BIOSYNTHESIS OF SOME PHENOLIC ACIDS AND LACTONES IN HIGHER PLANTS USING CARBON-14 By Ragai K. Ibrahim, M. Sc. A Thesis Submitted to the Faculty of Graduate Studies and Research in Partial Fulfilment of the Requirements for the degree of Doctor of Philosophy. Department of Botany, McGill University, Montreal. April , 19 61 . ACKNOWLEDGMENTS I wish to express my sincere thanks and indebtedness to Dr. G. H. N. Towers, for his continua! help and valuable criticisme in directing this work. Thanks are also tendered to Professer M.V. Roscoe, Cbairman of the Department of Botany, for making the facilities of the department available for this study; and to Dr. W. Boll, for useful criticism. The writer is grateful to Dr. P.I. Abell, of the Chemistry Department, University of Rhode Island, for the determination of the infra-red spectra of hydrangenol and a new coumarin derivative ; and to Dr. B.A. Bohm, N.R.C. Post Doctoral Fellow in this laboratory, for the interpretation of the infra-red spectrum of the latter compound and the preparation of its acetyl derivative. 14 The generous gifts of cinnamic acid-ring & -3-C , from Dr. E. Conn, University of California ; of an enoculum of Escherichia coli 83-24, from Dr . B. Davis, Harvard Medical School,Mass.;of scopoletin and iso-ferulic acid, from Dr. V. Runeckles, Imperial Tobacco Company, Montreal ; of a sample of scopoletin, from Dr. W.A. Andreae, Canada Department of Agriculture; and of the gydrangea flowers, from Mr. A. Bisaillon, Montreal Botanical Gardens, are also acknowledged. I wish also to thank the World University Service of Canada for a scholarship in 1958-1959 ; the authorities in McGill University for the awa~d of a Graduate Scholarship in 1959-1960 ; and the National Research Council of Canada for grants to Dr . Towers , for financial support of this work. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE ON PHENOLIC ACIDS AND PHENOLIC LACTONES 3 I. Naturally occurring phenolic acids and phenolic lactones in higher plants. 3 II. Origin of benzene rings in phenolic compounds. 12 1. The shikimic acid pathway. 12 2. The acetate pathway. 15 III. Sorne biosynthetic pathways of pbenolic compounds. 17 REVIEW OF METHODS OF SEPARATION AND IDENTIFICATION OF PHENOLIC ACIDS AND PHENOLIC LACTONES 20 MATERIALS AND METHODS 27 1. Plant material. 27 2. Chemicals. 27 3· Carbon-14 compounds. 28 4. Preparation of radioactive shikimic acid. 28 5. Preparation of plant extracts. 29 6. Chromatography. 31 7. Spray reagents. 33 8. Elemental analysis. 34 9. Spectropbotometry. 34 10. Techniques for administering carbon-14. 34 11. Radioautography and radioactivity determination. 35 EXPERIMENTAL AND RESULTS 38 A. PHENOLIC ACIDS 38 I. Chromatographie separation and identification of pbenolic acids. 38 II. The phenolic acid patterns of sorne common plants. 44 III. Identification of the phenolic acids of Hydrangea macrophylla. 56 Compound No. 8. 59 IV. Isolation and identification of three dihydroxybenzoic acids from Gaultheria procumb.ens and Primula acaulis. 65 1. ~-Pyrocatechuic acid. 65 2. Gentisic acid. 66 3. 2-Hydroxy-5-methoxybenzoic acid. 66 v. Biosynthesis of C6-Cl acids from c14-labelled compounds in higher plants. 67 1. Formation from benzoic acid. 67 2. Formation from salicylic acid. 70 3. Administration of ~-pyrocatechuic acid-c14 and gentisic acid-c14 to Gaultheria leaf disks. 70 4. Formation from phenylpropanoid compounds. 73 B. PBENOLIC LACTONES 77 I. Identification of the phenolic lactones of Hydrangea. 77 1. Isolation and identification of hydrangenol glucoside. 77 2. Isolation and identification of hydrangenol. 78 TABLE OF CONTENTS (Continued) Page 3. Isolation and identification of umbelliferone 85 4. Isolation and identification of compound No. 15. 89 Alkaline hydrolysis of compound No. 15. 96 II. Biosynthesis of phenolic lactones from cl4_labelled compounds in Hydrangea. lOO 1. Biosynthesis of hydrangenol. 101 2. Degradation of hydrangenol and identification of the degradation products. 105 3. Degradation of hydrangenol-c14. 109 4. Biosynthesis of ether phenolic constituants of Hydrangea from c14-labelled compounds. 112 5. Administration of umbelliferone-c14 to §ydrangea leaf disks. 117 6. Administration of L-phenylalanine-u-cl4 to aydrangea leaf disks. 117 DISCUSSION 121 I. Phenolic acids, their identification by chromatography and their distribution in plants. 121 II. The biosynthesis of sorne hydroxybenzoic acids in higher plants. 127 III. The phenolic constituants of Hydrangea. 137 IV. Biosynthesia of the phenolic constituents of Hydrangea. 