Ascorbic Acid - American Chemical Society

5 Dehydroascorbic A c i d BERT M. TOLBERT and JONI B. WARD

Downloaded by UNIV OF GUELPH LIBRARY on May 18, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch005

Department of Chemistry, University of Colorado, Boulder, CO 80309

Dehydroascorbic acid (DHA) is the first stable oxidation product of L-ascorbic acid (AA). D H A can be easily and quantitatively prepared by air oxidation of A A over char coal in ethanol. D H A is stable for days in aqueous solution of p H 2-4. H N M R and 13C N M R studies show that the principle species of D H A is the bicyclic hydrate, 3,6-anhydro-L-xylo-hexalono-l,4-lactone hydrate. This finding is confirmed by synthesis and spectral studies of related com pounds. D H A contains equilibrium concentrations of vari ous dehydrated and open side-chain forms, but these species are too small to detect using N M R spectroscopy. The D H A dimer is converted to the monomer when it is dissolved in water. The chemistry of D H A is reviewed, in cluding the hydrolysis to diketogulonic acid and the reac tions of the 2- and 3-oxo groups. D H A readily forms Schiff bases and undergoes a Strecker reaction with amino acids. The known enzymatic reactions of D H A are reviewed. 1

Thefirstchemically stable product in the oxidation of L-ascorbic acid (AA) is L-dehydroascorbic acid ( D H A ) . It is normally prepared from A A using a variety of oxidizing agents such as the halogens (1-5), oxygen (6), quinones (7), and potassium iodate (8). D H A is present in biological tissue and is a part of the A A / D H A oxidizing/reducing couple. In addition, D H A or A A / D H A ratios may be related to cell division and, therefore, may have a critical role in growth regulation. The oxidized form of A A was first detected when Zilva (9) noticed that freshly oxidized solutions of AA retained their nutritional or physio logical activity. At the same period Szent-Gyorgyi also recognized that the oxidized form of AA could be regenerated to AA (10). Further inves tigations of these discoveries led to conclusion that AA could be reversibly oxidized to D H A (11) without loss of nutritional activity (12). On the basis of the structure of AA, the structure of D H A was postulated as a 2,3-diketolactone with possibly one or more of the keto groups hydrated (2). 0065-2393/82/0200-0101$06.75/0 © 1982 American Chemical Society In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

102

ASCORBIC ACID

D u r i n g t h e last 20 years a b e t t e r u n d e r s t a n d i n g of t h e s t r u c t u r e a n d c h e m i c a l n a t u r e of D H A a n d t h e free r a d i c a l i n t e r m e d i a t e t h a t m a y b e f o r m e d d u r i n g the o x i d a t i o n of A A has d e v e l o p e d . ments w e r e b a s e d N M R and

1 3

These

develop

on modern instrumental techniques including * H

C N M R spectroscopies a n d p u l s e d r a d i a t i o n e l e c t r o n s p i n

resonance ( E S R ) spectroscopy.

T h e c h e m i s t r y a n d p r o p e r t i e s of m o n o -

d e h y d r o a s c o r b i c a c i d ( A A " ) , a free r a d i c a l i n t e r m e d i a t e t h a t m a y f o r m e d i n t h e o x i d a t i o n of A A , is c o v e r e d elsewhere i n this v o l u m e . chapter

concerns

D H A , its

reactions,

structure, a n d

be

This

physiological


chemistry. T h e fact t h a t D H A possesses v i t a m i n C a c t i v i t y w a s

recognized

e a r l y i n studies of A A a n d w a s s t u d i e d b y s e v e r a l g r o u p s

(13,14).

E n z y m e s t h a t c a t a l y z e t h e r e d u c t i o n of D H A to A A h a v e b e e n d e m o n strated i n m a n y systems a n d are d i s c u s s e d later i n this c h a p t e r . D i m e r i c D H A , or b i s d e h y d r o a s c o r b i c a c i d ( B D H A ) , a n d D H A b o t h are i m p o r tant i n n u t r i t i o n ; this i m p o r t a n c e has b e e n t a k e n for g r a n t e d i n t h a t the common

assay of a s c o r b i c a c i d , the d i n i t r o p h e n y l h y d r a z i n e ( D N P H )

m e t h o d , does not d i s t i n g u i s h b e t w e e n these forms. T h e n o m e n c l a t u r e of the o x i d i z e d forms of A A is b a d l y i n n e e d of r e v i s i o n . N o t o n l y is d e h y d r o a s c o r b i c a c i d a l o n g a n d c u m b e r s o m e n a m e , b u t it is also c o n f u s i n g i n i n f e r r i n g t h a t the c o m p o u n d is a n a c i d . A s is d i s c u s s e d l a t e r i n this c h a p t e r , the p r i n c i p l e s t r u c t u r e is a b i c y c l i c c o m p o u n d c o n t a i n i n g b o t h l a c t o n e a n d h e m i k e t a l groups.

Names such

as a s c o r b i t o n e or d e h y d r o a s c o r b i t o n e w o u l d be better t r i v i a l r e p r e s e n tations.

Preparation of DHA and BDHA D H A has b e e n p r e p a r e d f r o m A A u s i n g a great v a r i e t y of o x i d i z i n g agents a n d c o n d i t i o n s . T h e o x i d i z e r s i n c l u d e the h a l o g e n s , C l , B r , a n d 2

I

2

(1-5);

the q u i n o n e s ( 7 ) ; i o d a t e ( 8 ) ; a n d o x y g e n ( 6 ) as w e l l as other

reagents.

S i n c e A A is a g o o d r e d u c i n g agent t h a t r e a d i l y reacts w i t h

2

o n e - e l e c t r o n or t w o - e l e c t r o n o x i d i z i n g agents, the p r o b l e m i n the p r e p a r a t i o n of D H A is to find reagents that d o not o v e r o x i d i z e A A a n d t h a t g i v e r e a c t i o n p r o d u c t s that are easily s e p a r a t e d f r o m D H A . D H A is o n l y stable u n d e r c e r t a i n c o n d i t i o n s i n w a t e r s o l u t i o n a n d is also easily o x i dized.

I n g e n e r a l , e q u i v a l e n t a m o u n t s of A A a n d t h e halogens d o not

g i v e q u a n t i t a t i v e y i e l d s of D H A because of p a r t i a l o v e r o x i d i z a t i o n , a n d a c o m p l i c a t e d p u r i f i c a t i o n p r o c e d u r e is r e q u i r e d to g i v e p u r e D H A ( J ) . A l s o , the p u r i f i c a t i o n p r o c e d u r e s often l e a d to m o r e d e c o m p o s i t i o n t h a n purification. O n e of t h e m o r e extensive reports o n t h e p r e p a r a t i o n of D H A is b y Pecherer ( I ) oxidation.

w h o w o r k e d out a large-scale p r e p a r a t i o n u s i n g i o d i n e

N e i t h e r i n this s t u d y , n o r i n a n y other has D H A b e e n

ob

t a i n e d i n c r y s t a l l i n e f o r m , a l t h o u g h there does not seem to b e a n y g o o d

In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

5.

T O L B E R T A N D WARD

103

Dehydroascorbic Acid

reason t o b e l i e v e i t i m p o s s i b l e .

D H A is u s u a l l y o b t a i n e d as a t h i c k

s y r u p o r as a n a m o r p h o u s o r m i c r o c r y s t a l l i n e s o l i d b y solvent p r e c i p i t a t i o n or l y o p h i l i z a t i o n . B e c a u s e D H A is v e r y easy to p r e p a r e f r o m A A , i t is best p r e p a r e d as n e e d e d u s i n g t h e f o l l o w i n g m e t h o d . A p a r t i c u l a r l y u s e f u l p r e p a r a t i o n of D H A , d e s c r i b e d b y O h m o r i a n d T a k a g i , uses o x y g e n o x i d a t i o n o v e r a c h a r c o a l c a t a l y s t ( 6 ) . T h e u s e of o x y g e n a n d c h a r c o a l t o c o n v e r t A A t o D H A is a w e l l - k n o w n r e a c t i o n t h a t has b e e n u s e d i n A A assays f o r m a n y years. T h e o x i d a t i o n c a n b e m a d e i n e t h a n o l , m e t h a n o l , w a t e r , o r v a r i o u s m i x t u r e s of these solvents.

