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Biosynthetic studies on the p-benzoquinones produced by Shanorella Spirotricha Benjamin Wat, Chi-Kit 1969

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BIOSYNTHETIC STUDIES ON THE p-BENZOQUINONES PRODUCED BY SHANORELLA SPIROTRICHA BENJAMIN by CHI-KIT WAT B.S.P., University of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of BOTANY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1969 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f Botany  The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date Dec. 29, 190. i i ABSTRACT From the culture medium of Shanorella s p l r o t r l c h a Benjamin, four p-benzoquinones have been i s o l a t e d and I d e n t i f i e d . The major pigment i s shanorellin (2,6-dimethyl-3-hydroxymethyl-5-hydroxy-1,4-benzoquinone). The other three pigments are the acetate, the,ethyl ether and the dimer of shanorellin. Shanorellin was found to be synthesized v i a the acetate-polymalonate pathway by tracer experiments with ^ C - l a b e l l e d compounds and by chemical degradation of the l a b e l l e d shanorellin produced. A study was made of the optimal conditions required fo r the production of these benzoqulnones. S i g n i f i c a n t factors were pH, temperature and sources of nitrogen and vitamins of the nutrient medium. i i i TABLE OF CONTENTS Page Introduction 1 I. Natural D i s t r i b u t i o n of p-Benzoqulnones 1 I I . Established Biosynthetlc Pathways for 6 p-Benzoquinones I I I . Acetate-Polymalonate Pathway 9 A. H i s t o r i c a l Development 9 B. The Role of Acetate-Polymalonate 12 Pathway i n Fungal Benzoqulnone Biosynthesis Shanorella splrotrlcha t A p-Benzoqulnone- 35 Producing Ascomycete Part A. Environmental E f f e c t s on Pigment 36 Production and Growth i n Shanorella s p l r o t r l c h a I. E f f e c t s of pH 38 I I . E f f e c t s of Temperature 42 I I I . E f f e c t s of Nitrogen Source 43 IV. Effects of Vitamins 47 Part B. Production of E x t r a c e l l u l a r Colored 52 and Phenolic Compounds i n Relation to pH and Glucose Concentration of the Medium and Changes i n Mycelial Dry Weight i v TABLE OF CONTENTS (cont'd) Page Part C. Elucidation of the Chemical Structures 62 of Shanorellin, i t s Monoacetate (Comp-ound A) and i t s Ether Derivatives (Compound B and Compound C) I. Shanorellin 65 I I . Compound A 7^ I I I . Compound B 80 IV. Compound C 86 Part D. The Biosynthesis of Shanorellin 92 Part E. General Discussion 112 Bibliography 117 V LIST OF TABLES Table I. Changes i n pH Values of Media Inoculated with S. s p l r o t r l c h a a f t e r 1 7 Days of Incubation I I . L i s t of Nitrogenous Compounds and t h e i r Concentrations Used i n the Culturing of S. s p l r o t r l c h a . I I I . Concentrations of the Vitamins Used as Supplement to Czapek-Dox Medium IV. L i s t of Vitamins Used i n the Supplementation of Czapek-Dox Medium V. Results of Mycelial Growth and E x t r a c e l l u l a r Pigment Formation on Various Vitamin Sources i n Cultures of S. s p l r o t r l c h a . 1 4 VI. Incorporation of A c e t a t e - 1 - C into Shanorellin a f t e r D i f f e r e n t Periods of Growth of S. s p l r o t r l c h a . I k VII. Incorporation of A c e t a t e - 1 - C into Shanorellin by _S. s p l r o t r l c h a VIII. Incorporation of Various C-Labelled Compounds into Shanorellin & by S. s p l r o t r l c h a Page 4 0 4 4 4 8 50 9 6 9 7 9 9 v i LIST OF TABLES (cont'd) Page Table IX. Results of the Kuhn-Roth Degradation 10k of Shanorellin Labelled from Acetate-1 - ^C, a c e t a t e - 2 - C and Methionine-X" Schmidt Degradation of Acetate I s o l - 1 0 6 ated from Kuhn-Roth Oxidation of Shanorellin Labelled from Acetate-2 - l l fC and Methionine-^CH, v i i LIST OF FIGURES Page Fig. 1. Structure of Shanorellin 1 2. Examples of Naturally Occurring 2 Benzoquinones i n Higher Plants 3 . Examples of Fungal Blbenzoquinones 4 and Terphenylquinones 4 . New Fungal Monobenzoquinones 5 5. Proposed Scheme f o r Benzoquinone 6 Formation from Cinnamic Acid i n Wheat Seedlings 6. Fungal Degradation of Lignin Model 7 Compound Sy r i n g y l - g l y c o l - (3 -Guaiacyl Ether 7. Hypothetical Scheme f o r the Formation 8 of Thymoquinone as Proposed by Zenk 8. Hypothetical Reaction Scheme f o r the 11 Synthesis of Polyketo-Chain 9. Fungal Metabolites Biosynthesized from 11 Acetate-Polymalonate Pathway 10. Fungal Benzoquinones Derived from 1 4 6-Methylsalicylic Acid 11. Fungal Benzoquinones Derived from 15 O r s e l l i n i c Acid 12. Structures of Benzoquinones Produced 16 by Gliocladlum roseum v i i i LIST OF FIGURES (cont'd) Page Fig. 13. Biosynthetic Pathways Proposed for 17 the Quinones from Gliocladium roseum 14. Mechanism for Formation of the Enzyme- 20-22 Bound, Metal-Stabilized, Polyketide •Loop* as Proposed by Bu'Lock 15. Condensation Reactions Proposed f o r 25 C y c l i z a t i o n of Cg Chain i n Polyketide Biosynthesis 16. C y c l i z a t i o n of Cg Chain i n Polyketide 26 Biosynthesis with Reductive Steps 17. Formation of Fumigatin from O r s e l l i n i c 28 Acid and Orcinol 18. Modes of Decarboxylation i n the Bio- 30 synthesis of Fungal Toluquinones 19. Proposed I n t e r r e l a t i o n of Quinones 34 and Hydroquinonesfrom Gliocladium rpseum 20. Appearances of S. s p l r o t r l c h a Cultures 39 with I n i t i a l pH 4.2 to 9*0 a f t e r 12 Days of Incubation 21. Appearances of S. s p l r o t r l c h a Cultures 39 with I n i t i a l pH 4.2 to 9.0 a f t e r 17 Days of Incubation i x Page Pig. 22. Appearances of S. s p i r o t r l c h a Cultures 4 2 Incubated at Different Temperatures 23» Appearances of 12 Day Old Cultures of 4 6 S. s p i r o t r l c h a Grown on Different Nitrogen Sources 2 4 . Appearances of 17 Day Old Cultures 4 6 of S. s p i r o t r l c h a Grown on Different Nitrogen Sources 25. Appearances of 17 Day Old Cultures of 51 S. s p i r o t r l c h a Grown on Different Vitamin Sources 26. Extent of Mycelial Growth of 51 S. s p i r o t r l c h a on Plate #3 27. Absorption Curve of Shanorellin i n 55 CHCl^ at 272nm 28. Changes i n pH, Glucose and Shanorellin 57 Concentrations i n the Medium and Mycel-i a l Dry Weight with Growth i n S. s p i r o t r l c h a 29;-34 Detection of Colored and Phenolic 60 Compounds i n the Culture Medium During Growth of S. s p i r o t r l c h a by Thin-Layer Chromatography X LIST OF FIGURES (cont'd) Page Fi g . 35. Similar to Fig . 29-34 but without 61 spraying with p - n i t r o a n i l i n e reagent 36. Typical Thin-Layer Plate I l l u s t r a t i n g 61 the Relative Positions of the Compounds Present i n Conspicuous Amount 37. Mass Spectrum of Shanorellin 66 38. U l t r a v i o l e t Spectrum of Shanorellin 67 i n CHCI3 39« Infrared Spectrum of Shanorellin i n 68 KBr Disc 40. N.M.R. Spectrum of Shanorellin i n CDCl^ 69 41A and 4lB. Some of the Possible Bond Cleavages i n 72-73 Shanorellin upon Electron Impact 42. U l t r a v i o l e t Spectrum of Compound A i n 74 CHCl^ 4 3 . Infrared Spectrum of Compound A i n 75 KBr Disc 44. N.M.R. Spectrum of Compound A i n CDCl^ 76 45. Mass Spectrum of Compound A 77 46. U l t r a v i o l e t Spectrum of Compound B i n 80 CHC1„ x i LIST OF FIGURES (cont'd) Page Fig. 47 . Infrared Spectrum of Compound B i n 8 1 KBr Disc 4 8 . N.M.R. Spectrum of Compound B i n CDCl^ 8 2 4 9 . Mass Spectrum of Compound B 83 50. U l t r a v i o l e t Spectrum of Compound C 89 i n CHCl^ 51. Infrared Spectrum of Compound C l n 90 KBr Disc 52. N.M.R. Spectrum of Compound C i n CDCl^ 91 53» Mass Spectrum of Compound C 91 54. Possible Origins of Carbon Atoms i n 101 Shanorellin Based on O r s e l l i n i c Acid 55. Possible Origins of Carbon Atoms i n 102 Shanorellin Based on 6 - M e t h y l s a l i c y l i c Acid 56. Origins of Carbon Atoms i n Shanorellin 105 57. The Relative Positions of the Benzo- 110 quinones and Phenolic Compounds Detected l n the Medium and the Aromatic Standards 58. Suggested Biogenetic Scheme f o r the 116 Synthesis of Shanorellin and i t s Derivatives i n S. s p i r o t r l c h a x i i ACKNOWLEDGEMENT I wish to express my deepest gratitude to Dr. G.H.N. Towers f o r the Invaluable advice and i n s p i r a t i o n given to me throughout the course of my work and f o r his patience i n reading and correcting t h i s thesis. To my committee members, I would l i k e to express my appreciation f o r t h e i r comments made on t h i s work and especially to Dr. R.J. Band-oni f o r his advice on the cul t u r i n g of the fungus and Dr. E.B. Tregunna for his advice on the l i q u i d s c i n t i l l a t i o n technique. I would l i k e also to thank Dr. T. Bisalputra f o r allowing me to use the photographic f a c i l i t i e s i n his laboratory, to Miss Margaret Shand for her help i n the i s o l a t i o n of single spore from the fungal culture, to Miss Anne Loh f o r her help i n typing part of t h i s thesis, to the Chemistry Department of the University of B r i t i s h Col-umbia fo r the u t i l i z a t i o n of the spectroscopic instruments, and to the National Research Council of Canada f o r a post-graduate scholarship. 1 INTRODUCTION I # Natural D i s t r i b u t i o n of p-Benzoqulnones p-Benzoquinones are d i s t r i b u t e d widely l n Nature. Two groups can be distinguished by t h e i r chemical structures: those with a polyprenyl side chain and those without a poly-prenyl side chain. To the f i r s t group, which i s also referred to as the isoprenoid quinones, belong the ubiquinones, plasto-qulnones, tocopherolquinones and rhodoquinones ( 1 - 3 ) . The second group consists of a large number of variously s u b s t i -tuted compounds, Including a new benzoquinone, shanorellin (Fig. 1 ) , produced by the fungus Shanorella s p i r o t r l c h a Benjamin, with which t h i s thesis i s concerned ( 4 ) . The following discussion concentrates on t h i s second group of benzoquinones only. Benzoquinones without a polyprenyl side chain have been iso l a t e d from fungi (5)» lichens ( 6 ) , higher plants (7 ,8) and arthropods ( 9 , 1 0 ) . No compound of t h i s type has been reported from animals other than the arthropods, gymnosperms, lower plants or bacteria. Some examples of benzoquinones occurring o o F i g . 1. Structure of Shanorellin. 2 OCHr 2,6-dimethoxy-Benzoquinone Ranunculaceae  Mellaceae  Slmarubaceae  Apocynaceae Y Y o Thymoqulnone Umbelllferae  Cupressaceae Labiatae Composltae R = C 1 1 H 2 3 Embelln Myrslnaceae R - C-L^Hgr, Rapanone Myrslnaceae  Connaraceae Oxalidaceae R* — ^*]_2^/Lj,^ — (CHg )- 3^CH=CHC^H^ Polygonaquinone Maesaquinone Polygonaceae Myrslnaceae F i g . 2 . Examples of Naturally Occurring Benzoquinones l n Higher Plants. 3 i n the angiosperms are i l l u s t r a t e d i n F i g . 2. The presence of the benzoquinones embelin, rapanone and maesaquinone i n four genera of the Myrsinaceae distinguishes t h i s family from the neighbouring family, Primulaceae ( 8 ) . Ogawa and Natori, i n t h e i r studies on the hydroxybenzoquinones of Myrslnaceous species from Japan, also concluded that the d i s t r i b u t i o n of these benzoquinones i s a chemotaxonomical c h a r a c t e r i s t i c of the Myrsinaceae (11). Fungi produce the largest number of benzoquinones, and a l l of them are found i n the Deuteromycetes, Ascomycetes and Basidiomycetes. These fungal metabolites can further be divided into three groups: monobenzoquinones, bibenzo-quinones and terphenylqulnones ( 5 t l 2 ) . While the monobenzo-quinones and bibenzoquinones (Fig. 3) occur from a l l of the above three groups of fungi, the terphenyl derivatives (Fig.3 ) are found p r i n c i p a l l y i n the Agaricaceae and Polyporaceae of the Basidiomycetes. The structures of fungal quinones isola t e d before 1963 are shown i n the book ' L i s t of Fungal, Products' by Shibata, Natori and Udagawa ( 5 ) . Since then, additional monobenzoquinones, apart from shanorellin, have been i d e n t i f i e d (13-17) and they are indicated i n F i g . 4 . From the lichens, polyporic acid has been isol a t e d from S t l c t a and thelephoric acid from Lobarla. Blbenzoquinones Phoenicin Oosporein Terohenylqulnones Theleporlc Acid F i g . 3» Examples of Fungal Blbenzoquinones and Terphenylqulnones. 5 Aspergillus fumlgatus Fres. O HO . ^ \ ^ C H , Fumlgatln epoxide (13) Lentlnus degener Kalchbr, CH3O' H. 'OH 4-methoxy-6-hydoxyTQ Hellcobasldlum mompa Tanaka O :H0 H P e n l c l l l l u m splnulosum Thorn HO. HO-O Li + Fumlgatln Deoxyhelicobasidln (15) 3,4-dlhydroxyTQ (16) Gllocladium roseum Bainer HO HO CH-HO, CS3O-•CH-*CH, o o 3,4-dlhydroxy-6-methylTQ 3-hydroxy-4-methoxy-6methylTQ (17) F i g . k. New Fungal Monobenzoqulnones (TQ=Toluqulnone). 6 I I . Established Blosynthetlc Pathways for p-Benzoqulnones There are three pathways established for the biosynthesis of benzoquinones. One of these, based on shikimic acid and i t s transformation products, leads to the biosynthesis of the quinone nucleus of the isoprenoid quinones ( 1 8 ) . Two fungal benzoquinones, au r a n t i o g l l o c l a d i n ( F i g . 9 ) and coprinin (Fig. 1 0 ) have also been reported to be formed v i a t h i s route ( 7 , 1 9 ) . Bolkart and Zenk ( 2 0 ) , i n t h e i r studies on the biosynthesis of methoxy-hydroquinone and 2 ,6-dlmethoxy-hydro-quinone glucosides i n wheat seedlings, have shown that these two compounds (presumably the precursors for the corresponding quinones In wheat flour) are synthesized through oxidative decarboxylation of the s i m i l a r l y substituted benzolccacids as Indicated i n F i g . 5 » HC-C00H CH H p-oxldatlon OH (I) C00H H OCH. OCH OCH* (I) (II) (III) F e r u l l c acid V a n i l l i c acid Methoxy-HQ Sinapic acid Syringic acid 2 , 6-dimethoxy HQ F i g . 5 . Proposed Scheme for Benzoquinone Formation from Cinnamlc Acid i n Wheat Seedlings (HQ=Hydroquinone). 7 On the other hand, Kirk, Harkln and Cowling (21), using model compounds clos e l y related to l i g n i n , have demonstrated that cultures of wood r o t t i n g fungi, as well as p u r i f i e d p-diphenol oxidases from culture f i l t r a t e s , are able to cleave the a l k y l - a r y l bond i n substrates to form benzoquinone d e r i -vatives ( F i g . 