Crystallography of the Ascorbates - American Chemical Society

2 Crystallography of the Ascorbates

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JAN HVOSLEF University of Oslo, Department of Chemistry, Blindern, Oslo 3, Norway

A review of the various ascorbates is given in terms of the results from crystal structure determinations. The acid itself and the simple, monovalent salts consist of roughly planar γ-lactone rings with side-chains in a more or less staggered conformation. The acidity of the compound is associated with the proton at O3 because of the conjugated O1=C1—C2=C3—O3 system in the ring. By esterification the location of the ester group can be at either C2 or C3, but the C2 position is presumably the stabler site. By oxi dation of the vitamin dehydroascorbic acid is produced in different isomeric forms, depending on the solvent, on the time, and on a possible substituent in the lactone ring. The usual crystalline compound is a dimer comprising five fused rings, whereas monomeric derivatives are bicyclic, except in cases where the substituents induce sp hybridi zation at C3. 2

In the mid 1930s x-ray crystallographers (1) attempted to assist chemists (2) in the elucidation of the chemistry of vitamin C. The trial was destined to be unsuccessful, but could nevertheless support the brilliant chemical works that eventually lead to a correct chemical formula and to the synthesis of the vitamin (3). This relatively small and simple molecule, comprising only 20 atoms, has intrinsic properties that diversify its chemical behavior and mask its constitution. Features such as acidity without a carboxyl group, an unusually stable γ-lactone ring, reducing power, and two chiral carbon atoms form a combination with enigmatic biological functions that still puzzle chemists and biologists in the 1980s. With the advent of modern methods in crystallography the detailed geometries of ascorbates became accessible, and to date a number of crystal structures are known (Table I). The motivation for these investi gations was originally not any doubt about the gross structures of the ascorbates arrived at by chemical means; the x-ray results also generally 0065-2393/82/0200-0037$06.25/0 © 1982 American Chemical Society Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

38

ASCORBIC

Table I.


Crystallographic D a t a on

Formula

Compound L-Ascorbic acid

CQHLSOQ

Sodium L-ascorbate

Na C H 0 -

Calcium L-ascorbate dihydrate

Ca

T h a l l i u m (I) - L - a s c o r b a t e

T1 C H 0 -

L-Serine-L-ascorbic acid

C H N0

L-Arginine L-ascorbate

C H N 0

Barium 2-0-sulfonato-Lascorbate d i h y d r a t e

Ba

3 - 0 [ (bismorpholino) phosphinyl]-5,6-0-isopropylideneL-ascorbate

C17H27N2O9P

Dehydroascorbic acid dimer

C12H12O12

+

6

(6-8)

7

6

7

7

7

6

5

2 +

(10)

6

6

+

3

Reference

(C H 0 ) " • 2H 0

2 +

6

2

(12-W

2

(11)

6

•C H 0

3

4

6

2

8

(9)

6

• C H 0 -

+

6

7

(15)

6

(C H 0 S) - • 2 H 0 6

6

2

9

(18)

2

(21)

(4)

p - B r o m o p h e n y l h y d r a z i n e of CiaHnOsN^r monomeric dehydro-L-ascorbic acid

(25)

M e t h y l g l y c o s i d e of 2 - C - b e n z y l 3-keto-L-fa/xo-hexulosonic a c i d lactone

CuHieOe

m)

D-Isoascorbic acid

CeHgOe

(26)

Sodium D-isoascorbate-monohydrate

Na C H 0«- •H 0 +

6

ACID

7

(27)

2

°The technique used is indicated b y : F, film; D , diffractometer; or N , neutron diffraction spectrometer. confirmed

the predicted

chemical

formulae

and bonding

properties.

Surprises came, however, w h e n the v i t a m i n s oxidation product,

dehydro

a s c o r b i c a c i d , w a s e x a m i n e d b y x - r a y a n d N M R m e t h o d s (4,5).

Virtually

a l l textbooks

are s h o w n

t o b e m i s l e a d i n g i n t h e i r assessment

s t r u c t u r e of t h i s c o m p o u n d .

of t h e

This should actually not be surprising, for

t h e f o r m u l a c o m m o n l y u s e d is u n a c c e p t a b l e f o r v a r i o u s reasons. details o f t h e o x i d a t i o n a r e s t i l l left to b e sorted o u t , b u t t h e y difficult tasks b e c a u s e of t h e c o m p l i c a t e d r e a c t i o n m e c h a n i s m .

Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

Many present

2.

HVOSLEF

39

Crystallography of the Ascorbates

L-Ascorbic A c i d and Some Derivatives

Space Group

Unit Cell Dimensions


P2i

P2t

Average e.s.d. in Bond Lengths [Method (A) and Angles (°)]*

a = 1 7 . 2 9 9 , b == 6.353, c = 6.411 A ; 0 = 102.18°

0.003 A

0.2°

F,N

a = 19.051, b == 4.490, c = 8.516 A

0.006

0.4

F

a = 8.335, b = 15.787, c = 6.360 A ; j8 = 107.48°

0.004

0.2

D

0.040

—

D

P2 2i2

1

a =

P2 2 2

1

a = 5.335, b = 8.769, c = 25.782 A

0.005

0.3

D

P2i

a = 5.060, b = 9.977, c = 0 = 97.5°

0.006

0.4

D

PI

a = 5 . 2 0 1 , 6 = 6.951, c = 8.732 A a= 9 9 . 5 4 ° , / ? == 93.92°, y = 109.12°