We


c a r r y o u t this p r o c e d u r e as f o l l o w s : T e n grams of a s c o r b i c a c i d is d i s s o l v e d i n 300 m L of solvent, a n d 15 g of a c t i v a t e d c h a r c o a l is a d d e d .

O x y g e n o r a i r is b u b b l e d t h r o u g h

the s o l u t i o n at a flow rate of 20 m L / m i n f o r 3 0 - 6 0 m i n w h i l e t h e s o l u t i o n is g e n t l y s t i r r e d w i t h a m a g n e t i c stir b a r . A t t h e c o m p l e t i o n o f t h e r e a c t i o n t h e s o l u t i o n is filtered, first t h r o u g h a W h a t m a n # 2 filter p a p e r a n d t h e n b y s u c t i o n t h r o u g h a fine glass

filter.

T h e solvent is r e m o v e d

b y a r o t a r y e v a p o r a t o r w i t h a b a t h t e m p e r a t u r e of 3 0 ° C .

T h e resulting

s y r u p is p u r e D H A w i t h traces of t h e o r g a n i c solvent u s e d i n t h e p r e p a r a t i o n . A d d i t i o n of a s m a l l a m o u n t of w a t e r a n d r e p e a t e d l y o p h i l i z a t i o n w i l l r e m o v e t h e traces of o r g a n i c solvent. B e c a u s e t h e i n i t i a l r o t a r y e v a p o r a t i o n is faster w i t h t h e o r g a n i c solvent, w e h a v e u s u a l l y p r e p a r e d D H A i n 9 5 % e t h a n o l . I n m e t h a n o l the r e a c t i o n gives u p to 1 0 - 2 0 % of a m e t h a n o l c o m p l e x of D H A t h a t is o n l y p a r t l y r e c o n v e r t e d t o free D H A o n r e p e a t e d evaporations water.

from

E x t e n s i v e r o t a r y e v a p o r a t i o n w i t h r e p e a t e d a d d i t i o n s of d i e t h y l

ether, f o l l o w e d b y l y o p h i l i z a t i o n , y i e l d s a m o r e m a n a g e a b l e , s e m i s o l i d product.

D H A i n t h e s y r u p or s e m i s o l i d f o r m is stable f o r m a n y w e e k s

w h e n s t o r e d at — 1 0 ° t o — 2 0 ° C . A n a l y s i s of t h e p r o d u c t s p r e p a r e d as d e s c r i b e d a b o v e w a s done b y

1 3

C N M R , o n e of t h e f e w a n a l y t i c a l

t e c h n i q u e s t h a t gives u n a m b i g u o u s results o n t h e p u r i t y a n d i d e n t i t y of this c o m p o u n d . C r y s t a l l i n e B D H A c a n b e p r e p a r e d f r o m D H A b y t h e m e t h o d of D i e t z ( 1 5 ) : 10 g of D H A s y r u p , p r e p a r e d b y t h e m e t h o d

described

a b o v e , is d i s s o l v e d i n 30 m L of n i t r o m e t h a n e . A f t e r t h e s y r u p h a s d i s s o l v e d , 100 m L m o r e of i c e - c o l d n i t r o m e t h a n e is a d d e d . T h e s o l u t i o n is h e a t e d t o b o i l i n g . A w h i t e p r e c i p i t a t e of B D H A is f o r m e d a n d c a n b e filtered

off a n d d r i e d . H v o s l e f has p r e p a r e d m a c r o c r y s t a l l i n e B D H A f r o m

this m a t e r i a l f o r x - r a y c r y s t a l l o g r a p h i c studies

(16).

T h e d i m e r is stable i n s o l i d d r y f o r m . S e v e r a l c o m m e r c i a l firms s e l l " d e h y d r o a s c o r b i c a c i d " t h a t m a y or m a y n o t b e i d e n t i f i e d as B D H A .

We

h a v e a n a l y z e d several o l d c o m m e r c i a l a n d p r i v a t e l y p r e p a r e d samples of B D H A u s i n g

1 3

C N M R , a n d h a v e f o u n d l a r g e a m o u n t s of d e c o m p o s i

t i o n p r o d u c t s i n a l l of t h e m . H o w e v e r t h e p u r i t y of t h e o r i g i n a l p r o d u c t a n d storage c o n d i t i o n s w e r e n o t k n o w n .


104

ASCORBIC A d D

Chemical

Reactions of DHA

T h e e n e - d i o l system of A A a n d its d i k e t o o x i d a t i o n p r o d u c t a r e w e l l - k n o w n structures i n o r g a n i c c h e m i s t r y .

T h e lactone group i n A A

is q u i t e stable i n a c i d o r a l k a l i n e s o l u t i o n ; i n contrast, t h e lactone g r o u p i n D H A is r a p i d l y h y d r o l y z e d i n a l k a l i n e o r a c i d s o l u t i o n a n d is stable only i n a l i m i t e d p H range a r o u n d 2 - 4 . A s a n effective ketone, D H A reacts r e a d i l y w i t h h y d r a z i n e s t o f o r m a v a r i e t y of 2- a n d 2,3-hydrazones. Downloaded by UNIV OF GUELPH LIBRARY on May 18, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch005

the 2 , 3 - b i s k e t o b u t y r o l a c t o n e

T h e s e reactions a r e c h a r a c t e r i s t i c of

group.

T h e s t a n d a r d assay of A A takes

a d v a n t a g e of t h e r e a c t i o n b e t w e e n D N P H a n d D H A t o g i v e a b i s h y d r a z o n e t h a t e x h i b i t s a d i s t i n c t i v e a b s o r p t i o n b a n d at 516 n m i n d i l u t e s u l f u r i c a c i d s o l u t i o n ( 1 7 ) . T h i s r e a c t i o n is n o t q u a n t i t a t i v e ; a m i x t u r e of the h y d r a z o n e a n d d e c o m p o s i t i o n

p r o d u c t s is f o r m e d .

W e have esti

m a t e d t h a t t h e d e r i v a t i v e is f o r m e d i n a b o u t 3 5 % y i e l d . T h e success of the D N P H r e a c t i o n as a q u a n t i t a t i v e assay f o r A A is d e p e n d e n t o n t h e use of a d e q u a t e

controls, r e p r e s e n t i n g a significant weakness

of t h e

method. The

bishydrazones

of D H A a n d r e l a t e d c o m p o u n d s

s t u d i e d a n d u s e d t o synthesize a n u m b e r DHA

(18-23).

have

been

of n i t r o g e n d e r i v a t i v e s of

T h u s t h e b i s p h e n y l h y d r a z o n e of D H A is r e d u c e d b y

h y d r o g e n / p l a t i n u m to 2,3-diamino-2,3-dideoxyascorbic

acid, which i n

t u r n c a n b e c o n v e r t e d t o a v a r i e t y of a c y l d e r i v a t i v e s . T h e s t r u c t u r e of D H A p h e n y l o s a z o n e is a hexenonelactone

(24).

U n d e r p r o p e r c o n d i t i o n s D H A reacts w i t h a m i n e s t o f o r m Schiff bases.

Dahn and Moll

describe

this r e a c t i o n b e t w e e n

o-phenylene-

d i a m i n e a n d D H A as w e l l as w i t h other 2,3-diketobutyrolactones

(25).

W i t h a l i p h a t i c a m i n e s a n d a m i n o a c i d s , t h e Schiff base is n o t f a v o r e d i n aqueous

solution.

