THE BIOSYNTHESIS OF PHENOLIC PLANT PRODUCTS

CHAPTER

12

THE B I O S Y N T H E S I S OF P H E N O L I C PLANT PRODUCTS T. A . GEISSMAN Department of Chemistry, University of California, Los Angeles, California

CONTENTS INTRODUCTION

743

EARLIER STUDIES

745

GENETIC EVIDENCE

746

T H E BIOSYNTHESIS OF THE C 6 — C

3

UNIT

748

750

Cinnamic acids T H E U S E OF T W O - C A R B O N U N I T S IN AROMATIC BIOSYNTHESIS

751

T H E ACETATE HYPOTHESIS

751

T H E BIOSYNTHESIS OF PARTICULAR G R O U P S OF COMPOUNDS

Phenols, resorcinol derivatives, salicylic acid derivatives Extension of acyl residues by C 2 units The lichen compounds Anthraquinones Naphthoquinones Compounds derived from l-phenylpropane precursors Flavonoid compounds Isoflavones

755

755 760 764 769 770 112 772 777

Stilbenes and dihydroisocoumarins Carbon alkylation Isoprenoid substituents The furan ring Coumarins Hydroxylation of aromatic rings Oxidative metabolism in biosynthesis

779 781 784 787 788 791 192

SUMMARY

796

REFERENCES

797

INTRODUCTION

The phenolic compounds elaborated by plants range in structural type over 743

744

T. A.

GEISSMAN

a wide spectrum, from such simple phenols as hydroquinone (1), /?-hydroxyacetophenone (2) and anethole (3), to such complex compounds as emodin (4) and the dianthrone hypericin (5). OH

OH

OH

C0CH

Hydroquinone (I)

ρ -Hydroxyocetophenone (2) ΟΗ

Ο

Emodin

(4)

Present-day knowledge of the main routes by which phenolic compounds of these and many other types are synthesized in the plants, is based in part upon direct experimental evidence obtained with the use of radioactive tracer technics and in part upon systematic and compelling speculations that possess an impressive degree of coherence and consistency. While the origins of some plant phenols are open to alternative interpretations, a large number of them can be described with a high degree of assurance ; but much remains to be done to establish speculative routes of biosynthesis by experimental verification. In particular, little is known about the nature of the multitude of individual steps through which small structural details are elaborated: the addition and removal of hydroxyl groups, the introduction and saturation of multiple bonds, the introduction of "extra" carbon atoms, the occasional skeletal rearrangements; these and other changes can often be recognized as undoubted steps in biosynthetic sequences without it being possible to specify the point or the order in which they occur. In the following pages the biosynthesis of the plant phenolic compounds will be dealt with largely from the point of view of the theories of biosynthesis that have received experimental support. While experiment has established routes that may be regarded as prototypes for many as yet uninvestigated biosyntheses, a great many classes of substances have not yet

THE BIOSYNTHESIS OF P H E N O L I C P L A N T

PRODUCTS

745

come under direct experimental scrutiny. Of these, many can be accommodated into a few simple but compelling hypotheses about the essential soundness of which there can be little doubt. In some cases alternative hypotheses exist. Occasionally these can be seen to be but different expressions of a single idea; in others, they represent quite different viewpoints.

EARLIER STUDIES

Even before the advent of modern technics for the thorough study of plant constituents and of tracer methods using radioactive compounds, theories of biosynthesis were not wanting. The lack of experimental methods necessitated approaches that were based upon hypothesis a n d speculation, and before 1930 a considerable body of literature h a d been built u p around the theme of plant biogenesis. Much of this dealt with the biosynthesis of plant alkaloids and included the fruitful and prophetic ideas of Robinson and 2) Schöpf, < some of which received support in elegant in vitro syntheses under conditions comparable to those obtaining in living cells. While theories of the biogenesis of terpenoid compounds are not within the scope of this chapter, it is to be recalled that the possible role of isoprene in the formation of 3 terpenes has been a recurring theme, <> and a number of terpenoid hydro4) carbons were early synthesized < in the course of attempts to demonstrate that isoprene was at least a plausible, if not obligatory, precursor of this class of substances. Schemes have been devised to account for the biogenesis of 5 6 7 8) carbohydrates, <> steroid sapogenins, <> cinnamic acids, <> flavones,< and 9 other aromatic compounds ;< > but these early speculations can now be regarded as part of the historical development of the subject, and attention can be turned to the more sophisticated theories of recent years. Before leaving consideration of these early speculations, it is appropriate to "recall the part they played in the development of what is now known about 10 plant biogenesis. Schöpf < > gave the clearest expression of a point of view regarding an attack upon the problem by using a reconstructive approach based upon a "comparative anatomy" of plant compounds. By considering the structures of groups of compounds closely related in their botanical occurrence, and assuming that certain common intermediates or precursors lie at the start of the synthetic sequences, Schöpf was able to devise rational schemes of biosynthesis that were based upon permissible grounds. The in vitro syntheses (under "conditions possible in the cell") of a group of quinon line alkaloids in Angostura bark< > was one example of the validity of this 12 approach. S c h o p f s studies, and those of Robinson, < > were the forerunners of many others in which highly reactive intermediates, whose presence in the living cell could be regarded as reasonably likely, were shown to condense to give naturally occurring compounds or products only a step or two away from natural substances.

746

T. A. GEISSMAN

This reconstructive approach lies at the base of much of our present-day knowledge of biosynthesis. The chief difference between modern investigations and those of thirty years ago is that the older theories were supported chiefly by the compelling weight of coincidence while at the present time it is possible to test theory by experiments on the living plant itself. The use of radioactive tracers and the separation of enzyme systems capable of carrying out distinct stages of a synthetic sequence have made it possible to put biosynthetic theory to direct test. GENETIC EVIDENCE

A fruitful avenue of investigation of the chemical processes occurring in plants has been the combined chemical-genetical approach. Studies of this 13 kind were first carried out by Wheldale and Bassett < > and later extended by a 14 school of geneticists and chemists at the John Innes Institution. < > Evidence from the study of chemical structures of the anthocyanin pigments of plants of known genetic composition showed clearly that single factors ("genes") controlled specific chemical reactions in the elaboration of the anthocyanins. Genes specific for the establishment of nuclear hydroxylation patterns, for methylation of phenolic hydroxyl groups, for the attachment of sugar residues and for the control of the amount of pigment, were recognized. Evidence from the effects of interaction and competition between genes suggested that anthocyanins of various structural types were synthesized from common precursors. After the advent of methods that permitted the systematic study of noncyanic plant pigments, genetic studies were extended to the examination of 15 all of the flavonoid constituents of selected genotypes. < > These studies showed that the genetic control of the positions of hydroxylation in the anthocyanin pigments was exerted as well upon the hydroxylation patterns of the accompanying flavones. These findings offered additional support for the concept of parallel synthetic paths for the flavonoid constituents, starting from a common early precursor. Numerous other chemical genetical studies have confirmed this general picture of flavonoid biosynthesis; but the genetical approach has so far failed to provide clear evidence for the detailed sequence of synthetic transformations by which flavones, aurones, flavanones, and anthocyanins are derived from their precursors, or whether these compounds are directly interconvertible by simple oxidation or reduction processes. A significant observation was made in the study of an albino mutant of Antirrhinum majus, the garden snapdragon. This mutant contains n o flavonoid (i.e. C 6 — C 3 — C 6 ) substances in the colorless flower petals. Instead, esters 16 17 1 8) of caffeic, /?-coumaric and ferulic acids are p r e s e n t . * ' > The conclusion from these observations that the mutant lacks the capacity for coupling of a C 6 — C 3 unit to a C 6 unit (6) to provide the flavonoid precursor has been borne

THE

B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

747

(6)

out in the subsequent development of what is today an accepted overall view of flavonoid biosynthesis. The nature of the six-carbon fragment to which the phenylpropane-derived unit became attached to form the C 1 5 unit of the flavonoids was not revealed in the studies on the mutant snapdragon. That these six carbon atoms are derived from three molecules of acetic acid is now known. Evidence for this conclusion will be described in the sequel. The extension of the C 6 — C 3 unit of phenylpropane-derived precursors (such as the cinnamic acids) by the attachment of two-carbon units can easily be discerned in the 6-styryl-a-pyrones, kawain (7) and methysticin (8).

Kawain

Methysticin

(7)

(8)

Kawain (7) is a methyl ether of the cyclized form of the hypothetical C 6 — C 3 — 18 C 2 — C 2 precursor (9). < > ^ CH

-CH

CH j OH

CH

CO

CH 2

COOH

(9)

Paracotoin (10) is similarly related to the C 6 — C 3 — C 2 precursor (11).

A summary of the evidence from the reconstructive and genetic approaches gave strong support for a view of biosynthesis of many plant phenols that contained two fundamental ideas: (a) a nine-carbon atom unit with a 1arylpropane skeleton is one of the widely occurring building units in plant

748

T.

A. GEISSMAN

biosynthesis; and (b) the extension of the three-carbon side chain by twocarbon units can account for the elaboration of eleven-, thirteen- and fifteencarbon atom compounds. This hypothesis was later enunciated with explicit application to compounds of widely differing structural types by Birch and 2 0) Donovan, < and now embodies the essential idea of a generally accepted view of plant phenol biosynthesis. Its elaboration to include modifications of the C 6 — C 3 unit and of pathways involving only C 2 condensations has led to the development of the comprehensive view of plant biosynthesis that will be dealt with in the following sections.