6 ) . Caldwell and Steelink (22) have further obtained evidence suggesting that the depolymerlzation of l i g -n in by these fungi i s the r e s u l t of a series of one-electron oxidation steps catalyzed by phenol oxidases. Thus, the wide d i s t r i b u t i o n of 2,6-dlmethoxy-benzoqulnone reported from plants might indeed partly be due to the action of fungi rather than to the I n t r i n s i c biosynthetic c a p a b i l i t y of the plants. F i g . 6 . Fungal Degradation of Lignin Model Compound Syringylglycol-/3-Guaiacyl ether (22). 8 The second established pathway for benzoquinones b i o -synthesis i s based on the head-to-tail linkage of acetate u n i t s . I t i s by t h i s route that most fungal monobenzoquinones are formed. The development of th i s acetate theory and the mechanisms of reactions proposed i n respect to i t w i l l be discussed i n d e t a i l l a t e r on. The t h i r d established pathway i s also based on acetate, but by way of mevalonate. The fungal benzoquinone, h e l i c o - -basidln, has been shown to be synthesized as a sesquiterpene which undergoes subsequent r i n g formation and oxidation processes ( 1 5 ) . Zenk has suggested a scheme for the formation of thymoqulnone which occurs i n the essential o i l of Umbelli-ferae, Labiatae and Gupressaceae ( 2 3 ) . A l l the intermediate compounds i n th i s proposed scheme have been i d e n t i f i e d i n the o i l of Monarda f l s t u l o s a (Fig.?). quinone V. Thymoqulnone F i g . 7. Hypothetical Scheme fo r the Formation of Thymoqulnone as proposed by Zenk. 9 III. Acetate-Polymalonate Pathway (A) H i s t o r i c a l Development The p o s s i b i l i t y of the r o l e of acetate i n the biosyn-thesis of certain naturally occurring compounds was sugg-ested by C o l l i e i n 1907 (24), but i t was not u n t i l 1953 that Birch and Donovan proposed the hypothesis that these molecules were elaborated by head-to-tail linkage of acetate units ( 2 5 ) . These compounds are marked by the frequent occurrence of oxygen atoms i n fi -positions to each other and (i - to p o s i -tions of r i n g closure. The f i r s t experimental proof for t h i s hypothesis was the finding that sodlum-l-^C-acetate was Incorporated into 6-methylsalicylic acid by P e n l c l l l l u m grlseofulvum ( 2 6 ) . Since then, the studies on the biosynthesis 14 of many other fungal aromatic metabolites from 1- or 2 - C-acetate have tended to confirm t h i s o v e r a l l pathway ( 2 7 - 3 2 ) . With the increasing understanding i n the biosynthesis of f a t t y acids and of s t e r o l s , Bassett and Tanenbaum (33) proposed that the activated form of acetate u t i l i z e d would be a c e t y l -CoA and they obtained a c e l l - f r e e extract from P e n l c l l l l u m  patulum which converted acetyl-CoA into p a t u l i n , a metabolite known to be derived from 6-methylsalicylic acid ( 3 4 , 3 5 ) « The idea that these aromatic compounds were formed by repeated condensation of acetyl-CoA was not e n t i r e l y s a t i s f a c t o r y due to the unfavourable equilibrium (37)• and Lynen suggested that t h i s d i f f i c u l t y could be overcome i f malonyl-CoA were involved rather than acetyl-CoA ( 3 6 ) . Lynen and Tada obtained from 10 Pe n l c l l l l u m patulum a soluble preparation which catalyzed the synthesis of 6 - m e t h y l s a l i c y l i c acid from 1 mole of la b e l l e d acetyl-CoA and 3 moles of malonyl-CoA i n the presence of NADPH ( 3 8 ) . Lynen also proposed that the enzyme catalyzing the formation of the aromatic structure resembled the enzyme complex encountered i n the studies of f a t t y acid synthesis i n yeast and that the intermediates were bound to the complex by way of a thioester linkage, the product being liberated only a f t e r the process was completed ( 3 6 ) . L a t e r t i n his investigations, Lynen found that at le a s t two d i f f e r e n t types of sulphydryl groups are involved i n the jyeast f a t t y acid synthetase ( 7 8 ) . If the i n i t i a l steps i n the polyketlde biosynthesis do occur l n steps p a r a l l e l to that of fat t y acid, the reaction sequence would occur as shown i n Fig. 8 . Priming Reaction HS HS C H 3 - C O S C 0 A + >Enzyme > ^Enzyme + HSCoA HS/ C H 3 - C O S 4 Chain Lengthening Reactions HS^ malonyl-CoA COOH ^Enzyme > CH 2 - C 0 S CHq-COS '•• ;Enzyme + HSCoA CH3-COS COOH CH2COS condensation CU^-C-CHg-COS ;Enzyme > /Enzyme CH3COS HS + c o 2 11 o CH~-C-CH2-C0S. ac y l transfer HS ^ c -Enzyme > O )Enzyme HS CRj-C-CHg-COS HS. + n malonyl-CoA HS, O }Enzyme > 0 ft Enz CH3-C-CH2COS CH3C-(CH 2C) n iCHaCOS F i g . 8. Hypothetical Reaction Scheme f o r the Synthesis of Polyketo-chain. 1 4 1 4 Experiments with C-acetate and C-malonate did show that a wide range of fungal metabolites were derived by repeated condensation of malonyl-CoA units with an a c e t y l -CoA " s t a r t e r " , e,g, 6 - m e t h y l s a l i c y l i c acid ( 3 5 . 4 0 , 4 2 ) , o r s e l l i n i c a c i d ( 3 9 ) , c l a v a t o l ( 4 4 ) and a u r a n t i o g l i o c l a d i n ( 4 3 ) , ( F i g . 9 ) . COOH HO 00H H 3 H CH3 6 - M e t h y 1 s a l i c y l i c Acid H O r s e l l i n i c Acid CH 3 CK>. OH CH3 CH- O v f ^ T v C CH OH Clavatol 3 c ^ N r > ^ C H 3 o Auranti o g l i ocladin F i g . 9. Fungal Metabolites Biosynthesized from Acetate-Polymalonate Pathway. 12 Crude enzyme preparations have recently been isol a t e d for the biosynthesis of 6-methylsalicylic acid from Penl-c l l l l u m patulum (45) and o r s e l l i n i c acid from P e n l c l l l l u m  madrlti (46 ) . For the 6-methylsalicyllc acid synthetase to function, a NADPH-generating system was required. With the crude o r s e l l i n i c acid synthetase system, i f NADPH was added, both o r s e l l i n i c acid and f a t t y acids were detected? the omission of NADPH from the reaction mixture resulted i n the l o s s of f a t t y acid synthesis, but that of o r s e l l i n i c acid was retained. Additional proof f o r the occurrence of the acetate-polymalonate pathway was presented by Gatenbeck and Mosbach ( 4 7 ) . By cu l t u r i n g Chaetomlum cochllodes with O-acetate, the content of the carboxyl group was found to be half that of each hydroxyl group, i n accordance with the idea that coenzyme A i s involved i n the biosynthesis and that hydrolysis of the f i n a l acyl-CoA intermediate would introduce the second oxygen from water. (B) The Role of Acetate-Polymalonate Pathway i n Fungal Benzo-quinone Biosynthesis The fungal benzoquinones derived from the acetate-poly-malonate pathway are characterized by the presence of at least one methyl group (or i t s various oxidation states) on the quinone nucleus, and invariably, t h i s carbon has been proven to be derived from the methyl carbon of acetate. Bentley (48) has further noted that 6-methylsalicylic acid and o r s e l l i n i c 13 acid, both formed by the condensation of 1 mole of acetate with 3 moles of malonate, could be Intermediates In the biosynthesis of other aromatic compounds. With regards to benzoquinone biosynthesis, a l l the experimental evidence reported so f a r indicates that these two acids are precursors of quinones i n which there i s no extra methyl group added to the quinone r i n g other than the one derived from the methyl group of acetate. Examples of these benzoquinones are coprinin, i t s hydroxylated deriv a t i v e , t e r r e i c acid, fumigatin and splnulosin. Coprinin ( 4 9 ) , i t s hydroxylated derivative (50) and t e r r e i c acid (51) have been shown to be synthesized from 6-methylsalicylic acid ( F i g . 1 0 ) , and splnulosin (16) and fumigatin ( 6 2 , 5 2 , 6 9 ) from o r s e l l i n i c acid ( F i g . 1 1 ) . Examples of quinones with an extra methyl group on the r i n g are those reported from Gliocladium roseumi g l i o r o s e l n , a u r a n t i o g l i o c l a d i n and rubro-g l i o c l a d i n ( F i g . 1 2 ) . 5-Methylorcylaldehyde has been found by Steward and Packter to be the f i r s t aromatic compound i n -volved i n the biosynthesis of the quinones by the fungus. Birch ( 7 , 4 9 ) and Pettersson (54) have studied the biosynthesis of a u r a n t i o g l i o c l a d i n i n t h i s fungus also, and t h e i r r e s u l t s are indicated;; In F i g . 13. The fact that 5-methylorcylald-ehyde was incorporated into g l i o r o s e l n to the extent of 36# (53) as compared with the low incorporation of o r s e l l i n i c a cid ( 0 . 0 7 $ ) and 2 ,4-dihydroxy -5 ,6-dimethylbenzoic acid (0,9%) Into a u r a n t i o g l i o c l a d i n ( 5 4 ) , and that the Introduction of a methyl group i s mechanistically favourable I f i t were 1 4 CH-: COOH OH 6-Methyl-S a l i c y l i c Acid Tyrosine Packter (92) O Birch ( 4 9 ) ) -CH. CH^ O Coprinin (Lentinus degener) O Pettersson(50) ^ CH3O-• CH-: OH 4-Methoxy -6-Hydroxy -2 ,5-TQ (Lentinus degener) Read & Vining(51^ ^ Terreic Acid (Aspergillus terreus) F i g . 10. Fungal Benzoquinones Derived from 6-Methyl-S a l i c y l i c Acid. (TQ=Toluquinone) 15 Pettersson(l6) Spinulosin (Penlcllllum splnulosum) HC CH3 •COOH OH O r s e l l i n i c Acid Packter(62) HO OH Pettersson(52) HO CH3O 2H-O Fumlgatln (Aspergillus fumlgatus) O HO. Yamamoto(69) CH3O CHr o O Fumlgatln Epoxide F i g . 11. Fungal Benzoquinones Derived from O r s e l l i n i c Acid. 16 F i g . 12;. Structures of Benzoquinones Produced by Gliooladlum roseum. 17 Birch 6 - M e t h y l s a l i c y l i c Acid ( 4 9 ) -Phenylalanine ( 7 ) Pettersson ( 5 4 ) COOH H- HO V Steward and Paokter ( 6 3 ) Acetyl-CoA + 3 Malonyl-CoA + S-Adenogylmethionine OOH -CH3 •CHo OH HO Auranti o g l i ocladin Aurantiogliocladln H , HO. Y r/ ^ r J 3 H 3 HO CO.S.Enzyme CO.S^Enzyme H C V r ^ ^ r C H 3 CH3O. G l i o r o s e i n 4* Quinol -I Bubrogliocladin Aurantiogliocladln F i g . 1 3 . Blosynthetlc Pathways Proposed f o r the Quinones from Gliocladium roseum. 1 8 to be added to a polyketo-chaln rather than to an aromatic rin g , render the pathway proposed by Steward and Packter more acceptable. Subsequent to the formation of these aromatic acids, other reactions such as decarboxylation, hydroxylation and O-methylation take place. The oxidation of the hydroqulnone to the quinone has generally been accepted as the f i n a l reaction i n the biosynthesis process. These reactions together with the proposed mechanisms for polyketide b i o -synthesis w i l l now be considered. Bu'Lock, i n accordance with the current concept of f a t t y acid biosynthesis, has postulated a mechanism for the conden-sation of acetate (bound to an acyl c a r r i e r protein as i n f a t t y acid biosynthesis) and malonate units ( s i m i l a r l y bound to acyl c a r r i e r protein) i n the formation of a /3 -polyketoacyl chain ( F i g . 1 4 ) . "The condensing enzyme," Bu'Lock s u g g e s t e d ( 5 5 ) » "carries a single -SH group and metal ion. The t h i o a c y l derivative of the enzyme i s formed f i r s t , and t h i s then accepts, by further coordination with the metal, the malonyl-CP (here written as the enol). Condensation-decarboxylation occurs within the complex, producing the a c y l a c e t y l product attached to the c a r r i e r protein by the thloester l i n k of the o r i g i n a l malonyl-CP, but s t i l l attached to the enzyme through the metal. For f a t t y acid synthesis, d i s s o c i a t i o n of t h i s complex gives acylacetyl-CP from which the higher acyl-CP i s generated, by reduction, etc. For polyketide synthesis, the 19 acylacetyl-CP-metal-thiolenzyme complex undergoes transacyla-t l o n i n t e r n a l l y , giving the acylacetyl-derivative of the enzyme, and t h i s reacts again, as shown, with further molecules of malonyl-CP. Each condensation i s followed by the i n t e r n a l transacylation, and each condensation e f f e c t i v e l y inserts a (CH2CO) group between the growing chain and the enzyme. When a c e r t a i n stage i s reached, t h i s growing loop of (CH2CO) units i t s e l f becomes capable of occupying the coordination p o s i t i o n of the metal ion; i n the simplest case t h i s i s attained with four C 2 units, and the resultant complex, i s of the types i l l u s t r a t e d i n ( F l g . 14)." Bu'Lock stated that the construction of models had shown that a l l the stereoelectronic requirements f o r these reactions were elegantly met. He also noted that polyketide assembly and polyketide c y c l i z a t i o n were d i s t i n g -uishable processes, each subject to genetic control. After the formation but before the aromatization of the ft -polyketoacyl chain, two reactions are found to be c l o s e l y integrated with polyketide biosynthesis ( 5 5 , 5 6 ) . These two reactions are a l k y l a t i o n arid deoxygenation. A l k y l a t i o n invo-lves the p a r t i c i p a t i o n of cations such as CKj* (from S-adenosyl-methionine) and (CH-^C^HCHg* (from isopentenyl pyrophosphate), and these cations w i l l undergo e l e c t r o p h i l i c substitution with compounds that would r e a d i l y y i e l d an anion. On the methylene carbons of the ft -polyketoacyl chain, the presence of active hydrogens adjacent to a carbonyl function renders these carbons very susceptible to e l e c t r o p h i l i c attack. So, i t i s expected 20 M SH Enz CH^CO.SR — a — : ) RSH + 0=G-/ CHo ,M •S Enz / CO; CH2C0SR 0=C / CH-j 0 — C nj CH 0-C-SR Enz C0C M CH, ? Entz^ CH=C-SR SH CH^COCHgCO.SR + Enz-SH Reduction etc. Polyketide Biosynthesis (continued on next page) Fatty Acid Biosynthesis 2 1 3 ^CH CH / \ CO c \ // Enz-SH C-SR CH ( A ) (continued on next page) 22 (A) Enz-SH O r s e l l i n i c Acid )H O 2-Ac e ty1-Phiorogluc i n o l F i g . 14. Mechanism f o r Formation of the Enzyme-Bound, Metal-Stabilized, Polyketide 'Loop' as Proposed by Bu'Lock. 2 3 that these methylene carbons are the s i t e of a l k y l a t i o n reactions. This prediction i s borned out by the observation that many polyketides are alkylated on the alternate, non-oxygenated atoms of the chain, e.g. c l a v a t o l i n F i g . 