T h e extent of t h e r e v e r s i b l e f o r m a t i o n of Schiff

bases of D H A h a s n o t b e e n extensively s t u d i e d , a n d c l e a r l y needs m o r e a t t e n t i o n . D H A i n b i o l o g i c a l fluids is p r o b a b l y i n r e v e r s i b l e e q u i l i b r i u m w i t h a m i n o groups of a m i n o a c i d s , p r o t e i n s , a n d o t h e r a m i n e s .

How

ever, t h e extent of a n y s u c h c o n j u g a t i o n is u n k n o w n . I f s u c h bases a r e f o r m e d , t h e y p r o b a b l y i n v o l v e d e r i v a t i z a t i o n of t h e 2 - p o s i t i o n of D H A . T h e browning reaction between

carbohydrates

a n d amino

acids

has a t t r a c t e d a t t e n t i o n f o r m a n y years. I n t h e presence of a m i n o a c i d s , D H A undergoes a b r o w n i n g r e a c t i o n t h a t w a s d e s c r i b e d i n 1956 ( 26) a n d l a t e r extensively s t u d i e d (27-^33).

A d i s t i n c t i v e e a r l y p r o d u c t of

this r e a c t i o n is t h e f o r m a t i o n of a r e d c h r o m o p h o r e , A x 515 n m , ( I , 3 1 ) , m a

believed to be t h e product of a Strecker reaction between the a m i n o a c i d a n d D H A . T h i s r e a c t i o n p r o d u c t seems t o b e q u i t e specific f o r D H A .


5.

105



T h e r e a c t i o n itself is analogous to the r e a c t i o n of n i n h y d r i n w i t h a m i n o acids:


H

CHOHCH OH

+ C0 K u r a t a et a l . (32)

2

2

+

RCHO

h a v e i s o l a t e d this c o m p o u n d a n d o b t a i n e d a H N M R 1

s p e c t r u m w i t h shifts as f o l l o w s : 3.74 p p m ( d o u b l e t ) , 4.19 p p m ( t r i p l e t ) , a n d 4.88 p p m ( d o u b l e t ) .

W e h a v e also e x a m i n e d a l o w p u r i t y s a m p l e

of this r e d c h r o m o p h o r e a n d o b t a i n e d a s i m i l a r * H N M R s p e c t r u m as w e l l as a

1 3

C N M R spectrum.

(33)

T h e results o b t a i n e d s h o w t h a t t h e

c h r o m o p h o r e has a n o p e n s i d e - c h a i n a n d does n o t exist i n a h e m i k e t a l f o r m analogous to t h a t o b s e r v e d i n D H A a n d B D H A . T h i s c o l o r r e a c t i o n has n o t b e e n u s e d to a n y extent i n t h e assay of DHA.

W e h a v e u s e d i t to i d e n t i f y D H A i n t h i n - l a y e r c h r o m a t o g r a p h y

( T L C ) w i t h excellent results. T h e p l a t e is s p r a y e d w i t h 1 M

glycine

a n d h e a t e d i n a n o v e n at 9 0 - 1 0 0 ° C for 4r-5 m i n . A d i s t i n c t i v e p i n k spot d e v e l o p s , a n d s l o w l y turns b r o w n i n 1-2 d . V a r i o u s q u a l i t a t i v e studies w e r e d o n e to i m p r o v e t h e s e n s i t i v i t y of this assay f o r D H A . d i d not give the chromophore. different a m i n o acids. methanol

solution

There was

little difference

Amines between

H e a t i n g D H A a n d g l y c i n e i n either w a t e r

gives

the

chromophore.

Although a

better

or

yield

m a y b e o b t a i n e d i n w a t e r , p r o b l e m s result i n w a t e r d u e t o t h e i n s t a b i l i t y of the c h r o m o p h o r e i n this solvent. B D H A also gives this r e a c t i o n i n s o l u t i o n , p r e s u m a b l y because i t d e c o m p o s e s to D H A u n d e r t h e c o n d i tions of t h e r e a c t i o n .

T h e y i e l d of the c h r o m o p h o r e

is l o w , a n d t h u s

o n l y m o d e r a t e sensitivities for D H A assays w e r e a c h i e v e d .

If the y i e l d

i n this reaction c o u l d be substantially i m p r o v e d b y appropriate choices of solvent a n d r e a c t i o n c o n d i t i o n s , t h e r e a c t i o n has t h e p o t e n t i a l f o r a g o o d assay p r o c e d u r e .

T h e r e a c t i o n is specific a n d t h e p r o d u c t

d i s t i n c t i v e a n d easy to q u a n t i t a t e b y

spectroscopy.


quite

106

ASCORBIC ACID

A s s o c i a t e d w i t h t h e b r o w n i n g r e a c t i o n a r e a n u m b e r o f f a i r l y stable free r a d i c a l c o m p o u n d s i n w h i c h t h e u n p a i r e d e l e c t r o n is often associated w i t h a nitrogen atom.

A b l u e substance t h a t d i s p l a y s a n E S R t r i p l e t

s p e c t r u m c a n b e i s o l a t e d b y T L C ; a r e d c h r o m o p h o r e c a n also b e i s o l a t e d M a n y of t h e r a d i c a l c o m p o u n d s

(34-39).

red chromophore phore

either m a y be related to t h e

o r a r e i n t e r m e d i a t e s i n t h e synthesis o f t h e c h r o m o

(34-39).

M a n y decomposition

p r o d u c t s of D H A are p r o b a b l y t h e same as

those o b s e r v e d i n t h e d e c o m p o s i t i o n of A A (40).

Fifteen products from


D H A d e c o m p o s i t i o n i n aqueous s o l u t i o n w e r e r e p o r t e d (41). fifteen,

O f these

the five m a i n v o l a t i l e p r o d u c t s w e r e 3 - h y d r o x y - 2 - p y r o n e , 2 - f u r a n -

c a r b o x y l i c a c i d , 2 - f u r a l d e h y d e , acetic a c i d , a n d 2 - a c e t y l f u r a n . D H A also u n d e r g o e s a b e n z i l i c a c i d r e a r r a n g e m e n t i n a l k a l i n e s o l u t i o n (42). T h e r e d u c t i o n of D H A t o A A is a c c o m p l i s h e d

b y a v a r i e t y of

reagents. H y d r o g e n sulfide, cysteine, a n d other t h i o l s w i l l r e d u c e D H A . H y d r o g e n sulfide is f r e q u e n t l y u s e d since t h e excess reagent c a n b e p u r g e d from the reaction solution a n d sulfur, the oxidized product, c a n be removed b y

filtration.

B e c a u s e t h e r e a c t i o n w i t h h y d r o g e n sulfide is

d i s a g r e e a b l e t o use because of t h e t o x i c a n d o d o r o u s h y d r o g e n sulfide, this r e d u c t i o n has b e e n u s e d most often i n d i f f e r e n t i a l assay p r o c e d u r e s f o r D H A . S o d i u m d i t h i o n a t e r a p i d l y a n d q u a l i t a t i v e l y reduces D H A t o AA.

S o d i u m borohydride a n d l i t h i u m a l u m i n u m h y d r i d e give

complex

m i x t u r e s of p r o d u c t s w i t h D H A . Structural

Studies of DHA and

BDHA

T h e c r y s t a l l i n e d i m e r of D H A w a s a n a l y z e d b y x - r a y c r y s t a l l o g r a p h y (17), a n d t h e s t r u c t u r e p r o p o s e d b y e a r l i e r c h e m i c a l studies w a s c o n firmed

(43).

H

OH


5.

107



T h e d i m e r is n o t r e a d i l y s o l u b l e i n w a t e r , a l t h o u g h D H A is v e r y s o l u b l e i n water. BDHA

T h u s some questions h a v e a r i s e n as t o t h e exact n a t u r e of

i n water.

