Enzymology of Gallotannin Biosynthesis - American Chemical Society

It was proposed that glucose and gallic acid initially combined to give /?-glucogallin .... Figure 4. Chemical synthesis of galloyl coenzyme A thioest...
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Chapter 7

Enzymology of Gallotannin Biosynthesis Georg G. Gross Universität Ulm, Abteilung Allgemeine Botanik, Oberer Eselsberg, D-7900 Ulm, Federal Republic of Germany

Ample knowledge available to date of the chemistry and natural distribution of a wide variety of galloylglucose esters has led to the view that both gallotannins and the structurally related ellagitannins are derived from one common precursor, 1,2,3,4,6-O-pentagalloyl-β-D­ -glucose. Recent investigations with enzymes from oak or sumach leaves have shown that the biosynthesis of this polyphenolic ester is initiated by the formation of βglucogallin (1-O-galloyl-β-D-glucose) from UDP-glucose and free gallic acid. This monoester, in turn, was found to serve as the activated acyl-donor in a series of sub­ sequent transacylation steps, yielding specifically substi­ tuted di-, tri-, tetra-, and finally pentagalloylated glu­ cose derivatives. In contrast, the biosynthesis of gallic acid itself is still largely obscure, and this applies also to the final steps of the entire biogenetic sequence, i.e., the mechanisms involved in the formation of meta-depside linkages and hexahydroxydiphenoyl residues characteriz­ ing gallotannins and ellagitannins, respectively. Vegetable t a n n i n s are c o m m o n l y classified into condensed t a n n i n s (or p r o a n t h o c y a n i d i n s ) , w h i c h are o f flavonoid o r i g i n , a n d h y d r o l y z a b l e t a n n i n s , w h i c h are characterized by a c e n t r a l p o l y o l moiety ( m o s t l y /?-D-glucose) whose h y d r o x y 1 groups are esterified w i t h gallic a c i d . These p o l y p h e n o ­ lic c o m p o u n d s c a n be further s u b s t i t u t e d w i t h a d d i t i o n a l g a l l o y l residues a t t a c h e d v i a meta-depside linkages, g i v i n g rise t o the f o r m a t i o n o f the g a l ­ l o t a n n i n s proper ( F i g u r e 1). A l t e r n a t i v e l y , d i m e r i z a t i o n o f adjacent g a l l o y l groups c a n o c c u r , y i e l d i n g t h e h e x a h y d r o x y d i p h e n y l residues c h a r a c t e r i s ­ tic o f the related e l l a g i t a n n i n s ( F i g u r e 2 ) ; after h y d r o l y t i c release, these b i p h e n y l derivatives rearrange spontaneously t o give the stable n a m e - g i v i n g d i l a c t o n e , ellagic a c i d . 0097-6156/89/0399-0108$06.00/0 © 1989 American Chemical Society

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Enzymobgy of GaUotannin Biosynthesis

OH

OH

HO

OH

F i g u r e 1. S t r u c t u r e of a g a l l o t a n n i n t y p i c a l of Chinese g a l l o t a n n i n , f o u n d , e.g., i n galls of Rhus semialata. N o t e the c h a r a c t e r i s t i c m e t a - d e p s i d e l i n k ages of the g a l l o y l residues at C - 2 .

OH

OH F i g u r e 2. S t r u c t u r e of an e l l a g i t a n n i n , l - 0 - g a l l o y l - 2 , 3 : 4 , 6 - d i - 0 - h e x a h y droxydiphenoyl-/?-D-glucose (casuarictin).