0.004

0.2°

D

—

—

D

a = 15.728, b == 5.530, c = 9.453 A ; y3 = 130.56°

0.006

0.5

F

0.008

0.6

D

0.004

0.3

D

a = 5.165, b = 14.504, c = 4.724 A ; 0 = 99.50°

0.005

0.3

D

a = 8.307, 6

0.007

0.4

D

1

1

P2

1

a =

X

C2

10.883, b == 18.598, c = 8.066 A

9.487, b = 13.570, c = 8.355 A

P2 2 2

1

a =

P2 2 2

1

a = 6.339, b = 9.739, c =

1

1

1

1

P2x P2 2 2 1

1

1

15.330,

18.020, b == 12.859, c = 5.754 A

= 9.049, c =

22.484 A

11.181

X - r a y analyses h a v e also p r o v e d u s e f u l i n u n r a v e l i n g o t h e r c o m p l e x s t r u c t u r a l p r o b l e m s i n this field, a n d w e s h a l l r e v i e w some w o r k d o n e o n ascorbates b y d i f f r a c t i o n m e t h o d s .

Ascorbic Acid and Its Salts Ascorbic A c i d .

V i t a m i n C ( L - a s c o r b i c a c i d ) w a s the first ascorbate

t o b e s t u d i e d i n d e t a i l b y d i f f r a c t i o n m e t h o d s (6,7,8).

It crystallizes in

t h e m o n o c l i n i c space g r o u p P 2 i w i t h f o u r m o l e c u l e s i n t h e u n i t c e l l as


40

ASCORBIC

ACID

s h o w n i n F i g u r e 1. T h e r e are t w o m o l e c u l e s , A a n d B, i n t h e a s y m m e t r i c u n i t , b u t t h e y are r e l a t e d i n p a i r s b y p s e u d o s c r e w axes a l o n g [010] i n t h e p o s i t i o n s (x — 1/4, z =

5/8)

a n d (x — 3 / 4 , z — 3 / 8 ) .

I n the crys

tals t h e i n d e p e n d e n t m o l e c u l e s are s l i g h t l y different, m a i n l y b e c a u s e of a s m a l l difference i n t h e o r i e n t a t i o n of t h e a l m o s t i d e n t i c a l s i d e - c h a i n s . T h e d i s t i n c t i o n is d e f i n e d b y a + / — 7 . 9 ° t w i s t a b o u t t h e C 4 - C 5 b o n d to e a c h side of a n i d e a l l y s t a g g e r e d c o n f o r m a t i o n w h e r e 0 5

is s i t u a t e d


a b o v e t h e r i n g a n d w h e r e 0 6 is + 8 . 3 ° f r o m anti to 0 5 . T h e i n t e r a t o m i c distances a n d angles, h o w e v e r , are a l m o s t i d e n t i c a l i n t h e i n d e p e n d e n t m o l e c u l e s , w h i c h h a v e the a v e r a g e v a l u e s s h o w n i n F i g u r e 2. T h e C - C a n d C - O b o n d lengths s h o w some i n t e r e s t i n g v a r i a t i o n s , a n d explain i n a striking w a y the observed

chemical properties.

The

l o c a l i z a t i o n of t h e p r o t o l y t i c p r o t o n t o 0 3 w a s o b v i o u s f r o m t h e b o n d i n g p r o p e r t i e s of t h e c o n j u g a t e d 0 1 = C 1 — C 2 = C 3 — 0 3 — H

system, a n d i t

has l a t e r b e e n c o n f i r m e d b y t h e s t r u c t u r e of a n u m b e r of salts. W h e r e a s t h e e n e - d i o l i c g r o u p is e n t i r e l y p l a n a r , t h e l a c t o n e g r o u p shows m i n o r v a r i a t i o n s f r o m p l a n a r i t y . T h e s e d e v i a t i o n s are p r e s u m a b l y c a u s e d p a c k i n g effects b e i n g different i n the t w o i n d e p e n d e n t m o l e c u l e s . ever, t h e w h o l e

r i n g system c a n b e

adequately

described

by

How

as p l a n a r

b e c a u s e t h e best p l a n e s t h r o u g h the l a c t o n e a n d t h e e n e - d i o l g r o u p i n e a c h m o l e c u l e are at o n l y 0.6° to e a c h other.

Figure I . View of the structure of "L-ascorbic acid along [010] and the conesponding three-dimensional electron density. (Reproduced, with permission, from Ref. 7.)



2.

HVOSLEF

Figure 2.

41


Average values of bonding distances and angles in L-ascorbic acid. (Reproduced, with permission, from Ref. 7.)

A l l t h e o x y g e n atoms, except 0 4 , are e n g a g e d i n h y d r o g e n

bonds,

a n d t h e i n t e r a c t i o n s i n v o l v i n g t h e e n e - d i o l o x y g e n atoms are p a r t i c u l a r l y s t r o n g a n d a b o u t 0.15 A shorter t h a n t h e u s u a l a l c o h o l i c i n t e r a c t i o n s . T h e h y d r o g e n b o n d s y s t e m f o r e a c h of t h e t w o i n d e p e n d e n t m o l e c u l e s is s h o w n i n F i g u r e 3, a n d i t is n o t e w o r t h y t h a t t h e p a t t e r n is s i m i l a r f o r A a n d B a l t h o u g h t h e e n v i r o n m e n t s are different a n d t h e d o n o r - a c c e p t o r sequence is r e v e r s e d , except for 0 3 . T h e a s c o r b i c a c i d m o l e c u l e has also b e e n s t u d i e d i n t h e c r y s t a l l i n e complex w i t h L-serine [ C H ( O H ) C H ( N H ) C O O H ] , recently reported 2

2

by Sudhakar, Bhat, and Vijayan

(9).