T H E B I O S Y N T H E S I S O F T H E C 6— C 3 U N I T

The origin of the aromatic ring of the many natural phenylpropanoid compounds is now regarded to be the cyclohexane derivatives that arise by carbohydrate cyclization. The key compound in this series is shikimic acid (14), which is formed as shown in the following diagram: COOH

COOH

C-L-O-POs

CO

I

CH,

CH2

I

CH^O ÇHOH

CÇHH00HH

CH0H

CHOH

I C H 20 - P 0 5 .COOH

V

0H

Dehydroquinic (12)

I COOH

C H 20 - P O |

COOH

^OH

acid

Dehydroshikimic

acid

(13)

This synthetic pathway from carbohydrate has been established in microorganisms (E. coli mutants), from which extracts have been obtained that are capable of carrying out the combination of phosphoenolpyruvate with erythrose-4-phosphate to give shikimic acid (by way of dehydroquinic (1 21 23 and dehydroshikimic (13) acids. < - )) The synthesis of aromatic compounds of the general pattern C e — C — C — C , such as phenylalanine and tyrosine, takes place in microorganisms by the following pathway:

T H E B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

749

CH aC0C00H -CO2

Reductive amination

T

^CH 2CHCOOH

-HoO '

The formation of tyrosine from prephenic acid (15) appears to take place 24 by way of /7-hydroxyphenyllactic acid (16):< > CH 2CH0HC00H

HOOCv^

^CHoCOCOOH

Τ

OH

+ C02

Prephenic acid

/?-Hydroxyphenyllactic acid

(15)

(16)

Tyrosine is formed from phenylalanine in mammals, but that the hydroxylation of phenylalanine is the route by which tyrosine is formed in plants is ( 2 5) Hownot indicated by the results of experiments to test this suggestion. ever, that shikimic acid is the precursor in plants of the C 6 — C 3 compounds 2 5) that occur so widely, and indeed of phenylalanine and tyrosine, < has been well substantiated by a number of investigators. Early experiments to test this hypothesis were directed to the study of the biogenesis of lignin. Lignin, a polymeric substance of still undefined structure, is the product of the condensation of nine-carbon units—for example, coniferyl alcohol—into a high molecular weight substance. When wheat and maple cuttings were fed with radioactive (carbon-14) L-phenylalanine and shikimic acid, it was found that b o t h of these precursors were incorporated into lignin (26 with about equal facility. > Subsequent experiments showed that the specific 14 labeling patterns were preserved: cinnamic acid-3-C was incorporated into lignin in such a way as to provide formyl-labeled vanillin and syringaldehyde 27 on degradation. < > The biosynthesis of lignin is discussed further in Chapter 15.

750

T. A. GEISSMAN

A generalized scheme that has been proposed for the biosynthesis of 28 shikimic acid-derived compounds in higher plants is represented as follows : < > Shikimic acid

Phenylalanine

Prephenic ocid

Phenylpyruvic ocid

p-hydroxyphenylpyruvic

Phenyl lactic acid

/p-hydroxyphenyllactic

Cinnamic acid

/7-hydroxycinnamic acid

ΗΟχ HO

J \

C H O H C H 2C O O H +

ocid

tyrosine

acid

flavones, etc. (see p. 772)

Caffeic

HO HO—f

> — C O C H 2C O O H

Ferulic acid

>

Lignin (via A r y l — C H = C H C H 2O H )

H 0

\

HO—\/—COCH3

Sinapic acid J

(Pungenin aglycon)

While certain details of this scheme remain to be demonstrated, it represents a working hypothesis that embodies the present information about the chief synthetic role of the cinnamic acids and their precursors in plant metabolism.

Cinnamic Acids The conversion of phenylalanine into caffeic acid in plants, with specific retention of the position of isotopic labeling, was first found in experiments 2 9) on tobacco. < The biosynthetic relationships between caffeic and /?-coumaric (25 ferulic and sinapic acids were studied in Salvia splendens, > with findings that led to the conclusion that the course of synthesis is the following: /7-coumaric -> caffeic -> ferulic -> sinapic. Salvia could convert the simpler members of this series into the more complex compounds. The reverse process did not occur readily (but see p . 791).

THE BIOSYNTHESIS

OF P H E N O L I C P L A N T P R O D U C T S

751

When shikimic acid (labeled generally) was fed to Salvia, caifeic acid labeled only in the ring was obtained. This and other results have disposed of the possibility that an important pathway to the cinnamic acids is by extension of a substituted benzoic acid by the addition of a two-carbon unit. THE U S E OF TWO-CARBON UNITS IN AROMATIC BIOSYNTHESIS

The discoveries in the past ten years of the importance of acetic acid, in the form of its thioester, acetyl CoA, as a building unit in cellular synthesis have clarified and pointed the way toward the ultimate solution of a host of questions concerning biosynthetic pathways. We can distinguish three distinct routes of synthesis in which "acetate" is involved: (I) The condensation of acetate fragments into the branched chain compound mevalonic acid (17) and the ultimate conversion of this into such building units as (18) and (19): " 0 6P 2— 0 C H 2C H o C ^

Steroids Cartenoids Terpenes O - a n d C- Prenylations

C H 2

(18) H 0 . c/ C H CHUC00H —

3

Ctfo ^ C H

2

COOH CH 2OH Mevalonic

acid

(17)

^CH3 " ~ 0

6P 2—

0C0CH=C\

-

( 0 - a n d C-acylations)

CHj

(19)

(II) The linear condensation of acetate fragments into chains, with or without ultimate cyclization into benzene, naphthalene or anthracene derivatives. (Ill) The addition of two-carbon units t o a precursor that may or may not be acetate-derived. This route is most commonly recognized in the addition of — C H 2 C O — units to a carboxyl group (20) : R — C O O H -> R — C O C H 2 C O O H -> R — C O C H 2 C O C H 2 C O O H (20) The first of these routes will not be discussed here. Although numerous plant phenolic compounds are terpenoid, and may be regarded as arising by route I, a discussion of their biogenesis is to be found in Chapter 14. THE ACETATE HYPOTHESIS

Plant biosynthesis by way of routes II and III was suggested by Birch and 2 0) Donovan < as a general hypothesis. Collie, in 1907, was the first t o propose a role for polyketomethylene intermediates in the biosynthesis of aromatic 9 compounds ; <> and the possible role of two-carbon atom units in the biosynthesis of the fatty acids, characteristically even-numbered chains of carbon atoms, has been a prevalent theme for many years.

752

T.

A.

GEISSMAN

Many instances of biosynthesis by the acetate route—the linear condensation of two-carbon units into (hypothetical) poly-ß-ketomethylene chains followed by cyclization in one or another way—have now been studied, with the use of labeled compounds, in microorganisms, fungi and higher plants. The results of these studies leave no doubt about the essential validity of the broad outlines of the theory, although many details remain to be explained. The general outlines of the theory, with respect to both routes II and III, may be presented first as a prelude to a more detailed description of individual examples. In the simplest formulation, the polycondensation of C 2 units could proceed as follows (21): C H 3 C O O H -> C H 3 C O C H 2 C O O H -> C H 3 C O C H 2 C O C H 2 C O O H -> (21 ) C H 3C O ( C H 2C O ) nC H 2C O O H Cyclization of an intermediate of this kind could take place in many ways: pH3 Oj£

(a)

COOH

C 0

C H 2 - COOH

(22) CH,CO.

COOH

(b) CH3CO-ÇH2 X H

CO . C O TH2

2

(c) ÇH2-CO CO

C H rC p CO C H .

r

" C H 3- C 6

(d)

CH ? ,CHp CH 2 N

H OcOHCr-cCoH ^ 1:6 C H ^ Ccd H z ^ CcoH S

CO

C0

Q

a

2

(e) Q

C H 3 C H 2C H 2C H 2C H 2C 0 - C ^ ^ C H 2 C H 2 COOH CO% CO CO CHo CHo Î t f ?

/ C7

H

" 5 "

CO

f The formation of resveratrol (24) by the condensation and ring closure illustrated here is but one possible route to this compound. The occurrence in nature of other stilbenes with different hydroxylation patterns suggests an alternative course of synthesis in which one ring is phenylpropane-derived, the other acetate-derived. This will be discussed in the sequel.

THE BIOSYNTHESIS

OF P H E N O L I C P L A N T P R O D U C T S

753

(f) C H 3C H 2C H 2C H 2C H 2C O C H 2 CO ChL

X* C

C H 2— C 0 0 H + C 0

C H

'

^ruP° CH2

2C0 1

%

\ç

I

•

C

•

C H 2C 0 0 H 0

H0

Λ-CsH,,

C H 2C H 2C H 2C H 2C H 3 (27)

Examples of route III are typified by the biosynthesis of compounds in which one structural element is the 1-arylpropane-derived unit which will be referred to as C 6 — C — C — C , or C 6 — C 3 . Combination of this with a single C 2 unit can lead to the intermediate (28); addition of successive C 2 units provide, in turn, the precursor substances (31) and (34). As is shown in the following generalized schemes, various natural substances have structures that can be directly related to these intermediates :f

t The presence or absence of 4- and 3,4-hydroxylation in the final products used as illustrations is irrelevant to the point in question. This structural feature relates to the Q—Ca portion of the molecules.

754

T. A. GEISSMAN

/

V _

e

— C

COOH + 3 C 2

7 — C — C

- CO — CH 2CO

C H 2C O - CH^COOH

C—c—CO

(35)

The elaboration of a phenolic ring as a terminal unit on what is clearly a fatty acid-derived side chain, as in (36), presents a question to which n o unequivocal answer can be given. The short polyketomethylene " e n d " could be added to the preformed fatty acid (37), or could conceivably be the unreduced residue of a long polyketomethylene chain (38). The former of these is certainly the most likely, but the question remains open. For example, anacardic acid (39) is related to the intermediate (36):

*

C H 3( C H 2) 5 C H = C H (CH 2) 7COCH 2 C O C H 2C 0 C H 2C 0 0 H

(36)

i3C2

\

C H 3( C H 2) 5C H = C H (CH 2) 7COOH

\ C H , (CH 2) 5 C H = C H - ( C H 2) 7

(37) \

1/C00H

\

X

- ~ C H 3 ( C O C H 2 ) 7 COOH

"OH

(39)

Anacardic Acid

(38)

The foregoing presents an overall view of the main aspects of the role of acetate ( C 2 units) in phenolic biosynthesis.f In what follows, a more detailed examination will be made of the application of these hypotheses to the biosynthesis of particular groups of compounds. In some cases the evidence will be found to be direct and conclusive; in others, the conclusions are inferential and depend upon the inner consistency of hypothesis and structure, or upon the successful solution of a structural problem by the use of the hypothesis as a predictive device. t See p. 760.