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PLANT C E L L W A L L P O L Y M E R S

I n n u m e r a b l e v a r i a t i o n s of these f u n d a m e n t a l s t r u c t u r a l p r i n c i p l e s have been detected i n higher plants; discussion of w h i c h , however, lies b e y o n d the scope of t h i s article [for recent relevant reviews see, e.g., (1-7)]. T h i s well d o c u m e n t e d detailed knowledge of the c h e m i c a l c o n f i g u r a t i o n a n d n a t u r a l d i s t r i b u t i o n of h y d r o l y z a b l e t a n n i n s s t i m u l a t e d t h o u g h t s o n their b i o s y n t h e sis (cf. 5-9). It was proposed t h a t glucose a n d g a l l i c a c i d i n i t i a l l y c o m b i n e d to give /?-glucogallin ( l - 0 - g a l l o y l - / ? - D - g l u c o s e ) w h i c h , i n t u r n , u n d e r w e n t a series of further a c y l a t i o n reactions l e a d i n g to pentagalloyl-/?-D-glucose. T h i s l a t t e r ester was t h o u g h t to f u n c t i o n as the c o m m o n precursor b o t h of the g a l l o t a n n i n s a n d the related e l l a g i t a n n i n s . T h i s p l a u s i b l e p a t h w a y was u n t i l a few years ago t o t a l l y u n p r o v e n . It now comprises several i m p o r t a n t aspects, n a m e l y (1) the f o r m a t i o n of gallic a c i d , (2) the n a t u r e of the e n e r g y - r i c h intermediates required for the synthesis of / ? - g l u c o g a l l i n , (3) the t r a n s a c y l a t i o n m e c h a n i s m s l e a d i n g to pentagalloylglucose, a n d (4) the secondary t r a n s f o r m a t i o n s involved i n the synthesis of g a l l o t a n n i n s a n d ellagitannins. A s r e p o r t e d below, recent e n z y m a t i c studies have p r o v i d e d insight i n t o several of these p r o b l e m s ; i n p a r t i c u l a r , m a n y of the questions c o n c e r n i n g the f o r m a t i o n of /?-glucogallin a n d its subsequent conversion to p e n t a g a l loylglucose have been answered b y this technique. O t h e r aspects o u t l i n e d above r e m a i n rather obscure, b u t s t a r t i n g p o i n t s for t h e i r eventual c l a r i f i c a t i o n w i l l be discussed. Biosynthesis of Gallic A c i d In spite of numerous investigations d u r i n g the past decades, the b i o s y n thesis of gallic a c i d r e m a i n e d one of the m a j o r enigmas of p l a n t p h e n o lics m e t a b o l i s m . B a s e d o n feeding experiments w i t h p u t a t i v e precursors, three different p a t h w a y s , as depicted i n F i g u r e 3, were proposed [references i n ( 2 , 1 0 , 1 1 ) ] . E v i d e n c e was presented t h a t gallic a c i d was p r o d u c e d b y direct a r o m a t i z a t i o n of d e h y d r o s h i k i m i c a c i d (or s h i k i m i c a c i d ) . A l t e r n a tively, two routes v i a the p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y were p o s t u l a t e d a l o n g w h i c h gallic a c i d was f o r m e d , either b y /^-oxidation of 3 , 4 , 5 t r i h y d r o x y c i n n a m i c a c i d (never detected i n plants) or b y the sequence caffeic —» protocatechuic —• gallic a c i d . L a t e r , two short reports ( 1 2 , 1 3 ) o n w o r k w i t h cell-free systems were p u b l i s h e d suggesting a sequence d e h y d r o s h i k i m i c —• protocatechuic —• gallic a c i d ; u n f o r t u n a t e l y , these observations s t i l l await c o n f i r m a t i o n b y more detailed studies. T h e o r e t i c a l l y , m a n y of the above discrepancies c o u l d be s e t t l e d b y e x p e r i m e n t s w i t h c a r b o x y l - l a b e l e d s h i k i m i c a c i d because this f u n c t i o n a l group w o u l d be lost i n the f o r m a t i o n of p h e n y l a l a n i n e , b u t r e t a i n e d i n the case of a direct conversion to gallic a c i d . O n l y a m b i g u o u s evidence was o b t a i n e d , however, f r o m such efforts (10), a n d i t was concluded t h a t at least two p a t h w a y s for gallic acid biosynthesis must exist (14), w i t h the preferential route d e p e n d i n g o n leaf age a n d p l a n t species investigated (15,16). A different a p p r o a c h to d i s c r i m i n a t e between the two p r i n c i p a l routes to gallic a c i d , i.e., v i a C e C a - i n t e r m e d i a t e s vs. a direct conversion was de-