140 SUMMARY 150 CLAIM TO ORIGINALrrY OR CONTRIBUTION TO KNOWLEDGE 153 REFERENCES 154 INTRODUCTION One characteristic feature of higher plants is their ability to synthesize phenolic compounds in great variety and quantity. Lignin, which constitutes 20-30 ~ of the dry weight of plants, is formed of phenolic polymers. Phenolic compounds appear to be metabolically inert substances and are considered to be stable and characteristic end products in living plant tissues. No general function can be ascribed to these compounds and no explanation bas been given to their extraordinary diversity. This is possibly one of the reasons why little attention has been given to them by plant physiologiste and biochemists in the past. With the advent of paper chromatographie techniques, it was shown that many simple phenolic compounds are widely distributed in plants. The work of Bate-Smith (1954 a, 1956 a, b) and Williams (1955, 1956, 1957) provided a great deal of information on the distribution of certain phenolic acide (cinnamic acid derivatives). However, c6-c3 a large number of phenolic compounds which can be detected on paper chromatograms of plant extracts have not been identified. As chromatographie methode for the separation of c -c and c -c 6 1 6 3 phenolic acids bad not been fully explored, it was a prominent part in the planning of the present work to develop a suitable method for the 1 2 separation and identification, by two-directional paper cbromatography, of the plant phenolic acide. The application of this method bas been found useful in the distribution of phenolic acide in higher plants. The use of carbon-14 labelled compounds and the discovery of the pathvays of synthesis of the aromatic amino acide, phenylalanine and tyrosine, from carbohydrates (Davis, 1955, 1958), and of certain benzenoid compounds from acetate (Birch and Donovan, 1953), stimulated interest in the problems of biogenesis of aromatic compounds in plants. A significant discovery was tbat of Brown and Neish (1955 a, b) who showed that lignin, inspite of the uncertainty of its structure1 is formed of aromatic monomers of the type. c6-c3 It was also the aim of this work to include an investigation of the biosynthesis and metabolic relationships of certain phenolic acide, since early in the course of a survey of the plant phenolic acide, it was discovered that hydroxylated benzoic acide are widely distributed in plant tissues. As nothing appeared to be known of their relation­ ships to one another, a study of this problem was undertaken. Later, in the co~se of these etudies, attention was given to aydrangea macrophylla Ser., a plant which was found to be a particularly rich source of phenolic compounds. Of these constituents, three were found to be phenolic lactones , and the novel structure of one of them, the phenyl iso-coumarin,hydrandenol,prompted a study of its biogenesis. 3 REVIEW OF LITERATURE ON PHENOLIC ACIDS AND PBENOLIC LACTONES I. Naturally Occurring Phenolic Acide and Phenolic Lactones in Bigher Plants The most common, naturally occurring, phenolic acide are the c -c (benzoic) and the c -c (cinnamic), acide. Although these 6 1 6 3 acide show a variety of bydroxylation and methoxylation patterns (see Figure 1), bydroxylation is usual in the -o- and -p-position. Table 1 is a list of some of the well known, naturally occurring benzoic acid and cinnamic acid derivatives and their distribution in bigher plants. The development of paper partition cbromatograpby bas proved to be an excellent tool for the characterization of phenolic acids in plant extracts. Most of these acide show different fluorescence character- istics in ultraviolet light and give different colours witb diazotized pbenolic sprays. Bate-Smitb (1954 a, 1956 a) and Williams (1955) gave an account of the identification, by cbromatography and UV-fluorescence, of the more common phenolic constituents of plants. A survey of tbeir system- atic distribution was given by Bate-Smitb (1956 a). Griffiths (1958) studied the distribution of gentisic acid among dicotyledonous families and sbowed its wide spread occurrence. Prior to his survey, this acid was only reported to occur in microorganisme." K~ves and Varga (1959) found that benzoic acid and cinnamic acid derivatives are widely distributed among dry fruits. Figure 3 Pathways of synthesis of phenylalanine and tyrosine from carbohydrates in Escherichia coli, (see Davis, 1958). -- (*P) Stands for orthophosphate. I. ~-Keto-3-deoxy-D-arabo-heptonic acid-7-phosphate. II. Dehydroquinic acid. III. Dehydroshikimic acid. IV. Shikimic acid. V. 5-Phesphoshikimic acid. VI. Intermediate compound. VII. Prephenic acid. VIII. Phenylpyruvic acid. IX. p-Hydroxyphenylpyruvic acid. x. Phenylalanine. XI. Tyrosine. ..::t COOH COOH COOH COOH ~ OH OH ~O H ~ ~ OH HO OH Benzoic Salicylic P- Hydroxybenzoic o_Pyrocatechuic Genti sic "0 COOH c . COOH COOH COOH a1 en +" tJ c ..-1 a1 0 .-l N p, c (]) c p •.-l OCH HO OH 3 "0 bO (]) c OH OH OH +" ...-! OH :::1 J..< +" J..< Protocatechuic Vanillic Syringic Gallic •.-l :::1 +> tJ en tJ '§ 0 en >, CH=CH-COOH .-l CH = CH-COOH CH= CH-COOH CH= CH-COOH q...., c 0 s0 (]) s H a1 0 .-l tJ :::1 s en J..< "0 0 ..-1 OH q...., tJ al .-l OH a1 tJ OH J..< ..-1 Cinnamic o- Coumaric p-Coumaric :::1 s Caffeic +> a1 tJ c :::1 c J..< •.-l +' tJ Cl) CH2CH2COOH CH= CH- COOH CH= CH-CO OH .