THE BIOSYNTHESIS

OF P H E N O L I C P L A N T P R O D U C T S

755

THE BIOSYNTHESIS OF P A R T I C U L A R G R O U P S OF C O M P O U N D S

Phenols, Resorcinol Derivatives, Salicylic Acid Derivatives Perhaps the simplest of the natural acetate-derived phenols is 6-methylsalicylic acid (40), the derivation of which can be written as follows:

CM I

CH3

3

COOH

CO ÇH2 C H 2C 0 0 H CO

CO CH2

OH 6-Methylsalicylic acid (40)

That the sequence of carbon atoms indicated by this mode of formation is 31 correct was shown < > by the study of 6-methylsalicylic acid produced by 14 Pénicillium griseofulvum supplied with carboxyl-C acetic acid. Degradation of the salicylic acid gave results that were fully in accord with the following scheme :

CH3 *COOH

CH3

s t

£oOH^°^- ^-->

*

CH3COOH

*COOH *COOH CH3

Ba C 0 3 (9-9 cpm/mg)

__ Decarbox.

^ B a C 0 3( l 8 c p m / m g )

Combustion ^

aBC 0 3( 6 - 9 cpm/mg)

OH

CH3 N 0 2^ C B r 3N 0 2«

^/N02 ]|

^[

(inactive)

"-,

Q H

NO?

Further transformations, probably starting from 6-methylsalicylic acid, are brought about by Pénicillium strains to produce a variety of related compounds:^

756

T. A. GEISSMAN

OH

OH

Ι

rr X

HOCH.

COOH

COOH

ι

J

CHO

- ~

r C H 2O H

.

0

I

r CHO

—

.

r

r

LU

1 J r r ^0"" (40a)

OH

Patulin

The formation of various related aromatic compounds by various strains ( 3 3) of the ascomycete Daldinia concentrica is illustrated by the following :

THE B I O S Y N T H E S I S

OF P H E N O L I C P L A N T P R O D U C T S

757

To the foregoing examples can be added many others drawn from studies on the biosynthetic activities of microorganisms. Griseofulvin (41) is formed 34 from acetate units according to the scheme : < >

7C2

c

„C0 X

N x

c

£0N

CO

coCO

co-c I c

C H 3o

Griseofulvin (41)

Another mold metabolite, curvularin, has the structure (42), and has been 35 shown to be derived by head-to-tail linkage of eight acetate units : < >

758

T. A.

GEISSMAN .0.

CO

I

c. 8C2

CO C,

CO

CO ,C

HO-

c CO

CH3

ÇO CO G\

,C CO HO

O Curvularin (42)

The formation of compounds derived from head-to-tail combination of acetate units in higher plants has received far less attention. Much of what is postulated about phenolic biosynthesis in plants is based largely upon the ability of the acetate hypothesis to assist in structural investigations by predicting which of two or more plausible structures is the most likely. A n early 3 6) example of this is found in the revision < of the structure of eleutherinol (43), 37 first formulated < > as (44):

Structure (43) can be constructed of a regular chain of eight acetate units in head-to-tail linkage (45). While (44) can be constructed from acetate units, its irregularity casts doubt upon its correctness; and subsequent chemical evidence furnished support for (43). It is also to be noted that eleutherol (46) and eleutherin (47), which occur along with eleutherinol in Eleutherine bulbosa, follow the "regular" pattern of head-to-tail linkage of acetate units (with modifications due to oxidation and reduction reactions):

THE BIOSYNTHESIS OF PHENOLIC PLANT PRODUCTS

759

CH3 CO CO CO CH2 CH2 CH2 CO

XO CO CH2 CH2 CH 2COOH Eleutherol

(46)

CH,

I

CH^O

^CO. JZO ^CO CH2 CH2 CH2 C

Q

ßO, .CO. / C O CH 2COOH CH2 CH2 CH2

Similar consideration led to the alteration of the structure (48) first pro38 posed < > for α-sorigenin to (49), which was subsequently confirmed by chemical means. The hypothetical precursor (50) would lead more readily to (49) than to (48):

CH,0

C H 30

OH

OH

(48)

c6

C H

V°V-

C H 3

CH2 CH2 CH2 XO' CO "COOH

(50)

It is to be noted that a feature of the hypothesis, as expressed in the structures of compounds 43, 46, 47 and 49, is the placing of phenolic hydroxyl groups in the positions called for by the arrangement of acetate residues in the postulated precursors. A case that demonstrates the usefulness of this feature of the theory is that of flaviolin (51), for which the alternative structure (52) was at first also in consideration:

760

T. A. GEISSMAN

The placing of the oxygen atoms (*) in the ten-carbon atom precursor (53) is in better accord with (51). The synthesis of flaviolin trimethyl ether has 40 41 established (51) as the correct structure. < > ) It must be admitted that because of the ability of living systems to introduce and remove oxygen atoms in compounds of the types being considered here, arguments such as the ones that have been presented are not always compelling. They have, however, served as useful guides in directing degradative and synthetic experiments in one of several alternative directions, and have been of value in furnishing working hypotheses in structural investigations.

Extension of Acyl Residues by C 2 Units The synthesis of fatty acids by the reverse of the pathways of oxidative degradation can be summarized as follows (54) : R C H 2 C O — S C o A + C H 3 C O — S C o A ^ R C H 2 C O C H 2 C O — S C o A + HSCoA R C H 2 C O C H 2 C O — S C o A + D P N H + H+ ^ R C H 2C H O H C H 2C O — S C o A + D P N + TPNH + H

R C H 2C H 2C H 2C O — S C o A <

+

t

R C H 2C H = C H C O — S C o A (54) ( 4 2)

where the various steps are catalyzed by specific e n z y m e s . That this simple picture may not represent the only, or even the most important, route to long-chain fatty acids is suggested by the recent observation that an essential primary step in fatty acid biosynthesis involves the reaction of acetyl-CoA with carbon dioxide to form malonyl-CoA which is then converted into long-chain acids. The overall scheme that has been suggested 43 is the following (55) :< >

THE BIOSYNTHESIS OF P H E N O L I C PLANT P R O D U C T S

C H sC O — S C o A + C 0 2 + A T P

761

CH.CO—SCoA COOH

CH3CO—SCoA +

C H 2C O — S C o A

COOH

I

COOH

OH TPNH

CH3COCH—CO—SCoA

C H 3C H — C H — C O — S C o A COOH C H , C H 2C H — C O — S C o A COOH

C H 3C H = C — C O — S C o A

I

COOH C H 3C H 2C H 2C O — S C o A + C 0 2 ψ malonyl-CoA etc.

(55) The acetate hypothesis of the biosynthesis of plant substances implies a reaction such as the following, without, however, the necessary intervention of the reduction of — C O — to — C H 2 — between successive acylations (56) : R C H 2C O O H + C H 3 C O O H — > R C H 2C O C H 2C O O H R C H 2C O C H 2C O O H + C H 3 C O O H — • R C H 2C O C H 2C O C H 2C O O H (56) It should be emphasized, however, that in many of its applications the "acetate" theory has so far remained as a structural hypothesis, and none of its proponents have spelled out the details (as in (56)) of the process of chain extension. If a process such as (56) is to be assumed, the use of coenzyme A derivatives instead of the free acids would, of course, be implied. For the present the simpler scheme (54, 56) will be used for the purposes of this discussion. The extension of the fatty acid chain by the addition of a single C 2 unit has been shown by the incorporation of radioactivity into arachidonic acid (58) by rats fed with y-linolenic acid (57) and acetate: C H 3( C H 2) 4C H = C H C H 2C H = C H C H 2C H - C H ( C H 2) 4C O O H (57) COOH-labeled (57) + C H 3 C O O H -> C H 3( C H 2) 4( C H = C H C H 2) 4C H 2C H a C O O H unlabeled (57) + C H 3 C O O H -> C H 3 ( C H 2 ) 4 ( C H = C H C H 2 ) 4 C H 2 C H 2 C O O H (58) A group of compounds whose biosynthesis may be represented by an extension of this process is that which includes a number of phenols found in

T. A. GEISSMAN

762

the Anacardiaceae. These are urushiol (59), anacardic acid (60), cardol (61) and cardanol (62):f

(59)Urushiol

( 6 0 ) A n a c a r d i c acid

(61) Cardol

( 6 2 ) Cardanol

R = / 7 - C l5

The biosynthesis of these phenols may be formulated by the following scheme : .(OH)

C H fC 0 C H 3 ( C H 2) 5C H = C H

(CH 2) 7CO

/

CH 2

>• R

CHrCO I COOH

/

(/

(62)

—

J O x i d o t i ve

/

-co2 (61)

\

y

(60)

> = Λ / \ COOH OH d e c a r b o x y l o t i on

\ (59)

A most striking example of biosynthesis by this route is found in a group of compounds isolated from the oil of the anacardiaceous plant Campno( 4 4) sperma brevipetiolata ( T i g a s o ) . The phenol campnospermonol (63) was the first phenolic compound found in this oil:

C H 3( C H 2) 7C H = C H ( C H 2) 7C 0 C H 2— Ü

\

OH Campnospermonol

(63)

Later studies showed the presence in Tigaso oil of oleyl methyl ketone (64) C H 3( C H 2) 7C H - C H ( C H 2) 7C O C H 3 (64) and the cyclic ketone (65). f The side chains (R) in these natural phenols are mono-, di- and tri-unsaturated, with double bonds at 8, 8 : 11, or 8 : 11 : 14. For the present purposes, the mono-olefins will be chosen for discussion; this side chain is that of the natural fatty acid palmitoleic acid, CH 3(CH 2) 5CH=CH(CH 2) 7COOH.

