psilocybin . even after a 15-day-period of incubation and the cell extracts gave an UV spectrum of ergosterol (Figure 17) instead of that.of an indole. When fungus was kept on MYP agar plates, A-medium without yeast extract and A-medium without glycine were tried. However, there was no indole metabolites visual• ized from chromatograms and the UV absorption spectra. However, this does not preclude the possibility that the fungus when grown on MYP agar will eventually produce psilocybin if a longer incubation is allowed.

Catalfom6 and.Tyler (1) as well as Leung (12) maintained their cultures on potato-dextrose agar plates (PDA, 200. gm—15 gm) and succeeded in obtaining psilocybin. The sugar contents of either Sabouraud or PDA is larger that that of MYP. Amici e_t al. (48) found that the capacity to produce ergot alkaloids is correlated with the utilization of large amounts of sucrose and citric acid.

Amici suggested that the simultaneous utilizationcolf sucrose and citric acid is favorable to the accumulation of "primary precursors" as, for instance, acetyl

CoA and phosphoenolpyruvic acid. Catalfomo and Tyler found that higher levels of glucose resulted in higher yields of psilocybin in Psilocybe cubensis. Abe was also successful in promoting alkaloid synthesis in Claviceps by using a medium containing high concentration of sucrose and mannitol (49, 50).

The nitrogen source is also important in revealing alkaloid-producing strains (49). The differences in nitrogen sources in MYP and Sabouraud may also account for the alkaloid production.

After trying cellulose and silic acid TLC.and paper chromatography for

56 quantitative analyses, paper chromatography was finally selected because of reproducibility, although the recoveries were low (47+5%). Psilocybin is sensitive to shorter wavelength UV light and authentic psilocybin on paper turned pale yellow (without changing its UV spectrum) if it was exposed to

UV light for more than 1 minute. Silic acid plates, although they gave better resolution, led to the decomposition of the indole compounds when they were developed in the solvent systems used here. In conclusion, methods of purifying psilocybin are too long to allow rapid screening of different media. Recently, a sensitive GLC-Mass spectral analysis of psilocybin and psilocin was reported by Repke et al. (51) and this appears to be the method of choice. Unfortunately the instrumentation was not available for this study. '.'VV • ."-lifftoudc&d

Psilocybin was produced early in the development of the culture in both

A and B media. This might be expected because its formation does not i involve condensation with other carbon compounds such as amino acids or isoprene units which are involved in ergot alkaloid biosynthesis (52). The formation of the latter type of compound requires concerted condensations and perhaps the formation or the action of such enzymes is suppressed by primary metabolites (52) so that the biosynthesis of these compounds occurs only in the idiophase of growth.

Fluctuating rates of psilocybin production could be seen in both A and

B media. Rahacek et al. (53) suggested in Claviceps that alkaloid production commences while tryptophan synthetase activity is increasing. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan levels in balance. This could partially explain the fluctuation of psilocybin production in A and B media. Another reason for these fluc• tuations could be the heterogenous growth of the mycelium. Comparing the . 57 yield of psilocybin on two different media, fungus from B-medium gave a better yield but a poorer growth rate. Abe and co-workers stress that the fact that slow growth is required for alkaloid production (54). Demain (55) suggested that rapid growth during trophophase involves a balanced assimi• lation of all essential nutrients with a minimum of accumulation of meta• bolites.

By visual comparison of color intensities and size of spots on chromato• grams, the pellets from A-medium were found to contain more psilocin than those from B-medium. The pellets from the B-medium were less bluish green than those from the A-medium. The activities of acid phosphatases from these two cultures were quite different. Enzyme extract from A cultures dephosphorylated psilocybin very rapidly and gave a blue color after five minutes incubation. The enzyme preparation from B cultures gave a blue color only after fifteen minutes. The cause of the blue color has not been explained satisfactorily. It is suggested to be an enzymic oxidation (32,

34, 35, 37, 38, 44, 56) by some while, others indicate that oxygen is not required (57). The blue color can be elicited without enzyme, in the presence of ferric ion (35) and it can be blocked by EDTA.

It has been shown for several bacterial enzymes that the rates of enzyme synthesis are subject to regulation either by specific "inducer" substances or by specific "repressor" substances. The amount of alkaline phosphatase in E_. coli was markedly reduced when there was an excess of phosphate available (75, 76) and phosphate was demonstrated to beraprepressof of alkaline phosphatase formation (75, 76, 77). The amount of acid phosphatase formed in _E. coli was independent of phosphate concentration (75).

That phosphate ion inhibits various acid and alkaline phosphatases has been reported (72, 75, 77). Neal et al. (27) who worked on the relationships 58

between phosphate nutrition and accumulated psilocybin found excess phosphate

in the mycelia grown in phosphate-rich medium. Comparing the phosphate .

content of two media used here, B-medium had more than A-medium. The

difference in phosphatase activity could be due to higher endogenous phosphate

concentration in B culture inhibiting the enzyme activity.