T h e d i m e r disassociates t o a m o n o m e r

f o r m a m i d e , d i m e t h y l a c e t a m i d e , a n d p y r i d i n e (44) e a r l i e r studies (45-47).

i n dimethyl

i n agreement

with

A series of studies of A A a n d B D H A h a v e n o w

c l a r i f i e d m a n y aspects of this p r o b l e m .

1 3

C N M R studies of A A w e r e

p u b l i s h e d (48-51) t h a t s h o w t h e s t r u c t u r e of A A i n s o l u t i o n is essentially as p r o p o s e d b y c l a s s i c a l c a r b o h y d r a t e c h e m i s t r y a n d is t h e same as t h e structure f o u n d i n crystalline ascorbic


studies.

Recent

1 3

a c i d b y x-ray crystallographic

C N M R studies of B D H A

i n dimethyl sulfoxide-d

6

( D M S O - d ) s h o w t h a t i n this solvent B D H A is a m i x t u r e of t w o f o r m s , 6

a s y m m e t r i c a n d a n a s y m m e t r i c d i m e r ( 5 2 ) . T h e a s y m m e t r i c f o r m is thermodynamically favored. T h e best a p p r o a c h to t h e s t r u c t u r e of m o n o m e r i c D H A w a s t h r o u g h *H N M R and

1 3

C N M R studies. O n t h e basis of * H N M R , D H A w a s

p r o p o s e d t o exist i n aqueous s o l u t i o n as a b i c y c l i c h y d r a t e d species, t h a t is, 3,6-anhydro-L-xt/Zo-hexulono-l,4-lactone h y d r a t e (53).

W e have made

f u r t h e r studies o n this s t r u c t u r e u s i n g D H A p r e p a r e d b y o x y g e n o x i d a t i o n i n e t h a n o l , o r m e t h a n o l o r w a t e r u s i n g c h a r c o a l as a catalyst. T h e m e t h o d is d e s c r i b e d e a r l i e r i n this c h a p t e r . 1 3

1 3

C N M R Studies.

D H A was dissolved i n deuterium oxide a n d the

C N M R s p e c t r u m w a s o b t a i n e d f r o m a J O E L P F T - 1 0 0 spectrometer

u s i n g a n e x t e r n a l d i o x a n e reference. tained i n D M S O - d Results o n and B D H A Pederson's

1 3

6

T h e s p e c t r u m of B D H A w a s o b

i n s t e a d of d e u t e r i u m o x i d e .

C N M R shifts i n parts p e r m i l l i o n f r o m M e S i f o r D H A 4

are presented i n T a b l e I. results (52)

F o r comparison, Hvoslef a n d

are g i v e n , i n c l u d i n g assignments of shifts to

specific s y m m e t r i c a n d a n t i s y m m e t r i c structures f o r B D H A .

Hvoslefs

results a r e r e a d i l y r e p r o d u c e d u s i n g t h e m a t e r i a l p r e p a r e d b y t h e O h m o r i oxidation and the D i e t z dimerization procedure.

T h e spectra f r o m t h e

t w o laboratories s h o w a consistent difference i n shifts, p r o b a b l y a r i s i n g f r o m differences i n reference standards. T h e difference i n shifts of C 4 a n d C 5 is c a u s e d b y a difference i n assignment of these shifts, a n d is discussed later. The and

1 3

C N M R shifts f o r D H A , p r e s e n t e d i n T a b l e I , w e r e s t u d i e d

carbon

decoupling

assignments experiments

made

b y t w o methods:

a n d b y comparison

of

1 3

by

proton-carbon

C N M R s p e c t r a of

various derivatives. * H N M R Decoupling Experiments.

D H A was dissolved i n deu

t e r i u m oxide a n d the spectra were recorded

from a Nicolet NT-360

spectrometer w i t h a n internal s o d i u m 2,2-dimethyl-2-silapentane-5-sulfonate ( D S S ) s t a n d a r d . T h e shifts f o r t h e p r o t o n s of D H A w e r e

deter

m i n e d f r o m t h e * H N M R s p e c t r u m ( F i g u r e 1 ) : C 4 , s i n g l e t a t 4.76; C 5 ,


108

ASCORBIC ACID

T a b l e I. Sample

I 3

C N M R D a t a for D H A and

Identification

D H A (52) D H A p r e p a r e d i n absolute methanol" D H A prepared i n 9 5 % ethanol* D H A prepared i n H 0 * B D H A s y m m e t r i c (52) B D H A a n t i s y m m e t r i c (52)" 2


h

B D H A symmetric' B D H A antisymmetric*

Cl

C2

BDHA

CS

C4

C5

C6

174.2

92.0

106.3

73.5

88.6

76.2

173.6 173.3 173.5 169.1 168.1 168.7 169.3 168.4 168.8

91.4 91.1 91.3 91.6 99.1 104.2 91.9 99.8 104.0

105.7 105.7 105.6 105.6 103.4 113.9 105.9 103.7 114.1

87.6 87.3 87.4 73.0 73.2 73.4 89.6 88.4 89.4

73.0 72.6 72.8 90.3 88.3 89.1 73.3 73.3 73.8

76.2 76.2 76.1 76.3 74.5 76.7 75.6 74.7 76.6

• Solvent, D2O; reference, external dioxane. * Solvent, DMSO-cfo; reference, internal Me4Si. 'Solvent, DMSO-cfo; reference, external dioxane.


5.


109


singlet a t 4.6; C 6 , m u l t i p l e t a t 4.2. T h e i n t e r p r e t a t i o n of t h e * H N M R s p e c t r u m as a n A B C D p a t t e r n suggests t h a t t h e protons o n C 6 a r e differ ent f r o m o n e another; these protons s p l i t o n e another. T h e f a c t t h a t these p r o t o n s a p p e a r as different p e a k s i n t h e * H N M R suggests t h a t t h e r e is n o t free r o t a t i o n of C 6 . I n t h e p r o t o n - p r o t o n d e c o u p l i n g e x p e r i m e n t s ( F i g u r e 2 ) , t h e s p l i t t i n g o f t h e p r o t o n o n C 4 b y t h e p r o t o n o n C 5 is v e r y s m a l l . T h i s o b s e r v a t i o n suggests t h a t t h e o r b i t a l s f o r these p r o t o n s are s e p a r a t e d b y a n a n g l e of a p p r o x i m a t e l y 9 0 ° , a n d therefore

cannot

i n t e r a c t w i t h e a c h other to cause s p l i t t i n g . Downloaded by UNIV OF GUELPH LIBRARY on May 18, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch005

Proton-Carbon Decoupling Experiments.

H a v i n g obtained correct

v a l u e s f o r t h e p r o t o n shifts, a series of p r o t o n - c a r b o n d e c o u p l i n g e x p e r i ments

were

performed

(Figures

3-5).

T h e experiments

i n v o l v e d c o m b i n i n g t w o types of d e c o u p l i n g : off-resonance frequency.

I n off-resonance

d e c o u p l i n g experiments

X

performed a n d single-

H i r r a d i a t i o n is

k e p t at h i g h p o w e r levels. T h e center of f r e q u e n c y is m o v e d 500-1000 H z a w a y from the protons to be irradiated, a n d the excitation b a n d w i d t h generator is s w i t c h e d off. C a r b o n s h a v i n g zero, o n e , t w o , o r t h r e e

1—•— —•—|— —'—'—i—'— — —i—'—«— —|— —'— —I— 5.0 4.8 4.6 4.4 4.2 4.0 P P M 1

Figure 2.

1

1

1

1

1

1

r

H NMR spectrum of DHA. The proton on C5 has been decoupled.

1


110

ASCORBIC ACID


C-4 C-6 C-5

C-1' EtOH C-2' EtOH C-3 C-2

C-1

-i

1

1

i

T"

r —

150

0

50

100

i

PPM

Figure 3. C NMR spectrum of DHA dissolved in D 0. The spectrum was recorded on a Nicolet NT-360 spectrometer with an internal DSS reference. 13

2

protons b o n d e d n o w a p p e a r as singlets, d o u b l e t s , t r i p l e t s , a n d q u a r t e t s , respectively.