THE BIOSYNTHESIS

OF P H E N O L I C P L A N T

PRODUCTS

763

C H 3( C H 2) 7C H — C H ( C H 2) 7C O C H 2

(65)

The latter appears to be the aldol derived by ring closure of the intermediate (66): CH "CH C H , ( C H ?) 7C H 5

,

7

CH(CH),COCH2

2

/

\\ \pH CHpCO

,_ f

-C02 ^

(«J

I COOH (66)

The significance of this observation is two-fold: the cyclic ketone (65) is only a reduction-dehydration step removed from the oxidation stage of the tetraketo precursor (66a) : C H 3( C H 2) 7C H = C H ( C H 2) 7C O C H 2C O C H 2C O C H 2C O C H 2C O O H (66a) and the presence of the double-bond in the ring at a stage prior to that of complete aromatization indicates that the phenol (63) is not derived by removal of a nuclear hydroxyl group from the resorcinol that corresponds to cardol (61). Thus, removal of the phenolic hydroxyl group is probably not a stage in the synthesis of the monophenols (60) and (62). In another species of Campnospermum are found the related compounds (67)

67)

in which R represents unsaturated side-chains that can be reduced to the 4 5) «-C 1 9H 3 9-residue. The positions of unsaturation are unknown. < The modifications of structure in the phenols of the Anacardiaceae include catechols and resorcinols, with C 1 5, C 1 7 and C 1 9 side chains. Moreacol and thitsiol are 4-alkyl (or alkenyl) catechols (68) and may arise by hydroxylation OH

(68)

of the " p a r e n t " 3-substituted phenols, of which (62) and (63) are prototypes. It is clear that these phenols form a group of compounds that provide excellent support for the hypothesis of synthesis by the acetate route. While alternative hypotheses, for example that a crotonyl fragment is incorporated

764

T.

A.

GEISSMAN

in the course of formation of (66), may be entertained, these are obvious variants of the acetate hypothesis. Experimental studies to establish the steps in these involved syntheses remain to be carried out. There can be little doubt that the positions of labeling that would result from the experimental incorporation of, say, methyl-labeled acetic acid into the phenols of one of these plants could be predicted with considerable assurance. Such demonstrations, while valuable confirmation of the main course of synthesis, would add little to the understanding of the details of the sequence of steps involved.

The Lichen Compounds The depsides and depsidones that are found widely distributed and in great structural variety in the lichens provide another closely integrated group of compounds, many of whose structures fit into the general scheme of biosynthesis by linear combination of C 2 units. The depsides are esters formed between the carboxyl group of one, and a phenolic hydroxyl group of another di- or tri- hydroxybenzoic acid. The simplest of the depsides is lecanoric acid (69) :

(69)

Lecanoric

acid

Orsellinic acid (22), into which lecanoric acid is converted by hydrolysis, could be formed by the cyclization of a precursor derived from four acetate units. The resemblance of this scheme to that proposed for the synthesis of COOH

O r s e l l i n i c acid

6-methyl-salicylic acid (40) is apparent. The formation of (22) is more direct; all of the oxygen atoms, except the one lost in the ring closure, are retained. Divaricatic acid (70) is an example of a compound formed from a 5 X C 2 precursor :

Divaricatic (70)

aCH2CH2 cid

CH-j

THE BIOSYNTHESIS OF PHENOLIC PLANT PRODUCTS

765

The theory provides no basis for deciding whether the unit C H 3 C H 2 C H 2 C O — forms the starting point, or whether the carbonyl group in a hypothetical compound C H 3 C O C H 2 — A r y l is later reduced to a — C H 2 group. Anziaic (71) and perlatolic (72) acids possess structures similar to that of divaricatic acid, but with C H 3 C H 2 C H 2 C H 2 C H 2 — residues on the aromatic rings. Sphaeropherin contains a methyl group in one ring, a w-heptyl group in the other (73) : R

M

(71)

R=R

(72)

R = R" =/7-C 5H u;R' = CH3;Perlatolic

(73)

R* R

1

sfl-CsHnjR' = H;

Anziaic acid acid

= C H 3 ; R"v?-C 7H l 5; Sphaeropherin

Numerous additional compounds with the same fundamental structure as lecanoric and similar acids, varying in the nature of the R groups (as in 71-73), are known. That these esters are formed by combination of the separate aromatic monomers is indicated by the occurrence of triesters typified by gyrophoric acid (74): CH, CO

COOH

0

CO —

0"

OH

(74) Gyrophoric acid

Ring closure at a different point in the hypothetical linear precursor can account for the depsides of the type exemplified by olivetoric acid (75): CH3 CH2 CH2 CH2 CH2 C0CH2 / C O — 0

HO

OH

/OH

COOH C H ? C H , C H , C H :X H 3

(75) Olivetoric acid

The precursor (76) can account for the formation of the left-hand ring of

766

T. A. GEISSMAN C H 5 C H r - C H ? C H 2 ~ C H 2 CO

CH2 ^CO CH2 C H 2 COOH CO

(76)

(75); the other ring would be derived by ring closure of the orsellinic acid type. Although little has yet been done in the experimentally difficult field of 46 lichen biosynthesis, Mosbach< > has observed that gyrophoric acid (74) is formed in Umbilicaria pustulata by the usual acetate-malonate pathway. The three orsellinic acid units in (74) had identical specific activities when radioactive ethyl malonate was utilized by the lichen. Shibata a n d his col4 7) laborators < have found that the acetate-malonate pathway can account for the formation of lecanoric acid and atranorin in Parmelia tinctorum. 14 In this case it was also observed that C -formate is incorporated into atranorin (and chloroatranorin), presumably to provide the — C H 3 and — C H O groups in these depsides. 48 49 Mosbach < > and Maas, Towers and Neish < > have shown that pulvinic and vulpinic acids are formed by way of polyporic acid, which is derived from a Q - C 3 precursor in the shikimic acid-phenylalanine pathway of aromatic biosynthesis. Cinnamic acid was not utilized by the lichen (Pseudo-

cyphellaria crocatd). A group of depsides whose structures depart from the simple ( C 2 ) x pathway contain an additional carbon atom in each ring. The phenolic acids that are found as component parts of compounds in this group include the following (77-79):

It is not possible to make a categorical statement about the origin of the "additional" carbon atom in the compounds 77-79. It is found in the position at which carbon-alkylation would be most likely to occur; and, as will be seen in the sequel, carbon-methylation is a common biosynthetic process. A n alternative possibility is that a single propionic acid unit is incorporated during the process of chain-extension. Propionic acid is known to participate in biosynthetic processes in a manner very similar to that in which acetate

THE BIOSYNTHESIS OF PHENOLIC PLANT PRODUCTS

767

units take part. Erythromycin (80), for example, is synthesized by Streptomyces erythreus from propionic acid units, and not from acetate units with 50 accompanying C-methylation. < > The oxidation of — C H 3 to — C H O and CH.

CH3 CH3CH2 C H

- C - C H O H - -CHCH3— CO -CHCH3—CH2-.1

OH

OH O R

R=(CH3) 2N-( w

0 X

;

R

CH3

(80)

OR'

CO

=

Ο N y-\ OCH, C H / OH

Erythromycin

— C O O H , as in 77-79 is unexceptional ; many comparable examples are known (see, for example, patulin (40a)). The occurrence of depsides containing the trihydroxytoluic acid residue C81) represents another departure from strict adherence to an acetate "rule".

J . / C 0 0 H H O ^ V ^ O H OH (81)

However, the introduction and removal of hydroxyl groups are processes that are not necessarily relevant to considerations of the biosynthesis of the carbon skeleton. The relationship between (79) and (81) is reminiscent of that between anacardic acid (60) a n d urushiol (59). In this connection, evidence concerning the origin of the "extra" carbon atoms (in — C H 3 , — C H O and — C O O H ) in compounds (77-79) may be drawn from studies on a product of mold metabolism, mycophenolic acid (82):

(82) Mycophenolic acid

T. A.

768

GEISSMAN

Mycophenolic acid may be regarded as being a nuclear dialkylation product (see 83) of an acetate-derived derivative of orsellinic acid (o;* its precursor). C O OH

Terpenoid side-chain

(83)

The following labeling pattern (84) has been observed, using carboxyl51 labeled acetic acid:< > HO CH3 CHjCOOH

•

HOOC-CH2 — C H

2

— C=

CH —

CH2

(84) The labeling in the isoprenoid side-chain does not concern us here (other 51 experiments < > establish its origin as from mevalonic acid), but the fact that it was found to represent an introduced substituent is of importance, since it corresponds in position to the — C H 3 , — C H O and — C O O H groups in (77-79). The additional methyl group in mycophenolic acid is derived from i52 methionine (as is the methyl group of the C H 3 0 — ) , > and thus is the result of carbon-alkylation by a methyl donor (see p . 781). The conclusion that the depsides are acetate-derived, and that orsellinic acid and such related dihydroxy-benzoic acids as those found in (70) and (75) are the primary substances which may undergo further elaboration, seems to be well supported by both direct labeling studies and by the structural correlations that exist with these groups of compounds. Alternative schemes have, however, been proposed. Aghoramurthy and 53 Seshadri < > have put forward a theory of biosynthesis of compounds of these types in which the recognition of a C 8 unit as the fundamental unit of the depsides and depsidones forms the basis of the proposal. The derivation of orsellinic acid by the condensation of a hexose and a biose is formulated in the following way (85). The lengthening of the side chain by further conCHOH CHOH ^CHOH

I

I

CH 2OH CHOH — C H O CHO

CH 2OH

I

C

°

H

Y

"COOCH3

C H . OH

(85)

(and other compounds)

THE BIOSYNTHESIS

OF P H E N O L I C

PLANT

769

PRODUCTS

densations of the methyl group of orsellinic acid (or a derivative, such as an ester) with additional biose residues accounts for the production of the nalkyl derivatives described earlier. The frequent occurrence of erythritol in lichens both as the free sugar and i 3 0) and as in erythrin, the erythritol ester of lecanoric acid, has led R o b i n s o n (54 Asahina and Shibata > to suggest that two four-carbon units combine (86) to form the fundamental orsellinic acid (22) : H O C H 2( C H O H ) 2C H 2O H

•

C H 3C O C H 2C O O H CH, COOH

C H 3C 0 C H 2C 0 0 H

2

>- |

(96)

|f

H O ^ ^ ' "

^OH

(22)

It is apparent that these older theories bear a similarity to the acetate hypothesis, and it is worthy of note that they were devised at a time when the recognition of the role of two-carbon units in biosynthesis was gaining widespread acceptance. They are still worth noting, however, because, despite the satisfactory manner in which the acetate hypothesis can be used to account for the synthesis of these lichen acids, its very facility can tend to quiet critical inquiry. Until further experimental demonstration of the ways in which acetate units are utilized in the formation of these complex substances, alternatives or modifications of the theory must be held as worthy of consideration.