In the present studies, 8-day-old cultures still had a creamy white

appearance. After eight days, the pellets in A-medium gradually turned blue.

The blue color would not be produced after eight days, however, if shaking

of the culture was stopped. Acid phosphatases have been reported to occur

in fungicsuch as Aspergillus (58). Wakao et al. (58) have shown that two

types of acid phosphatases in Aspergillus oryzae located either in the cell

wail or the cytoplasmic membrane. The pellets used here for enzyme prepara•

tions were white before extraction. After homogenization the insoluble

residue was quite greenish. It would seem therefore that some of the acid

phosphatases are located on the cell wall. The occurrence of acid phosphatases

could possibly explain the decrease of psilocybin production after nine days.

Psilocin aqueous solution should be prepared freshly or d'ferturnsn. brown

easily.

L-Tryptophan does not appear to have a clear-cut role in regulating

psilocybin biosynthesis in all species which produce it. Several workers

have shown (1, 12) that there is no consistent relationship between tryptophan

addition and psilocybin production. In this work, L-tryptophan stimulated

psilocybin production by almost a factor of two in the very beginning in B'-

jmedium. On the other hand, in- Aiamedium, the stimulation was not so signi•

ficant.

In the study of Claviceps strain 47 A (78), the results indicated that additions of D,L-phehylalanineTfavorablyfinfluenced3ergot alkaloid accumulation. 59

Sucrose was also noted to stimulate the accumulation of alkaloids in strain

47 A. Additions of sucrose and D,L-phenylalanine were synergistic in their

influence if the phosphate concentration was high (0.1%), but added amino

acid had little influence in the presence of a low phosphate concentration

(0.01%). Brady and Tyler (78) suggested that at least two limiting factors

are involved in clavine alkaloid biosynthesis. One of the limiting factors

is apparently an aromatic amino acid or a related metabolic substance; the

other appears to be a component associated closely with carbohydrate meta• bolism.

Floss and Mothes (59) showed that for the maximum effect on ergot alkaloid

production of adaaftfg tryptophan, it must be done at the beginning of the

fermentation long before alkaloid synthesis begins. They concluded that

this "training" phenomenon involved some kind of inducer action and suggested de• repression or activation of the enzyme system as the most likely mechanism

(59, 60). Whether or not tryptophan acts as an'inducef ofetheVehzymei necessary

for psilocybin production has not been investigated. B-medium seems to be a

promising medium to grow Psilocybe cubensis from the point of view of low acid

phosphatase activity and tryptophan enhancement in early stage.

It was observed (see Figure 12) that the residual tryptophan concentration

in the medium fluctuated with the age of the culture. Teuscher reported (61)

that there exists a proportionality between uptake and degradation of trypto- .

phan. Besides the participation in alkaloid biosynthesis, many other path• ways of tryptophan metabolism are operative in fungi and plants as shown in

Scheme IV (62). In A and A' media, the fungus metabolized tryptophan mainly

through kynurenine and anthranilic acid; while in B'-medium instead of kynurenine, tryptamine was formed. Kaplan et al. (63) concluded that the

degradation of tryptophan by the kynurenine pathway did not play a signifi- 60

Aromatic NAD pathway Quinaldine pathway pathway jSiAD

Scheme IV. Outline of Tryptophan Metabolism in Plants and Microorganisms. 61 cant role in the metabolism of tryptophan, under condition of high alkaloid production in Claviceps strain SD 58. There is some.evidence (49) that the fate of tryptophan added to culture is different in ergot alkaloid producing and non-producing strains. The formation of anthranilic acid was observed in a culture which failed to produce alkaloid. The appearance of kynurenine and anthranilic acid in the later stage of growth might also explain the decrease in psilocybin.production.

After feeding labeled D,L-tryptophan to Psilocybe cubensis grown on

B-medium, three radioactive spots were identified by radioautograpfty as tryptophan, psilocybin and psilocin although the last one was only faintly discernible. The percentage incorporated into psilocybin was 0.062 here and

0.43 in the report of Agurell (exposed for 5-6 days) (29) and 7.97 in the work of Brack et al. (exposed for 60 days) (23).

The diamine, putrescine (NH2CH2CH2CH2CH2NH2) is well-established as a constituent of microorganisms, animals and plants (64). The level of putre• scine is greatly increased in potassium-deficient plants (65, 66). Coleman et al. (66) suggested that basicity may conceivably help to explain why this particular substance accumulated in potassium starvation for if alkali metal deficiency shifts the internal balance between inorganic anions and cations in the direction of. increased acidity, basic metabolites maiy become important as compensatory factors and their metabolic stability increased. The feeding of hydrochloric acid also caused significant increases in amines such as arginine and putrescine content (67) .