C o u p l i n g i n f o r m a t i o n is therefore r e t a i n e d w i t h o u t m u c h

loss of s e n s i t i v i t y

(54).

T h e c a r b o n s of interest i n D H A are C 4 , C 5 , a n d C 6 . C a r b o n s - 1 , - 2 , a n d -3 a p p e a r as singlets. C a r b o n s - 4 a n d -5 a p p e a r as d o u b l e t s , a n d C 6 a p p e a r s as a t r i p l e t .

Because

C 4 a n d C 5 b o t h a p p e a r as

doublets,

a s s i g n i n g c h e m i c a l shifts to these c a r b o n s w i t h o u t f u r t h e r i n f o r m a t i o n w o u l d b e difficult. I t w a s i m p o r t a n t , therefore, t o p e r f o r m a series of single-frequency

proton-carbon

t a i n i n g t h e off-resonance

decoupling

experiments, w h i l e

main

decoupling.

S i n g l e - f r e q u e n c y p r o t o n d e c o u p l i n g , also k n o w n as selective coupling, depends

u p o n p r o p e r a s s i g n m e n t a n d i d e n t i f i c a t i o n of

de the

p r o t o n resonances f o r a g i v e n m o l e c u l e . O n c e the p r o t o n resonances are i d e n t i f i e d i n a n * H N M R s p e c t r u m , i t is p o s s i b l e to i r r a d i a t e specific


5.

111



p r o t o n s a t l o w r a d i o f r e q u e n c y p o w e r . T h e r e s u l t as seen i n t h e

1 3

CN M R

s p e c t r u m is t h e c o l l a p s e to a s i n g l e t of t h e m u l t i p l e t associated w i t h t h e carbon attached to that proton. some C - H c o u p l i n g

T h e o t h e r p r o t o n a t e d carbons

I n these e x p e r i m e n t s , e a c h p r o t o n resonance the resulting

1 3

retain

(54). was irradiated, a n d

C N M R s p e c t r u m w a s o b s e r v e d t o see w h i c h m u l t i p l e t

c o l l a p s e d t o a singlet. P r o p e r a s s i g n m e n t of t h e c h e m i c a l shifts c a n b e made for C 4 , C 5 , a n d C 6 (Figures 4 a n d 5 ) . T h e assignment

of p r o t o n

c h e m i c a l shifts is also s u p p o r t e d

by


c a r b o n - 1 3 c h e m i c a l shifts o b t a i n e d f o r t h e 5 , 6 - i s o p r o p y l i d e n e d e r i v a t i v e s of D H A , D-erythro-DHA

a n d 6-bromo-6-deoxy-L-DHA. A l l the dehydro

compounds were prepared b y oxidation using oxygen over charcoal i n 9 5 % ethanol. T h e preparation of the isopropylidene derivatives follows. L-5,6-0-Isopropylidene

AA.

I n a 5 - L r e a c t i o n flask e q u i p p e d w i t h

a n efficient p a d d l e stirrer a n d a w a t e r - c o o l e d c o n d e n s e r w i t h a d r y i n g t u b e , a n d h e a t e d b y a steam b a t h , w a s p l a c e d 250 g o f L - A A (1.4 m o l ) ,

i— —'— —•—r 1

100 Figure 4.

1

90

— —r~ 1

80

n— — ~ 1

r

70 P P M

C NMR spectrum of DHA obtained when the proton on C4 was selectively decoupled.

13



112

ASCORBIC

— i — > -

100 Figure 5.

C

13

i

i

I

i

T 90

i

ACID

i

80

70

PPM

NMR spectrum of DHA obtained when the proton on CS was selectively decoupled.

437 g of 2 , 2 - d i m e t h o x y p r o p a n e

(4.2 m o l ) , 880 m L of p - d i o x a n e ,

and

6 m L of t r i f l u o r o a c e t i c a c i d ( T F A ) . T h e r e a c t i o n is s t i r r e d a n d h e a t e d to reflux. cipitate.

A f t e r 15 m i n , t h e w h i t e v o l u m i n o u s p r o d u c t b e g i n s to H e a t i n g a n d s t i r r i n g are c o n t i n u e d f o r

pre

a n o t h e r 45 m i n , at

w h i c h t i m e t h e r e a c t i o n has s o l i d i f i e d i n t o a c o p i o u s w h i t e mass.

The

r e a c t i o n m i x t u r e is c o o l e d , s l u r r i e d i n s e v e r a l liters of p e t r o l e u m ether, and

filtered.

T h e p r o d u c t is d r i e d i n a v a c u u m desiccator.

The product

is o b t a i n e d as a w h i t e s o l i d i n 9 5 - 1 0 0 % y i e l d . D-5,6-0-Isopropylidene IsoAA.

T h e p r o p o r t i o n of reactants a n d the

r e a c t i o n c o n d i t i o n s are t h e same for p r e p a r i n g this i s o m e r as f o r p a r i n g A A . H o w e v e r , t h e r e a c t i o n takes 2 - 3 h to c o m p l e t e .

pre

T h e r>iso-

p r o p y l i d e n e i s o A A does n o t p r e c i p i t a t e f r o m the r e a c t i o n m i x t u r e , e v e n o n c o o l i n g to r o o m t e m p e r a t u r e . evaporation

(water

T h e solvents are r e m o v e d b y r o t a r y

aspiration, bath temperature

of

35°C)«

and

the

r e s u l t i n g r e d - b r o w n o i l is p o u r e d i n t o f o u r t i m e s its v o l u m e of efficiently s t i r r e d p e t r o l e u m ether to p r e c i p i t a t e the p r o d u c t as a t a n p o w d e r .


The

5.

powder must be

filtered

113



a n d q u i c k l y d r i e d under v a c u u m to prevent

reconversion into a n o i l . T h e D-isopropylidene i s o A A c a n be obtained as w h i t e flakes b y p r e c i p i t a t i n g w i t h p e t r o l e u m ether f r o m a c h l o r o f o r m acetone s o l u t i o n . Discussion NMR

of Structural Studies.

Collected

data from

the

1 3

C

spectra are shown i n T a b l e I I . L - D H A a n d D - i s o D H A have similar

c h e m i c a l shifts, s u g g e s t i n g

a similar structure.

They

both

show a n

a s s i g n e d shift f o r C 6 t h a t is f u r t h e r d o w n f i e l d t h a n f o r t h e C 6 of t h e respective s t a r t i n g m a t e r i a l s . O n e w o u l d expect t h e C 6 t o b e s h i f t e d


d o w n f i e l d i f t h e c o m p o u n d is i n t h e h e m i k e t a l f o r m . T h i s fact, w i t h t h e i n f o r m a t i o n f r o m t h e p r e c e d i n g p r o t o n e x p e r i m e n t s , supports a p r o p o s e d h e m i k e t a l structure. T h e a s s i g n e d shift f o r C 2 of L - D H A a n d D - i s o D H A is f u r t h e r u p f i e l d than w o u l d be expected i f C 2 were a keto group, indicating hydration at this c a r b o n . For

L - i s o p r o p y l i d e n e - D H A , D-isopropylidene i s o D H A , a n d 6-bromo-

6 - d e o x y - L - D H A , t h e a s s i g n e d shifts f o r C 2 a n d C 3 a r e u p f i e l d values e x p e c t e d f o r k e t o g r o u p s .

from

T h i s o b s e r v a t i o n i n d i c a t e s t h a t these

c a r b o n s a r e h y d r a t e d . I n these c o m p o u n d s t h e C 6 h y d r o x y g r o u p h a s either b e e n

d e r i v a t i z e d o r r e p l a c e d , p r e v e n t i n g t h e f o r m a t i o n of t h e

h e m i k e t a l . T h e s e c o m p o u n d s r e a d i l y f o r m a n o p e n - c h a i n f o r m of D H A , suggesting a reasonable stability for the h y d r a t e d diketo structure. D - I s o D H A , w h i c h r e a d i l y forms the h e m i k e t a l , has a n e n d o - 5 - h y d r o x y l g r o u p , b u t L - D H A is a n e x o - 5 - h y d r o x y l c o m p o u n d .