T h e crystals a r e

orthorhombic

w i t h Z = 4, a n d t h e m o l e c u l e s are m a i n l y h e l d t o g e t h e r b y bonds.

hydrogen

T h e i n d i v i d u a l c o m p o n e n t s are b o t h n e u t r a l , a n d t h e m o l e c u l a r

d i m e n s i o n s i n t h e a s c o r b i c a c i d m o l e c u l e are n o t s i g n i f i c a n t l y different f r o m those i n t h e p u r e a c i d .

T h e side-chain conformation varies

by

r o t a t i o n of 0 6 a b o u t the C 5 - C 6 b o n d to a n e a r gauche a r r a n g e m e n t , t h u s b r i n g i n g i t close to t h e s i t u a t i o n i n s o d i u m ascorbate

(10).

The

h y d r o g e n b o n d s y s t e m is, of course, different, a n d i t is i n t e r e s t i n g to n o t e t h a t t h e c a r b o x y l g r o u p of L - s e r i n e is t i e d to t h e e n e - d i o l g r o u p of L - a s c o r bic a c i d ( F i g u r e 4)

t h r o u g h r a t h e r short h y d r o g e n b o n d s

(2.542 a n d

2.642 A ) . Salts o f A s c o r b i c A c i d .

I n salt f o r m a t i o n t h e i o n i z a t i o n of L - a s c o r b i c

a c i d is c l o s e l y associated w i t h t h e b e h a v i o r of t h e e n e - d i o l g r o u p a n d t h e conjugated 0 1 = C 1 — C 2 = C 3 — 0 3

system. A l l t h e salts w e k n o w so f a r

are f o r m e d b y d i s s o c i a t i o n of t h e p r o t o n at 0 3 monovalent.

o n l y , a n d are

hence

B i v a l e n t anions p r e d i c t e d f r o m studies of solutions h a v e

not been isolated a n d characterized. C o m m o n to t h e i n v e s t i g a t e d ascorbate

anions a r e t h e s i g n i f i c a n t

l e n g t h e n i n g of t h e d o u b l e , s h o r t e n i n g of t h e s i n g l e b o n d s i n t h e c o n j u -



42

ASCORBIC

ACID

Figure 3. Environment and hydrogen bonding for molecules A and B. Oxygen atoms in neighboring molecules are indicated by triple circles. (Reproduced, with permission, from Ref. 7.) g a t e d s y s t e m , a n d changes i n t h e a n g u l a r d i s t r i b u t i o n , e s p e c i a l l y at C 2 and C3. S e l e c t e d v a l u e s of b o n d lengths a n d angles i n t h e ascorbate a n i o n are g i v e n i n T a b l e I I a n d are c o m p a r e d w i t h those of t h e free a c i d . T a k i n g t h e b o n d i n g p r o p e r t i e s of the l a c t o n e g r o u p i n t o a c c o u n t also, t h e observations s u p p o r t t h e v i e w t h a t t h e a n i o n m a y b e t h o u g h t of as a resonance h y b r i d of I, II, a n d III, b u t w i t h a d o m i n a t i n g c o n t r i b u t i o n f r o m I.

(XII)

0(16) Figure 4. The hydrogen-bonded interaction between the ene-diol group of ascorbic acid and the carboxylate group of serine. The primed atom belongs to a neighboring molecule. (Reproduced, with permission, from Ref. 9.)



107.8 109.5 124.6 133.5 117.1 112.7 114.8

1.216 1.361 1.326 1.355 1.444 1.427 1.431 1.338 1.452 1.493 1.521 1.521 109.5 105.8 121.6 131.3 122.9 110.1 116.1

109.8 107.5 124.4 130.7 121.7 110.9 115.3

1.200 1.340 1.329 1.365 1.437 1.416 1.428 1.331 1.446 1.493 1.531 1.520

107.1 110.1 124.5 132.8 116.3 112.5 114.0

1.222 1.364 1.301 1.381 1.465 1.420 1.441 1.362 1.418 1.516 1.521 1.513

1.233 1.384 1.287 1.358 1.448 1.410 1.423 1.373 1.416 1.516 1.536 1.503

D-Isoascorbic Acid (26)

(°)

109.4 106.9 121.1 131.9 121.7 115.6 119.6

1.229 1.366 1.288 1.374 1.451 1.431 1.425 1.370 1.421 1.518 1.528 1.508

1.231 1.375 1.275 1.365 1.453 1.436 1.422 1.371 1.417 1.516 1.533 1.514 109.8 106.2 120.1 130.9 122.8 114.6 112.4

(B)

(A)

(14:)

in Ascorbate

Ca L-Ascorbate

and Angles

Na Isoascorbate (27)

(A)

Na L Ascorbate no;

Selected Bond Distances a

109.2 106.9 125.9 130.8 122.1 111.9 113.6

1.218 1.364 1.275 1.361 1.440 1.424 1.401 1.364 1.423 1.499 1.510 1.497

Arginine L Ascorbate (15)

Anions

111.2 ( ° ) 105.7 122.2 131.4 122.8 111.2 112.4

1.218 1.389 1.265 1.376 1.455 1.421 1.422 1.367 1.422 1.515 1.523 1.524

(W

(A)

Ba 2-0SulfonatoL-ascorbate

• The mean values of the L-ascorbic acid molecules and of D-isoascorbic acid are given for comparison. The values for C a L-ascorbate hydrate are weighed means of Refs. 12 and 18.