In this work, potassium deficient condition decreased the psilocybin yield without changing growth or.medium pH. Potassium supplement had no effect at all. Vining and Nair, however, showed (50) that increasing the concentration of potassium caused an increased in alkaloid production in 62

Claviceps. Steinberg (68) reported that insufficiency of potassium caused a disturbance in protein metabolism resulting in a relative increase in amino acid nitrogen and a decrease in protein. In Psilocybe cubensis, the decrease of psilocybin caused by potassium deficiency could be due to a disturbance of protein synthesis especially of enzymes necessary for psilocybin production. 63

LITERATURE CITED

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6. Worthen, L. R., Stessel, G. J. and Youngken, H. W. Jr. (1962). The occurrence of indole compounds in Coprinus species. Economic Botany 16: 315-318.

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8. Turowska, I., Kohlmuenzer, S. and Molik-Wegiel, J. (1969). Search for physiologically active constituents of some domestic species of higher fungi. II Chromatographic examination of further species of the fungi Diss. Pharm. Pharmacol. 21: 417-423.

9. Willaman, J. J. and Li, H. L. (1970). Alkaloid-bearing plants and their contained alkaloids. Lloydia 33 (supplement): 1-286.

10. Smith, T. A. (1977). Tryptamine and related compounds in plants. Phytochem. 16: 171-175.

11. Leung, A. and Paul, A. G. (1967). Baeocystin, a mono-methyl analog of psilocybin from Psilocybe baeocystis saprophytic culture. J. Pharm. Sciences 56^: 146.

12. Leung, A. (1967). Investigations on psilocybin and its analogs in certain fungi. Ph.D. Thesis, Univ. of Michigan.

13. Leung, A. Y. and Paul, A. G. (1968). Baeocystin and norbaeocystin: new analogs of psilocybin from Psilocybe baeocystis. J. Pharm. Sciences _57: 1667-1671.

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16. Weber, H. P. and Petcher, T. J. (1974). Crystal structures of the

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17. Petcher, T. J. and Weber, H. P. (1974). Crystal structures of the

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18. Ott, J. and Guzman, G. (1976). Detection of psilocybin in species of Psilocybe, Panaeolus and Psathyrella. Lloydia 39: 258-260.

19. Robbers, J. E., Tyler, V. E. and Olah, G. M. (1969). Additional evidence supporting the occurrence of psilocybin in Panaeolus foenisecii. Lloydia 3_2: 399-400.

20. Hofmann, A., Heim, R., Brack, A. and Kobel, H. (1958). Psilocybin, , ein pJsxG'K'0.t.r.qp:er Wirkstof f aus dem mexikanischen Rauschpilz Psilocybe mexicana Heim. Experimentia 14: 107-109.

21. Heim, R. (1959). Old and new investigations on the hallucinogenic mushrooms of Mexico. Action and active principles. Actualite's Pharmacol. 12: 171-192. Cited from Chemical Abstracts (1960) 54: 15716a.

22. Olah, G. M. et Heim, R. (1967). Une nouvelle espece nord-americaine de psilocybe hallucinogene: Psilocybe quebecensis G. Olah et R. Heim. C. R. Acad. Sci. Paris 264: 1601-1604.

23. Brack, A., Hofmann, A., Kalberer, F., Kobel, H. und Rutschmann, J. (1961). Tryptophan als biogenetische Vorstufe des Psilocybins. Arch, der Pharmazie 294: 230-235.

24. Picker, J. and Richards, R. W. (1970). The occurrence of the psycho- tomimetiee agent psilocybin in an Australian agaric, Psilocybe subaeruginosa. Aust. J. Chem. 23: 853-855.

25. Leung, A. Y., Smith, A. M. and Paul, A. G. (1965). Production of psilocybin in Psilocybe baeocystis saprophytic culture. J. Pharm. •Sc Sciences 54: 1576-1579.

26. Yokoyama, K. (1976). A new hallucinogenic mushroom, Psilocybe argentipes K. Yokoyama sp. Nov. from Japan. Transactions of the Mycological Society of Japan ^7: 349-354.

27. Neal, J. M. , Benedict, R. G. and Brady, L. R. (1968). Interrelation• ship of phosphate nutrition, nitrogen metabolism, and accumulation of key secondary metabolites in. saprophytic cultures of Psilocybe cubensis, Psilocybe cyanescens and Panaeolus campanulatus. J. Pharm. Sciences 57: 1661-1667. 65

28. Kitamoto, Y., Horikoshi, T., Hosoi, N. and Ichikawa, Y. (1975). Nutritional study of fruit-body formation in Psilocybe panaeoliformis. Transactions of the Mycological Society of Japan 16: 268-281.