T h e endo-hydroxyl

does n o t cause sufficient steric h i n d r a n c e to p r e v e n t t h e f o r m a t i o n of t h e hemiketal ring. T h e most i m p o r t a n t i n f e r e n c e to b e d r a w n f r o m t h e d a t a is t h a t DHA

c a n exist as a m i x t u r e of v a r i o u s structures.

A n e q u i l i b r i u m of

m a j o r a n d m i n o r f o r m s of D H A i n a q u e o u s s o l u t i o n u n d o u b t e d l y exists, w i t h the hydrated hemiketal being the favored form

( S c h e m e 1, A ) .

M o s t of these forms h a v e b e e n p o s t u l a t e d f o r m a n y years ( 2 ) . T h e o n l y f o r m detected

by

1 3

C N M R s p e c t r o s c o p y i n a q u e o u s s o l u t i o n is t h e

hydrated hemiketal form DHA

( S c h e m e 1, A ) .

A s s u m i n g t h a t 9 9 % of t h e

is t h e h y d r a t e d h e m i k e t a l f o r m , this f o r m is c a l c u l a t e d t o b e

favored

b y 2.5 k c a l / m o l .

When

t h e s i d e - c h a i n is d e r i v a t i z e d as i n

L - i s o p r o p y l i d e n e - D H A , D-isopropylidene i s o D H A , or 6-bromo-6-deoxyL-DHA,

the open-chain

dihydrate

is seen.

This

finding

suggests

a

r e a s o n a b l e s t a b i l i t y f o r t h e h y d r a t e d h e m i k e t a l f o r m a n d is e v i d e n c e f o r an

open

side-chain compound

i n t h e e q u i l i b r i u m m i x t u r e of D H A

( S c h e m e 1, C ) . T h i s c o m p o u n d has b e e n suggested as a m a j o r p r o d u c t i n a g e d solutions of D H A ( 5 2 ) . A l t h o u g h s m a l l c o n c e n t r a t i o n s ( < 1% ) m a y exist i n s o l u t i o n , t h e p r i n c i p l e c o m p o u n d f o r m e d i n a g e d solutions of D H A has

1 3

C N M R shifts t h a t c o r r e s p o n d t o d i k e t o g u l o n a t e ( D K G ) .



Forms

Identification

Forms

Note: Shifts in ppm from Me4Si.

L-DehydroAA L-ISODHA Isopropylidene-L-DHA Isopropylidene-D-isoDHA 6-Bromo-6-deoxy-L-DHA

Oxidized

L-AA D-ISOAA Isopropylidene-L-AA Isopropylidene-D-isoAA 6-Bromo-6-deoxy-L-AA

Reduced

Compound

173.6 173.5 173.9 173.9 173.6

170.6 173.1

91.4 91.5 91.3 91.3 91.3

105.7 104.8 96.0 95.7 96.0 87.6 83.1 84.6 84.4 83.5

76.7 74.4 74.9 74.7 76.8

156.1 155.6 152.9 152.6 155.2

118.3 118.2 118.2 118.7 118.1

73.0 71.9 74.2 73.9 68.2

69.4 71.0 73.3 73.9 69.0

C5

76.2 69.5 64.4 65.2 34.2

62.6 61.4 64.8 65.3 32.8

C6

—

—

110.5 110.4

—

26.1 25.8

26.2

109.4

—

— — 25.0

S

-CH

— — 109.8

Carbonyl Isopropylidene

and Related Compounds

173.8

on D H A

C4

Data

C3

C NMR

C2

1 3

Cl

Table II.


—

25.0 24.6

25.7

— — 24.4

3

-CH




Scheme 1.

Equilibrium mixture of the various forms of DHA.


116

ASCORBIC ACID

T h e e v i d e n c e for t h e other forms of D H A s h o w n i n S c h e m e

1 is

b a s e d o n t h e o r y of r e a c t i o n m e c h a n i s m s . T h e f a c t t h a t D H A reacts w i t h D N P H to f o r m a h y d r a z o n e is s u p p o r t i v e e v i d e n c e f o r t h e existence of a 2- or 3 - m o n o k e t o c o m p o u n d .

A 3-monoketo c o m p o u n d

is r e q u i r e d b y

a n y r e a s o n a b l e m e c h a n i s m f o r the f o r m a t i o n of the h y d r a t e d h e m i k e t a l f o r m . T h e 2,3-diketo c o m p o u n d w o u l d b e v e r y u n s t a b l e d u e t o t h e h i g h p o s i t i v e c h a r g e associated w i t h the c a r b o n y l carbons. w o u l d b e v e r y s u s c e p t i b l e to n u c l e o p h i l i c attack.

These

carbons

However, a small

c o n c e n t r a t i o n of the 2,3-diketo f o r m s h o u l d exist i n e q u i l i b r i u m m i x t u r e s . Downloaded by UNIV OF GUELPH LIBRARY on May 18, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch005

D i r e c t o x i d a t i o n of A A s h o u l d give the 2,3-diketo f o r m . A

proposed

mechanism

for

the

acid-catalyzed

h y d r a t e d h e m i k e t a l is s h o w n i n S c h e m e

2.

formation

O n the basis of

of

the

analogy

w i t h h e m i k e t a l r i n g f o r m a t i o n a n d m u t a r o t a t i o n i n sugars, o p e n i n g a n d c l o s i n g of the h e m i k e t a l r i n g is expected to b e fast c o m p a r e d w i t h other reactions of D H A , s u c h as h y d r o l y t i c cleavage to D K G . H y d r a t i o n of the c a r b o n y l groups s h o u l d also b e r a p i d . It thus appears t h a t there is a r a p i d e q u i l i b r i u m of a l l the p o s t u l a t e d forms of D H A i n aqueous s o l u t i o n . W h i c h of these forms are a c t i v e i n b i o l o g i c a l reactions is u n k n o w n . T h e f a c i l e f o r m a t i o n of the h y d r a t e d a n d h e m i k e t a l forms of D H A , as w e l l as the a l c o h o l complexes, opens the c r i t i c a l q u e s t i o n of w h e t h e r D H A i n b i o l o g i c a l fluids is to a n y great extent c o n j u g a t e d

with

other

c o m p o u n d s s u c h as a m i n o acids a n d p r o t e i n s . A t p H 7, D H A is r a p i d l y converted fluids

to D K G , b u t there is no e v i d e n c e t h a t D H A i n b i o l o g i c a l

is r a p i d l y h y d r o l y z e d .

It is therefore

appropriate

to

question

w h e t h e r D H A , as s u c h , exists i n a n y significant c o n c e n t r a t i o n i n b i o l o g i c a l fluids.

F u r t h e r studies of D H A i n tissue are n e e d e d to c l a r i f y the n a t u r e

of this c o m p o u n d i n b i o l o g i c a l systems. T h e U V spectra of t h e D H A u s e d i n the experiments

described

shows a w e a k b r o a d t r a n s i t i o n at 225 n m l e a d i n g i n t o a strong a b s o r b a n c e b e l o w 200 n m . T h e t r a n s i t i o n at 225 n m c a n be u s e d i n l i q u i d c h r o m a t o g r a p h y of D H A i f the s a m p l e is f a i r l y p u r e . compounds

Unfortunately, many

absorb i n this r e g i o n , so d i r e c t s p e c t r o p h o t o m e t r i c

D H A b y U V w i t h h i g h pressure l i q u i d c h r o m a t o g r a p h y

assay of

(HPLC)

prob

a b l y is not possible i n most experiments. Stability o f D H A and B D H A . s o l u t i o n (33,44). it appears Because

to d e c o m p o s e s l o w l y to

BDHA

80-100%

BDHA

is n o t stable i n

aqueous

I n the s o l i d f o r m d r y B D H A is q u i t e stable, a l t h o u g h a complex

dissociates to the m o n o m e r

m i x t u r e of

products.

i n w a t e r , a n d D H A has

of the v i t a m i n C a c t i v i t y of A A , B D H A is a n i n t e r e s t i n g f o r m

of this v i t a m i n w i t h properties of l o w s o l u b i l i t y i n w a t e r a n d g o o d resist ance to a i r o x i d a t i o n . D H A is h y d r o l y z e d i n aqueous solutions to y i e l d D K G . T h e r e a c t i o n v e l o c i t y is p H d e p e n d e n t , subject to b o t h a c i d a n d base catalysis.