9« Traceristudies with 6 - m e t h y l s a l i c y l i c acid and o r s e l l i n i c acid have shown that these two acids were not converted into g l i o r o s e i n by Gliocladlum roseum ( 1 9 ) , but the methylated o r s e l l i n i c d e r i v a t i v e was ( 5 3 ) « Labelled o r s e l l i n i c acid has been shown to be the precursor of lecanoric acid but not of atranorin which possesses an a d d i t i o n a l unit on each r i n g of the l i c h e n depside ( 5 7 ) ' S i m i l a r l y , resaceto-phenone was not incorporated into c l a v a t o l by Aspergillus  c l a vat us ( 4 4 ) . Mutant studies i n the tetracycline-sproducing Streptomyces aurefaclens has demonstrated that the wild type could not transform the non-methylated cy c l i z e d intermediate into t e t r a c y c l i n e ( 5 8 ) . Recently, Gatenbeck, Eriksson and Hannson have is o l a t e d a crude protein f r a c t i o n from Aspergillus  f l a v l c e p s . a mould which produces a series of aromatic com-pounds such as 5 - m e t h y l o r s e l l i n i c acid, f l a v i p i n , and i n low y i e l d , o r s e l l i n i c a c i d ( 5 9 ) « Their r e s u l t s indicated that® methylation occurred on a t e t r a a c e t i c a c i d structure bound*to the protein Isolated, and not on the o r s e l l i n i c a c i d . They suggested also that the protein to which the t e t r a a c e t i c acid was attached might be involved i n the formation of the polyketo-chain from acetyl-CoA and malonyl-CoA units. Thus, a l l the evidence would seem to e s t a b l i s h that i n polyketide synthesis 24 a l k y l a t i o n processes occur on the ft-polyketoacyl structure p r i o r to c y c l i z a t i o n and aromatization. Mention must also be made, however, of an enzyme system f o r non-specific C-methylation of an aromatic nucleus from Streptomyces rlmosus which was shown to convert resacetophenone and 3-methylres-acetophenone to c l a v a t o l (44). Before considering the deoxygenation process, an exam-inat i o n of the mechanism proposed for the c y c l i z a t i o n and subsequent aromatization of /3-ketoacyl chain would indicate that deoxygenation occurs before aromatization. Two organic reaction mechanisms have been suggested f o r the formation of an aromatic r i n g ( 5 6 , 6 0 ) : (1) Clalsen-condensation involving an ester group and an activated CFL, leading to ft -polyketoesters or ft -polyketones, and (2) Aldol-conden-sation involving a reactive methylene and a carbonyl function (Fig. 15) . These two reactions would lead to the formation of two basic forms of Cg polyketides, the o r s e l l i n i c and the 2-acetyl-phloroglucinol types. There are two other groups of Cg polyketides which can be formed s i m i l a r l y but which have one oxygen function missing. These are the 6-methylsalicylic and 2-acetyl-resorcinol types of compounds (Fig. 16). As can be seen i n F i g . 14 and 15, the p o s i t i o n at which deoxygenation occurs i s at the carbonyl that plays no part i n the c y c l i z a t -ion of the ft-polyketoacyl chain. Birch has proposed that t h i s absence of an oxygen function could r e s u l t from reduction of a carbonyl i n an open-chain or c y c l i c , but non-aromatic, 25 o o o CRyC-(CH2-C-)2CH2-C-SR 2-Acetylphloroglucinol O r s e l l i n i c Acid Fi g . 15. Condensation Reactions Proposed f o r C y c l i z a t i o n of Co Chain i n Polyketide Biosynthesis. 26 o o o CH3-C-(CH2-G-)2CH2-C-SR Claisen-Condensatlon I NADPH + H HO ^^<^0H 2-Acetyl-Resorcinol Aldol-Condensation Me H J<OH H Y T" C 0 S R O ^ < ^ 0 H NADPH + E H M ^ ° H H ' i i T C 0 S R HO i v ^ O H ITSH " H 20 Me dr )H Me ^ S ^ C O O H 6-Methylsalicylic Acid Pig. 16. C y c l i z a t i o n of Cg Chain i n Polyketide Biosynthesis with Reductive Steps. 27 precursor, aromatization being accomplished by dehydration rather than by enolization (60). No experimental proof has been put f o r t h f o r t h i s suggestion, but evidence f o r the synthesis of aromatic compounds from shikimate pathway does give support to t h i s proposal. Dehydroshikimic dehydrase has been i s o l a t e d for the formation of protocatechuic acid from Neurospora crassa (75) and prephenic acid aromatase f o r phenylpyruvic a c i d from Escherichia c o l l (76). Both studies have shown that these two acids were formed by a dehydration process involving the respective c y c l i c but non-aromatic precursors. Also, i n microorganisms and higher plants, the keto-acids that give r i s e to phenylalanine and tyrosine from shikimic acid are known to branch from each other a f t e r prephenic acid (77)» Similar types of reactions would be quite l i k e l y to occur l n the biosynthesis of the above described oxygenated arid deoxygenated types of compounds derived from the acetate-polymalonate pathway. For the formation of the aromatic r i n g , Birch has sugg-ested that proper a c t i v a t i o n of the appropriate carbonyl function together with an enzyme to bind the intermediate c o r r e c t l y would bring about r i n g closure at the appropriate carbon atoms (56). As i l l u s t r a t e d e a r l i e r , Bu'Lock has proposed another mechanism involving a chelated metal complex for these reactions. Two other mechanisms have been post-ulated f o r the formation of an aromatic r i n g from acetate units (32,47), but due to lack of supporting evidence, they are generally disregarded. 28 Decarboxylation i s a common process i n the secondary metabolism of molds ( 7 4 ) , and i s involved i n a l l fungal benzoqulnone biosynthesis. Pettersson (52) showed that o r s e l l i n i c a c id, s p e c i f i c a l l y l a b e l l e d at C"2 was converted to fumigatin, presumably by a process of non-oxidative decarboxylation (Fig. 17)• That t h i s i s indeed the process i s supported by the f i n d i n g that o r i n c o l was a good precursor of fumigatin (62). H C Y T ^ ^ V C H o 00H H CH^ O' OH O r s e l l i n l Acid O Fumigatin HO. OH Orcinol F i g . 17. Formation of Fumigatin from O r s e l l i n i c Acid and Orcinol. 29 With Gliocladium roseum. the formation of g l i o r o s e l n involves decarboxylation and oxidation at the same carbon atom. l ,3-Dihydroxy -4 ,5-dimethylbenzene was found not to be incorporated into g l i o r o s e l n (54,63) but 5-methyl-orcylal-dehyde, which presumably was converted to the acid derivative i n the organism, was incorporated (53,63). An oxidative decarboxylation reaction was thus implied to have occurred, though', a non-oxldatlve decarboxylase has been i s o l a t e d from the same organism (64). Pettersson has reported the f a i l u r e of incorporation of fumigatin into splnulosin by a certain s t r a i n of P e n i c i l l i u m spinulosum which synthesizes both fumigatin.and spinulosin and both quinones have been shown to be derived from o r s e l l i n i c acid (65). This f a i l u r e can be explained i f t h e i r metabolic pathways depart from each other at the stage of decarboxylation. An oxidative decarboxylation would give r i s e to splnulosin and a non-oxidative decarboxyl-ation would give r i s e to fumigatin (Fig. 18A). A si m i l a r explanation could account f o r the unsuccessful conversion of coprinin hydroquinone to 4-methoxy-6-hydroxy-2,5-toluquinone by Lentinus degener (65) (Fig. 18B). Hydroxylation i s another common metabolic process. It occurs widely i n microorganisms, e s p e c i a l l y i n the degradation of aromatic compounds (66). The conversion of 6-methylsali-c y l i c acid to o r s e l l i n i c acid has been reported by Packter to occur i n Aspergillus fumlgatus. though 6-methylsalicylic acid has never been detected i n normal cultures (67). The 30 A. Non-Oxidative Decarboxylation ^ HO ^ X <v^ rCH3 ^^S:OOH O r s e l l i n i c Acid Oxidative Decarboxylati on Fumigatin Spinulosin B. Non-Oxidative Decarboxylatlon 00H 6 - M e t h y l s a l l c y l i c i Add Oxidative Decarboxylati on OH L V ^ A ) H OH 4-Methoxy-6-Hydroxy-2,5-Toluquinone F i g . 18. Modes of Decarboxylation i n the Biosynthesis of Fungal Toluquinones. 31 conversion of o r s e l l i n i c acid to 6-methylsalicylic acid has been mentioned by Glover, but his r e s u l t s have not been published ( 6 8 ) . The o r i g i n of an epoxide oxygen on a benzoquinone r i n g i s of p a r t i c u l a r Interest. Read, Westlake and Vining ( 5 D have established that atmospheric oxygen i s the source of the epoxide oxygen i n t e r r e i c acid, and the oxygen was introduced into an intermediate derived from 6-methylsalicylic a c i d . Yamamoto et al.,,have presented evidence that fumigatin epoxide i s converted to fumigatol and fumigatin but not vice versa by a s t r a i n of Aspergillus  fumlgatus ( F i g . 11) and that splnulosin arises as an a r t i -f act from t h i s epoxide during i s o l a t i o n . The methyl group of the methoxy function i n fungal benzoquinones has been shown to originate from methionine and formate ( 2 8 , 6 3 ) . The active methyl donor i n t h i s reaction i s regarded as S-adenosylmethionine, the formate carbon being converted to the methyl carbon of methionine before incorp-oration. Steward and Packter have made a study of the 0- and C?»methylation processes i n g l i o r o s e l n biosynthesis with (Me-V*C) and («Me-%)-methionine ( 6 3 ) . They reported that the methyl group was transferred Intact i n O-methylation but only two t r i t i u m atoms from the o r i g i n a l methyl group was transferred i n C-methylation. Similar r e s u l t s with respect to C-methylation have also been observed i n ergosterol and tuberculostearic acid biosynthesis (70). In the same study. Steward and Packter also reported that the s p e c i f i c a c t i v i t i e s 32 of the l a b e l l e d 0-methyl and C-methyl groups showed an appreciable difference. The percentage difference between C-methyl and 0-methyl was l k % i n a methionine-^CH^ feeding, and 15$ when formate-^C was used. To explain t h i s d i s c r e -pancy, they suggested two p o s s i b i l i t i e s . The f i r s t one was that two methyl group pools were involved. The second one was that, i f the C-methylated precursor was already present i n a much larger amount at the time when the l a b e l l e d compounds were added, the 0-methyl groups formed subsequently would have a higher s p e c i f i c a c t i v i t y . It i s generally agreed that the f i n a l step i n fungal benzoquinone biosynthesis i s the oxidation of the correspond-ing hydroquinone. Ktlster and L i t t l e (71) had proposed that fumigatin and spinulosln might be formed from the corresp-onding hydroquinones by the action of an endocellular phenol-ase system, but t h e i r experimental re s u l t s have been disproved by Pettersson (72). In Pettersson's view, these quinones are derived from t h e i r respective hydroquinones by a non-enzymatic a i r oxidation process i n the culture medium (65). He arrived at t h i s conclusion by (1) determining the r e l a t i v e proportion of oxidized to reduced quinone i n the medium during growth of the mold by potentiometry, (2) studying the k i n e t i c s of autooxidation of hydroquinones to quinones, (3) i s o l a t i n g and i d e n t i f y i n g the hydroquinones i n the medium. On the basis of these c r i t e r i a , his re s u l t s from studies of cultures of Aspergillus fumlgatus (72), Gliocladlum roseum (5^ )» 33 P e n l c l l l l u m spinulosum (16) and Lentinus degener (14) have shown that a l l the quinones produced were present In the reduced forms during the larger part of the production phase, being converted into the quinone forms at a l a t e r stage i n the development of the molds. Packter and Steward, by c o r r e l a t i n g the r e s u l t s obtained from the production curve of each metabolite concerned with the s p e c i f i c a c t i v i t i e s of these compounds from radioactive tracer experiments, came to a somewhat d i f f e r e n t conclusion than that suggested by Pettersson ( 7 3 ) . After feeding Gliocladium roseum with acetate-l- l 2 +C, they i s o l a t e d aurantiogliocladin only from the mycelium, and g l i o r o s e l n , the quinol and quinhydrone of aur a n t i o g l i o c l a d i n from the medium. The amounts and s p e c i f i c a c t i v i t i e s were found to increase i n the order l i s t e d . Moreover, when la b e l l e d a u r a n t i o g l i o c l a d i n was added to the medium, 52$ of i t was found to enter the mycelium, and labelled g l i o r o s e l n and .the quinol were obtained on i s o l a t i o n . Also, g l i o r o s e l n has been found to isomerize to the quinone aurantiogliocladin (via the quinol and quinhydrone) when allowed to incubate alone i n autoclaved medium. From these r e s u l t s , Packter and Steward proposed a scheme (Fig. 19) to indicate the i n t e r r e l a t i o n s h i p among these compounds and t h e i r probable s i t e s of formation ( 1 9 ) . With s i m i l a r type of studies with Aspergillus fumigatus. Packter (62,67) concluded that although most of the fumigatin present i n the medium could ari s e from oxidation of fumigatol, some fumigatin was secreted as such, but his int e r p r e t a t i o n of his r e s u l t s was strongly refuted by Pettersson (50)« 34 Mycelium tautomerase Gliorosein < » Quinol rapid spontaneous Glioroseln > Quinol—> Quinhydrone—>• Quinone slow Medium Fig. 19. Proposed Interrelationship of Quinones and Hydroquinone from Gliocladium roseum (Quinol = aurantiogliocladin hydroquinone Quinhydrone = rubrogliocladin Quinone = aurantiogliocladin See F i g . 12 f o r structures) 35 Shanorella s p i r o t r l c h a : A p-Benzoquinone-Produclng Ascomycete Shanorella s p i r o t r l c h a was described by R.K. Benjamin who c l a s s i f i e d i t i n the Gymnoascaceae of the Ascomyeetes (79). The culture used i n this study (UBC 2k0) was kindly provided by Dr. R.J. Bandoni (Dept. of Botany, U.B.C). Shanorellin (2,6-dimethyl-3-hydroxymethyl-5-hydroxy-1,4-benzoquinone) was f i r s t detected as an e x t r a c e l l u l a r purple pigment when S. s p i r o t r l c h a was cultured on malt extract agar. On examination of the medium, the most conspicuous pigment (shanorellin) was found to behave s i m i l -a r l y to the hydroxybenzoquinones, fumigatin and spinulosin, i s o l a t e d by R a i s t r i c k et al. ifrom species of Aspergillus and P e n l c i l l i u m (80,81). The physical, chemical and spectro-scopic properties of shanorellin were determined, and i t s derivatives were prepared. From these data, two isomeric structures were found to be possible. Chemical syntheses were carried out f o r the two isomeric forms, and simultane-ously, the chloro-derivative was prepared and subjected to X-ray crystallographic analysis. Experiments on environmental effects and n u t r i t i o n a l requirement of the culture were performed. The biosynthetic pathway by which shanorellin was produced was studied with various -^C-labelled compounds. From the mixture of pigments present i n the medium, a second compound was i s o l a t e d and i d e n t i f i e d as the acetyl-derivative of shanorellin, a t h i r d and a fourth were found to be ether derivatives of shanorellin. 