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Enzymofogyof Gallotannin Biosynthesis

v e l o p e d . T h i s uses g l y p h o s a t e [ N - ( p h o s p h o n o m e t h y l ) g l y c i n e , a n i n h i b i t o r of s h i k i m i c a c i d u t i l i z a t i o n ] a n d L - 2 - a m i n o o x y - 3 - p h e n y l p r o p i o n i c a c i d ( L A O P P , a n i n h i b i t o r of p h e n y l a l a n i n e d e a m i n a t i o n ) i n feeding e x p e r i m e n t s w i t h labeled s h i k i m i c a c i d . W h i l e A O P P h a d no effect, g l y p h o s a t e g r e a t l y enhanced the a m o u n t a n d r a d i o a c t i v i t y of the i s o l a t e d g a l l i c a c i d (17), i n ­ d i c a t i n g t h a t the direct conversion of s h i k i m i c to g a l l i c a c i d represented at least a significant, i f not the m a j o r , route. Biosynthesis of /?-Glucogallin T h e n a t u r a l l y o c c u r r i n g depside /?-glucogallin (l-0-galloyl-/?-D-glucose) was considered the p r i m a r y m e t a b o l i t e i n the biosynthesis of h y d r o l y z a ble t a n n i n s ( 5 , 7 , 8 ) . For t h e r m o d y n a m i c reasons, the p a r t i c i p a t i o n of a n a c t i v a t e d i n t e r m e d i a t e has to be p o s t u l a t e d i n the f o r m a t i o n o f such a n ester. T h i s requirement can be met i n two ways, either b y r e a c t i o n of a n e n e r g y - r i c h g a l l o y l derivative w i t h free glucose, or b y u s i n g a nucleoside d i p h o s p h a t e - a c t i v a t e d glucose (most likely U D P G ) a n d the free a c i d . T h e results of o u r recent work o n these aspects are presented below. Galloyl-Coenzyme A. N u m e r o u s e n z y m a t i c studies o n the f o r m a t i o n o f the u b i q u i t o u s p l a n t phenolic chlorogenic a c i d ( 3 - O - c a f f e o y l - D - q u i n i c acid) a n d related depsides have showed t h a t c i n n a m o y l - C o A thioesters were u t i l i z e d as a c t i v a t e d intermediates i n these esterification reactions ( T a b l e I). B y analogy, i t appeared conceivable t h a t g a l l o y l - C o A m i g h t be i n v o l v e d i n the biosynthesis of g a l l o t a n n i n s . T o test t h i s hypothesis, t h i s t h e n u n k n o w n thioester was synthesized (26), v i a the ΛΓ-succinimidyl d e r i v a t i v e of 4 - 0 - / ? D - g l u c o s i d o g a l l i c a c i d (cf. F i g u r e 4), a n d characterized b y s p e c t r o p h o t o ­ m e t r i c m e t h o d s ; the U V - s p e c t r u m was perhaps the most p r o m i n e n t p r o p ­ e r t y ( F i g u r e 5). T a b l e I. P h e n o l i c A c i d Esters F o r m e d v i a I n t e r m e d i a t e A c y l - C o e n z y m e A Esters Donor

Acceptor

Product

Ref.