Anthraquinones Anthraquinones (and some of the related anthrones) are common metabolic products of higher plants, lichens, and microorganisms. The perfect adherence of certain of these compounds to the acetate hypothesis is striking indeed. F o r example, endocrocin (87, R = C O O H ) , a component of the lichen Nephromopsis endocrocea, can be related directly to the poly-ßketomethylene acid (88) :

CHXO

CH2

HOOC-CH2

CO

CH? CH> CO CO

CH2 CO"

CH2

CO"

CH

xo"

OH 2

OH (88)

Ο

OH

(87) R= C O O H ; Endocrocin R = H; Emodin

2C

770

T. A.

GEISSMAN

Emodin (87, R = H), an anthraquinone of a higher plant, lacks only the carboxyl residue of endocrocin, but bears the same pattern of oxidation. Alteration by oxidation (at some unknown stage in the overall synthesis) of the side-chain residue, and removal of nuclear hydroxyl groups (perhaps before cyclization), are processes that can be invoked to account for the structures of such anthraquinones as aloe-emodin (89) and rhein (90) :

HOCH,.

r

HOOC

.Κ

" ' V Y Y • : 'ί- , A Χ Ο

OH

OH

89) Aloe-emodin

In addition to these "regular" anthraquinones, there are a number whose derivation from a precursor such as (88) involves stages that cannot be so simply related to the acetate hypothesis. Alizarin (91), rubiadin (92) and morindadiol (93) are examples. OH

CH,

0

I

J ,

O

OH

(93) Morindadiol

Finally, the genesis of solorinic acid (94) from a precursor (95) represents an obvious extension of the synthetic scheme:

co^ / 7 - C 5H „ — C O

CH2 HOOC^

.co. ,αχ

CH2

CH2

CH2

.CO.

.CO.

JZO~

CH2

CH2

CH2

/ H 0 ^

v>. /

\

/

\ / /

^ ^0CH

Xf

3

0 <

9 5

( 9 4 ) Solorinic acid

)

Naph thoquinones Flaviolin, sorigenin and the naphthalene derivatives from Eleutherine species have been dealt with earlier. Besides these, numerous naphthalene derivatives are found in nature, many of them the products of mold metabolism. A particularly interesting group of compounds includes fusarubin (95) 0) and javanicin (96) from Fusarium solani'X fulvic acid (97) from Pénicillium griseofulvin; citromycetin (98) from Citromyces species; and purpurogenone (99) from Pénicillium purpurogenum.

THE B I O S Y N T H E S I S OH

OF P H E N O L I C

PLANT

PRODUCTS

771

0

C H 30 x

C H 2C O C H 3 H O . CHoR OH

0

( 9 5 ) R = O H Fusarubin (9 6 ) R- H

( 9 7 ) Fulvic acid

Javanicin

OH

( 9 8 ) Citromycetin

OH

( 9 9 ) Purpurogenone

The derivation of fusarubin and javanicin by the acetate pathway is strongly suggested by the presence of the acetonyl residue. If the following pathway is followed (100) the — C H 3 (—CH 2 OH) group would be the result of reduction of the terminal — C O O H group : CH CO'

C H

2

^ C O

2

^ C H

2—

I CH2 ^CO

C H

2C

O C H

3

• (95) (96)

CH2 / C H 2C O O H ^ C O

(100) 55

5 6)

It has been suggested < » that the relationship of javanicin and fusarubin to the compounds (97) and (98) is to be found in the hypothetical structure (101): OH

CO

/ C H 2C 0 C H

3

XH CO'

^

_

Γ 0

^ C H 2O H

\

CX>

l T =

H

C H — C H0 H

2

C O — C H

2C

0 C H

3

y ^ ^ C O O H OH

COOH

(ΙΟΙ)

The relationship of (101) to (95) and (96) could be through the cleavage of one aromatic ring; or, as Whalley has suggested, (101) could be the primordial precursor from which (95) and (96), as well as (97), (98) and (99) are

£0^

I f?

CH^T

V"

CO*

(96)

^ C H 20 H

-CHXOCH

_^leoye^ o,(o)

| 0 |( )

T. A. G E I S S M A N

772

formed despite the fact that it does not conform to a "regular" acetate route of formation. It will be noted that in both schemes, the reduction of — C O O H to — C H 2 O H is involved: in the route via (96), the — C O O H group of (100); in the route via (101), the — C O O H group of (101).

Compounds Derived from l-Phenylpropane Precursors Flavonoid Compounds The biosynthesis of compounds that possess the structural unit (102) has been discussed earlier (p. 747), where it was remarked that numerous compound classes can be related to hypothetical precursors derived by extension of the C 6 — C 3 unit by two-carbon fragments.

^3^c—C-C (102) The largest and most varied class of substances of this kind is that of the flavonoid compounds. These are described as C 6 — C 3 — C 6 compounds, and may be regarded as being formed by the combination of the shikimic acidderived C 6 — C 3 fragment with a six-carbon atom unit formed by the linear combination of three acetate units (103):

— C—CO-C—CO-C—COOH

(103) The substantiation of this course of synthesis has been accomplished by a 57 60 number of studies. ( ~ > Numerous phenylpropanoid precursors serve as the source of the B-ring (see 103); shikimic acid, phenylalanine, /7-hydroxycinnamic, cinnamic and caifeic acids were good precursors of quercetin in buckwheat plants. Tyrosine and such other cinnamic acids as sinapic, ferulic and m-methoxycinnamic acid were utilized, but poorly. The most revealing result of these studies was that the C 6 — C 3 skeleton of the precursor was incorporated as a unit; the labeling pattern shown in (104) was estab57 59 lished : < > >

THE B I O S Y N T H E S I S

OF P H E N O L I C

PLANT

PRODUCTS

773

OH

H CK

OH

OH

Y

^OH

ο

(104)

When uniformly-labeled phenylalanine was fed to buckwheat plants, sevenninths of the activity of the precursor was recovered in the veratric acid formed by methylation and cleavage of the quercetin; two-ninths was found in the acetophenone derivative formed along with veratric acid 57 (105):< >

cleave

k

OH

C H 30 ,

" ΌΗ v

OH

C O C H 2O C H 3

0CH3

0

Quercetin (105) 0CH3 C H 30

14

Surprisingly, Grisebach observed that coniferin-3-C did not give radioactive cyanidin in red cabbage seedlings, although a flavone present in the plants was radioactive. Experiments with labeled sodium acetate showed that this was incorporated exclusively (except for slight randomization) into the Α-ring of the flavone (106) OH

HO. "OH

C H 3C 0 0 N a U~or2-C

1 4

:

OH

(106)

When cyanidin is pioduced by red cabbage seedlings fed with labeled sodium acetate, the Α-ring labeling is consistent with the view that this ring

774

T. A.

GE1SSMAN

is formed by head-to-tail union of acetate units followed by cyclization (107): («ο

S h i k i m ic - d e r i v ed

(107) While the biogenesis of the numerous flavonoid compounds related to the flavones, but differing only in the state of oxidation of the C 3 part of the molecule and in the number of hydroxyl groups on the aromatic rings, has not been established with equally definitive experiments, there can be little doubt that, so far as the source of the carbon skeletons is concerned, they are similarly derived. What has not yet been discovered in the sequence of changes that occur in the formation of so diverse a group of compounds, embracing at the one extreme the highly reduced catechins (e.g. 108) and at the other the flavonols (e.g. 109), with many different levels and kinds of oxidation in other compounds of the class.

HO

(108) (109) The most likely course of synthesis of the flavonoid compounds appears to be by way of a C 6 — C 3 fragment in the oxidation state of a cinnamic acid, and a C 1 5 intermediate in the oxidation state of a chalcone (110):f

CH=CH-COOH

+

3C2

CO

COOH

CH

II

- I CH v ,

CH? CH CO'" ^CO'"

„OH

(HO)

f Only the o-hydroxyl group of the Α-ring is shown in the final product of sequence (110) because it is only this position that is invariably hydroxylated. Other hydroxylation patterns are discussed further on.

THE BIOSYNTHESIS

OF P H E N O L I C

PLANT

PRODUCTS

775

Information as to the order in which hydroxyl groups are introduced or removed from rings A and Β is still lacking, and only speculation can be offered to define the stages in the overall synthesis at which such changes occur. Indeed, the evidence from many observations fails to provide a clear and compelling hypothesis. It might be argued that since baicalein (111) and wagonin (112) occur in the same plant, the common open-chain precursor (113) precedes them in the synthetic order:

HO

OH

i

(b)~ (113)

On the other hand, the co-occurrence in a single plant of quercimeritrin (114) and gossypitrin (115) suggests that hydroxylation occurs at a stage after ring formation has been completed. This suggestion depends upon the conclusion that glycosylation is a late development in flavone biosynthesis, a conclusion supported by the co-occurrence in single plants of numerous glycosides of a ( 6 1) given a g l y c o n .