29. Agurell, S. and Blomkvist, S. (1966). Biosynthesis of psilocybin in submerged culture of Psilocybe cubensis. Acta Pharm. Suecica 3_: 37-44.

30. Agurell, S. and Nilsson, J. L. G. (1968). Biosynthesis of psilocybin. Acta Chemica Scandinavica 22: 1210-1218.

31. Blaschko, H. and Levine, W. G. (1960). Enzymic oxidation of psilocin and other hydroxyindoles. Biochemical Pharmacology _3: 168-169.

32. Blaschko, H. and Levine, W. G. (1960). A comparative study of hydroxy- indole oxidases. British J. of Pharmacology 1_5: 625-633.

33. Horita, A. and Weber, L. J. (1960). Dephosphorylation of psilocybin to psilocin by alkaline phosphatase. Proceeding Society for Experi• mental Biology and Medicine 106: 32-34.

34. Weber, L. J. and Horita, A. (1963). Oxidation of 4- and 5-hydroxy- indole derivatives by mammalian cytochrome oxidase. Life Sciences U 44-49.

35. Levine, W. G. (1967). Formation of Blue oxidation product from psilocybin. Nature _215: 1292-1293.

36. Bocks, S. M. (1967). Fungal metabolism-IV. The oxidation of psilocin by p-diphenol oxidase (Laccase). Phytochem. 1629-1631.

37. Bocks, S. M. (1968). The metabolism of psilocin and psilocybin by fungal enzymes. Biochem. J. 106: 12p-13p.

38. Bocks, S. M. (1967). Fungal metabolism-II. Studies on the formation and activity of para-diphenol oxidase (Laccase). Phytochem. _6: 777- 783.

39. Hearn, M. T. W., Sir Jones, E. R. H., Pellatt, M. G., Thaller, V. and

Turner, J. L. (1973). Natural acetylenes. Part XLII, Novel G7> Cg,

Gjjciand C1f) polyacetylenes from fungal cultures. J. of Chem. Society Perkin I 42: 2785-2788.

40. Bohlmann, F., Burkhardt, T. and Zdero, C. (1973)). "Naturally Occuring Acetylenes". Academic Press.

41. Stoll, A., Brack, A., Kobel, H., Hofmann, A. and Brunner, R. (1954). Die Alkaloide eines Mutterkornpilzes von Pennisetum ITyphoideum Rich, und deren Bildung in saprophytischer Kultur. Helv. Chim. Acta 37: 1815-1825.

42. Spiess, J. R. and Chambers, D. C. (1948). Chemical determination of tryptophan. Analytical Chemistry J20: 30-39. 66

43. Udenfriend, S. , Weissbach, H. and Brodie, B. B. (1958). Assay of serotonin and related metabolites, enzymes and drugs. Methods of Biochemical Analysis 6_: 95-130.

44. Horita, A. and Weber, L. J. (1961). The enzymic dephosphorylation and oxidation of psilocybin and psilocin by mammalian tissue homogenates. Biochemical Pharmacology _7: 47-54.

45. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement,"-with"theofolintpheribl reagent. J. Biol. Chem. 193: 265-275.

46. Daniels, F. (1965). A simple microbiological method for demonstrating phototoxic compounds. J. of Investigative Dermatology 4_4: 259-263.

47. Shriner, R. L. and Fuson, R. C. (1948). "Identification of Organic Compounds". 3rd ed., John Wiley rand Sons, N. Y.

48. Amici, A. M. , Minghetti, A., Scotti, T., Spalla, C. and Tognoli L. (1967). Ergotamine production in submerged culture and physiology of Claviceps purpurea. Applied Microbiology 15_: 597-602.

49. Vining, L. C. and Taber, W. A. (1963). "Biochemistry of Industrial Microorganisms'.' Ed. by C. Rainbow and A. H. Rose, Academic Press, N. Y. pp. 341-378.

50. Vining,L. C. and Nair, P. M. (1966). Clavine alkaloid formation in submerged cultures of a Claviceps species. Can. J. of Microbiology 12: 915-931.

51. Repke, D. B., Leslie, D. T., Mandell, D. M. and Kish, N. G. (1977). GLC-mass spectral analysis of psilocin and psilocybin. J. Pharm. Sciences 66: 743-744.

52. Taber, W. A.„ (1967). Fermentative production of hallucinogenic indole compounds. Lloydia 3_0: 39-66.

53. Rehacek, Z., Kozova, J., Ricicova, A., Kaslik, J., Sajdl, P., Svarc, S. and Basappa, S. C. (1971). Role of endogenous tryptophan during submerged fermentation of ergot alkaloids. Folia Microbiology 16: 35-40.