C l — C 2 - -C3 C 2 — C 3 - -C4 C 1 — C 2 - -02 C 2 — C 3 - -03 0 3 — C 3 - -C4 C 4 — C 5 - -C6 C3—C4—C5

Cl=01 C2—02 C3—03 Cl—04 C4—04 C5—05 C6—06 C2=C3 Cl—C2 C3—C4 C4—C5 C5—C6

L-Ascorbic Acid (7)

Table II.


44

ASCORBIC

ACID

T h e effect of a n i m p a i r e d sp h y b r i d i z a t i o n a t C 2 a n d C 3 is c o n f o r m a


2

tional lability i n the y-lactone ring.

A s a result, t h e p r o x i m i t y o f a

m e t a l l i c c a t i o n causes t h i s r i n g t o d e v i a t e f r o m p l a n a r i t y ; i t also increases 02—C2=C3—03 N a ascorbate

d i h e d r a l angles.

(10), Tl(I)-ascorbate

T y p i c a l is t h e effect o n salts l i k e (11)

a n d C a ascorbate

dihydrate

(13,14) w h e r e t h e a n g l e b e t w e e n t h e m e a n p l a n e s d e f i n e d b y t h e l a c t o n e group ( 0 4 , C 4 , C 1 , 0 1 , C 2 ) a n d the e n e - d i o l group ( C 2 , 0 2 , C 3 , 0 3 )

occa

s i o n a l l y is as h i g h as 8 ° . A s o m e w h a t s m a l l e r a n g l e ( 5 ° ) is o b s e r v e d i n L - a r g i n i n e ascorbate

( 1 5 ) w h e r e t h e i n f l u e n c e o f a m e t a l l i c c a t i o n is

avoided. W h e r e a s t h e c o n f o r m a t i o n a l differences i n t h e r i n g s a r e m o d e r a t e , the side-chains are f o u n d to be more susceptible to t h e influence of packing, metal coordination, a n d hydrogen bonding.

I n m o s t cases t h e

ascorbate ions h a v e 0 5 i n a r o u g h l y s t a g g e r e d p o s i t i o n , b u t w i t h a p p r e ciable tolerance.

0 6 has e i t h e r a n e a r anti o r n e a r gauche o r i e n t a t i o n

as i l l u s t r a t e d i n F i g u r e 5.

* V Figure 5.

Dihedral angles (parentheses) U (gauche) and V (anti) for C6-O6 in ascorbates.

05-C5-

The gauche orientation occurs in: ascorbic acid in serine complex (66°), sodium ascorbate (70°), thallium ascorbate A (71.7°), arginine ascorbate (56°), barium 2-O-sulfonato-L-ascorbate dihydrate (73.3°). The anti orientation occurs in: ascorbic acid (8.3°), thallium ascorbate B (1°), p-bromophenylhydrazine of dehydroascorbic acid (1.2°).


2.

HVOSLEF

45


I n c e r t a i n cases, exceptions t o these r u l e s are o b s e r v e d . I n o n e of t h e t w o i n d e p e n d e n t a n i o n s ( B ) of C a a s c o r b a t e d i h y d r a t e , 0 5 ( F i g u r e 6 ) a d o p t s a r a r e , u n f a v o r a b l e ( p e r i ) i n t e r a c t i o n (16)

with 0 3 . In addi

t i o n , 0 6 is r o t a t e d a b o u t C 5 - C 6 to p e r m i t t h e t h r e e o x y g e n atoms 0 3 , 0 5 a n d 0 6 to f o r m a n i n t e r e s t i n g t r i d e n t a t e c o m p l e x w i t h C a .

Mole-


2 +

Figure 6. Perspective drawing of the independent ascorbate anions A and B. Distances and angles for the nonhydrogen atoms are included in the drawing. (Reproduced, with permission, from Ref. 13.)


46

ASCORBIC

cule A can be derived f r o m B b y rotating the side-chain b y a r o u n d C 4 - C 5 , a n d the o c t a h e d r a l c o o r d i n a t i o n a r o u n d C a

2 +

ACID

—117° is

com

pleted b y 0 5 a n d 0 6 f r o m A , t w o water molecules, a n d O l f r o m a s y m m e t r y e q u i v a l e n t of B . T h e s t r u c t u r e of C a ascorbate d i h y d r a t e w a s s i m u l t a n e o u s l y d e t e r m i n e d b y t w o i n d e p e n d e n t g r o u p s (12,13), were subsequently compared

a n d t h e i r d a t a a n d results

a n d a n a l y z e d b y A b r a h a m s et a l .

(14).