36 Part A. Environmental Effects on Pigment Production  and Growth i n Shanorella s p i r o t r l c h a Shanorella s p l r o t r i o h a was f i r s t cultured on a l i q u i d medium of modified malt extract. In about f i v e days, a mycelial mat was formed which was white i n i t i a l l y but changed to golden yellow and orange red as i t became established on the surface. About the eighth day, the medium started to turn purple and by the f i f t e e n t h day, the o r i g i n a l l y transparent yellowish brown medium was a dark purple. As the culture aged, the mycelial mat grad-u a l l y turned brown and abundant c l e i s t o t h e c i a were formed. At the same time, the purple pigment i n the medium started to disappear, and i n about two months, the color of the medium was brown. When cultured on Czapek-Dox medium, the mycelium grew very slowly. On addition of yeast extract (0.2$) to the medium, growth and pigmentation resembling that described with malt extract were observed. Czapek-Dox medium with the addition of 0.2$ yeast extract was used as the standard medium i n the investigations which followed. Observations were made of the effects on mycelial growth and pigment-production of pH, temperature and nitrogen and vitamin sources of the medium. 37 General Methods Culture Media (A) Modified Malt Extract - malt extract (Difco) 30g, soytone (Difco) 5g, yeast extract (Difco) l g , d i s t i l l e d water 1 l i t r e . (B) Modified Czapek-Dox Medium - glucose 50g, NaNO^ 2g, KgHPO^ l g , KC1 0 .5g , MgS04.7H20 0 .5g , FeSO^.7H20 O.Olg, yeast extract (Difco) 2g, d i s t i l l e d water 1 l i t r e . For agar plate s , 1.5% agar was added to the above media. Preparation of Inoculum An ascospore from an old culture grown on modified malt extract agar was i s o l a t e d with a micro-manipulator and transferred onto a modified Czapek-Dox agar plate. The culture which developed from t h i s single spore was used f o r subsequent subculturing. A stock culture was kept on a modified malt extract slant. Culture Conditions A l l cultures were maintained i n the dark at 25° i n a Sargent Incubator unless otherwise stated. 38 I. E f f e c t s of pH Method Each sample consisted of one 125ml f l a s k containing 50ml modified Czapek-Dox medium. The pH of the medium was adjusted by taking two autoclaved samples, t i t r a t i n g one with 0.5N HC1 (autoclaved) and the other with 0.5N NaOH (autoclaved), to f i n d the required volume of the acid or base to bring the medium to the pH values ranging from 4.2 to 9.0. The mycelial growth and pigment production were observed a f t e r 7. 12 and 17 days of incubation. Results and Discussion A f t e r 7 days of growth, surface mycelia began to appear i n the solutions with I n i t i a l pH 4.6 to 9.0. On the 12th day, purple pigmentation was apparent i n fl a s k s with i n i t i a l pH ranging from 6.7 to 7.6. In the cultures s t a r t i n g with pH 6.5» 8.1 and 9.0, the purple pigment was already present i n r e l a t i v e l y large amount by t h i s time (Pig. 20). By the 17th day, the media i n f l a s k s with the i n i t i a l pH ranging from 6.0 to 9.0 were opaque with purple coloration, and pigment was beginning to appear In the culture with the s t a r t i n g pH of 5 .3 . Only s l i g h t pigmentation was observed i n the two flasks with the lowest i n i t i a l pH (Fig. 21). The f i n a l hydrogen ion concentrations of these media a f t e r 17 days of growth were measured and are indicated i n Table I, 39 Fig. 20. Appearances of S. s p i r o t r i c h a Cultures with I n i t i a l pH 4.2 to 9.0 afte r 12 Days of Incubation. Fig. 21. Appearances of S. s p i r o t r l c h a Cultures with I n i t i a l pH 4.2 to 9.0 a f t e r 17 Days of Incubation. 40 I n i t i a l pH F i n a l pH 4 . 2 4 . 6 4 . 6 5 . 7 5 . 3 6 . 2 6 .0 6 . 4 * 6 . 5 6 . 6 6 .7 6 .8 7 .0 6 .9 7 .3 7 .0 7 .6 7 . 1 8 . 1 7 . 2 9 . 0 7 . 4 • Table I. Changes i n pH Values of Media Inoculated with S. s p i r o t r l c h a a f t e r 17 Days of Incubation. ( * - normal pH of modified Czapek-Dox Medium) 41 As can be observed from the color photographs, a l l the f lasks showed s i m i l a r mycelial development a f t e r 17 days of incubation, except the one with the normal pH of Czapek-Dox medium, i . e . pH 6.5» This f l a s k showed a more copious mycelial growth as well as an early onset of pigment production. An early onset of pigment production was also shown by the two flasks with i n i t i a l pH of 8.1 and 9« 0. Since the oxidation of hydroquinone to the quinone form by a i r In a buffer solution at pH 8 i s a standard procedure f o r the chemical synthesis of benzoquinone (82), one might expect an early appearance of quinoid pigment i n the medium at an a l k a l i n e pH. However, t h i s explanation does not account for the results observed i n the^culture with an i n i t i a l pH of 6.5/ It i s evident that more experiments must be performed before the e f f e c t s of d i f f e r e n t pH on mycelial growth and pigment production i n S. Spl r o t r l c h a could be explained. It was concluded from the above observations that a l t e r -ations of the pH of the modified Czapek-Dox medium do not give better r e s u l t s i n terms of mycelial growth or pigment production. 42 I I . E ffects of Temperature Method S. sp l r o t r l c h a was cultured i n 125ml f l a s k s with 50ml of modified Czapek-Dox medium per f l a s k . Each set consisted of three flasks and one set was placed i n one of the following temperature chambers! 5 ° , 1 5 ° . 24°, 28°, 32° and 3 5 ° . Mycelial growth and pigment production were observed a f t e r 2 weeks of incubation. Results and Discussion The appearances of the cultures a f t e r two weeks of incubation at d i f f e r e n t temperatures are shown i n F i g . 22. F i g . 22. Appearances of S. s p l r o t r l c h a Cultures Incubated at Different Temperatures. (Prom l e f t to r i g h t : 5°, 15°, 24°, 28°, 32° and 35°) 43 There was no mycelial growth i n flasks kept at 5 ° . A s l i g h t amount of surface mycelium was developed i n f l a s k s incubated at 15°» but no pigment was observed i n the media. The cultures grown at 24° and 28° c l e a r l y showed an abundant production of the quinoid pigment i n the media together with a normal development of mycelial mats. In f l a s k s incubated at 32° and 35°» the mycelia showed a scanty growth and were greyish brown i n color rather than reddish orange, and no e x t r a c e l l u l a r pigment was produced. Thus, the temperature range of 24° to 28° appeared to be most appropriate f o r the growth of and the pigment production by t h i s fungus. The Incubation temperature of 25° was maintained i n subse-quent work. III. Effects of Nitrogen Source Method The basic medium used was modified Czapek-Dox medium without sodium n i t r a t e . Nitrogen containing compounds used as substitutes for n i t r a t e nitrogen are l i s t e d i n Table II. Three agar plates were prepared f o r each compound. Mycelial growth and pigment production were observed on the 7 t h , 12 th and l ? t h day of growth. 44 Nitrogen Sources Concentration (g/1) 1. Ammonium acetate 1.81 2. Ammonium t a r t r a t e 2.16 3. Sodium n i t r a t e 2.00 4. Sodium n i t r i t e 1.62 5. Ammonium sulfa t e 1.55 6. Sodium azide 0.51 7. Urea 0.71 8. Asparagine,hydrated 1.76 9. Casein hydrolysate 2.00 10. Peptone 2.00 Table I I . L i s t of Nitrogenous Compounds and t h e i r Concentrations used i n the Culturing of S. s p l r o t r i c h a . (concentrations were calculated on the basis of percentage of nitrogen i n each compound, taking 2g of sodium n i t r a t e as standard) ^5 Results and Discussion One of the components of the basic medium used i n this i n v e s t i g a t i o n was yeast extract, and since yeast extract contains protein, i t can also serve as a source of nitrogen. I t must be pointed out, therefore, that the re s u l t s of th i s experiment only indicate the ef f e c t s on mycelial growth and pigment production of the substitution of n i t r a t e by other nitrogenous compounds i n the presence of yeast extract protein. That yeast extract (#11) can serve as a source of nitrogen i s indicated i n F i g . 23 and 24. The addition of n i t r a t e (#3). n i t r i t e (#4) and peptone (#10) to the medium gave s i m i l a r r e s u l t s i n respect to fungal growth and pigment production,(Fig. 23 and 24). As sodium azide (#6) i s a respiratory i n h i b i t o r , i t i s not surprising that no growth occurred on the medium prepared with t h i s compound. The cultures grown on urea (#7) and asparagine (#8) showed a thicker mycelial mat and the appearance of i n t r a c e l l u l a r and e x t r a c e l l u l a r pigments were delayed as compared to that observed with yeast extract (F i g . 23 and 24). The ammonium compounds gave variable r e s u l t s . With ammonium sul f a t e , mycelial growth and pigment production observed on the 12th day of culture were s i m i l a r to those observed with yeast extract, but the e x t r a c e l l u l a r pigment seemed to decrease from 12th day to 17th day of growth, i n contrast to the culture with yeast extract l n which i t increased. The mycelial development of cultures with ammonium acetate and ammonium ta r t r a t e was si m i l a r to that 4 6 Pig. 23. Appearances of 12 Day Old Cultures of S. s p i r o t r l c h a Grown on Different Nitrogen Sources, (numbers r e f e r to nitrogen compounds l i s t e d i n Table II, and # 11 Is the control) F i g . 24. Appearances of 17 Day Old Cultures of S. s p i r o t r l c h a Grown on Different Nitrogen Sources. (numbering of plates: 1 2 3 4 5 6 7 8 9 10 11 Numbers r e f e r to nitrogen compounds l i s t e d i n Table II, and #11 i s the control) 47 observed with urea and asparagine. On pigment production, i n t r a c e l l u l a r and e x t r a c e l l u l a r pigments were apparent on the 17th day of growth with ammonium acetate, and these pigmenations were not observed with ammonium t a r t r a t e . The general conclusions that may be drawn from these observations were that there seemed to be a c o r r e l a t i o n between i n t r a c e l l u l a r and e x t r a c e l l u l a r pigment formation, and that, f o r the culture of S. s p i r o t r l c h a , n i t r a t e nitrogen i s among those nitrogenous compounds that, together with yeast extract protein, give the best r e s u l t s i n terms of growth and pigment production. IV. E f f e c t s of Vitamins Methods Agar plates were prepared with Czapek-Dox medium with the addition of the various vitamins l i s t e d i n Table IV. The concentrations of the vitamins are indicated i n Table I I I . Spores from an old culture plate with abundant c l e i s -tothecia were transferred to the prepared plates with s t e r i l i z e d cotton swabs. Observations were made on the 2nd and 17th day of growth. 48 Vitamins Concentration (mg/100ml) Ni c o t i n i c Acid 0.060 Thiamine HC1 0.024 D-Biotin 0.500 X 10 -3 Pyridoxine HC1 0.100 Riboflavin 0.008 Table I I I . Concentrations of the Vitamins used as Supplement to Czapek-Dox Medium. Results and Discussion A comparison of the extent of mycelial growth and pigment production when Czapek-Dox medium was supplemented with the vitamins l i s t e d i n Table IV to that of yeast extract on the 2nd and 17th day of growth of S. s p i r o t r l c h a i s shown i n Table V. The plates i n F i g . 25 are representa-t i v e examples of the appearance of the cultures, and F i g . 26 shows the extent of mycelial formation a f t e r 17 days of incubation when yeast extract was substituted by other vitamins. These r e s u l t s c l e a r l y demonstrated that the substitution of yeast extract by the vitamins studied brought about a great retardation of mycelial growth. The scanty growth of mycelium i n these plates might be taken as an i n d i c a t i o n that there are some other growth factors l n the yeast extract that are required by the fungus f o r proper development. 49 Plate No. N i c o t i n i c Acid Thiamine HCl B l o t i n Pyrldoxine HC1 1 + 2 + 3 + 4 5 + + 6 + + 7 + + 8 + + 9 + + 10 + . + 11 + + + 12 + + + 13 + + + 14 + + + + 15 + + and Riboflavin + + 16 Y Yeast Extract Table IV. L i s t of Vitamins Used i n the Supplementation of Czapek-Dox Medium. 50 2nd Day 17th Day Plate No. Myoel. Growth Ex. Plgm Mycel. Growth Ex. Plgm 1 ± — + ± 2 + - + + 3 + - + + 4 + - + 5 + mm + + 6 + - + 7 + - + + 8 + - + + 9 + - + + 10 + - + + 11 + - + + 12 + - + + 13 + - + + 14 + MB + + 15 + - + + 16 + - + + Y ++ — +++++++ +++++ Table V. Results of Mycelial Growth and E x t r a c e l l u l a r Pigment Formation on Various Vitamin Sources i n Cultures of S. s p l r o t r l c h a . ( - no pigment observed; + s l i g h t growth and pigment observed; + growth and pigment observed) 51 A l l the media i n which pyridoxine was present possessed a greater amount of purple coloration as compared with those i n which other vitamins were added (Fig. 2 5 ) . Thus i t seemed that pyridoxine has some effect on the e x t r a c e l l u l a r pigment formation process. Fig. 25. Appearances of 17 Days Old Cultures of S. s p i r o t r l c h a Grown on Various Vitamin Sources, (number r e f e r to vitamins added as l i s t e d i n Table IV) Fig. 26. Extent of Mycelial Growth of S. s p i r o t r l c h a on Plate #3. 52 Part B. Production of E x t r a c e l l u l a r Colored and Phenolic  Compounds In Relation to pH and Glucose Concen- t r a t i o n of the Medium and Changes In Mycelial  Dry Weight Methods Culture Condition The organism was cultured i n Roux bottles with 100ml modified Czapek-Dox medium per bottle. General Procedure and Isolation of Shanorellin Each sample consisted of three Roux bottles. The medium was separated from the mycelial mat by f i l t r a t i o n through three layers of cheese c l o t h (preweighed). The pH of the f i l t r a t e was measured and the solution d i l u t e d to 500ml with d i s t i l l e d water. An aliquot of 10ml was taken for glucose determination. The remaining solution was a c i d i f i e d with d i l u t e HC1 to pH 2 and extracted with ethyl acetate. After the removal of ethyl acetate with a f l a s h evaporator, the residue was dissolved i n 1 ml ethanol. 