54. Taber, W. A. and Vining, L. C. (1957). A nutritional study of three strains of Claviceps purpurea (Fr). Tul. Can. J. of Microbiology .3: 1-11.

55. Demain, A. L. (1968). Regulatory mechanisms and the industrial produc• tion of microbial metabolites. Lloydia _31: 395-418.

56. Peisach, J. and Levine, W. G. (1966). A comparison of the enzymic activities of pig ceruloplasmin and Rhus vernicifera laccase. J. Biol. Chem. 240: 2284-2289. 67

57. Gilmour, L. P. and O'Brien, R. D. (1967). Psilocybin: reaction with a fraction of rat brain. Science 155: 207-208.

58. Wakao, N., Sakurai, Y., Shiota, H. and Tanimura, I. (1975). Cytochem- ical localization of acid and alkaline phosphatases in Aspergillus oryzae. J. General and Applied Microbiology 21_: 233-249.

59. Floss, H. G. and Mothes, U. (1964). liber den Einfluss von Tryptophan und analogen Verbindungen auf die Biosynthese von Clavinalkaloiden in saprophytischer Kultur. Arch. Mikrobiol. 48: 213-221.

60. Vining, L. C. (1970). Effect of tryptophan on alkaloid biosynthesis in cultures of a Claviceps species. Can. J. Microbiology 16: 473-480.

61. Teuscher, E. (1964). The relationship between active assimilation of tryptophan and alkaloid biogenesis in Claviceps purpurea. Flora Allg. Bot. Zeitung 155: 80-89. Cited from Biological Abstracts 46: 91266 (1965).

62. "Metabolic Map" ed. by Tanpakushitsu Kakusan Kohso Supplement, Japan 1968 pp. 43.

63. Kaplan, H., Hornemann, U., Kelly, K. M. and Floss, H. G. (1969). Tryptophan metabolism, protein and alkaloid synthesis in saprophytic cultures of the ergot fungus (Claviceps sp.). Lloydia 32: 489-497.

64. Smith, T. A. (1970). Putrescine, spermidine and spermine in higher plants. Phytochem. 9: 1479-1486.

65. Smith, T. A. (1973). Amine levels in mineral-deficient Hordeum vulgare leaves. Phytochem. 1_3: 2093-2100.

66. Coleman, R. G. and Richards, F. J. (1956). Physiological studies in plant nutrition XVIII. Some aspects of nitrogen metabolism in barley and other plants in relation to potassium deficiency. Annals of Botany 20: 393-409.

67. Smith, T. A. and Sinclair, C. (1967). The effect of acid feeding on amine formation in barley. Annals of Botany 3_1: 103-111.

68. Steinberg, R. A. (1951). Mineral nutrition of plants. Ed. by E. Truog,

Univ. of Wisconsin Press, pp. 359-386.

69. Yu, C. J. (1959). The Continental Magazine _19(8): 1-4. Taiwan, ROC.

70. Hoffer, A. and Osmond, H. (1967). The hallucinogens. Academic Press, pp. 480-500. 71. Hoffmann, A., Heim,-R. , Brack. A., Kobel, H., Frey, A., Ott, H., ... Petrzilka, Th. and Troxler, F. (1959). Psilocybin und Psilocin, zwei psychotrope Wirkstoffe aus mexikanischen Rauschpilzen. Helv. Chim. Acta 42: 1557-1572. Cited from Chemical Abstracts 54: 5830g (1960). 68

72. Colowick, S. P. and Kaplan, N. 0. (1955). "Methods in Enzymology". Vol. II, pp. 523-616. Academic Press, N. Y.

73. Brack, A., Brunner, R. und Kobel, H. (1962). Mikrobiologische Hydroxyl- ierungen an Mutterkornalkaloiden von clavine Typus mit dem rcexikanischen Rauschpilz Psilocybe semperviva Heim et Cailleux. Helv. Chim. Acta 45: 276-281. Cited from Chemical Abstracts 57: 6443e (1962).

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II. A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF

LENTINUS AND LENTINELLUS 69

INTRODUCTION

The Basidiomycete, Lentinus lepideus, is a frequent cause of brown rots in timber. It is able to attack many woods on account of its tolerance to comparatively high concentrations of creosote (1). In culture on wood or oh glucose it frequently produces a strong, sweet, aromatic odor due to the synthesis of methyl cinnamate (I), methyl p-methoxycinnamate (II) and methyl anisate (III).

Crystalline deposits of methyl p-methoxycinnamate are often obtained butiif the culture flasks in which the crystals have accumulated are continuously shaken, the amount of the deposit diminishes rapidly and after a few days, almost completely disappears. After 1 or 2 days of shaking, methyl p-coumarate (IV) was present in the medium (2). In surface as well as shaken cultures, the presence of methyl isoferulate (V) has also been

established%(3).