S o m e g e n e r a l c o n c l u s i o n s c o u l d b e d r a w n f r o m this i n v e s t i g a t i o n w i t h respect to the g i v e n values of s t a n d a r d d e v i a t i o n s , to t h e effect of a n o m a lous s c a t t e r i n g o n a t o m i c c o o r d i n a t e s , a n d to the a b s o l u t e c o n f i g u r a t i o n of the m o l e c u l e i n q u e s t i o n . A m o n g t h e salts i n t h e ascorbate series is also B a 2 - O - s u l f o n a t o - L ascorbate d i h y d r a t e t h a t is d e r i v e d f r o m t h e a s c o r b i c a c i d 2-sulfate ester. This biologically important compound

(17)

was m u c h debated because

i t w a s difficult t o d e c i d e w h e t h e r the sulfate g r o u p w a s a t t a c h e d t o C 2 o r C 3 . T h e s t r u c t u r a l a n a l y s i s b y M c C l e l l a n d (18)

p r o v e d t h e site t o

b e at C 2 as s h o w n i n F i g u r e 7. T h e b o n d l e n g t h s , angles, a n d resonance f o r m s are c l e a r l y s i m i l a r to those of t h e s i m p l e ascorbate anions i r r e s p e c t i v e of t h e effect of t h e sulfate g r o u p a t t a c h e d to C 2 . A s i m i l a r a n d e v e n m o r e c o m p l e x p r o b l e m arose for the

phosphate

ester of a s c o r b i c a c i d , a n d a g a i n x - r a y m e t h o d s p r o v e d u s e f u l i n u n r a v e l i n g t h e d i l e m m a of w h e r e to a s s i g n t h e p h o s p h a t e g r o u p . a s s i g n e d b y the o r i g i n a l authors (19)

T h e position

w a s at C 3 , b u t L e e et a l .

(20)

a n d others c o n t e n d e d t h a t w a s at C 2 . I n a recent, i n t e r e s t i n g p a p e r b y

Figure 7. Configuration, bond lengths (A), and bond angles (°) of the 2 - 0 sulfonato-L-ascorbate anion. (Reproduced, with permission, from Ref. IS.)


2.

47


HVOSLEF

J e r n o w et a l . (21) d e r i v a t i v e of

a b r i e f c r y s t a l l o g r a p h i c a n a l y s i s demonstrates t h a t a

t h i s ester, t h e

(3-0-(bismorpholino)phosphinyl)

5,6-iso-

p r o p y l i d e n e - L - a s c o r b a t e , i n d e e d has its p h o s p h a t e g r o u p a t t a c h e d to C 3 . T h e m o l e c u l a r s t r u c t u r e is s h o w n i n F i g u r e 8, b u t u n f o r t u n a t e l y n o d e tails of t h e s t r u c t u r e are g i v e n u n d e r t h e c i r c u m s t a n c e s ; o n l y t h e gross s t r u c t u r e w a s r e q u i r e d . N o i n f o r m a t i o n is therefore a v a i l a b l e w i t h respect to t h e state of c o n j u g a t i o n , b u t i t is e x p e c t e d to b e different f r o m t h a t i n


t h e s i m p l e salts a n d i n the 2-O-esters.

U p o n a c i d h y d r o l y s i s of t h e c o m

p o u n d , t h e authors c l a i m t h a t a n u n s t a b l e 3 - O - p h o s p h a t e

is i n i t i a l l y

f o r m e d , b u t t h a t i t r a p i d l y isomerizes t o t h e s t a b l e r 2 - O - p h o s p h a t e .

It

is c o n c l u d e d that t h e tris ( c y c l o h e x y l ) a m m o n i u m salt of the a s c o r b i c a c i d p h o s p h a t e , first p r e p a r e d b y C u t o l o a n d L o r i z z a (19)

a n d assigned t h e

3 - O - p h o s p h a t e , is i n fact the 2 - O - p h o s p h a t e as p r o p o s e d b y L e e et a l . (20). A l l the compounds

mentioned above have common

features

with

respect to m o l e c u l a r a n d c r y s t a l structures, a n d t h e results a r r i v e d at b y different authors are h e a r t e n i n g l y u n a n i m o u s . F o r t h e h y d r o g e n b o n d s y s t e m , f o r e x a m p l e , i t is e s t a b l i s h e d t h a t t h e distances associated w i t h t h e e n e - d i o l i c h y d r o x y l s are s i g n f i c a n t l y shorter t h a n t h e others

(Table

I I I ) . A v a l u e w e l l b e l o w 2.6 A f o r a 0 2 — H • • • 0 3 i n t e r a c t i o n is u s u a l , a n d i f 0 3 is a n a c c e p t o r e v e n a n a l c o h o l i c h y d r o x y l g r o u p p r o d u c e s

a

s t r o n g i n t e r a c t i o n . T h i s a t o m has a n u n u s u a l l y h i g h c a p a c i t y for h y d r o gen bonding. T h e o r i e n t a t i o n a n d c o n f o r m a t i o n s of t h e s i d e - c h a i n h a v e o n l y m i n o r

Figure 8.

Drawing showing the conformation of the ascorbate 3-O-phosphinate. (Refffj^^f^^^^^on, from Ref. 21.)