Caffeoyl-CoA

Quinate

Chlorogenate

18,20

p-Coumaroyl-CoA

Quinate

3-p-Coumaroylquinate

19,21

p-Coumaroyl-CoA

Shikimate

3-p-Coumaroylshikimate

22

p-Coumaroyl-CoA

Tartronate

p-Coumaroyltartronate

23

Caffeoyl-CoA

Isocitrate

Caffeoylisocitrate

24

HydroxycinnamoylCoAs

S u g a r acids (glucuronate, glucarate, galactarate)

Monoesters; exact p o s i t i o n of the O-ester group unclear

25

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PLANT C E L L W A L L P O L Y M E R S

COOH

COOH

F i g u r e 3. P r o p o s e d b i o s y n t h e t i c p a t h w a y s t o gallic a c i d (5). (1) D e h y d r o s h i k i m i c a c i d ; (2) caffeic a c i d ; (3) 3 , 4 , 5 - t r i h y d r o x y c i n n a m i c a c i d ; (4) protocatechuic acid.

F i g u r e 4. C h e m i c a l synthesis of g a l l o y l c o e n z y m e A thioester (4). (1) 4 - 0 /?-D-glucosidogallic a c i d ; (2) ΛΓ-succinimidyl 4-0-/? - D - g l u c o s i d o g a l l a t e ; (3) 4-0-/?-D-glucosidogalloyl-CoA.

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113

In e n z y m a t i c studies w i t h cell-free extracts f r o m higher p l a n t s , h o w ­ ever, no evidence has been f o u n d to date for g a l l o y l - C o A ' s i n v o l v e m e n t i n the biosynthesis of / ? - g l u c o g a l l i n , or i t s higher g a l l o y l a t e d d e r i v a t i v e s ; a n e v e n t u a l role i n the f o r m a t i o n of the c h e m i c a l l y different meta-depside b o n d s of g a l l o t a n n i n s has not been i n v e s t i g a t e d . Formation of β-Glucogallin. A b o u t the t i m e as the above m e n t i o n e d studies w i t h g a l l o y l - C o A were c a r r i e d o u t , i t b e c a m e evident t h a t glucose esters of p h e n o l i c acids c o u l d be f o r m e d b y a n a l t e r n a t e m e c h a n i s m , i.e., b y r e a c t i o n of the free a c i d w i t h U D P - g l u c o s e s e r v i n g as the e n e r g y - r i c h c o m p o n e n t . T h i s was because of a n u m b e r of findings (27), where i t was d e m o n s t r a t e d t h a t the c o n j u g a t i o n of glucose w i t h numerous benzoic a n d c i n n a m i c acids (28-33), a n d indole-3-acetic a c i d ( 3 4 , 3 5 ) , o c c u r r e d a c c o r d i n g to the general m e c h a n i s m s h o w n below ( E q u a t i o n 1): A c i d + U D P glucose — l - 0 - a c y l - / ? - D - g l u c o s e + U D P

(1)

It therefore appeared t h a t a general m e c h a n i s m for e n z y m a t i c e s t e r i fication of phenolic acids w i t h glucose was o p e r a t i v e , whereas the r e a c t i o n w i t h other alcoholic moieties proceeded v i a c a r b o x y l - a c t i v a t e d a c y l d e r i v a ­ tives. [In t h i s context i t s h o u l d be e m p h a s i z e d t h a t glucose esters must not be confused w i t h glucosides; different enzymes are i n v o l v e d i n the b i o s y n ­ thesis of these two types of phenolic glucose derivatives (36)]. It was t h u s not s u r p r i s i n g to establish t h a t /?-glucogallin was also s y n ­ thesized a c c o r d i n g to t h i s m e c h a n i s m ( F i g u r e 6) b y e n z y m e p r e p a r a t i o n s f r o m t a n n i n - p r o d u c i n g oak leaves ( 3 7 , 3 8 ) . S u b s t r a t e specificity studies re­ vealed t h a t t h i s g l u c o s y l transferase depended e x c l u s i v e l y o n U D P - g l u c o s e as d o n o r s u b s t r a t e , a n d t h a t i t e x h i b i t e d a c t i v i t y t o w a r d a great v a r i e t y of benzoic a n d c i n n a m i c acids as acceptor molecules. C o n s i d e r i n g the grow­ i n g i m p o r t a n c e of such esters w i t h i n the area of general p l a n t secondary m e t a b o l i s m , t h i s l a t t e r p r o p e r t y has recently been u t i l i z e d for the conve­ nient p r e p a r a t i o n a n d s p e c t r o s c o p i c c h a r a c t e r i z a t i o n ( U V , I R , H - N M R ) of differently s u b s t i t u t e d l - 0 - b e n z o y l - / ? - D - g l u c o s e s (39), t h u s p r o v i d i n g a convenient a l t e r n a t i v e to c h e m i c a l syntheses [cf., e.g., (40)]. 1