(I) (II) (III) (IV) (V)

When Lentinus lepideus was grown in media containing radioactive glucose, radioactivity was significantly incorporated into (II). It indicated that

this compound may be synthesized via shikimic acid (4) and that it is not

a product of the degradation of lignin (5).

Among seven species of Lentinus and Lentinellus, a closely related genus,

only Lentinus lepideus and Lentinus ponderosus were found to produce, either

in light or dark conditions, methyl esters of phenolic acids (6). Lentinus 70 ponderosus yielded compounds I, II, III and V. Neither L_. lepideus nor

_L. ponderosus produced detectable amounts of free p-OH-cinnamic acid (VI),

caffeic acid (VII) or isoferulic acid (VIII).

OH OH 0CH3

(VI) (VII) (VIII)

An enzyme, L-phenylalanine ammonia lyase (PAL) (E.C. 4.3.1.5) which

converts L-phenylalanine directly to cinnamic acid with the liberation of ammonia (7) and which often has a similar action on L-tyrosine (TAL) has been found in Basidiomycetes including Lentinus lepideus. In some fungi,

or in higher plants, light triggers the appearance of PAL (26). Cinnamic

acid is esterified enzymically to give methyl cinnamate, the methyl donor being S-adenosylmethionine. Methyl cinnamate may undergo one or two hydroxy-

lations and the phenols in turn undergo O-methylation.

Biological methylations in nature are sufficiently important that

special enzymes for carrying out these reactions have been developed during

the course of evolution (8). Keller et al. (9) gave a direct quantitative demonstration that the methyl group was transferred as an intact unit.

S-AdenosyMethionine (SAM) has been established as the direct methyl donor

and there are more than thirty reactions in which SAM has been demonstrated to serve as a methyl donor (8). The methyl group may be transferred to the nitrogen of primary, secondary or tertiary amines or of heterocyclic comr pounds, the sulfur of thioesters, the oxygen of phenols, carboxylate _ 71 structures and carbon atoms of a number of compounds.

The methyl transferases, enzymes catalyzing transmethylation reactions have been found in a wide range of biological forms. In general, each methyl- transferase catalyzes the transfer of a methyl group to only a specific acceptor. For example, Fales et al. (8, 10) showed that the ratio of para to meta methylation of the diphenol norbelladine was 22:1, when the reaction was catalyzed by a plant enzyme, whereas the ratio was 0.28:1, when the reaction was catalyzed by rat liver catechol O-methyltransferase.

A number of enzyme preparations from higher plants have been described which catalyze the transfer of the methyl group of ,SAM to the meta position of vicinal polyphenolic compounds (11, 12, 13, 14, 15, 16, 17). Enzymatic para-O-methylation by catechol O-methyltransferase from the rat was reported

(18). The partially purified preparation of norbelladine O-methyltransferase from Nerine bowdenii is the first reported cell-free system from a higher plant that catalyzes para-O-methylation predominantly (19). Using the cell- free enzyme system of Foeniculum vulgare, 4-OH-cinnamate is methylated to

4-methoxycinnamic acid (20). The partially purified enzyme from peyote

(Lophophora williamsii) Catalyzes O-methylation in the meta position but also in the para position depending upon the substrate (12). Partially purified catechol O-methyltransferase from pampas grass (Cortaderia selloana) catalyzes the methylation at both meta and para positions,bu6',vcwheh acting . on caffeic acid, the preparations catalyze methylation of the meta hydroxyl

(14). A phenolic O-methyltransferase from Lentinus lepideus (21) was shown to catalyze para-O-methylation but only with methyl esters of the hydroxy- cinnamic acids as substrates. The same enzyme preparations catalyze the formation of the methyl ester of cinnamic acid from the free acid. 72

There is little evidence for the participation of cofactors or other activators in transmethylation reactions. An occasional methyltransferase has been found to be stabilized by mercaptoethanol (13, 15, 21), but the activity of a partially purified methyltransferase from Vinca rosea was reduced by mercaptoethanol and enhanced by dithiothreitol (22). Requirement I | for Mg or other metal ions is not clear-cut (.'(21, 23).