Society Library 1155 16th St. N. W. Seib and Tolbert; Ascorbic Acid: Metabolism, and Uses Washington, D. Chemistry, C. 20036 Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


0 6 ' : 2.612 0 5 ' : 2.645

01:2.656 0 1 ' : 2.666

0 2 : 2.786 0 6 : 2.707

0 2 ' : 2.935 0 5 : 2.769

Donor

02—H 02— H '

03— H 03—H'

05—H 05— H '

06— H 06—H'

02 :

06 :

2.766

2.709

0 2 ( S ) : 2.542

0 1 ( S ) : 2.642

Ascorbic Acid in Serine Complex (9)

05:2.777

02:2.827

01:2.643

06:2.584

Isoascorbic Acid (26)

01:2.820

02:2.709

03:2.546

Na Ascorbate (10)

0 3 : 2.828 0 3 : 2.785

03:2.993

03:2.801

W : 2.699

Na Isoascorbate (27)

05': 02': 03 : 01:

02: 01:

2.972 2.921 2.707 2.782

2.715 2.786

03:

04: 01:

2.622

2.921 2.913

0 1 ( A ) : : 2.676

0 3 ' : 2.549 0 3 : 2.571

0 4 ' : 2.862 0 4 : 2.742 0 2 : 2.793

Arginine Ascorbate (15)

i n Ascorbates*

Ca Ascorbate Dihydrate (13)

Examples of H y d r o g e n Bond Distances ( A )

* If two independent molecules A and B are involved B is indicated by: S, serine; A, arginine; W, water.

0—H ) i n water I

Ascorbic Acid (7)

Table III.


W:

2.899

0 3 : 2.693

Ba 2-0-Suljo nato ascorbate (18)

2.

49


HVOSLEF

i n f l u e n c e o n t h e b o n d lengths i n t h e r i n g , b u t h a v e s o m e effect o n its planarity.

T h i s is reflected i n t h e a n g u l a r d i s t r i b u t i o n a r o u n d C 4 a n d

C 5 , p a r t i c u l a r l y i n C a - a s c o r b a t e d i h y d r a t e w h e r e d e v i a t i o n s u p to

7.2°

are o b s e r v e d .

Dehydroascorbic Acid and Derivatives


O x i d a t i o n of L - a s c o r b i c a c i d p r o d u c e s a v a r i e t y of c h e m i c a l species d e p e n d i n g o n t h e n a t u r e of t h e solvent, the t i m e , a n d t h e s t r e n g t h of t h e o x i d a t i o n agent.

B y c a r e f u l , m i l d o x i d a t i o n t w o h y d r o g e n atoms

are

g i v e n off i n successive steps. I n t h e first step, a n u n s t a b l e r a d i c a l " s e m i dehydroascorbic

a c i d " is f o r m e d .

I n the second, the biologically active

d e h y d r o a s c o r b i c a c i d results. T h e c o m m e r c i a l l y a v a i l a b l e i s o m e r of

dehydroascorbic

a c i d is a

c r y s t a l l i n e , s y m m e t r i c d i m e r c o m p r i s i n g a system of five f u s e d r i n g s 22),

(4,

I V , a n d n o t the t r a d i t i o n a l "textbook c o m p o u n d " w i t h t h e f o r m u l a

V . T h e l a t t e r has not b e e n i s o l a t e d a n d c h a r a c t e r i z e d , a n d is p r e s u m a b l y not present i n significant a m o u n t w h e n the v i t a m i n is o x i d i z e d .

I f the

o x i d a t i o n of L - a s c o r b i c a c i d takes p l a c e i n i n e r t solvents s u c h as d i m e t h y l formamide

or d i m e t h y l s u l f o x i d e , it has b e e n

shown by Hvoslef

and

P e d e r s e n ( 5 ) , u s i n g N M R m e t h o d s , t h a t at l o w t e m p e r a t u r e s a d i m e r i d e n t i c a l to t h e c r y s t a l l i n e c o m p o u n d is f o r m e d .

W h e n the t e m p e r a t u r e

IV


50

ASCORBIC

ACID


OH

VI

rises, this dimer gradually transforms to another dimer, V I , which is unsymmetrical, but closely related to the first. However, i n water a hydrated bicyclic monomer is the prime result. This is unstable in water, and with time the furanose ring opens to give a monocyclic molecule with an open side-chain. T h e Dehydroascorbic A c i d Dimer.

O f the possible versions of pure

dehydroascorbic acid, only the symmetric dimer has been obtained in crystalline state and investigated by diffraction methods.

However,

derivatives of monomers are known and will be discussed below. Monoclinic crystals of the symmetric dimer can be precipitated by successive addition of formic acid and solid oxalic acid (23)


to a d i -

2.

HVOSLEF

51


m e t h y l f o r m a m i d e s o l u t i o n of t h e u s u a l m i x t u r e of s y m m e t r i c a n d a s y m metric dimers.

T h e s o l u b i l i t y of t h e f o r m e r is s t r o n g l y d e c r e a s e d

by

these reagents, b u t leaves t h e l a t t e r i n s o l u t i o n . T h e space g r o u p

C 2 imposes t w o f o l d

symmetry on the dimeric

m o l e c u l e w i t h t h e d i m e n s i o n s s h o w n i n F i g u r e 9. T h e t w o h a l v e s are j o i n e d b y the 0 3 atoms o r i g i n a l l y associated b y the e n e - d i o l g r o u p , a n d establish a c e n t r a l d i o x a n r i n g i n the t w i s t e d b o a t c o n f o r m a t i o n .