0.05ml of the solution was subjected to thin-layer chromatography f o r detection of colored and phenolic compounds. The remainder of the ethanolic solution was evaporated to dryness and the residue dissolved i n CHCly This solution was passed through a s i l i c i c acid column with CHCl^ as the elutlng solvent. The shanorellin band, which appeared as the t h i r d colored one on the column, was collected. The residue obtained on removal of the solvent from th i s f r a c t i o n was 53 sublimed at 100° under vacuum. The sublimate was further p u r i f i e d by thin-layer chromatography with solvent system A and the product resublimed. Its concentration was determined by u.v. spectrophotometry. Determination of Absorption Curve of Shanorellin The absorption of shanorellin i n chloroform at 272nm at d i f f e r e n t concentrations was measured with a Unicam SP. 800 U l t r a v i o l e t Spectrophotometer. Solutions of shanorellin were found to obey the Beer-Lambert Law at concentrations of 0 to 0.9 X 1 0 " 2 mg per ml. (Fig. 2 7 ) . Determination of Mycelial Dry Weight. pH and Glucose The mycelial mat on the Btlchner funnel a f t e r separation from the medium i n the above procedure was dried overnight on a p e t r i d i s h i n an oven at 90° and weighed. The pH of the medium was measured with a Leeds & Narthrup pH meter. Glucose concentration was determined by the method of Nelson ( 8 3 ) . Detection of Colored and Phenolic Compounds The thin-layer plates were developed two-dimensionally with solvent systems B and C. After observation under u.v. l i g h t , the plates were sprayed with a reagent to detect the presence of phenolic compounds. C hromat o gr ap h.v (I) Column Chromatography S i l i c i c acid columns were prepared by mixing three parts of s i l i c i c a cid (Baker Analyzed reagent) to one part of Hyflo Super Cel (Fisher Laboratory Chemical). Chloroform 54 ( a n a l y t i c a l grade) was used as the eluent solvent. (II) Thin-Layer Chromatography Thin-layer plates were prepared by mixing 40g of s i l i c a gel G (Merck) with 100ml of d i s t i l l e d water. This mixture was applied with a spreader (Desaga) onto the plates to give a thickness of 0.5mm. The plates were activated at 110° f o r 1/2 hr and kept at 80° u n t i l used. The solvent systems used were as follows: (A) Benzene : Acetic Acid 9 « 1 (v/v) (B) Chloroform : Acetic Acid 500 : 37-5 (v/v) (C) Cyclohexane : Ethyl acetate : Acetic Acid 20:10:1 (v/v/v) Phenolic compounds were detected by spraying with a f r e s h l y prepared mixture of the following solution f i r s t : 0.3$ p - n l t r o a n i l l n e i n 8$ HC1 5ml 5$ sodium n i t r i t e 1ml 20$ sodium acetate 15ml The plates were then oversprayed with 2N NaOH. 0.4 0.8 1.2 1.6 2.0 Shanorellin Concentration 10"2mg/ml. Fig. 27. Absorption Curve of Shanorell in in CHCI3 at 272 nm. 56 Results and Discussion In the f i r s t eight days of growth, there was a rapid increase of mycelial dry weight, a r i s e i n pH and rapid u t i l i z a t i o n of glucose from the medium (Fig. 28). During t h i s period, only traces of phenolic compounds were detected by thin-layer chromatography. On the 8th day, shanorellin began to appear, the pH of the medium decreased, and the mycelial growth rate started to decrease* By the 18th day, the shanorellin concentration i n the medium was at i t s max-ium. Afte r t h i s , i t gradually disappeared i n d i c a t i n g that i t was being metabolized further to other compounds. The s l i g h t r i s e of pH between the 27 th and 45 th day suggests*? that there was a production of new metabolites by the organism. There were four other spots i n addition to shanorellin present i n conspicuous amounts on thin-layer plates (Fig. 29-35)• A number or l e t t e r , as shown i n F i g . 36, i s given f o r each spot to f a c i l i t a t e t h e i r i d e n t i f i c a t i o n on the plates and f o r the purpose of discussion. Spots A and C and shanorellin are yellow and e a s i l y observed on the plates (Fig. 3 5 ) . They appear dark purple under u.v. l i g h t . Spot A was found to consist of two compounds (Compound A and Compound B). These two compounds, together with Compound C (spot C), were isol a t e d . Their chemical properties w i l l be discussed i n d e t a i l i n Part C together with the s t r u c t u r a l determination of shanorellin. T l TO 00 ro CL f-C 3 l— A) H P 3" O CL fl) r t P fl) s TO ro o cn O ro l-h t—' H-H- P OJ fl) (—' T3 pa PC o o « c X VI O w 33 c o fD o r t H* o r t TO cn h-> p r ro o fB r t i-i CD fl) o p r t r t p r p r c/3 p r <D fl) ' \ p o o o fl) i-i V ! r t ro cn p r i — • •—' h- 1 H-P P C/D PT o 0) o P p O o i-i ro ro p r t h-' i-i DJ fl) r t cn h1' X) O H- P l-i cn O r t i-i P H-n r t PT p r fli ro OJ -1— On _1_ I VO _L_ PH.. G l u c o s e Cone, (g) o M y c e l i a l Dry Weight (g) p o © © 00 NJ 4> OJ J S h a n o r e l l i n Cone (mg) -p-o Li .1 58 Compounds I and II were the major compounds detected on the plates with the phenolic reagent spray. Compound I was present i n the medium before shanorellin on the 7th day of culture and persisted throughout the period studied. Compound II appeared l a t e r and i t s concentration seemed to increase and then decrease as the culture became older. E f f o r t s to i s o l a t e compound I and II f o r s t r u c t u r a l determinations were unsuccessful because they seemed to be absorbed by s i l i c a gel G and only trace amounts could be eluted from the plates. Similar studies on changes i n pH, glucose concentration and mycelial growth i n r e l a t i o n to benzoquinone formation i n fungi have been carried out by Pettersson with Aspergillus  fumlgatus (72, 84) and by Read, Westlake and Vining with Aspergillus terreus ( 6 l ) . Since;the medium used i n each culture was d i f f e r e n t , a comparison of the factors mentioned and benzoquinone formation i n these fungi could not be made. Fig. 30. Fig. 33 Fig. 29-31*. Detection of Colored and Phenolic Compounds i n the Culture Medium during Growth of S. s p i r o t r l c h a by Thin-layer chromatography, (number on plate indicates the days of incubation; a l l plates were sprayed with p - n l t r o a n i l i n e reagent) 61 Fig. 35. Similar to Fig. 29-3^ but without spraying with p - n l t r o a n i l i n e reagent. Fig. 36. Typical Thin-Layer Plate I l l u s t r a t i n g the Relative Positions of the Compounds Present l n Conspicuous Amount. ( S = Shanorellin, A and C = Benzoquinones, I and II = Phenolic Compounds, o = origin) 62 Part C. Elucidation of the Chemical Structures of  Shanorellin. i t s Monoacetate (Compound A)  and i t s Ether Derivatives (Compound B and  Compound C) The p a r t i a l s t r u c t u r a l determination of shanorellin was accomplished by chemical and spectroscopic studies (4). The f i n a l structure was determined by X-ray d i f f r a c t i o n crystallography carried out by Dr. E. Subramanian and Dr. J. Trotter (Chemistry Dept., U.B.C). Preliminary examination of Compound A, B and C indicated that they were clos e l y related to shanorellin and s u f f i c i e n t amounts of these three compounds were isola t e d for u.v., i . r . y n.m.r. and mass spectroscopic analyses. From these r e s u l t s , chemical structures can be proposed for them. Methods Culture Conditions For i s o l a t i o n of metabolites produced i n the medium, 1 l i t r e flasks with 400ml of modified Czapek-Dox medium per f l a s k were used. A l l the other conditions were the same as those described i n the general procedure of Part A. Iso l a t i o n of Metabolites The i s o l a t i o n and p u r i f i c a t i o n procedures as described i n Part B f o r shanorellin were followed. Compound A, B and C appeared together as the second yellow band on a s i l i c i c a c id column and were subsequently separated from one another 63 by t i c with solvent system A (Part B) and solvent system D (Chloroform.-acetic acid 9«1 v/v). A l l compounds were c r y s t a l l i z e d from benzene-light petroleum (b.r. 6 5 - 1 2 0 ° ) . Instrumentationa Melting Point : Thomas Hoover C a p i l l a r y Melting Point Apparatus. ( A l l readings were uneorredted). U.V. Spectrophotometry.' Unicam SP 800 U l t r a v i o l e t Spectrophometer. I.R. Spectrophotometry : Unicam SP. 200G Infrared S p e c -trophotometer. N.M.R. Spectroscopy : Varian Model HA-100 and T - 6 0 , Joel C60H. In a l l samples, CDCl^ was used as solvent with tetramethylsllane as i n t e r n a l standard (Chemistry Dept., U.B.C). Mass Spectroscopy : Associated E l e c t r i c a l Industries Model M89 Instrument (Chemistry Dept., U.B.C ). Elemental Analysis Elemental analyses were carried out by Dr. C. Daessle of Montreal. Preparation of Derivatives (1) Shanorellin dlacetate. To shanorellin (lOOmg) i n acetic anhydride ( 2 m l ) was added cone. H 2 S0^ (2 drops) and the solution warmed for a few seconds. The diacetate separ-ated out as a brownish yellow o i l on addition of i c e water and was extracted with ethyl ether. On evaporation 64 of the ether, a viscous brownish yellow o i l (93.8mg) was obtained which could not be c r y s t a l l i z e d from either methanol or l i g h t petroleum. Tic on s i l i c a gel G with solvent system A showed the presence of one compound at O . 5 5 , (n.m.r. spectrum: 2 CH^ at r 7 . 8 7 and 8 . 0 3 ; CH 2 at T 5 . 4 and 2 CH^CO at t 7 . 6 8 and 7 . 9 8 ) . (2) Shanorellin tetra-acetate. Shanorellin (lOOmg) i n ethanol (5™-l) was treated with NaBH^ u n t i l the yellow color disappeared. Water ( 2 0 m l ) was added, and the solution extracted four times with ethyl ether ( 2 0 m l ) . The ether extract was dried with anhydrous sodium sulfate and the solvent removed. Acetylation with a c e t i c anhyd-ride ( 2 m l ) and pyridine ( 2 m l ) overnight at room tempera-ture followed by addition of ethanol ( 10ml) and evaporat-ion In vacuo below 40° u n t i l the pyridine was removed gave a s l i g h t l y yellowish gummy residue. C r y s t a l l i z a t i o n from methanol afforded the col o r l e s s tetra-acetate. (m.p. 1 1 2 ° , y i e l d 47mg. n.m.r. spectrum: 2CH^ at T 7 . 8 7 and 8 . 0 5 ; CH 2 at T 4 . 9 5 ; 4 CH3CO at T 7 . 7 , 7 . 7 4 , 7 . 7 8 and 8 . 0 3 ) . (3) Shanorellin chloride. Thionyl chloride (65.45mg) was added dropwlse to shanorellin (91mg) i n chloroform (20ml) and pyridine (39.5mg) i n an ice bath. Afte r the addition the solution was warmed to room temperature and then refluxed f o r 2 . 5 hr. The solvent was removed i n vacuo and the residue chromatographed on s i l i c a gel G plates with solvent system A as the developing system. The chloride band had 65 an Rf of 0.6. I t was c r y s t a l l i z e d from hexane and chloroform giving orange-yellow needles, (m.p. 69°, y i e l d 46mg. MW 200.5. Founds C, 53.67? H, 4.4; C l , 17.66. C ^ O ^ C l required. C, 53.86; H, 4.48; C l , 17.71 per cent, n.m.r. spectrum: 2 CH-j at r 7.8 and 8.03; CH 2 at T 5.53; OH at T 2.985). Results and Discussion A l l hydroxybenzoquinones produced by fungi have been detected as purple pigments i n the medium (5, 80,81). The s o l u b i l i t y of these compounds i n water i s due to the disso-c i a t i o n of the a c i d i c hydrogen at the hydroxyl group situated at the carbon (A to the carbonyl function. Other character-i s t i c s of t h i s type of compound include the ease of sublima-t i o n and oxidation of the reduced form i n a buffer solution at pH 8 by aeration. Shanorellin, Compounds A, B and C were shown to possess these properties. The chemical and spectro-scopic studies on each compound w i l l be discussed i n d i v i d u a l l y . I. Shanorellin Shanorellin c r y s t a l l i z e s as bright orange-yellow needles, m.p. 121°, and sublimes at 100° under vacuum. Its solutions are decolorized by sodium borohydride or sodium d i t h i o n i t e , and on adjusting the pH to 8 and aeration, a purple color appears i n d i c a t i n g the quinone i s formed. I t i s unstable i n highly a l k a l i n e solution, e.g. 2N NH^OH or 0.5N NaOH, turning brown a f t e r standing f o r 15 minutes. From the elemental analysis (Found: C, 58.81; H, 5.56 66 per cent) and the molecular weight of 182 from the mass spec-trum (Fig. 37)» the molecular formula of shanorellin i s c a l -culated to be C H 0^ (Required: C, 59.3; Ht 5.5 per cent). ill | i ! I .J i M IJ ; F i g . 37. Mass Spectrum of Shanorellin. The u.v. absorption spectra of hydroxybenzoquinones variously substituted with methyl groups show two regions of absorption: (1) a strong band i n the range of 263-280nm (loge ca 4) and (2) a weak band i n the range of 379-409nm (loge. 2-3) (85). Shanorellin with absorption bands at 272nm (log £ 4.05) and 406nm (log€ 2.07) i n chloroform (Fig. 38) shows that i t has si m i l a r absorption properties to the hydroxy-alkylbenzoquinones. In ethanol, the band with ^Max 3?2 l n chloroform i s shifted to 269.5nm and the one 2.0 ~~300 325 350 400 WAVELENGTH mji Fig. 38. U l t r a v i o l e t Spectrum of Shanorellin i n CHCl^. at 406nm remains the same. On addition of NaBH^, the band at 269.5nm disappears, and,;.if the spectrum i s taken immediately, a band at 355nm appears, and on standing, this band i s gradually replaced by another at 300nm. The i . r . spectrum of p-benzoquinones have been studied by Yates, Ardao and Fieser (86). Two bands are observed i n the carbonyl stretching region i n these compounds, and i n shanor-e l l i n , they appear at 1660 and 1640 cm Other bands i n the spectrum of shanorellin are the 0-H stretching vibration at 3180 and 3^50 cm"1, C=C stretching at 1620 cm"1, and alkane C-H stretching i n the region of 2900-3000 cm"*1 (Fig. 39). 68 F i g . 39 . I n f r a r e d S p e c t r u m o f S h a n o r e l l i n i n K B r D i s c . The n.m.r. s p e c t r u m o f s h a n o r e l l i n r e v e a l s t h e p r e s e n c e o f f i v e t y p e s o f p r o t o n s i n t h e p r o p o r t i o n o f l i 2 : l : 3 : 3 ( T , 3 . 0 6 , 5 .475. 7 .75. 7.885 a n d 8 . 0 7 5 . F i g . 