The objective of this study was to survey the existence of the phenol-

O-methyltransferase in eight species of Lentinus and Lentinellus and also to study the effect of light on the levels of enzyme activity. 73

MATERIALS AND METHODS

A. Chemicals: The source of the chemicals used was as follows:

Chemical Source

Cinnamic acid Aldrich Chemical Company, Inc

p-Hydroxybenzoic acid Eastman Organic Chemicals

Methyl p-OH-cinnamate

Methyl 3,4-dimethoxycinnamate All these methyl esters were obtained Methyl caffeate ^from Dr. C. K. Wat, Dept. of Botany, U. B. C., Vancouver Methyl ferulate

Methyl isoferulate

Sephadex G-25 Pharmacia Fine Chemicals

Polyclar AT Sigma Chemical Company

) (polyvinylpolypyrrolidone)

(NH4)2S04 BDH, Chemicals LTD

NaHoP0.. H„0 Fisher 2 4 2

Na2HP04 Fisher

Mercaptoethanol Calbiochem

Soytone Difco

Omnifluor New England Nuclear

Dithioerythritol (DTE) Sigma

Dithiothreitol (DTT) Sigma

Glutathione (reduced form) Sigma ,14 S-Adenosyl-L-methionine,methyl-CJ

Specific activity 51.8 mCi/m mole

(10 uCi, 0.11 ml) ICN Pharmaceuticals Inc. 74

Medium and culture

Medium:

Seven gm of malt extract, 0.5 gm of yeast extract and 1 gm of

soytone were dissolved in 1 liter of distilled water. Two hundred ml

of the medium in a 500 ml Erlenmeyer flask was sterilized at 15 lb,

o

121 C for 15 minutes and used for culturing the fungi.

Culture:

Lentinus edodes (UBC 767) and L_. lepideus (UBC 718) were obtained

from Dr. R. J. Bandoni, Dept. of Botany, Univ. of British Columbia,

Lentinellus vulpinus (177 A) from Dr. R. S. Smith, Western Forest

Products Laboratory, Vancouver, B. C., Lentinus tigrinus (RLG-9953-Sp),

L_. sulcatus (OKM-8302-Sp) and L_. ponderosus (OKM-3120-S) from Dr. J. G.

Palmer, USDA, Madison, Wisconsin, Lentinellus cochleatus (DAOM 22534)

and Lentinus kauffmanii (DAOM 11660, 17180) from Dr. J. H. Ginns,

Agriculture Canada, Ottawa.

The fungus was maintained on MYP (malt-yeast-soytone, 7 gm—0.5 gm-

1 gm) agar slants. After growth, the cultures were covered with sterile

mineral oil and stock cultures were stored in closed screw-cap tubes

at 4°C.

Mycelia from a slant were transferred to a MYP agar plate and

incubated at room temperature for approximately two weeks. After

growth, this plate culture served as a "working plate". Mycelial

tissuesffromiaaworkihggplalHeewereetransferred to a sterilized Waring

Blendor and homogenized for 30 seconds with sterile distilled water

(100 ml water for one plate). Ten ml of the resulting suspension was

pipetted under sterile conditions into a 500 ml Erlenmeyer flask

containing 200 ml medium. All cultures were kept at 25°C with 8 hours 75

of fluorescent light per day.

C. Extraction of enzyme

All steps were carried out in a cold room.

Three replicate cultures were sampled each time. The medium of a

3-4-week-old culture was decanted and the mycelial mat was washed three

times with distilled water. The wet weight was determined. The mycelial

mat was frozen in liquid ^ and ground. Polyclar AT was added (5% of wet

weight) and the mixture was stirred for 20 minutes in phosphate buffer

(0.1 M, pH 7.0 containing 2 mM mercaptoethanol).

The suspension was filtered through two layers of cheesecloth and

the filtrate centrifuged at 7,000 g (SORVALL RC 2-B) for 15 minutes.

The supernatant was further fractionated by addition of solid (NH^^SO^.

The 20 to 60% ammonium sulfate precipitate was dissolved in 2 ml buffer

and desalted by passage through Sephadex G-25 column. The protein was

eluted from the column and a 5 ml aliquot was used in the enzyme assay

and for the determination of protein.

D. Enzyme assay

The assay mixture consisted of 0.1 ml of substrate (0.1 u mole),

0.1 ml SAM^C1^ (0.1 uCi in 0.0525 u mole) and 0.5 ml of the above

enzyme (21). The reaction mixture was incubated at 30°C for 30 minutes

and terminated by the addition of 0.5 ml 5% HG1. The reaction mixture

was extracted with 10 ml anhydrous diethyl ether. A 2.5 ml aliquot of

ethereal extract was pipetted into a scintillation vial and placed in

a fume hood. The ether was evaporated and then 10 ml of scintillator

(6 gm Omnifluor dissolved in a mixture of 625 ml toluene and 375 ml

ethanol) was added. All readings (Searle Isocap/300) were corrected 76

using efficiency curves calibrated from standard quenching solution by

the channel ratio method. The substrate specificity was calculated from

the activity obtained and the specific activity of SAM.