The


f o u r t h v a l e n c e at C 3 closes t h e s i d e - c h a i n t o f o r m a n i r r e g u l a r f u r a n o s e m o i e t y w i t h C 6 0.55 A f r o m t h e best p l a n e t h r o u g h t h e o t h e r atoms. L o o k i n g at t h e l a c t o n e r i n g one observes t h a t t h e l a c t o n e g r o u p

(C4,04,

C 1 , 0 1 , C 2 ) is a l m o s t p l a n a r , b u t t h a t C 3 deviates b y 0.192 A , w h i c h is m o d e r a t e f o r f u r a n o s e o r f u r a n o i d l a c t o n e r i n g s . T h e g e o m e t r y of t h i s m o d e r a t e l y s t r a i n e d r i n g s y s t e m is n o r m a l f o r a c o m p o u n d w i t h c o v a l e n t bonds.

O n e m a y , h o w e v e r , n o t i c e t h a t t h e C 2 - 0 2 b o n d is v e r y s h o r t

(1.352 A ) a n d t h a t t h e t w o C - O b o n d lengths i n t h e d i o x a n r i n g are u n e q u a l . O n r e a c t i o n w i t h w a t e r t h e d i o x a n r i n g is s p l i t a t t h e l o n g e r C 2 - 0 3 b o n d , a n d t w o b i c y c l i c , h y d r a t e d monomers are f o r m e d ( 5 ) .

It

is n o t u n l i k e l y t h a t t h e short C 2 - 0 2 b o n d is r e s p o n s i b l e f o r t h e a c i d i t y of t h e c o m p o u n d . T h e r e a l l y i n t e r e s t i n g a s p e c t of this s t r u c t u r e is t h a t i t is d i m e r i c . T h i s f a c t o b v i o u s l y has r e l e v a n c e t o t h e c o m p l i c a t e d r e a c t i o n m e c h a n i s m a s s o c i a t e d w i t h t h e o x i d a t i o n of t h e v i t a m i n i n i n e r t solvents.

Whether

t h e d i m e r is f o r m e d as a s h o r t - l i v e d i n t e r m e d i a t e b y o x i d a t i o n i n w a t e r is s t i l l a n o p e n q u e s t i o n . A t t e m p t s t o d e t e r m i n e t h e c r y s t a l s t r u c t u r e of t h e a s y m m e t r i c d i m e r are n o w b e i n g m a d e i n o u r l a b o r a t o r y .

Figure 9.

Bonding distances and angles in dehydroascorbic acid. (Reproduced, with permission, from Ref. 4)


52

ASCORBIC ACID

D e r i v a t i v e s of Dehydroascorbic

Monomers.

Monomeric

dehydro

a s c o r b i c a c i d p r e s u m a b l y is f o r m e d o n l y i n solvents t h a t p r e v e n t t h e f o r m a t i o n of

dimers, for example, water a n d alcohols.

T h e unstable

e v a s i v e n a t u r e of t h e c o m p o u n d i m p e d e s its c r y s t a l l i z a t i o n , a n d i n t r a c t a b l e s y r u p s are o f t e n t h e r e s u l t . H o w e v e r , d e r i v a t i v e s of t h e c o u l d be

monomer

o b t a i n e d w i t h satisfactory c r y s t a l l i n e q u a l i t y f o r a r e g u l a r

x-ray study.


T h e q u e s t i o n of w h e t h e r a b i - o r m o n o c y c l i c m o l e c u l e is p r e s e n t i n these crystals w a s of m a j o r interest, b u t w e h a v e also s o u g h t

possible

factors t h a t w o u l d cause a n o p e n s i d e - c h a i n i n t h e m o n o m e r . I n t h e m e t h y l g l y c o s i d e of 2 - C - b e n z y l - 3 - k e t o - L - & / x o - h e x u l o s o n i c a c i d l a c t o n e ( F i g u r e 10) a b i c y c l i c ascorbate i s o m e r w a s f o u n d (24). s u g a r m o i e t y bears great r e s e m b l a n c e t o t h e a s y m m e t r i c u n i t of crystalline dimeric dehydroascorbic acid. B o t h

five-membered

The the

rings have

irregular envelope conformations w i t h the c o m m o n C 3 atom deviating f r o m t h e best p l a n e s t h r o u g h t h e f o u r o t h e r atoms i n t h e r i n g s .

The

u n i q u e r o l e of C 3 is p r e s u m a b l y c a u s e d b y r e p u l s i o n b e t w e e n t h e b e n z y l a n d m e t h o x y g r o u p s , w h e r e b y a n e c l i p s e d c o n f o r m a t i o n for 0 3 a n d C 8 is a v o i d e d b y a + 3 8 . 8 ° t w i s t a b o u t t h e C 2 - C 3 b o n d .

T h e free

OH

g r o u p at C 2 has t h e same o r i e n t a t i o n r e l a t i v e t o t h e l a c t o n e r i n g as i t has i n t h e d i m e r , b u t t h e C - O b o n d is s o m e w h a t l o n g e r .

Besides this,

t h e b o n d lengths a n d angles are g e n e r a l l y s i m i l a r t o those i n t h e d i m e r , e x c e p t for t h e c o n f o r m a t i o n a l angles i n t h e furanose m o i e t y . O n the other h a n d , a monocyclic derivative was obtained w h e n a s o l u t i o n of

dehydroascorbic

acid and p-bromophenylhydrazine i n d i -

m e t h y l a c e t a m i d e w a s a l l o w e d t o s t a n d o v e r n i g h t at r o o m t e m p e r a t u r e (25).