/î-Glucogallin to

Pentagalloylglucose

A c c o r d i n g to recent suggestions, /?-glucogallin s h o u l d undergo a series of position-specific g a l l o y l a t i o n steps to y i e l d pentagalloylglucose (5-9). E l u c i d a t i o n o f t h i s h i t h e r t o u n k n o w n biogenetic sequence was established w i t h e n z y m e p r e p a r a t i o n s f r o m y o u n g oak leaves (41); i t was t h u s d e m o n s t r a t e d t h a t , by analogy w i t h the preceding synthesis of / ? - g l u c o g a l l i n , g a l l o y l - C o A was not required. Instead, b o t h d i - a n d trigalloylglucose were f o r m e d i n the presence o f / 3 - g l u c o g a l l i n as the sole s u b s t r a t e , p r o v i n g t h a t t h i s ester served also as a n a c y l donor (41). S u p p o r t i n g evidence f r o m several l a b o r a t o r i e s also i n d i c a t e d t h a t phenolic a c i d esters were not necessarily m e t a b o l i c a l l y i n e r t c o m p o u n d s b u t c o u l d be e m p l o y e d as a c t i v a t e d i n t e r m e d i a t e s for seco n d a r y t r a n s a c y l a t i o n reactions. A s s u m m a r i z e d i n T a b l e I I , t h i s new view was c o r r o b o r a t e d b y m a n y detailed enzyme studies.

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250

355

350

WAVENLENGTH (nm) F i g u r e 5. U V s p e c t r u m o f g a l l o y l c o e n z y m e A . ( ) Spectrum of the thioester; ( ) s p e c t r u m after h y d r o l y s i s i n 0 . 1 N N a O H or h y d r o x y l a m i n o l y s i s i n 1 M N H 0 H , p H 6. B o t h s p e c t r a were recorded i n 0 . 1 M p o t a s s i u m phosphate buffer, p H 7.0. 2

1

3 0 F i g u r e 6. E n z y m a t i c synthesis o f /?-glucogallin.

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Enzymology of Gallotannin Biosynthesis

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T a b l e II. B i o s y n t h e s i s o f A r o m a t i c M o n o a c y l Esters b y from Higher Plants

Acyltransferases

Donor

Acceptor

Product

Ref.

1-O-Sinapoylglucose

L-Malate

Sinapoyl-Lmalate

42,43

1-O-Sinapoylglucose

Choline

Sinapoylcholine (Sinapine)