4 0 ) . on a d d i t i o n o f D 2 0 t h e p e a k a t T 3.06 a n d t h e b r o a d b a n d a r o u n d f 7.75 d i s a p p e a r i n d i c a t i n g t h e p r e s e n c e o f two e x c h a n g e a b l e p r o t o n s . Of t h e r e m a i n i n g p e a k s , t h a t a t T 5*475 c o u l d be a s s i g n e d t o m e t h y l e n e p r o t o n s a n d t h e o t h e r t w o , a t 7 7.885 a n d 8,075» t o p r o t o n s o f two m e t h y l g r o u p s . On e x p a n s i o n o f t h e sweep w i d t h o f t h e s p e c t r u m ( 1 0 X ) , t h e m e t h y l e n e p r o t o n s a p p e a r a s a q u a r t e t , a n d t h e m e t h y l p r o t o n s a t T 7.885 a s a t r i p l e t . T h e s e two g r o u p s o f p r o t o n s a r e t h u s c o u p l e d w i t h e a c h o t h e r ( c o u p l i n g c o n s t a n t J = 0 . 5 H z ) . 69 F i g . 40. N.M.R. Spectrum of Shanorellin i n CDCl^, The coupling constant i s too small f o r a CH^CHg function but could account f o r a methyl and methylene group on adjacent carbons, on the same side of the quinone nucleus. The n.m.r. spectrum of tauranin ( I ) , a benzoquinone produced by Oospora aurantia (87), i s of p a r t i c u l a r relevance to shanorellin because th i s compound possesses an hydroxy as well as a hydroxymethyl function. The T values of the hydroxyl protons and the methylene protons are very close to those observed i n shanorellins 2.95r O 5.47r HCX^^NYCHaOH R = (I) 70 Thus, the band with 7 at 3•06 could be assigned to the nuclear hydroxyl proton while the band at 7 .75 to the hydroxyl proton of the hydroxymethyl group. A diacetate and a tetra-acetate of shanorellin were prepared and the n.m.r. spectra obtained. The preparation of these derivatives confirmed the presence i n the molecule of two hydroxyl functions i n addition to the two carbonyl function of the benzoquinone nucleus. With th above information, i t i s possible to assign either of the two isomeric structures shown below to shanorellin: o CH20H CBjyf^^ CH20H H 0 ' s v > < ^ C H 3 O (III) The mass spectrum of shanorellin ( F i g . 37) could not be used to d i s t i n g u i s h between these two isomeric forms either, From the known fragmentation pattern of benzoquinones ( 8 8 ) , these two isomers would give s i m i l a r ions: R ^  \ Compound (II) (III) 71 A common feature of the mass spectra of benzoquinones i s the occurrence of the ions corresponding to the loss of one and of two molecules of carbon monoxide ( 8 9 ) . In shanorellin, they are represented by the peaks at masses 15^ and 126. The base peak at mass 83 could be accounted for by the 1 ,2-and 4,5-bonds cleavages with the loss of a hydride ion (Fig. 4lA). The intense peak of mass 80 could be derived from the ion with mass 15^ by dehydration and further cleavage of the 5 membered r i n g (Fig. 4lB). The dehydration of benzyl alcohol with an ortho methyl group has been studied by Aczel and Lumpkin ( 9 0 ) . In thi s type of compound, the peak due to ( p — H 2 0) ion i s often one of the most prominent ones i n the spectra, and i t i s possible due to the formation of a hydrogen bond p r i o r to fragmentation. To account f o r the appearance of some of the major peaks i n the mass spectrum of shanorellin, the fragmentation patterns shown i n Fig. 4 l A and 4lB are proposed. The f i n a l structure of shanorellin (II) was obtained by X-ray crystallographic analysis of the chloro-derivative of shanorellin ( 2 , 6-dimethyl - 5-hydroxy - 3-chloromethyl-l , 4 -benzoquinone). This was determined by Dr. E. Subramanian and Dr. J. Trotter of the Chemistry Dept., U.B.C. 72 o HO > * \ ^ > X : H 2 0 H o m/e 182 •I CH3 c m/e 84 - H-t II! CH3. 0^ m/e 83 -CO CH 3Y c - c o * CH3C+ m/e 27 . ^ H 3 C-^0H->0H II ? m/e 98 - • C H 2 O H H3 0 + m/e 67 -CO 3 H 3 + m/e 39 0' m/e 55 F i g . 41A. Some of the Possible Bond Cleavages i n Shanorellin upon Electron Empact. 73 |CH3 HO «=CH m/e 108 C H 3ln _H-cr m/e 56 ^ C H 2 m/e 136 J ^CH 2 C > C H 2 ' 0 m/e 80 CH3 HO -Cr-CH3 CH20H| m/e 126 CH3. H O ^ m/e 56 L ^CH2OHJ m/e 70 CH 3^ III. c + m/e 39 ( l f C H 3 ^ C H 2 m/e 53 CH3 m/e 55 ,^CH2 ^CH 2 m/e 52 F i g . 41B. Some of the Possible Bond Cleavages i n Shanorellin upon Electron Impact. 74 I I , Compound A Compound A c r y s t a l l i z e s as bright orange-yellow needles, m.p. 128° and sublimes at 100° under vacuum. As with shanor-e l l i n , i t s solutions are decolorized by sodium borohydride .•: and sodium d i t h i o n i t e , and on adjusting the pH to a l k a l i n i t y , a purple color appears on aeration. The u.v, spectrum of Compound A i n chloroform shows a strong absorption band at 269nm with a shoulder at 274nm and a weak one at 395nm (Fig. 42). These bands are within the regions of absorption shown by hydroxybenzoquinones and with sim i l a r extinction c o e f f i c i e n t s ( 8 5 ) . In ethanol, these bands are shifted to 267.5nm and 297nm. The shoulder that appears i n chloroform i s not observed. On addition of NaBH^, as i n shanorellin, the band at 267.5nm i s f i r s t replaced by one at 360nm then by one at 295nm. F i g . 42. U l t r a v i o l e t Spectrum of Compound A i n CHC1 75 Fi g . 43. Infrared Spectrum of Compound A i n KBr Disc. 76 The i . r . spectrum of Compound A (Fig. 43) indicates the following functional groups: quinone C=0 stretching at 1665 and 1645 cm - 1, C=C stretching at 1635 cm"1, alkane C-H stretching i n the region of 2900-3000 cm"1, ester C=0 s t r e t -ching at 1740 cm"1 and ester C-0 stretching a t 1255 cm"1. A comparison of thi s spectrum with that of shanorellin shows that apart from the ester function bands, Compound A contains bands that are c h a r a c t e r i s t i c of shanorellin. Thus an ester of shanorellin i s indicated. That th i s compound i s an ester derivative of shanorellin i s confirmed by the n.m.r. spectrum and mass spectrum. The n.m.r. spectrum (Fig. 44) reveals that there are 5 types of protons i n the proportion of 1 : 2 : 3 : 3 : 3 at T 3.04, 5 .00 , 7 .86 , 7.97 and 8 .07 . F i g . 44 . N.M.R. Spectrum of Compound A i n CDC1 77 The peak at r 3.04 disappears on addition of DgO. The value of the three protons at*7.86 indicates that i t could be due to an ace t y l function as i n shanorellin diacetate, whose acetyl protons give s i m i l a r T value. The mass spectrum of Compound A (Fi g . 45) gives the molecular peak at mass 224, the molecular weight of the monoacetate derivative of shanorellin, with the acetate ion (mass 43) as the base peak. The peaks prominent i n shanorellin are also observed i n t h i s spectrum: masses at 53, 55. 6?, 80, 83, 108, 136, 154 and 182. F i g . 45. Mass Spectrum of Compound A. 78 The peaks of masses 164 and 60 could be accounted f o r by the following fragmentation! • C H ^ C H J HO I O8SC-CH3 m/e 60 To confirm the presence of the ester function, a hydrolysis reaction was carr i e d out on Compound A with d i l u t e NaOH f o r one minute. The r e s u l t i n g compound gave the same u.v. spectrum and Rf values on thin-layer plates as shanorel-l i n (Rf values of Compound A i n solvent system A i> 0.55, system B» 0.79, system C« 0.45, system D 0 .79). Thus, the following structure i s proposed f o r Compound A : C H 3 M ^ N V C H 3 HO ><X^>^CH20-C-CH3 O Since ethyl acetate was used i n the i s o l a t i o n procedure, i t was considered possible that Compound A was an a r t i f a c t of the extraction procedure used i n i s o l a t i n g shanorellin. The extraction procedure was repeated with a sample of shan-79 o r e l l i n . The residue obtained a f t e r removal of the solvent was chromatographed on thin-layer plate and developed i n solvent system A. In addition to shanorellin, a samll amount of another yellow spot with the same Rf as Compound A was present on the plate. This yellow compound turned to a purple color on exposure to ammonia vapour and had the same u.v. spectrum as Compound A. Similar r e s u l t s was also observed when shanorellin (lOmg) i n ethyl acetate (20ml) i n the presence of cone. HC1 (2 drops) and anhydrous sodium sulf a t e was allowed to stand f o r one week at room temperature. Thus Compound A could be an a r t i f a c t . One way to avoid t h i s complication a r i s i n g from solvent i s to substitute ether f o r ethyl acetate. I t should be pointed out that Compound A, as well as Compounds B and C were not produced by every batch of culture. 80 I I I . Compound B Compound B c r y s t a l l i z e s as f i n e d u l l orange yellow needles, m.p. 170°,and sublimes at 145° under vacuum. It i s soluble i n alk a l i n e medium giving a purple coloration and on a c i d i f i c a t i o n and extraction with chloroform, the o r i g i n a l compound i s recovered as shown by i t s u.v. spectrum and Ef values on thin-layer plates (Rf values of Compound B i n solvent system A: 0.4-9, system Bt 0 .77, system C: 0 . 4 4 , system Di O .76) . Compound B possesses two bands i n the u.v. spectrum (Fig. 46), with \ i n chloroform at 268nm ( l o g * 4.48) and at 405nm (loge. 3 . 1 0 ) . In ethanol, these bands are sh i f t e d to 267nm and 406nm. On addition of NaBH^, the band at 267nm i s displaced f i r s t to 362nm, and a f t e r one hour, t h i s band i s further replaced by one at 305nm. l.s 1.6 i.i 1.2 1 .0 I 0 . 8 2 3 0 . 6 O A 0 . 2 0 . 0 200 225 250 275 300 325 350 400 Tw W»T« length, c run • Fig. 46. U l t r a v i o l e t Spectrum of Compound B i n CHC1,.. 81 F i g . 47. Infrared Spectrum of Compound B i n KBr Disc. 82 The i . r . spectrum of Compound B (Fig. 47) shows the following bands: 0-H stretching at 3 3 3 0 cm"1, C=C stretch-ing at 1620 cm""1, quinone C=0 stretching at 1660 and 1640 cm"1, and ether C-0 stretching at IO85 cm"*1. A comparison of the fi n g e r - p r i n t region of thi s spectrum with that of shanorellin indicates that Compound B possesses a number of bands that are present also i n shanorellin, e.g. 0-H bending and C-0 stretching of the t e r t i a r y alcohol at 1180 and 1 3 5 5 cm""1, the C-H alkane bending bands i n the region between 1400 and 1 5 0 0 cm"*1. The n.m.r. spectrum of Compound B (Fig. 48) shows four types of protons at 1 2.95» 5.5, 7.825, 8.02 i n the proportion of 1:2 : 3 : 3 . The band at T 2.95 disappears on addition of DgO. These T values c l o s e l y resemble those given by shanorellin. . . - / v . . J 1 1 . 1 1 , 1 , •-] ; 1 I 1 I . I _. L5 6 7 8 T L F i g . 48. N.M.R. Spectrum of Compound B i n CDCl 83 Using t h i s information, the following structure i s pro-posed f o r Compound B: CH 2— 0 — C H 2 > \ f i ^ x ) : The proposed structure of Compound B i s confirmed by-i t s mass spectrum which shows the molecular peak at mass 346 (Fig. 4 9 ) . F i g . 49 . Mass Spectrum of Compound B. 84 The peak at mass 181 could be assigned to the ion produced from the cleavage of the ether linkage ( 9 0 ) ; H A 2 T G H 2 R RCH2 m/e 181 The base peak at mass 83 and those at masses 53» 55» 67, 108 and 136 also occur i n shanorellin's spectrum. They could be accounted f o r by the subsequent fragmentation of the ion derived from the cleavage of the ether bond by processes s i m i l a r to that described f o r shanorellin. The peak at mass 166 could be derived by si m i l a r bond cleavage and hydride transfer as proposed f o r bibenzyl ether (90): CH HO 3 ^ A ^ Y ° H 3 o C H ^ ^ ^ ^ CH3 + OHCR and the further loss of a carbon monoxide from t h i s ion of mass 166 would give the peak at mass 1 3 8 . Additional loss of a hydride ion and a carbon monoxide would give the peak at mass 109 and the loss of a *CH2 would give the mass 9 5 . 85 The a c e t y l derivative of Compound B had been prepared by the method described f o r shanorellin diacetate. The product was p u r i f i e d on s i l i c a gel G plate with solvent system D ( Rf 0.85, m.p. 105°. n.m.r. spectrum: CR"2 at T 5.16} 2 CH3 at T 7.83 and 8.00; CH3CO at T 7.60). 86 IV. Compound C Compound C i s a viscous, orange-yellow l i q u i d which evaporates r e a d i l y at 78° under vacuum. It i s soluble i n a l k a l i n e medium to give a purple solution,,and, on a c i d i -f i c a t i o n and extraction with chloroform, i s recovered unchanged as indicated by i t s u.v. spectrum and Rf values on thin-layer plates (Rf values of Compound C i n solvent system A: 0 .6 ,.system B: 0 . 8 3 , system C: 0 .51 , system D: 0 . 8 5 ) . The u.v. spectrum of Compound C (Fig. 5°) i n chloro-form shows the two bands t y p i c a l of benzoquinones : \ 269.5nm (log£ 4 . 4 9 ) and 404nm (log<£ 3»10). In ethanol, these bands are s h i f t e d to 267.5nm and 407nm. On addition of NaBH^, as i n previously described compounds, the band at 267.5nm i s replaced f i r s t by one at 359nm and l a t e r by another at 279nm. The i . r . spectrum (Fig. 51) indicates the presence of 0-H stretching v i b r a t i o n at 3400 cm"1, alkane C-H stretching at 2895, 2940 and 2985 cm"1, quinone C=0 stretching at 1655 and 1645 cm"1, C=C stretching at 1630 cm"1 and the ether C-0 stretching at 1105 cm"1. The n.m.r. spectrum (Fig. 52) indicates the presence of an ethyl function i n addition to those closely resembling that of Compound B. The methylene protons at T 6.43 (quartet, J = 7Hz) and the methyl protons at * 8.80 ( t r i p l e t , J = 7Hz) 87 are evidently coupled with each other. The band at T 2 . 8 6 could be assigned to a nuclear hydroxyl proton, and this assignment i s further supported by i t s disappearance from the spectrum a f t e r the addition of D2O. The band at T 5 . 5 8 could be due to methylene protons and the ones at 17 7 . 8 3 and 8 . 0 7 to methyl protons. Thus the following structure i s proposed f o r Compound Ci C H g - O — C H g C H ^ The mass spectrum (Fig. 53) gives the molecular peak at mass 210, the molecular weight of the structure shown above. The general grouping and some of the major mass peaks of t h i s spectrum show great s i m i l a r i t i e s to that of shanorellin, e.g. peaks at masses 3 9 , 53, 55, 6 7 , 80, 8 3 , 108 and 1 3 6 . The base peak of mass 31 could be derived as i n ethyl butyl ether by a «o, (J cleavage with a single hydrogen transfer: (91) CHg- V C H 2 - C H 3 CHq + ~* C H g s O H + » C H 3 + HO m/e 31 88 The peaks at masses 181, I65, 153, 45 and 29 could be due to ions formed by the following cleavages» CH3 ' j m/e 45 CH27O-CH2CH3 m/e I65 / ! m/e 29 CH2-0|CH2CH3 G m/e 181 -CO m/e 153 89 ~2io 2i5 250 2^ 5 3& 325 350 400 ^50 Wavelength, o l l l ia lcrone Fig. 50. Ultraviolet Spectrum of Compound C in CHC1 90 Wavanunbar, cn"l Fig. 51 . Infrared Spectrum of Compound C in KBr Disc. 91 Fig. 5 3 . Mass spectrum of Compound C. 92 Part D. The Biosynthesis of Shanorellin As stated i n the introduction, three metabolic pathways have been shown to lead to the biosynthesis of p-benzoquin-ones. From the structure of shanorellin, the absence of any lsoprene unit precludes i t s formation from mevalonate. The biosynthesis of the benzoquinone au r a n t i o g l i o c l a d l n (7) and coprinin hydroquinone (92) have been reported to be derived from compounds v i a the shikimic acid pathway. 