The

T O L B E R T AND WARD


117


5.

Scheme 2.

Acid-catalyzed formation of the hydrated hemiketal form of DHA.


118

ASCORBIC ACID

k i n e t i c s w e r e r e p o r t e d t o b e first o r d e r w i t h respect to H a K i of 7.6 X 1 0 " at p H = 4

v e

+

with

(55,56)

7.2. T h e specific r a t e constant f o r this

r e a c t i o n w a s r e p o r t e d as 2.08 X 1 0 " s" a t 3 0 ° C . 5

1

I n n e u t r a l a n d a l k a l i n e aqueous

solutions, D H A is v e r y r a p i d l y

h y d r o l y z e d to D K G . I f D H A is a d j u s t e d to p H 7.0 i n b u f f e r e d s o l u t i o n a n d i m m e d i a t e l y assayed b y T L C o r

1 3

C N M R , o n l y D K G is o b s e r v e d .

I n u n b u f f e r e d s o l u t i o n t h e c o n v e r s i o n is s l o w because h y d r o l y s i s o f D H A p r o d u c e s a n a c i d , l o w e r i n g t h e p H to a p p r o x i m a t e l y 2.5. M a n y o f t h e 1 3

C N M R spectra of D H A d e s c r i b e d i n this c h a p t e r w e r e r u n o n samples


at 3 0 ° C over 1 2 - 2 4 h , a n d these spectra c o u l d b e r e p e a t e d 3 d l a t e r i f t h e samples w e r e stored at 4 ° C . Hvoslef

suggests

that t h e h e m i k e t a l f o r m

of D H A is c o n v e r t e d

s l o w l y to t h e o p e n s i d e - c h a i n f o r m i n w a t e r . W a r d ( 3 3 ) shows t h a t t h e p r e s u m e d o p e n s i d e - c h a i n f o r m is a c t u a l l y D K G , w h i c h has

1 3

C NMR

shifts as s h o w n i n T a b l e I I I . I n t h e N M R spectra of D K G t h e shifts of C 2 a n d C 3 a r e c h a r a c t e r i s t i c of gera-diols a n d thus t h e p r i n c i p a l f o r m of D K G i n aqueous

s o l u t i o n is w i t h f u l l y h y d r a t e d c a r b o n y l s o n C 2

and C 3 . Assay of D H A .

T h e r e is n o c o m p l e t e l y satisfactory assay o f D H A

a v a i l a b l e at this t i m e . T h e t w o most c o m m o n l y u s e d p r o c e d u r e s a r e t h e D N P H reaction, done under conditions i n w h i c h the oxidation of A A to D H A is m i n i m i z e d ( 5 8 , 5 9 ) a n d t h e d i f f e r e n t i a l d i c h l o r o i n d o p h e n o l m e t h o d (60,61).

B o t h m e t h o d s are subject to i n t e r f e r e n c e a n d r a t h e r

l a r g e r a n d o m errors. D H A c a n b e s e p a r a t e d f r o m A A a n d most i o n i c c o m p o u n d s b y i o n exchange columns.

S u c h c o l u m n s d o n o t separate D H A f r o m other A A

metabolites a n d neutral carbohydrates. these c o m p o u n d s

D H A c a n be separated

from

b y reverse p h a s e H P L C u s i n g w a t e r o r w a t e r - a c e t o -

n i t r i l e eluants. G o o d separations of D H A f r o m b i o l o g i c a l samples c a n be expected to be achieved b y H P L C .

D e t e c t i o n is a p r o b l e m since U V

a b s o r p t i o n is i n a d e q u a t e . T h e r e d c h r o m o p h o r e w i t h a m i n o acids is n o t v e r y sensitive. P e r h a p s D H A c o u l d b e r e d u c e d t o A A after s e p a r a t i o n a n d d e t e c t e d b y t h e s t r o n g 2 6 3 - n m a b s o r p t i o n of A A o r b y a n e l e c t r o c h e m i c a l detector.

D H A levels a n d D H A / A A ratios a r e p r o b a b l y q u i t e

i m p o r t a n t i n b i o l o g y a n d m e d i c i n e , a n d g o o d p r o c e d u r e s f o r these assays are of c o n s i d e r a b l e interest.

Table III.

1 3

C N M R Shifts of D i k e t o g u l o n i c A c i d

Cl

pH7.0 pH2.0 tt

174.5 171.2

C2,C3

a

94.7,94.4 94.5,96.1

C4

74.6 74.3

C5

68.6 68.0

Shifts for C2 and C3 are too similar to assign to specific carbons.


C6 62.5 62.4

5.

119



Biochemistry of DHA A n u m b e r of e n z y m e s h a v e b e e n c h a r a c t e r i z e d t h a t c a t a l y z e reactions i n v o l v i n g D H A . I n a d d i t i o n , o t h e r aspects of D H A b i o c h e m i s t r y c a n be

deduced

from

metabolic

studies

of

ascorbic

acid.

Experiments

d e m o n s t r a t i n g t h e b i o l o g i c a l o x i d a t i o n o f A A a n d r e d u c t i o n of D H A w e r e first m a d e i n 1928 ( J O ) a n d d u r i n g t h e next d e c a d e several g r o u p s s t u d i e d these reactions.

B y 1941 C r o o k (62) w a s a b l e to separate t h e

a s c o r b i c a c i d oxidase a n d D H A r e d u c t a s e a c t i v i t i e s a n d t o s h o w t h a t


glutathione was used i n the reductase reaction. T h e best c h a r a c t e r i z e d of t h e e n z y m e s i n v o l v i n g D H A is a s c o r b i c a c i d oxidase ( L - a s c o r b a t e : 0

2

o x i d o r e d u c t a s e , E C 1.10.3.3).

e n z y m e catalyzes t h e r e a c t i o n of A A a n d o x y g e n

This plant

to g i v e D H A a n d

1 m o l of w a t e r ( 6 3 ) . H COH 2

A s c o r b a t e oxidase is a d i m e r c o n t a i n i n g t w o i d e n t i c a l s u b u n i t s , a n d appears to b e a c c o m p a n i e d (64-66). colored.

This compound

the K

m

forms

a n d is b l u e

T h e c o p p e r m a y b e r e m o v e d to g i v e a n i n a c t i v e a p o e n z y m e .

A s c o r b a t e oxidase 67,68).

b y s m a l l e r a m o u n t s of o l i g o m e r i c

contains 8 - 1 0 atoms o f c o p p e r

contains C u

2 +

i n three different e n v i r o n m e n t s

(63,

T h e e n z y m e also c a t a l y z e d t h e o x i d a t i o n of o-catechols, a l t h o u g h is less f a v o r a b l e t h a n t h a t f o r A A : Km, ( + ) - c a t e c h i n , 3.08 m M ;

Km, L - A A , 0.24 m M (69).

T h e r o l e o f ascorbate o x i d a s e i n p l a n t s is

not k n o w n . T w o other c o p p e r e n z y m e s possess ascorbate oxidase a c t i v i t y , h u m a n c e r u l o p l a s m a n d Polyporus laccase (70,71). as a n A A oxidase i n v i v o .