44-46

1-O-Indolylacetylglucose

myo-Inositol

Indolylacetylmyo-inositol

35

1-O-Caffeoylglucose

D-Quinate

Chlorogenate

47,48

1-O-p-Coumaroylglucose

D-Quinate

p-Coumaroylquinate

49

1-O-p-Coumaroylglucose

meso-Tartarate

p-Coumaroylmeso-tartarate

50

Chlorogenate

Glucarate

Caffeoylglucarate

51

W i t h respect to g a l l o t a n n i n s , s u b s t a n t i a l evidence i n d i c a t e d t h a t βg l u c o g a l l i n was also m e t a b o l i c a l l y a c t i v e , as the a c y l donor for g a l l o y l t r a n s ferase reactions; some details related to t h i s aspect are described below. Galloyl-Exchange between β-Glucogallin and Glucose. Cell-free e x t r a c t s f r o m oak leaves p r o d u c e d labeled /^-glucogallin w h e n i n c u b a t e d w i t h u n ­ labeled g l u c o g a l l i n a n d [ C ] g l u c o s e (41). T h i s u n e x p e c t e d result was ex­ p l a i n e d b y the existence of an acyltransferase c a t a l y z i n g the exchange re­ a c t i o n s h o w n below: 14

/ 2 - G l u c o g a l l i n + * glucose ^

*/?-glucogallin + (*)glucose

(2)

where the asterisk indicates an a p p r o p r i a t e l a b e l (e.g., C ) t o a l l o w m e a ­ surement of the r e a c t i o n . Studies w i t h the p u r i f i e d e n z y m e showed t h a t it was active w i t h various 1-O-benzoylglucose esters as d o n o r s u b s t r a t e s , w h i l e D-glucose f u n c t i o n e d very specifically as the acceptor molecule. T h e p h y s i o l o g i c a l significance of t h i s reaction is s t i l l u n k n o w n ; however, i t was successfully e m p l o y e d for the facile a n d rather e c o n o m i c p r e p a r a t i o n of l a ­ b e l e d g l u c o g a l l i n a n d related esters, t h u s a v o i d i n g m a n y p r o b l e m s i n v o l v e d i n the c h e m i c a l synthesis of such c o m p o u n d s (52,53). 1 4

Biosynthesis of Digalloylglucose. Besides the above m e n t i o n e d a c y l t r a n s ­ ferase, oak leaves also c o n t a i n e d a completely different t y p e of a c y l t r a n s ­ ferase t h a t c a t a l y z e d the f o r m a t i o n of digalloylglucose (41). It b e c a m e e v i ­ dent t h a t t h i s ester was synthesized by a new reaction m e c h a n i s m i n w h i c h /^-glucogallin was u t i l i z e d as b o t h a c y l d o n o r a n d acceptor; t h i s c o n c l u ­ s i o n was s u p p o r t e d b y the i s o l a t i o n of analogous acyltransferases r e l a t e d to other m e t a b o l i c p a t h w a y s (cf. T a b l e III). Recent studies (54) have s h o w n , i n accordance w i t h previous proposals ( 5 , 7 , 8 ) , t h a t 1,6-O-digalloylglucose was p r o d u c e d by the e n z y m e , a n d t h a t the s t o i c h i o m e t r y of the r e a c t i o n

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was f u l l y consistent w i t h the assumed m e c h a n i s m (cf. F i g u r e 7). S u b s t r a t e specificity studies w i t h numerous 1-O-benzoylglucoses revealed t h a t the e n ­ z y m e was most active w i t h its n a t u r a l s u b s t r a t e , /?-glucogallin ( G r o s s , G . G . ; Denzel, K . ; Schilling, G . , unpublished data). T a b l e I I I . B i o s y n t h e s i s of M u l t i p l e - s u b s t i t u t e d P h e n o l i c A c i d E s t e r s b y Acyltransferases from Higher Plants Donor

Acceptor

Product

Ref.

1 - O - G alloy lglucose (/?-glucogallin)

1-O-Galloyl­ glucose

1,6-0-Digalloylglucose

54

1 - O - G alloy lglucose

1,6-O-Digalloylglucose

1,2,6-O-Trigalloylglucose

55

1-O-Galloylglucose

1,2,3,6-0Tetragalloylglucose

1,2,3,4,6-0Pentagalloylglucose

a

1-O-Sinapoylglucose

1-O-Sinapoyl­ glucose

1,2-0-Disinapoylglucose

56-58

Chlorogenate

Chlorogenate

3,5-Dicaffeoylquinate (Isochlorogenate)

59,60

a

C a m m a n , J . ; Denzel, K . ; Schilling, G . ; Gross, G . G . , unpublished data.