14 Therefore, C-labelled shikimic acid, phenylalanine and tyrosine were fed to S. s p i r o t r l c h a and t h e i r incorporation into shanorellin determined. Propionate has been shown to be u t i l i z e d by Streptomyces i n the biosynthesis of macrolide a n t i b i o t i c s (93) and by P e n i c l l l i u m baarnense to give homoorsellinic acid (94). Since the number of carbon atoms i n shanorellin i s a multiple of three, the p o s s i b i l i t y of the p a r t i c i p a t i o n of propionate i n shanorellin production, though remote, was tested. The roles of acetate, malonate and the donors, methionine and formate, were also investigated. The o r i g i n s of the carbon atoms of shanorellin were determined by the chemical degradation of s h a n o r e l l i n - ^ C obtained i n feeding experiments. Based on the r e s u l t s obtained, a possible route to i t s biosynthesis i s suggested. 93 Methods Culture Conditions Cultures of S. s p l r o t r l c h a were grown i n 125ml flasks or i n Roux bottles as stated i n each experiment. The preparation and conditions of cultures were the same as described i n the general methods i n Part A. Preparation of Radioactive Compounds 14 A l l C-labelled compounds were dissolved i n water to give the appropriate concentrations and a c t i v i t i e s . For short-term feeding, i . e . less than 6 hr, the solutions were added without being autoclaved. For feedings of two days duration, the solutions were autoclaved before being used, except for malonic acid and ox a l i c acid which were s t e r i l i z e d by f i l t r a t i o n through M i l l i p o r e f i l t e r s (0.22u pore s i z e ) . I s o lation of Shanorellin For comparative studies on the incorporation of various l a b e l l e d precursors, the i s o l a t i o n procedure described i n Part B was followed up to column chromatography. The shanorellin band from the column (3?cm X 2jcm) was sublimed and the a c t i v i t y of the sublimate was determined. Shanorellin that was used i n degradation reactions was further p u r i f i e d on preparative thin-layer plates, developed f i r s t with solvent system B and then with system G (Part B), and sublimed again. The sublimate was c r y s t a l l i z e d repeatedly from benzene-petroleum ether u n t i l constant s p e c i f i c a c t i v i t y was obtained. 94 Determination of the A c t i v i t i e s of -^C-labelled Compounds A l l -^C-labelled compounds were counted with a Nuclear Chicago Liquid S c i n t i l l a t i o n System 724 and 725 i n 15ml of s c i n t i l l a t o r (PPO 6g/l, POPOP O.lg/1, toluene 250ml, ethanol 150ml). A l l readings were corrected with an e f f i c i e n c y curve ca l i b r a t e d from standard quenching solutions by the channel r a t i o method. A l l shanorellin samples were dissolved i n 10ml of CHCl-j and an aliqu o t of 0.01ml was used i n the determination of i t s a c t i v i t y . The quenching e f f e c t of shan-o r e l l i n (0.01ml) had been tested with the following concent-rations j 40mg, 20mg, and lOmg per 10ml of CHCl^. The a c t i v i t y of 0.01ml n-hexadecane-l-^C was used as the stand-ard l n the above s c i n t i l l a t o r . The e f f i c i e n c i e s calculated from the readings by the channel r a t i o method coincided with those obtained from the standard quenching solutions. The quenching due to 2-phenylethylamin and a c e t i c a c i d i n aqueous solutions i n the amounts used i n the experiments had also been s i m i l a r l y tested. The e f f i c i e n c i e s of the countings were found to be very close to the re s u l t s obtained from the standard quenching solutions. Degradation of x ^ C - l a b e l l e d Shanorellin The p u r i f i e d shanorellin was f i r s t degraded by the Kuhn-Roth method as described by Eisenbraun, McElvain and Aycock (95). The acetate obtained was subsequently degraded by the Schmidt reaction as described by Phares (96). The carbon dioxide evolved i n the l a t t e r reaction was trapped i n an 95 aqueous solution of 80$ 2-phenylethylamine instead of 0.5N NaOH as described. Autoradiography The developed thin-layer plates were allowed to be in contact with Kodak Blue Brand Medical X-ray films for two weeks. Results and Discussion To find the most appropriate age of the culture and duration of feeding of labelled compounds, two preliminary experiments were performed. The f i r s t experiment was to find the % incorporation of acetate-1- C into shanorellin when i t was added to the medium of different stages of growth of the organism for 2 days. The use of acetate-l-^^C was chosen because i t was considered to be the most; li k e l y precursor of shanorellin, and the duration of feeding was arb i t r a r i l y taken as 2 days. After finding the approximate stage of growth of the organism when acetate was best u t i l i z e d to form shanorellin, the length of feeding time was again 1 4 tested with acetate-1- C. The results are shown in Table VI and Table VII. Table VI indicates that the specific activity of shano-r e l l i n decreased as the culture aged, but the f, activity incorporated into shanorellin was highest around the eight-eenth day of growth (approximately 13$). Thus, the most appropriate time to add the labelled compounds In order to obtain a high incorporation i s about one week after the 96 Shanorellin Growth (Days) pH of Medium Mycelial Dry Weight (mg) Amount \*S) A c t i v i t y <uo) Sp e c i f i c A (uc/mg) 10 6.75 31.50 3.19 0.70 0.175 11 6.70 30.40 6.29 1.00 0.160 12 6.60 32.46 11.30 1.85 0.160 13 6.71 34.95 13.70 1.68 0.123 14 6.69 32.86 11.43 1.60 0.139 16 6.61 43.52 20.81 2.27 0.109 18 6.58 33.11 22.00 1.90 0.086 20 6.55 40.18 11.80 1.08 0.091 23 6.40 52.87 16.30 1.59 0.095 26 6.30 36.95 11.50 0.9^ 0.081 Table VI. Incorporation of A c e t a t e - l - ^ C into Shanorellin a f t e r D i f f e r e n t Periods of Growth of S. s p l r o t -r l c h a . Each sample consisted of three Roux b o t t l e s . To each b o t t l e , 5uc of acetate-1- C was added on the day of growth as indicated. Incubation period was 2 days. The res u l t s are expressed as t o t a l of three Roux b o t t l e s . 97 Shanorellin Duration of Incubation Amount A c t i v i t y S p e c i f i c A c t i v i t y  (Days) (mg) (uc) (uo/mgT 1 4.15 0.30 0.073 2 4.50 0.42 0.093 3 5.85 0.45 0.077 5 11.30 0.58 0.052 8 17.50 0.47 0.027 14 Table VII. Incorporation of Acetate - 1 - C into Shanorellin by S. s p l r o t r l c h a with Different Incubation Times- Each sample consisted of three 125ml f l a s k s with 5 uc of a c e t a t e - l - ^ C per f l a s k added on the 15th day of growth. Results are expressed as the t o t a l of three f l a s k s . 98 appearance of shanorellin. The s p e c i f i c a c t i v i t y of shanorellin i s o l a t e d during t h i s time, though lower than that i s o l a t e d from e a r l i e r stages, i s s u f f i c i e n t l y high f o r degradation reactions. From the r e s u l t s shown i n Table VII, i t i s apparent that the shanorellin i s o l a t e d on the second day a f t e r the addition of a c e t a t e - l - ^ C to the medium gave the highest s p e c i f i c a c t i v i t y . So, f o r t e s t i n g the incorporation 14 of other possible C-labelled precursors, the experiment was carried out one week a f t e r the appearance of shanorellin i n the medium, and the incubation period was two days. The r e s u l t s of t h i s experiment are shown i n Table VIII. The r e s u l t s l i s t e d i n Table VIII c l e a r l y show&that shanorellin i s derived from the acetate-polymalonate pathway, with methionine as a one-carbon donor. The s l i g h t a c t i v i t y of shanorellin l a b e l l e d from phenylalanine and tyrosine could come from the acetate produced during the degradation of these two amino acids (97). Sodium propionate and 2-methylmalonic acid were d e f i n i t e l y not precursors of shanorellin. The a c t i v i t y of shanorellin l a b e l l e d from malonic acid was 1 4 lower than that from acetate-2- C, but s i g n i f i c a n t enough to be taken that i t was u t i l i z e d i n shanorellin production. 1 4 Sodium formate- C had been fed to another batch of S. s p l r o t r l c h a . and as with methionine, i t was e f f i c i e n t l y incorporated into shanorellin (6.9% incorporation). As stated i n the introduction of t h i s thesis, there are four prototypes of aromatic compounds formed from the acetate-polymalonate pathway. The absence of any acetyl function on 99 14 Shanorellin C-Compound Amount(mg) S p e c i f i c Activity(uc/mg) a c e t a t e - l - ^ C 8.26 0.036 acetate-2- 1^C 11.93 0.050 malonic-^-^C 9.10 0.019 s h i k i m i c - U - 1 " 4 ^ 9.00 0.000 phenylalanine-U - ^ C 9.00 0.002 t y r o s l n e - U - ^ C 7.40 0.005 l it. propionate-1- C 8.70 0.000 2-methyl - 1 " *C-malonic 9.20 0.000 methionine - ^ C R o 8.70 0.140 Table VIII. Incorporation of Various -^C-labelled Compounds Into Shanorellin by S. s p i r o t r l c h a . Each sample consisted of three Roux b o t t l e s . The s p e c i f i c a c t i v i t y of a l l the compounds were adjusted to 2uc/uM/ml, and 4uc were added to each b o t t l e of a 14 day old culture. Incubation time was 2 days. A l l r e s u l t s are expressed as the t o t a l of three b o t t l e s . 100 the nucleus of shanorellin eliminates i t s formation by way of the acetyl-phloroglucinol and a c e t y l - r e s o r c i n o l types of r i n g closure. Processes s i m i l a r to those i n the biosynthesis of o r s e l l i n i c a c i d and 6-methylsalicylic acid are more probable i n shanorellin biosynthesis. The l a b e l l i n g patterns of o r s e l l i n i c a c i d and 6-methylsalicylic a c i d from acetate have been well established (41, 47, 26, 98), and the carbon atoms i n shanorellin could s i m i l a r l y be l a b e l l e d . The pathways shown i n F i g , 54 and F i g . 55 are designed to indicate the possible sources of the carbon atoms i n shanor-e l l i n without considering the In vivo biosynthetic sequence. The Kuhn-Roth oxidation reaction i s used routinely i n the tracer studies of methylated benzoquinones (51, 5^, 63, 67, 92), the r i n g methyl group becoming the methyl group of a c e t i c a c i d . Shanorellin, when subjected to such a reaction would give two moles of a c e t i c acid f o r each mole of shanorellin used, the other carbon atoms would most probably be l o s t as carbon dioxide i o 2 101 O H O ..^methionine! a l l 1- C -acetate* none 2- 1'*C -acetate: 1/2 F i g . 54. Possible Origins of Carbon Atoms i n Shanorellin Based on O r s e l l i n i c Acid. ( A = non-oxidative decarboxylation B = oxidative decarboxylation • s carbon from methyl carbon of acetate + = carbon from C-, donors) 102 .OH H CH .0 CH20H H, .^methioninei a l l 1- f v c -acetate: none 2- 1 4 C -acetate: 1/2 COOH 6-Methyl-s a l i c y l i c Acid Er •.+ HO 0 O CH. -.^methionine: a l l 1- *r7C -acetate: 1/2 2- 1 4 C -acetate: 1/4 CH20H +CH, H0H2C O B HO OH IH. 3 ,^methionine: 1/2 1- t j c -acetate: 2/3 2- C -acetate: 1/4 OH H-HO methionine: 1/2 O' l - J f c -acetate: 2/3 +CH 2OH 2-1^C -acetate: 1/4 F i g . 55. Possible Origins of Carbon Atoms i n Shanorellin Based on 6-Methylsalicylic Acid. ( C = no decarboxylation. For other symbols, see F i g . 54) 103 T r i a l experiments with unlabelled shanorellin did give such a y i e l d of a c e t i c acid (9&-99%) on t i t r a t i o n with 0.05N NaOH. I f shanorellin, separately l a b e l l e d from 14 14 14 a c e t a t e - 1 - C, a c e t a t e - 2 - C and methionine- CH^ are degraded i n t h i s way, the proportion of a c t i v i t y recovered i n the a c e t i c acid would give an i n d i c a t i o n of the o r i g i n of the carbon atoms. The t h e o r e t i c a l values of the proportion of a c t i v i t y recovered i n the acetic acid from shanorellin l a b e l l e d with these three precursors and derived from the d i f f e r e n t pathways proposed, are shown also l n F i g . 5^ and:55* The degradation r e s u l t s of shanorellin l a b e l l e d from 14 1 4 14 acetate - 1 - C, a c e t a t e - 2 - C and methionine - GH^ by Kuhn-Roth oxidation are shown l n Table IX. The percentage of recovery of a c t i v i t y i n the acetic acid from shanorellin 14 l a b e l l e d from a c e t a t e - 2 - C could be taken as 50%, In one experiment a recovery of k9% had been obtained. The excess 5% could be due to the presence of unlabelled acetyl-CoA and short chain polyketo intermediate already formed l n the mycelium. On addition of the l a b e l l e d acetate, i t would be incorporated to the l a t t e r part of the molecule, thus, giving higher a c t i v i t y l n the degradation r e s u l t s . S i m i l a r l y , the percentage of recovery of a c t i v i t y l n the acetic acid from shanorellin l a b e l l e d from methionine- A CH^ could be taken as 1 0 0 $ . The 10% discrepancy could be accounted for i f the mono-methylated polyketo Intermediate was present i n the mycelium when thi s l a b e l l e d compound was added. In 104 A c t i v i t y of Shanorellin A c t i v i t y Recovered l n Acetic Acid 14 A. Prom Acetate-1- C 0.9?uc O.OOuc 1.13*'. 0.00 14 B. From Acetate-2- TC 0.23 0.11 0.33 0.19 1.31 0.72 14 C. From Methionine- CH^ 0.18 0.16 0.19 0.17 0.46 0.41 % Recovery* 0.00 0.00 48.7 57.1 55.7 90.2 86.4 91.3 Table IX. Results of the Kuhn-Roth Degradation of 14 Shanorellin Labelled from Acetate-1- C, Acetate-2- 1^: and Methionine-^CH^. ( +, a c t i v i t y of ac e t i c acid i s o l a t e d from 75ml of d i s t i l l a t e and d i l u t e d to 100ml solution: 0.5ml of the solution was used i n the radioactive assay and 75ml were pipetted for t i t r a t i o n with 0.05M NaOH. *, % recovery was calculated from the s p e c i f i c a c t i v i t y of shanorellin before degradation and the s p e c i f i c a c t i v i t y of shanorellin calculated from the a c t i v i t y and number of mmoles of acetic acid recovered) , 105 addition to these factors, the experimental errors due to technique and measurement should also be added. Shanorellin l a b e l l e d from a c e t a t e - l - ^ C gave unlabelled acetic acid. Thus, these degradation results and the predicted values shown i n F i g . 