Ceruloplasm may function

B o t h c e r u l o p l a s m a n d laccase a r e 1 0 t i m e s 4

less a c t i v e t o w a r d A A o x i d a t i o n t h a n is ascorbate

oxidase.

However,

t h e r e a c t i o n is definitely e n z y m i c , a n d w a t e r i s p r o d u c e d . In

r e c e n t years D H A r e d u c t a s e

has b e e n

purified from

several

sources a n d c h a r a c t e r i z e d . D H A r e d u c t a s e ( E C 1.8.5.1) p u r i f i e d f r o m carp hepatopancreas (72).

w a s specific f o r g l u t a t h i o n e as a r e d u c i n g

Km values w e r e 5.7 X

1 0 " M f o r D H A a n d 1.5 X 4

agent

IO" M for 3

g l u t a t h i o n e . T h e e n z y m e w a s n o t affected b y m e t a l i o n c h e l a t i n g agents. D H A r e d u c t a s e f r o m s p i n a c h leaves has a MW o f a b o u t 25,000 d a l t o n s


120

ASCORBIC ACID

a n d a p H o p t i m u m of 7.5. K

m

values w e r e 4.4 m M f o r g l u t a t h i o n e a n d

0.34 m M f o r D H A ( 7 3 ) . D H A r e d u c t a s e appears to b e w i d e l y d i s t r i b u t e d i n p l a n t a n d a n i m a l tissue, a n d to consistently u s e g l u t a t h i o n e as the r e d u c i n g agent (74, 7 5 ) . A D H A lactonase has b e e n d e s c r i b e d (76, 77) i n t h e ox, r a b b i t , r a t , a n d g u i n e a p i g . I n the ox t h e lactonase is present i n several tissues b u t is most a b u n d a n t i n t h e l i v e r .

T h e e n z y m e appears to b e absent i n

h u m a n a n d m o n k e y tissue. T h i s result is consistent w i t h t h e o b s e r v a t i o n that p r i m a t e s a n d fishes d o n o t c a t a b o l i z e l a b e l e d a s c o r b i c a c i d to c a r b o n Downloaded by UNIV OF GUELPH LIBRARY on May 18, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch005

d i o x i d e . A A a n d D H A a p p e a r t o b e m e t a b o l i z e d i n t o a series of w a t e r s o l u b l e p r o d u c t s that are excreted i n the u r i n e , b u t 2 , 3 - D K G is d e c a r boxylated a n d otherwise degraded and C

4

to i n t e r m e d i a t e s t h a t enter t h e C

5

c a r b o h y d r a t e pools (78).

D H A a n d A A c a n react to f o r m t h e free r a d i c a l i n t e r m e d i a t e , m o n o d e h y d r o a s c o r b a t e i o n ( A A " ) , also c a l l e d s e m i d e h y d r o a s c o r b a t e . that r e d u c e A A " w e r e d e m o n s t r a t e d i n a n i m a l s (79,80), and microorganisms

Enzymes

plants

(81),

(82).

O t h e r t h a n t h e three enzymes

( A A oxidase, D H A reductase, a n d

D H A lactonase) n o other enzymes h a v e b e e n d e m o n s t r a t e d t h a t d i r e c t l y i n v o l v e D H A . D H A m a y b e p r o d u c e d b y a n u m b e r of oxygenases t h a t use A A as a cofactor, a n d i t seems reasonable that m u c h of t h e D H A f o r m e d i n v i v o is p r o d u c e d b y these reactions. Intravenous

i n j e c t i o n o f D H A i n rats at 4 0 - 6 0

mg/kg

produces

e x c i t a t i o n , s a l i v a t i o n , l a c r i m a t i o n , a n d e l e v a t e d b l o o d pressure M o s t of t h e responses

h i g h e r doses, r e s p i r a t o r y arrest occurs a b o u t 300 m g / k g .

(83-85).

o r i g i n a t e d w i t h t h e c e n t r a l nervous system. A t (86).

The L D

5 0

appears to b e

W h e n r e p e a t e d injections of D H A are g i v e n to rats

at a b o u t 20 m g / k g , m a r k e d h y p e r g l y c e m i a is o b s e r v e d i n m a n y of t h e rats after 3 w e e k s

(87).

T h i s diabetogenic

effect w a s c o n f i r m e d

(88)

a n d seems to b e associated w i t h a b n o r m a l i t i e s of t h e beta cells of t h e islets of L a n g e r h a n s of t h e pancreas. of t h e beta cells is n o t seen.

U n l i k e a l l o x a n diabetes,

necrosis

A l l o x a n a n d D H A a r e s t r u c t u r a l analogues

i n that b o t h have a p o t e n t i a l 1,2,3-triketo structure. T h e doses of D H A r e q u i r e d to p r o d u c e

these p h y s i o l o g i c a l effects are so l a r g e t h a t t h e

t o x i c i t y of D H A does n o t h a v e a n y n o t i c e a b l e significance i n t h e n u t r i t i o n a l use of A A . D H A is a m i n o r b y - p r o d u c t of storage a n d is a n o r m a l component

of b o t h foods a n d tissue. N o r m a l b l o o d levels o f D H A a r e

p r o b a b l y a r o u n d 0.2 m g / 1 0 0 m L , a n d tissue levels m a y b e c o m p a r a b l e . D H A is r a p i d l y t r a n s p o r t e d across c e l l m e m b r a n e s .

Autoradiographic

studies u s i n g l a b e l e d D H A s h o w t h a t D H A is m o r e r a p i d l y a b s o r b e d b y g u i n e a p i g s t h a n is A A (89). from the blood

(90-92)

I n j e c t e d D H A is n o t r a p i d l y a b s o r b e d

a n d t h e observations suggest that D H A is n o t

f a v o r e d as a p h y s i o l o g i c a l transport f o r m .

O n l y i n the brain a n d bone


5.


121


m a r r o w are D H A t a k e n u p m o r e r a p i d l y t h a n A A . B o t h leucocytes a n d erythrocytes are r e a d i l y p e r m e a b l e to D H A as w e l l as A A (93,94).

After

u p t a k e of D H A b y leucocytes, o n l y A A is f o u n d , s h o w i n g a n a c t i v e D H A reductase system. T h e r e d u c t i o n o f D H A i n r e d b l o o d cells i s less r a p i d a n d i n c o m p l e t e , s u g g e s t i n g t h a t a D H A r e d u c t a s e system is either absent or of l o w a c t i v i t y . A g e n e r a l belief, s u p p o r t e d b y a l i m i t e d a m o u n t of e x p e r i m e n t a l evidence,

is t h a t D H A levels

or alternatively, D H A / A A

ratios, a r e

sensitive i n d i c a t i o n s of c e l l p h y s i o l o g y , i n c l u d i n g p a t h o g e n i c states a n d


mitotic index

(95-98).

Because

glutathione

( G S H ) is t h e r e d u c i n g

agent f o r D H A reductase, t h e D H A / A A r a t i o m a y reflect t h e G S S G / G S H r a t i o , a n d this r a t i o w a s r e l a t e d to t h e N A D P H / N A D P r a t i o . A s s u m i n g the D H A / A A r a t i o reflects t h e o x i d a t i o n state o f t h e m e t a b o l i s m o f t h e c e l l , i n c l u d i n g t h e N A D P H / N A D P r a t i o , t h e c o r r e l a t i o n appears t o h a v e m e r i t . C e r t a i n l y m o r e e x p e r i m e n t a l studies a r e i m p o r t a n t i n this area.

Acknowledgments W e t h a n k P a u l S e i b f o r t h e samples o f acid a n d 6-bromo-6-deoxy-L-dehydroascorbic

D-en/£hro-dehydroascorbic

acid.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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5. tolbert and ward Dehydroascorbic Acid

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Ascorbic Acid - American Chemical Society

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