Higher Galloylated Glucose Derivatives. A s s u m m a r i z e d i n T a b l e I I I , the f o r m a t i o n of higher g a l l o y l a t e d derivatives occurs by the same sort of m e c h ­ a n i s m as above, i.e., b y the transfer of the g a l l o y l moiety of /?-glucogallin to the acceptor s u b s t r a t e . T h u s , the biosynthesis of 1 , 2 , 6 - t r i g a l l o y l g l u c o s e f r o m the 1 , 6 - d i s u b s t i t u t e d precursor was proven u n e q u i v o c a l l y w i t h a n e n ­ z y m e f r o m s u m a c h leaves (55), a n d recently a n enzyme f r o m oak leaves t h a t c a t a l y z e d the g a l l o y l a t i o n of 1 , 2 , 3 , 6 - t e t r a g a l l o y l g l u c o s e to 1 , 2 , 3 , 4 , 6 peηtagalloylglucose was characterized ( C a m m a n n , J . ; D e n z e l , K . ; S c h i l l i n g , G . ; G r o s s , G . G . , u n p u b l i s h e d d a t a ) . O n l y the conversion of t r i - to t e t r a galloylglucose has not yet been verified b y detailed e n z y m e studies, due to insufficient a m o u n t s of available s u b s t r a t e . T h e expected ester, 1 , 2 , 3 , 6 tetragalloylglucose, has, however, been isolated as a b y - p r o d u c t f r o m scaledu p enzyme assays designed for the p r e p a r a t i o n of 1,6-digalloylglucose [cf. (54)]. T h u s , the entire sequence of reactions f r o m /?-glucogallin to p e n t a g a l ­ loylglucose is s u m m a r i z e d i n F i g u r e 7. P r e l i m i n a r y evidence suggests t h a t the i n d i v i d u a l steps are c a t a l y z e d b y different enzymes, b u t t h i s question has to be answered by future detailed investigations. C o n c e r n i n g the s u b ­ s t i t u t i o n p a t t e r n of the m e t a b o l i t e s , the scheme presented here is i n accord w i t h recent results o b t a i n e d b y a n a l y z i n g the galloylglucoses p r o d u c e d i n callus cultures f r o m oak (61).

F i g u r e 7. B i o s y n t h e t i c p a t h w a y f r o m /^-glucogallin to pentagalloylglucose. T h e g a l l o y l residue i n t r o d u c e d i n each i n d i v i d u a l step is m a r k e d by an asterisk; as i n d i c a t e d by the dashed a r r o w , the e n z y m e c a t a l y z i n g the step f r o m t r i - to tetragalloylglucose has not yet been i s o l a t e d . / ? G , / ^ - G l u c o g a l l i n ; G l c , glucose.