54 and 55 indicate that the sources of carbon atoms of shanorellin are as follows: C H V ^ V C H HC 3 H20H O • = methyl carbon of acetate + = methyl carbon of methionine Fig. 56. Origins of Carbon Atoms i n Shanorellin. To confirm t h i s l a b e l l i n g pattern, the acetate isolated from Kuhn-Roth oxidation of shanorellin was further degraded by the Schmidt reaction: CH^COOH > CH^NH2 + COg The r e s u l t s of the Schmidt degradation of the acetates >m 14, 14 from shanorellin l a b e l l e d from acetate-2- C and methionine-CH-^are shown i n Table X. In t r i a l experiments with commercial a c e t a t e - l - ^ C and 14 acetate-2- C, i t was noted that when these solutions were subjected to l y o p h i l i z a t i o n to remove the water present, 15-30$ of the a c t i v i t y was l o s t during the process. The addition of 3 drops of 0.5N NaOH prevented such loss of a c t i v i t y . 1 4 The Schmidt degration of acetate-2- C gave over 95$ of the 106 A c t i v i t y of Acetate A. From Shanorellin Labelled with Acetate - 2- 1 4C 0.13 0.13 0.03 B. From Shanorellin Labelled with M e t h i o n i n e - 1 4 ^ 0.16 0.16 0.12 A c t i v i t y Isolated ($ Recovery) CH-jNHg 0.05K38JS) 0.049(37$) 0.014(46$) 0.158(98$) 0.157(98$) 0.116(97$) 0 . 0 6 2(46$) 0.058(43$) 0.009(30$) 0.000(0$) 0.000(0$) 0.000(0$) Table X. Schmidt Degradation of Acetate Isolated from Kuhn-Roth Oxidation of Shanorellin Labelled from A c e t a t e - 2 - 1 4 c and Methlonine-^CH^. To each sample, 0.2mmoles of cold sodium acetate, 3 drops of 0.5N NaOH were added. (*, a c t i v i t y of the residue remaining i n the reaction f l a s k ) 107 a c t i v i t y i n the methyl carbon ( a c t i v i t y remaining i n the flask) and none i n the carboxyl carbon ( a c t i v i t y i n the trapping solution)} but with a c e t a t e - l - ^ C , a range of 50-80$ of the t o t a l a c t i v i t y was is o l a t e d i n the trapping s o l -ution and 2-5$ remained i n the f l a s k . There was s t i l l 20-40$ of the a c t i v i t y which could not be accounted f o r . This may be due to the i n e f f i c i e n c y of the trapping solution f o r carbon dioxide, although phenethylamine has been reported to be one of the most e f f i c i e n t trapping reagents f o r carbon dioxide (99). Another reason may be due to the presence of sodium hydroxide i n the reaction mixture which was not present i n the reaction described i n the o r i g i n a l l i t e r a t u r e , and i t s presence may cause undesirable side react!on(s). When the data shown i n Table X was obtained, another source of error i n the degradation procedure was r e a l i z e d . A f t e r the acetic acid was Isolated by steam d i s t i l l a t i o n i n the Kuhn-Roth oxidation, i t was t i t r a t e d with 0.05N NaOH with phenol-phthalein as Indicator. Since phenolphthaleln contains two phenolic hydroxyl functions, i n the presence of 100$ H2S0^, i t could react with the ace t i c a c i d present to give esters, thus retaining some of the a c t i v i t y as acetate i n the f l a s k . The r e s u l t of Schmidt reaction with commercial 14 acetate-1- C with 3 drops of 0.5N NaOH, 2 drops of phenol-phthaleln confirmed t h i s source of errort 36$ of the a c t i v i t y was recovered i n the f l a s k and 40$ i n the trapping solution. Thus, another Kuhn-Roth degradation of shanorellin l a b e l l e d from acetate-2- 1^C was ca r r i e d out. The ace t i c a c i d i s o l a t e d 108 was assayed f o r i t s a c t i v i t y (55% of the t o t a l a c t i v i t y i n shanorellin was present i n the acetic acid solution) and the solution was then made alk a l i n e with 0.05N NaOH to pH 12 as tested with litmus paper. The Schmidt degradation of t h i s sample gave 4.7$ a c t i v i t y remaining i n the f l a s k and 56.9$ a c t i v i t y i n the trapping solution. This r e s u l t i s within the range of a c t i v i t i e s recovered with commercial acetate-1- A C. Thus, i t could be taken that most of the a c t i v i t y i n the a c e t i c a c i d i s o l a t e d from shanorellin l a b e l l e d from 14 acetate-2- C resided i n the carboxyl carbon. Together with the r e s u l t obtained from the degradation of the acetic acid 14 derived from shanorellin l a b e l l e d from methionine- CH^ , i t i s conclusive to state that the l a b e l l i n g pattern shown i n F i g . 56 i s correct. 14 14 The metabolism of acetate-1- C and acetate-2- C, and the incorporation of the r a d i o a c t i v i t i e s into compounds of the pool have been observed by Birch et a l . (28) and by Bentley and Lavate (43) i n the biosynthesis, of aurantioglio-cladln. Birch suggested that the incorporation of a c t i v i t y i4 from acetate-1- C into the methoxyl group of aurantioglio-cladln occurred v i a ^CO^ The f i x a t i o n of C 0 2 v i a (3 -carb-oxylatlon of pyruvate to oxaloacetate by the. enzyme systems of phosphoenolpyruvlc carboxylase and phosphoenolpyruvic carboxykinase have been demonstrated to occur i n fungi (100). The entry of oxaloacetate into the t r i c a r b o x y l i c acid cycle and glyoxylate cycle would eventually l a b e l the glyoxylate. The conversion of glyoxylate to formate v i a oxalate has been 109 observed i n bacteria and fungi (101). The involvement of 14 acetate-2- C i n the t r i c a r b o x y l i c acid cycle and glyoxylate cycle would also lead to the l a b e l l i n g of the pool. For the two d i f f e r e n t l y l a b e l l e d acetate species to enter the pool, oxalate would be the common precursor. Thus, to test the p o s s i b i l i t y of the occurrence of these pathways 14 i n S. s p i r o t r l c h a . oxalic-1,2- C-acid was fed to three cultures grown i n Roux bottles f o r 2 days (5uc/bottle). No a c t i v i t y was detected i n the shanorellin i s o l a t e d . This r e s u l t may be taken as an in d i c a t i o n that the randomization of l a b e l of acetate by eventual incorporation into the C-^  pool v i a the above routes does not occur i n S. s p l r o t r i c h a . or, i f i t does occur, i t i s only to an i n s i g n i f i c a n t extent. As indicated i n Fi g . 54 and 55» three routes (pathway A i n F i g . 5$, pathways A and B i n Fi g . 54) to the biosynthesis of shanorellin are possible. Since the methylation process occurs most l i k e l y at the polyketo-chain l e v e l f o r reasons already discussed i n the introduction, the pathway from 6-methylsalicylic acid, i . e . pathway A i n F i g . 5j5» a n d "the one v i a non-oxidative decarboxylation of o r s e l l i n i c a c id, i . e . pathway A i n Fig. 54i, may be eliminated. The presence of 6-methylsalicylic acid and o r s e l l i n i c acid could not be detected i n the culture medium at any stage of growth by thin-layer chromatography. 3«5-Dimethylorsellinic acid has been isol a t e d from M o r t l e r e l l a ramannlan (102). A sample was kindly sent to us by Dr. W. W. Andres. It was not detected i n the fungal medium however. The r e l a t i v e positions of 110 some of the standard aromatic compounds to the two major phenolic compounds detected on thin-layer plates are shown In F i g . 57. Solvent System C Solvent System B F i g . 57. The Relative Positions of the Benzoquinones and Phenolic Compounds Detected i n the Medium and the Aromatic Acid Standards. 1 - 6-methylsalicylic Acid 2 - 3 , 5-dimethyl-orselllnic acid 3 - o r s e l l i n i c acid 4 - 3-methylorsellinic Acid I and I I - Phenolic Compounds 5 - 3,4-dimethyl-5-hydroxy-phenol 0 - o r i g i n S - shanorellin A - Compound A B - Compound B C - Compound C I l l These observations thus add support to the postulation that the methylation steps i n polyketide biosynthesis occur before the aromatization of the ring. The absence of 3 , 5 -d i m e t h y l - o r s e l l i n l c acid i n the medium might be taken as an ind i c a t i o n that the oxidation of the methyl group to hydroxy-methyl function occurs early i n the biosynthesis of shanorell-i n . A compound with structure IV might be the f i r s t aromatic compound formed: The pihenolic compound II (Pig. 57) had been detected 14 -by autoradiographs to be l a b e l l e d with C-acetate and methionine. Its Rf values on thin-layer plates are very close t o to that of 3 , 5-dlmethyl-orselllnlc acid (Fig. 57)* and on spraying with p - n i t r o a n i l i n e reagent, i t appears as orange brown, a color reaction also given by 3 i 5-dimethyl-orsellinic acid. That t h i s phenolic compound II has the structure IV shown above i s not unlikely. Subsequent oxidative decarbox-y l a t i o n of IV and oxidatiom of the hydroquinone formed would give shanorellin. COOH 112 Part E. General Discussion The occurrence of p-benzoquinones with structures s i m i l a r to shanorellin i s not uncommon i n fungi ( 5 . 1 3 - 1 7 ) . Tauranin produced by Oospora aurantlaca possesses a hydroxy-methyl and a hydroxyl function on the quinone ring, ( 8 7 ) and the oxidation of a methyl group to a primary alcohol has been shown to occur l n the biosynthesis of oosponol ( 1 0 2 ) . The aoetylatlon of a primary alcohol function has been reported, e.g. g l l o t o x i n acetate and cephalosporin C ( 1 0 3 ) . The structures of the ether derivatives of shanorellin (Compound B and C) are unique. Compounds with an ether linkage at two a l i p h a t i c carbons have not been reported from the fungi. Compound B could be regarded as a dlmer of shanorellin. Fungal metabolites such as the blbenzoquinones and terphenyl-quinones are linked through the r i n g carbons (104)1 the benzophenone derivatives, the spirans, the diphenyl ethers and the depsidones a l l have a carbonyl and/or oxygen function situated between the two rings ( 1 0 5 ) . The nearest equivalent to a structure l i k e Compound B i s the lignan pinoresinol which occurs i n Pinus and Picea (106) .!--.»Wlth^ rjeg«JWt>8&-ito Compound C, the ethyl ether of shanorellin, no such equivalent has been found l n fungi or i n plants. Glycerol ethers have been is o l a t e d from l i p i d s of animals and a bacterium ( 1 0 7 - 1 0 9 ) . The chain'!-length of these ethers ranges from 14 to 22 carbons. The biosynthesis of shanorellin has been discussed. The biosynthesis of Compounds A, B and C have not been studied 113 systematically, but some i n d i c a t i o n as to t h e i r mode of formation may be suggested from those studies c a r r i e d out on shanorellin. During the tracer studies of shanorellin, i t was part of the routine to take an al i q u o t of a, the ethyl acetate extract of the medium and subject i t to thin-layer chromatography and autoradiography. In experiments where the l a b e l l e d compounds were fed f o r 2 days, precursors that l a b e l l e d shanorellin were found to l a b e l Compound A, B and C al s o . So, Compounds A, B and C are synthesized from the acetate-polymalonate pathway. With short-term feedings with acetate-14 14 2- C and methionine- CH^ f o r 20 min, the aut©radiographs of the thin-layer plates developed two dimensionally with solvent systems B and C showed three radioactive areas» one f o r shanorellin, one f o r phenolic compound II (Fig. 57) and one i n the area of Compound A and B. Most probably the l a s t radioactive compound i s the acetate of shanorellin (Compound A), and i s an a r t i f a c t of the I s o l a t i o n procedure. The ethyl ether of shanorellin (Compound C) became l a b e l l e d i n experiments i n which the period of administration of radioactive substrates was 40 min or longer. That the appear-ance of the ethyl ether of shanorellin as a radioactive comp-ound f a l l s behind that of shanorellin suggests that i t Is formed from shanorellin. Labelled shanorellin was fed to a culture of S. splrotrlcha..but unfortunately,>that p a r t i c u l a r batch did not produce these derivatives of shanorellin. i 114 The a c e t y l carbons of s c l e r o t l o r l n have been shown to be derived from acetate ( 1 1 0 ) , and si m i l a r biosynthetic process i n S. s p l r o t r l c h a would give shanorellin acetate, i f thi s compound i s produced by the fungus and not present as-an a r t i f a c t of the i s o l a t i o n procedure. The reduction of the carbonyl function could then give r i s e to the ethyl ether of shanorellin (Compound C). Another p o s s i b i l i t y f or the biosynthesis of Compound C i s that the hydroxymethyl function of shanorellin i s methylated twice with methionine. Two successive methylations to give an ethyl function have been shown i n the biosynthesis of the plant s t e r o l s , e.g. /3 - s i t o s t e r o l and fucosterol ( 1 1 1 ) . Unfortunately, t h i s could not be tested as s u f f i c i e n t amounts of t h i s compound were not recovered from tracer experiments with methionine. For Compound B, i t i s most l i k e l y formed by the condensation of two moles of shanorellin or i t s hydroquinone or while shanorellin i s i n i t s quinhydrone form. I f Compound B i s synthesized i n a si m i l a r pattern to ligni n s , ( 1 1 2 ) , i t i s formed v i a a free r a d i c a l mechanism. In the presence of peroxidase and hydrogen peroxide, shanorellin, or i t s hydroquinone or quinhydrone, would react to give Compound C. Or, Compound C i s synthesized by a process s i m i l a r to those of g l y c e r o l ethers i s o l a t e d from animals. An enzyme has been i s o l a t e d from preputial tumors i n mice which i s cap-14 able of converting i n t a c t 1- C f a t t y alcohol, but not the acid or aldehyde, into a l k y l g l y c e r y l ethers ( 1 1 3 ) . 115 In conclusion, a biogenetic scheme i s proposed f o r the synthesis of shanorellin and i t s derivatives i s o l a t e d from S. s p i r o t r l c h a (Pig. 58). CGOH 1 mole Acetyl-CoA 3 moles Malonyl-CoA 2 moles S-Adenosylmethionine o o o II II fl CH3-C-CH2-C-CH2-C-CH2-COSR T T T Oxid? C x Ci H 0 Y i T T C H 2 0 " C ' CH3 O Compound A I i iReduction I 4r Acetyl-X CoA \ \ \ y-/ / / 2 C 1 \£ Units L Oxidation CH20H HO CH. :H20H ^H3 OH Oxidative 'Decarboxylation Shanorellin Fig. 58. Suggested Biogenetic Scheme for the Synthesis of Shanorellin and i t s Derivatives in S. spirotricha. 117 BIBLIOGRAPHY 1. Pennock, J.F. 1966. Occurrence of Vitamin K and Related Quinones. Vitamins and Hormones 24.307-329. 2. Redfearn, E.R. 1965« Plastoquinone. In: Biochemistry of Quinones. 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