( 1 1 4) Q u e r c i m e r i t r in

R ^ - g l u c o s yl

(11 )5 G o s s y p i t r ni

Genetic studies have given strong support to the view that the flavonoid 63 compounds of various levels of oxidation derive from a common precursor. < > Single genetic factors ("genes") are known to control whether 4'- or 3',4'hydroxylation of the B-ring occurs, the presence of absence of methoxyl

776

T. A.

GEISSMAN

groups, the position and degree of glycosylation. There is no doubt that the processes by which the simple precursors (ultimately, shikimic acid and acetate units) are converted into the many flavones, anthocyanins, aurones, etc., that are found in plants are very complex, and represent competing and interacting pathways of oxidation and reduction. The concept of the central role assigned to chalcones in flavonoid biosynthesis embraces dihydrochalcones as well, as seen by the demonstration that apple leaves contain a reductase / , , , into capable of converting 2 ,4 ,6 ,4-tetrahydroxychalcone-2 -glycoside 62 phloridzin. < > The view that the precursor at the C 6 — C 3 — C 6 level is indeed represented best by (110), that is, by the chalcone, finds support from a number of observations, none, however, of a compelling nature. Studies on the chemical genetics (63) of pigmentation in Antirrhinum flowers have disclosed the suggestive fact that anthocyanin formation, governed by a specific dominant gene (P), is always accompanied by flavonol formation; in acyanic flowers flavones, but not flavonols, are present. Moreover, the production of the aurone, aureusidin (as 116) takes place in all non-albino flowers, but in increased amount in the absence of the dominant gene Y. Finally, a gene M (dominant) produces 3',4'-dihydroxy flavones and anthocyanin; and in the absence of the dominant gene N , cinnamic acids, but no flavonoid compounds, are produced. Flavanones are also present in many genotypes. While any scheme that attempts to use these observations for the construction of a detailed scheme of the total path of pigment biosynthesis would be a very tenuous one, certain possibilities suggest themselves. The cooccurrence of flavonol and anthocyanin may mean that a step in pigment synthesis, governed by factor P, is the production of the flavanonol (117) from a primordial chalcone :

ο (116)

Aurone

T H E B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

777

It is worthy of comment that the flavanonol (117) is at the oxidation level of the anthocyanidin ; the transformation can be represented (formally only) as follows:f

H

+

+ H 20

The transformation of chalcones into aurones by hydrogen peroxide oxidation, presumably by way of the epoxy intermediates, is a well-known trans( 7 1) formation, although in the laboratory it is governed by rather strict structural requirements. In the following section another possible role of a chalcone epoxide or flavanonol in a different biosynthetic sequence will be considered.

Isqflavones 6 4)

The biogenesis of isoflavones has been found < to proceed by way of a Q — C 3 — C 6 intermediate and involves at some point in the sequence a rearrangement of the aryl B-ring to the adjacent carbon atom (117a):J

(117a)

The sequence (117a) is shown in outline only. It was demonstrated for the case 64 of formononetin (in clover) < > and for biochanin-A in Cicer arietinum (see refs. 59, 60 and footnote). It is not known with certainty at which point in the synthetic process the rearrangement occurs. It has been suggested that the chalcone oxide (117b) is the intermediate, a n d rearranges according to recognized mechanisms put forward to explain acid-catalyzed rearrangements of 6 7) α,β-oxidoketones. < t Again the hydroxyl groups normally present in the natural compounds are not shown that attention( 6may be confined to the particular processes under discussion. 5 66 % The report » > that a different course is followed was withdrawn after repetition of 64 the experiments confirmed the original observation of Grisebach.* )

778

T. A .

GEISSMAN

The mechanistic details of the rearrangement of the aryl group from position 2 to 3 are still a matter for conjecture. That the oxide (e.g. 117b) itself is not an obligatory intermediate is shown by the conversion of 3,4',768 trihydroxyflavanone (garbanzol) < > into formononetin in Cicer arietinum ( 6 9) seedlings. It should be noted, however, that the interconversion 2'hydroxychalcone epoxide 3-hydroxyflavanone is a clear mechanistic possibility, and so the question of the exact nature of the immediate natural progenitor of the isoflavone skeleton remains unsettled. The possible intervention of chalcone epoxides in flavonoid interconversions in vivo is rendered plausible by the results of many studies on the chemistry of compounds of this kind. The following reactions (in which only the prototype skeleton is used for illustration) show some of the alterations that have been observed (118):

(118)

No case of rearrangement of a chalcone epoxide of the kind found in nature to an isoflavone has been observed, however. 7 0) The occurrence in nature of angolensin (119) < suggests that the postulated rearrangement can occur before closure of the heterocyclic ring:

T H E B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

(119)

779

Angolensin

Stilbenes and Dihydroisocoumarins The naturally occurring stilbene derivatives form a group of compounds of which certain structural features suggest their biogenetic origins. Hydrangenol, a dihydroisocoumarin, may be regarded as the isomer, by a simple ring closure, of a stilbene-carboxylic acid, and, further, appears to be derived from a cyclization of a C 6 — C 3 — C 6 precursor (120):

(120)

(121)

Hydrangenol

( 7 2)

Early e x p e r i m e n t s supported this view, but were not conclusive. An alternative theory of stilbene and dihydroisocoumarin biosynthesis, put ( 7 3) forward by S e s h a d r i , suggests that the building units of hydrangenol are an orsellinic acid-derived fragment (122) and the C 8 fragment (123) formed by the degradation of a C 6 — C 3 precursor (phenylalanine, etc.): OH

(OH)

OH

/ Stilbene derivatives

• n

(

Since (122) would be derived from four acetate units (see 22), the labeling of the hydrangenol skeleton should distinguish clearly between these alterna14 tive paths of synthesis. For example, 2-C -labeled phenylalanine would give unlabeled hydrangenol according to the scheme 122 -> 123 -> 121, but 4labeled hydrangenol by the route 120 -> 121. Similar comments would apply to the natural stilbenes, the structures of several examples of which—pinosylvin (124), resveratrol (125), hydroxyresveratrol (24), (126) and (127)—are shown :f f A number of other natural stilbenes are methyl ethers of these. Compounds (126) and (127) are named systematically.

780

T.

A.

GEISSMAN

( 2 4 ) Hydroxyresveratro!

(126)

HO

OH

C H = C H

/

HO

^>—OH

OH (127)

7 4 75

Recent studies < ' > have confirmed that hydrangenol and the stilbenes are formed from a shikimic acid-acetate-derived C 6 — C 3 — C 6 precursor. The following scheme represents the manner in which hydrangenol and (probably by decarboxylation of (128)), the stilbenes are formed:

COOH

HO

COOH

CO C H 2 CH2

;CO

CH

CH

<

C O C H 2|

v.-..

—

•

Co —

C3 ex

Shikimic pathway (128)

acid

Stilbenes (121)

Again it should be pointed out that the inclusion or exclusion of hydroxyl groups from these formulas entails n o suggestion as to the point at which hydroxylation (or reduction) takes place in the synthesis of the end product. Much remains to be done in working out the many final details of the biosynthesis of the isocoumarins and stilbenes, and very little experimental work involving the feeding of labeled compounds to plants has yet been done.

THE BIOSYNTHESIS

OF P H E N O L I C

PLANT

PRODUCTS

781

Carbon Alkylation Prominent among the processes carried out in the course of biosynthesis in many organisms is the introduction, by the formation of carbon-carbon linkages, of substituents into one or another of the fundamental classes of compounds that have already been discussed. The two kinds of substituents most often encountered are (a) the methyl group and (b) alkyl groups of isoprenoid origin. In some cases the latter may undergo subsequent degradation to smaller fragments. Examples of these will be given in the discussion. It must be noted, however, that few experiments have been performed to provide positive evidence of the biological source of such "extra" carbon atoms. It is nevertheless very probable that living organisms possess the synthetic mechanisms for the carbon-alkylation of carbon atoms of at least two kinds: those alpha t o , or flanked by, carbonyl groups, and those in phenolic rings, especially when in the 2-position to 1,3-di-hydroxyl groups. It is evident that in many cases these two situations are mechanistically equivalent. 76 Numerous C-methylated chromones are known in nature. < > A n interesting group of compounds is that found in Eugenia caryophyllata; these are eugenin (129), eugenitin (130), isoeugenitin (131), and isoeugenitol (132). There also exists in nature, but in another plant (Backhousia angustifolia Benth.), the related compound angustifolionol (133).

OH

0

(132) Isoeugenitol

OH

0

(133) Angustifolionol

The origin of these compounds by the carbon-methylation of an acetatederived parent substance seems t o o probable to be doubted. Whether the introduction of the C-methyl group takes place after the chromone system (as in 129) has been formed, or at the earlier stage of the hypothetical polyketomethylene precursor, or at the state just prior to the closure of the heterocyclic ring, cannot be stated with assurance. The co-occurrence of (130) and

782

T. A.

GEISSMAN

(131) finds a ready explanation in the assumption that the precursor (134) 77 undergoes ring closure in two ways:< > /

( a)

OH

<

/)

OH

COCH 2COCH 3

-HO

-V

<

/

^(130) COCH 2

) OH

(b)

(131)

OH

CH3

(129)

COCH 3

( b)

(134)

Carbon methylation in 2-arylchromones (flavones) is also quite common in nature. Pinoquercetin (135) and pinomyricetin (136) occur in a species of

(135) R=H

Pinoquercetin

(137) R = OCH 3 Matteucinol

(136) R=OH Pinomyricetin

(138) R = H Demethoxy matteucinol

Pinus, while the C-methylated flavanones matteucinol (137) and demethoxymatteucinol (138) are components of Matteucia orientalis. A number of Cmethylated flavones and dihydroflavonols occur in various Pinus species, and 76 further examples are given in the review cited. < > An interesting example of C-methylation in a C 6 — C 3 — C 6 compound is found 78 in ceroptene (139), a constituent of a fern. < > In this compound alkylation of the phloroglucinol ring has proceeded to the stage of dimethylation and CH, C H , 0

o .

OCH, (139) Ceroptene

preservation of the triketo structure of the Α-ring. It is known that Cdimethylation of phloroglucinol derivatives can occur under treatment with methyl iodide a n d alkali; and the C-dialkylation of 1,3-diketones is a wellknown reaction. Thus, it is still open to future investigation to determine at which state the biological alkylation takes place.