OH

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Biosynthesis of Gallotannins a n d Ellagitannins A s m e n t i o n e d , recent biogenetic schemes suggest 1 , 2 , 3 , 4 , 6 - p e n t a g a l l o y l glucose to be the p r i n c i p a l c o m m o n precursor o f g a l l o t a n n i n s a n d e l l a g i ­ t a n n i n s . However, there are m a n y c o m p o u n d s o f these two classes o f n a t u ­ r a l p r o d u c t s t h a t c o n t a i n c o n t a i n one, or occasionally more t h a n one, free O H - g r o u p o n the glucose moiety, p a r t i c u l a r l y at the C - l p o s i t i o n . N o ev­ idence is c u r r e n t l y available as to whether such t a n n i n s are derived f r o m p a r t i a l l y g a l l o y l a t e d precursors o f pentagalloylglucose, or f r o m the result o f secondary d e a c y l a t i o n reactions. T h e l a t t e r c o u l d be caused b y a very a c t i v e esterase encountered i n cell-free extracts f r o m oak or s u m a c h leaves ( G r o s s , G . G . ; D e n z e l , K . , u n p u b l i s h e d results). L i t t l e knowledge also exists o n the m e c h a n i s m s involved i n the b i o s y n ­ thesis of the characteristic s t r u c t u r e s of g a l l o t a n n i n s a n d e l l a g i t a n n i n s , as w i l l be briefly discussed below. Gallotannins. V i r t u a l l y n o t h i n g is k n o w n about the f o r m a t i o n of the c h a r ­ acteristic meta-depside b o n d of these c o m p o u n d s . F o r t h e r m o d y n a m i c r e a ­ sons one has to p o s t u l a t e a n a c t i v a t e d g a l l o y l derivative i n t h i s r e a c t i o n . T h e o n l y s p e c u l a t i o n s possible at present are whether ^ - g l u c o g a l l i n serves as a n a c y l donor, or w h e t h e r , due to the m a r k e d l y differing n a t u r e o f the phenolic O H - g r o u p , other intermediates w i t h a m u c h higher group-transfer p o t e n t i a l (e.g. g a l l o y l - C o A ) are r e q u i r e d . Ellagitannins. M o r e t h a n 50 years ago i t was p o s t u l a t e d (62) t h a t the c h a r ­ acteristic h e x a h y d r o x y d i p h e n o y l residues of e l l a g i t a n n i n s o r i g i n a t e d f r o m the d e h y d r a t i o n of g a l l i c a c i d esters, e.g., depsidic t a n n i n s , a n d t h i s w i d e l y accepted view has been s u p p o r t e d b y recent feeding e x p e r i m e n t s (16). S u c h o x i d a t i o n reactions [reviewed, e.g., i n (63)] were c a r r i e d out by c h e m i c a l means [e.g. (64)], or by in vitro studies w i t h the f u n g a l e n z y m e laccase (65,66) or w i t h peroxidases f r o m higher plants ( 6 4 , 6 7 , 6 8 ) , u s i n g gallic a c i d , m e t h y l gallate, /7-glucogallin, 3,6-digalloyglucose a n d pentagalloylglucose as substrates. In a l l cases, ellagic a c i d was formed as a t y p i c a l p r o d u c t , i n ­ d i c a t i n g the expected i n t e r m e d i a t e synthesis o f h e x a h y d r o x y d i p h e n i c a c i d . U n f o r t u n a t e l y , t h i s l a t t e r c o m p o u n d has never been isolated i n its esterified f o r m i n these e x p e r i m e n t s , a n d t h u s i t remains questionable whether these e n z y m e systems (i.e., laccase or some other p o l y p h e n o l oxidase + O 2 , or peroxidase + H2O2) really reflect n a t u r a l c o n d i t i o n s . These doubts are s u p ­ p o r t e d by recent studies w i t h cell-free e x t r a c t s f r o m oak leaves ( H o f m a n n , Α.; G r o s s , G . G . , u n p u b l i s h e d results). In the presence of s u i t a b l e electron acceptors, the s u b s t r a t e , 1,2, 3 , 4 , 6 - p e n t a g a l l o y l g l u c o s e , was converted to a not yet f u l l y characterized new p r o d u c t ( a n d e v e n t u a l l y 1 , 4 , 6 - t r i - O - g a l loy 1-2,3-O-hexahydroxydiphenoylglucose), together w i t h m i n o r q u a n t i t i e s of a c o m p o u n d t h a t c o c h r o m a t o g r a p h e d w i t h p e d u n c u l a g i n ( 2 , 3 : 4 , 6 - d i O - h e x a h y d r o x y d i p h e n o y l g l u c o s e ) . These p r e l i m i n a r y results i n d i c a t e t h a t rather specific oxidoreductases, a n d not the abovementioned very unspecific oxidases, are i n v o l v e d i n the biosynthesis of e l l a g i t a n n i n s .

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