Identification of reaction products

The remainder of the above ethereal extract (7.5 ml) was pipetted

to a vial and the ether was evaporated in the fume hood. The residue was redissolved in 0.2 ml ether and the solution was spotted on a 10 x 2

10 cm silic acid plate (Eastman Si gel chromatogram with fluorescent indicator) with reference compounds. The plate was developed two- dimensionally in benzene-acetone (9:1) followed by benzene-acetic acid- isooctane (90:10:15). After drying, the plate was examined under shorter wavelength UV lamp for the fluorescent spots. A radioautograph was prepared by exposing the chromatogram to a Kodak X-ray film for one week.

The film was developed in Kodak X-ray developer for 4-5 minutes, washed with dilute acetic acid solution and transferred to a fixative solution for about 10 minutes. After washing.in running tap water for

20-30 minutes it was dried and examined. The dark spots on the radio• autograph represented the radioactive substances on the chromatogram.

Protein concentration

Protein concentration was determined by the method of Lowry et al.

(24). The concentration of protein was calculated from a standard curve of Bovine Serum Albumin (BSA). 77

RESULTS AND DISCUSSION

Among eight species (Lentinus ponderosus, L_. edodes, L_. kauffmanii

(DAOM 11660, 17180), L_. tigrinus, L_. sulcatus, L_. lepideus, Lentinellus vulpinus and Lentinellus cochleatus), in addition to Lentinus lepideus, only Lentinus ponderosus was found to contain the phenol-O-methyltransferase.

The data are indicated in Table 1 (columns 5 and 7).

Of six substrates tested, methyl p-OH-cinnamate had the highest activity.

The para-specificity of this fungal enzyme was clearly demonstrated in its ability to methylate methyl caffeate and methyl ferulate but not methyl isoferulate. Cinnamic acid was reported to be the only free acid to serve as substrate for the enzyme from Lentinus lepideus (21). In this investiga• tion, however, it did not show activity, and the radioautograph did not give any corresponding spot either.

Radioautograms of the methylated reaction products distinctly identified the labeled main products as methyl p-methoxycinnamate *f(#2) , methyl isoferu• late (#3) and methyl 3,4-dimethoxycinnamate (#4). This is shown in Figure 1.

Lentinus lepideus was reported to produce methyl p-methoxycinnamate either in light (21) or in dark (25). Lentinus ponderosus was also reported to produce methyl p-methoxycinnamate in light and dark (6). In the study of

Polyporuss (26) PAL and other enzymes associated with styrylpyrone biosyn• thesis are influenced by light. In this case, the effect of light and dark on phenol-O-methyltransferase activity in these two species was also studied.

The data shown in Table 1 indicates there is no difference between light and dark grown cultures of Lentinus ponderosus and _E_. lepideus. Light, therefore, does not seem to play a role in the regulation of cinnamic metabolism in these two species, although the increase in PAL activity occurs when certain

Basidiomycetes are cultured in light (7). Table 1. Substrate Specificity of Methylating Enzymes from Lentinus poridero.siusaaridlLenfcinus lepideus

Rf Values Enzyme Specific Activity*

Benzene Benzene L. s.poriderosus L. lepideus # Substrate Product Acetone Acetic acid Isooctane light dark light dark

1. Cinnamic acid - - - 0 0 0 0

2. Methyl pj^OH-cinnamate Methyl p-methoxycinnamate 0.66 0.62 37.63 36. 60 37.80 36.80

3. Methyl caffeate Methyl isoferulate 0.32 0.40 66:19 10. 80 6.50 7.04

4. Methyl ferulate Methyl 3, 4-dimethoxy- 0.54 0.59 6.89 6. 93 5.88 6.96 cinnamate 5. Methyl isoferulate - - - 0 0 0 0

6. p-Hydroxybenzoic acid — — _ 0 0 0 0

Specific activity expressed as the number of n mole product/mg protein/hr. Figure 1. Radioautographs of the chromatographed methylated cinnamate products

#2 methyl p-methoxycinnamate

#3 methyl isoferulate

#4 methyl 3,4-dimethoxycinnamate 80

The addition of 0.05 mM DTE (dithioerythritol), DTT (dithiothreitol) and glutathione did not increase the enzyme activity.

From this study and from previous reports, it seems that only species which produce methyl p-methoxycinnamate or methyl isoferulate have the phenol-

O-methyltransf erase. Although both Lentinus ponderosus and L_. lepideus produce methyl anisate, none of the benzoic acids or their esters were active substrates for these enzyme preparations (6, 21). Thus it would seem that in Lentinus the methylating enzyme for benzoic acid derivative must be a different one to that for cinnamic acid (21) or that methyl - anisate is formed from methyl p-OH-cinnamate.

Lentinus ponderosus ,aL. lepideus and L_. kauffmanii belong to a class of fungi, members of which cause brown rots in wood, and which bring about the preferential decomposition of cellulose. Although the wood is destroyed when attacked by this type of mold, it is interesting that the metabolism

of some members of the group differs from that of others despite the fact

that the overall process, the decay of the wood is the same-. 81

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