THE BIOSYNTHESIS

OF P H E N O L I C

PLANT

PRODUCTS

783

The occurrence of C-methylated and C-dimethylated phloroacetophenones is common in nature. Angustione (140) a n d dehydroangustione (141) occur in the same plant along with angustifolionol (133). CH,

CH, 0

CH,

CH,

,

CH,

j COCH,

|j 0

(141) Dehydroangustione

(140) A n g u s t i o n e

Essential oils of Leptospermum species contain leptospermone (142); and male fern contains albaspidin (143) a n d flavaspidic acid (144):

(142)

CH3 CH,

C H 3C H 2C H 2C O "

γ

Leptospermone

CH, CH3

ZW{

j

CH3 CH3

" C O C H 2C H 2C H ,

CH3 CH3

C H 3C H 2C H 2C O '

COCH 2CH 2CH3 Ο

( 1 4 3 ) Albaspidin

0

( 1 4 4 ) Flavaspidic acid

Usnic acid (145) is formed by the oxidative coupling of two molecules of Cmethylphloroacetophenone : C H 3C 0 -OH

H CK

COCH,

CH^

CH CH,

OH

OH

r

COCH,

OH

(145) U s n i c acid

The source of both O-methyl and C-methyl groups is probably the same as that of the N-methyl groups so common in plant alkaloids, namely, the

784

T.

A.

GEISSMAN

methyl-transfer systems that involve methionine and the compounds associated with it. The active methylating agent, at least in animal organisms, is S-adenosylmethionine (146): +

C H 3- — S

Adenosine

CH2

CH2 CH — COO" NH2 (146) S-Adenosylmethionine

The attack of an anionic carbon, oxygen or nitrogen atom upon the methyl group brings about its attachment to the attacking atom. The ultimate genesis of the methyl groups can take place by a number of routes, in which, among other substances, formate can serve as a precursor. Biogenetic studies have shown that formate can often serve to introduce single methyl groups; the best examples of this are found in the field of alkaloid biosynthesis, which is discussed in Chapter 17. The incorporation of a C-methyl group of mycophenolic acid (82) by feeding of methyl-labeled ( 5 2) methionine has been d e m o n s t r a t e d .

Isoprenoid Substituents™ The widespread occurrence in plants and other organisms (e.g., see 84) of compounds that contain, as both C— and Ο— linked substituents, the typical isoprenoid residue (147) strongly suggests that there exists in many living

(147) Isoprenoid residue

organisms a source of this (and higher isoprenoid) grouping in a biological form that permits its introduction by alkylation or acylation. The ultimate source of these isoprenoid units is mevalonic acid (17) (via acetate), which eventually gives rise to the active synthetic fragment,

THE

BIOSYNTHESIS

OF

PHENOLIC

PLANT

PRODUCTS

785

isopentenyl-pyrophosphate (148). The elaboration of this fragment into terpenes and polyterpenes is beyond the scope of the present discussion. However, the availability of (148) or its isomeric 3-methyl-2-butenyl fragment, as CH3 Η 0 χ

j

^ C H , •

)C' CH2

C H 2

H00C

C

C H 2

C H 2— 0

Ρ Ο 2

-0

POf

C H 2

C H 20 H

·

(17) Mevalonic acid

(148) Isopentenyl-pyrophosphate

an alkylating agent provides a ready solution t o the question of the biosynthesis of such compounds as, for example, suberosin (149), peucenin (150) and brayleyanin (151): CH3

(149) Suberosin

(150) Peucenin

CH3

CH3

C \

ο

CH c H2

•O-) |i"

KJ

:

Lz

λ r

0

τ

C H 30

^

(151)

Brayleyanin

j

0

3

0

OX/ V

/ C H 3 C H 2C H — C .

M

[Γ ~

CM CH

II

OH Τ

l.J

CHjCH-^C

*

CH

R,

*U

V C H 2C H = C 3

c CH3

CH3 (152)

C-Prenylation

Attack by anionic oxygen, to produce prenyl ethers (e.g. 151) can proceed by a completely analogous course. Numerous derived forms of the prenyl residue occur in nature. A few respresentative examples are visamminol (153), lapachenol (154), xanthyletin (155) and the flavone, amurensin (156):

C

H

786

T. A.

GEISSMAN

CH3

CH3 (153) Visamminol

(154) Lapachenol

(155) Xanthyletin

OH

OH (156)

O Amurensin

The biosynthetic steps involved in these hydrations and hydroxylations (or oxidations) of the prenyl residue are not known; while speculation can lead to some obvious possibilities for the formation of the 2-isopropylfuran and 2,2-dimethylchrome rings of (153) and (155), no direct information exists regarding the course of these transformations. The senecioyl residue, as in the C-acylated glabra lactone (157) and the O-acylated samidin (158), appears frequently in natural compounds. C H 3. C

;

0

C H . 0

0

CH CH

:

0

CO

C00

r

C H,

ι C H,

Ο

O C H3

(157)

(158)

Senecioic acid (β,β-dimethylacrylic acid) is of natural occurrence, and its availability as an acylating agent, perhaps as the CoA ester (159), is a plausible supposition: CHw C~-

CH

CO

SCoA

CHY (159)

Senecioyl

CoA

Indeed, the structure of leptospermone (142) invites the supposition that the senecioyl residue (or its dihydro derivative) can serve as a starting point for elaboration by the linear condensation of acetate units:

THE B I O S Y N T H E S I S

OF P H E N O L I C

PLANT

PRODUCTS

787

CHpx

CH :CHCH 2COOH + 3 C 2

Ring closure methylation

In concluding this discussion of carbon-alkylation it must be emphasized that most of what can be said about biosynthetic pathways rests upon structural correlations. In very few instances (e.g. mycophenolic acid (82), where the seven-carbon-atom side chain has been shown to be derived from mevalonic acid, and the C-methyl group from methionine) has the source of the alkylating agents, or the biosynthetic stage at which alkylation occurs, been demonstrated by the use of labeled precursors. It seems very probable that the course of synthesis described in this section represents the essential nature of these processes but direct confirmation is still to be desired.

The Furan Ring Numerous natural substances, of which dictamnine (160) and psoralene (161) are simple examples, contain the unsubstituted furan ring: 0CH3

(160)

(161)

Dictamnine

Psoralene

7 9)

The origin of this ring has been discussed by Aneja et α/.,< who take the very reasonable view that it represents a degradation of a C 5 unit, possibly by way of a dihydroxyisopentyl substituent. The high incidence, especially in the Umbelliferae and Rutaceae, of furano compounds that contain, as C— and Ο— substituents, prenyl or modified prenyl groups, makes this suggestion an attractive one. A notable example of the association of compounds related in this way is found in the occurrence in Flindersia species of such compounds as flindersine (162) and maculosidine (163):

(162)

Flindersine

(163)

Maculosidine

788

T. A.

GEISSMAN

In other cases, both furano rings and iso-C 5 substituents are found in the same molecule; for example, evoxine (164) and oxypeucedanin (165):

(164) Evoxine

(165) Oxypeucedanin

Coumarins The biosynthesis of the coumarin ring system has only recently been investigated experimentally. A n early proposal of Haworth's was that the fundamental reaction in coumarin biosynthesis is the oxidative ring closure (166) of /?-coumaric acid. This hypothesis served to account for the common occurrence in coumarins of the 7-hydroxyl group and for the introduction of the lactonic oxygen a t o m :

OH

OH

7-Hydroxycoumarin

A similar scheme, involving the intermediate formation of a spirolactone, ( 6 9 a) has also been p r o p o s e d . Recent studies have defined the course of the biosynthesis of several natural coumarins, and have shown that a key step in the formation of the ( 8 0 8 9) heterocyclic ring is the öri/jo-hydroxylation of a cinnamic acid. ~ Cinnamic acid, formed in the shikimic acid-phenylalanine pathway, appears to be the common intermediate in the biosynthesis of substituted coumarins. In the biosynthesis of the many 7-hydroxylated coumarins, the first step is the conversion of irajw-cinnamic acid into trans-p-co\\max\c acid. The

T H E B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

789

succeeding steps in the synthesis of umbelliferone in Hydrangea macrophylla can be represented as follows :

.COOH

Umbellic acid

diglucoside

Skimmin (*GL-0

Umbelliferone = /3-D-glucosidoxy)

Coumarin itself is formed from cinnamic acid administered to Melilotus albus. The presence in higher plants of cinnamic acid-c/s-iraws-isomerase is indicated by the efficiency of trans-cmnaxmc and frws-/?-coumaric acids as precursors for natural coumarins, and by the demonstration that a heatlabile extract prepared from M. alba was able to carry out the conversion of 9 0) trans- into c/s-cinnamic acid. < The biosynthesis of furocoumarins appears to follow a similar course. Radioactive feeding experiments using Pimpinella magna led to the conclusion that furocoumarins are formed by way of /7-coumaric acid and not by 9 1) the further elaboration of coumarin itself. < It is interesting to note that both isopimpinellin (167) and khellin (168) are found in umbelliferous plants (although not, so far as has been reported, in the same plant).

790

T.

A.

GEISSMAN

OCH3

OCH^ Ο

(167) Isopimpinellin

(168) Khellin

The similarity between the coumarin (167) and the 2-methylchromone (168) is striking, and suggests that the two heterocyclic rings have a common origin. Extension of the cinnamic acid side-chain, possibly at the stage of the tf/7/70-hydroxylated intermediate (as the glucoside), by the addition of a two-carbon fragment, could take place in one of the following ways:

C O CHoCOOH

O-GL

/COOH

.CO,

O-GL

/COOH CH2

O-GL

α coumarin

.CO.X a

2-methylchromone

ÇH2

O-GL

CO

C H 2C 0 0 H

(169) The addition of a two-carbon fragment (from malonate-CoA) to either — C H = C H C O S C o A or — C O C H 2 C O S C o A can be viewed as unexceptional. It is worth recalling in this connection that the glucoside pungenin (p. 750) has been shown to be formed from such C e — C 3 compounds as caffeic, /?-coumaric, cinnamic, phenyllactic and shikimic acids and phenyl9 2) alanine. < This finding indicates that the α,β-unsaturated acids, the ahydroxy acid and the amino acid can be converted into the ß-keto acid and thence into the acetophenone :

THE

B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

791

A r C H 2C H C O O H - - » A r C H 2C H C O O H — > A r C H - C H C O O H OH

NH2 - - - > A r C H C H 2C O O H — * A r C O C H 2C O O H — > A r C O C H 3 OH (170)

Hydroxylation of Aromatic Rings The conversion of cinnamic acid into p-coumaric acid, a reaction which plays a fundamental role in the biosynthesis of naturally occurring coumarins, appears to be a process common to many biosynthetic sequences in higher plants. The efficiency with which phenylalanine, cinnamic acid and their congeners are utilized in the synthesis of lignins, flavonoid compounds, coumarins, and other substances containing the C e — C 3 unit or products derived from it by chain elongation or degradation, shows clearly that the introduction of hydroxyl groups into the aromatic nucleus is a general biosynthetic process. The hydroxylation of other aromatic compounds has been observed as well. Stem tissue of Avena sativa converts phenoxyacetic acid into 4( 9 3) hydroxyphenoxyacetic a c i d . Salicylic acid can be formed in higher plants either by ör/Ao-hydroxylation

of benzoic acid (in Helianthus anuus, Solanum tuberosum, and Pisum sativum, 9 4)

but not in wheat coleoptiles), < or of cinnamic acid (and subsequent side96 9 7) chain degradation) in Gaultheria procumbens^* > and in other plants. < The phenylalanine-cinnamic acid-/?-coumaric acid route, followed by sidechain degradation, leads to the formation of /?-hydroxybenzoic acid in

Catalpa ovata and C. bignonoides.

(98)

Many cases of what is surely hydroxyl group introduction into aromatic nuclei are found in the flavonoid compounds, in which 5 and 8-hydroxylation in the Α-ring and 2'-hydroxylation in the B-ring are probably the result of the late introduction of oxygen into the fundamental 5,7-dihydroxy Α-ring and the 4'-hydroxy B-ring. Although the removal of hydroxyl (or methoxyl) groups from aromatic rings is not a common or general process in phenolic biosynthesis, it has been observed in a number of cases. Sinapic acid is converted into coniferyl 99 1 0 0 lignin as well as into sinapyl lignin, < > > and Hordeum, Triticum and 97 Oryza can demethoxylate sinapic acid to form ferulic acid. < > (see also Chapter 15). There is no indication that flavonoid compounds possessing a resorcinolderived Α-ring (butein, formononetin, etc.) are formed by way of the corresponding phloroglucinol-derived compounds by loss of the 5- (or corresponding) hydroxyl group. Indeed, the loss of oxygen where there are

792

T. A . G E I S S M A N

hydroxyl groups "missing" from the normal acetate-derived aromatic rings is probably to be accounted for by the following sequence: —COCH2CO—

—>

—C H — C H 2 C O

—>

— CH=CHCO—

at the polyketomethylene level of elaboration (see p p . 760-761).

Oxidative Metabolism in Biosynthesis The elaboration of complex compounds by the oxidative coupling of simpler molecules has been proposed by many investigators to account for the biosynthesis of compounds of a number of different classes. The origin of such compounds as hypericin (5) and the perylene quinone (40b) found in Daldinia concentrica from the corresponding anthracene and naphthalene compounds is readily explicable. Simple oxidative changes that lead to compounds in which the carbon skeletons of their progenitors are immediately apparent are exemplified by chebulic acid (172), derived from ellagic acid 101 (171); and the bisflavones, such as sotetsuflavone (173);< >

OH

0

(173) Sotetsuflavone

A widely occurring class of natural phenols whose origins and interrelationships are readily inferred on structural grounds are the lignans. (See also Chapter 15.) Lignans are C 1 8 compounds that may be regarded as C 6 — C 3 — C 3 — C 6 compounds in which the bond between the two phenylpropanederived residues is at the point shown in (174):

THE

B I O S Y N T H E S I S OF P H E N O L I C P L A N T P R O D U C T S

793

Q^c-L-l-c-Q (174)

These are known in a variety of modifications, including diarylbutanes such as guaiaretic acid (175), tetrahydrofurans such as olivil (176), and a-phenyltetralins such as conidendrin (177):

V

C H 3

H 0

~W^

CH3 CH3

C H r

/°

^ ~^ ^3" H

C H

C H

>

'°\

0 H

C H

HO-/~VÇH ÇH 0 N

= /

^ ^

CH-CH I

H0CH 2 (175) G u a i a r e t i c acid

(176)

(177)

'

I

CH 2OH Olivil

Conidendrin

A general theory of the biosynthesis of the lignans is that first outlined 102 103 by Erdtman, < > and reinforced by experimental findings of Freudenberg. ( > The essential features of the hypothetical scheme of the biosynthesis of the lignans can be represented in the following somewhat simplified form. It can be seen that the process of condensation depends for its initiation upon an oxidation of a 4-propenylphenol, of which a natural prototype may be coniferyl alcohol (178). In the equations below a generalized /7-hydroxystyrene (179) is used as the example, and a two-electron oxidation is chosen ( 1 0 2) for convenience in representation. ( E r d t m a n discusses these reactions in terms of a one-electron oxidation.)

(178)

Coniferyl a l c o h o l

(179)

(180)

^-Hydroxystyrene

derivative

794

T. A.

GEISSMAN

The following examples illustrate the processes that can lead to representative lignan structures: (a) tf

0

0

0

CH

CH

Y

CH

J CH

T/

CH-W

CH-

I

I

R

R

7

A -£ χ > Η

1

CH

CH R

R (181)

OH

(b)

(181)

+

OH

i

CH

Λ HO-

Τ/ Ύ

CH I I CH

CH I

CH I

CH I

-CH

1

R (182)

(c)

or (d) if (181) already contains the o-dihydroxy grouping: XH — R

I

CH —

R

HO"

Y X

CH

OH

( 181 a)

1

I

R

^CH

ί

0

R

HO..

I

^1

H 20 -

I! CH

(d)

OH

j

(184)

CH -

R

/ C H - R

\

T H E B I O S Y N T H E S I S OF P H E N O L I C P L A N T

PRODUCTS

795

Freudenberg discovered that if coniferyl alcohol (178) is oxidized in the presence of an enzyme preparation there are formed, in addition to a polymer, "synthetic lignin", a number of intermediate oxidation products. These are (185-188): C H C H 20 H HO^

HOCHp—CH

I

CH —

/0 CH CH2 I I CH — CH I I

CH3O"

0CH3

0

C

^2

/CH

.OCH,

CHCHO CH — C H C H 20 H

OCH,

The derivation of these by way of an initial oxidation of the type ( 179 -> 180) is readily understood; for example: 0CH3

C H = C H C H 20 H

(185)

(179a)

The attack upon (179a) by the anionic oxygen of (178) (rather than by the ring) leads to (187); and (186) is derived as described earlier for (182), with the modification that the furan ring closure occurs by attack of the terminal — C H 2 O H groups. A continuation of processes of oxidation and condensation such as these outlined above can lead to complex, polymeric molecules. Freudenberg

796

T.

A.

GEISSMAN

takes the view that lignin owes its formation to such a course, a view that is supported by the observations that (a) coniferyl alcohol can be enzymatically oxidized to a polymer that is indistinguishable from lignin, (b) coniferyl 14 alcohol—C is incorporated into lignin in spruce saplings, (c) the number of hydroxyl groups in lignin is less than one per C 9 unit, and (d) the production of as much as 25 per cent of vanillin by the oxidation of spruce wood indicates that the 3-methoxy-4-hydroxyphenyl residue makes up a large part of the structure of the wood. It must be recognized, however, that it is difficult to prove the identity of natural and synthetic lignins, and that in nature the exact point at which O-methylation occurs need not necessarily be at the level of coniferyl alcohol (or coniferin). The evidence that coniferyl alcohol is indeed the real precursor is however, substantial, and no alternative hypothesis has been put forward with the support of equally cogent evidence. The ultimate origin of lignin from the shikimic acid pathway seems clear from the evidence: all that is still in need of further clarification is the nature 2 6 1 0 4) of the steps at and beyond the C 6 — C 3 level of elaboration. ( » SUMMARY

In the foregoing pages is found a summary of the results of experimental findings, structural correlations, and mechanistic speculations as they have led to the development of present-day views concerning the origins of phenolic substances in higher plants. The chief processes are, basically, few in number: (a) The synthesis, from carbohydrate and via shikimic acid, of a C 6 — C 3 (a 1-phenylpropane derivative) unit; (b) the joining of C 2 (acetate) units into linear chains and the cyclization of these, by aldolization, into carbocyclic rings. Other kinds of ring closures, leading to oxygen heterocyclic rings, also occur: (c) the lengthening of the C 6 — C 3 unit by addition of C 2-units, or shortening by degradation to C 6 — C 2 and C 6 — Q structures; (d) the modification of the basic structures derived as in (a), (b) and (c), by oxidation or reduction, or by the attachment, by C-alkylation or C-acylation, of side chains; (e) the incursion into the shikimic- or acetate-derived synthetic pathways of terpenoid fragments derived from ß-methylcrotonyl residues produced through the mevalonic acid pathway. For most of the types of synthetic processes, direct substantiation of the route of synthesis has been obtained by experiments involving labeled precursors introduced into living organisms (higher plants or microorganisms), but most of the enormous number of individual substances have not been studied in detail. Existing views as to the biosynthesis of most of the known plant phenolics depend upon the structural kinship to those few whose biosynthesis has been established by experiment. Nevertheless, the remarkable degree of structural coincidence that exists within this large and diverse group of compounds makes it very likely that the fundamental synthetic processes are relatively few in number. The least that can be said is that to

THE BIOSYNTHESIS

OF P H E N O L I C

PLANT

PRODUCTS

797

undertake the study of the biogenesis of most of the plant phenolics would be to start with a working hypothesis that would in most cases turn out to be correct. Concerning the many details of structural alteration, such as the oxidation of methyl groups, the reduction of carbonyl and carboxyl groups, the introduction a n d removal of hydroxyl groups, the cleavage of rings a n d the formation of new ones, there is little but speculation. The time has come t o penetrate below the level of the intact plant and to seek the enzymatic systems that bring about the sequential stages in synthesis. Until this is accomplished our knowledge of the processes of biosynthesis will remain confined to knowledge of the origin of the carbon atoms of the skeletons of the molecules.

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