The compound

was precipitated w i t h water a n d recrystallized

f r o m a b s o l u t e a l c o h o l ; its m o l e c u l a r s t r u c t u r e is s h o w n i n F i g u r e 11. T h e l a c t o n e r i n g is of t h e e n v e l o p e t y p e w i t h C 4 d e v i a t i n g b y 0.126 A f r o m the plane defined b y C l , O l , C 2 , a n d 0 4 .

T h e s i d e - c h a i n is v e r y

n e a r l y s t a g g e r e d a n d w i t h 0 5 a n d C 6 anti. A s i g n i f i c a n t feature of this c o m p o u n d is t h e p r o p e r t y of t h e C 7 N 2 - N 1 - C 2 - C 3 m o i e t y w i t h its p l a n a r , e x t e n d e d c h a i n of atoms a n d its b o n d l e n g t h s a n d angles t y p i c a l of sp

2

hybridization. The

resonance

structures t h a t c o u l d d e s c r i b e t h e ^ - e l e c t r o n d e r e a l i z a t i o n a r e :



Figure 10.

ORTEP

plot of the methyl glycoside of the 2-C-benzyl-3-keto-L-\yxo-hexulosonic acid lactone molecule. (Reproduced, with permission, from Ref. 24.)



Figure 11.

ORTEP

plot of the p-bromophenylhydrazone of dehydroascorbic acid. (Reproduced, with permission, from Ref. 25.)


5

w o >

8

o

2.

HVOSLEF

55


O b v i o u s l y these are p r e f e r r e d to a s y s t e m w i t h a b i c y c l i c r i n g as i n d i cated b y


O H

T h e c o n c l u s i o n f o l l o w s t h a t a n y s u b s t i t u e n t at C 2 t h a t i n d u c e s ^ - e l e c t r o n d e r e a l i z a t i o n into the region w i l l open the furanoid ring. is s i m p l y t h a t sp

2

T h e reason

h y b r i d i z a t i o n at C 3 p r e c l u d e s t h e necessary f o u r t h

valence for r i n g formation.

It has r e c e n t l y b e e n s h o w n t h a t solutions

c o n t a i n t w o isomers i n e q u i l i b r i u m w i t h 180° difference i n o r i e n t a t i o n of the hydrazine group.

T h e N - H group hence can establish hydrogen

b o n d s to e i t h e r O l or 0 3

(26).

Ascorbate Isomers O f substances c l o s e l y r e l a t e d to v i t a m i n C are isomers w i t h i n v e r t e d substituents at C 4 a n d C 5 . T h e so c a l l e d D-isoascorbic a c i d is a stereo i s o m e r w i t h i n v e r s i o n of t h e O H g r o u p at C 5 o n l y . A n x - r a y i n v e s t i g a t i o n b y A z a r n i a , B e r m a n , a n d R o s e n s t e i n (27)

revealed that i n this c o m p o u n d

t h e distances a n d angles are q u i t e s i m i l a r to those f o u n d i n L - a s c o r b i c a c i d ( T a b l e I I ) . T h e p l a n e r e l a t i o n s h i p is also c o n g r u o u s , b u t t h e p r o p e r t i e s of t h e s i d e - c h a i n are, of course, different as a r e s u l t of t h e i n v e r s i o n at C 5 . C 4 - C 5 - C 6 - 0 6 f o r m a roughly planar z i g z a g c h a i n where C 6 adopts a p o s i t i o n a p p r o x i m a t e l y c o r r e s p o n d i n g t o t h a t of 0 5 i n L - a s c o r b i c a c i d . T h e d i h e d r a l C 3 - C 4 - C 5 - C 6 a n g l e is + 7 8 ° vs. + 5 8 ° f o r t h e C 3 - C 4 - C 5 0 5 a n g l e i n the v i t a m i n . R e l a t i v e to 0 5 , 0 6 is i n t h e gauche c o n f o r m a t i o n t h a t takes i t farthest a w a y f r o m t h e r i n g ; 0 6 is t i e d t o 0 2 i n a n e i g h b o r i n g m o l e c u l e b y a short h y d r o g e n b o n d (2.584 A ) .

A l s o , the hydrogen

b o n d s y s t e m d u p l i c a t e s one of t h e schemes f o u n d i n the L - a s c o r b i c a c i d . K a n t e r s , R o e l o f s e n , a n d A l b l a s (28)

have compared

D-isoascorbic

a c i d a n d L - a s c o r b i c a c i d w i t h respect t o t h e i r b e h a v i o r o n i o n i z a t i o n . T h e y o b s e r v e d t h a t a l m o s t t h e same changes i n b o n d l e n g t h s a n d angles o c c u r i n t h e t w o c o m p o u n d s , e x e m p l i f i e d b y t h e i r s o d i u m salts ( T a b l e I I ) . E v e n the d i s t o r t i o n s i n t h e r i n g s are s i m i l a r ( K a n t e r s et a l / s a d v e r sary c o n c l u s i o n w a s b a s e d u p o n a m i s p r i n t i n T a b l e I of R e f . 1 0 ) . is i l l u s t r a t e d b y t h e a l m o s t i d e n t i c a l angles b e t w e e n

This

t h e best p l a n e s

t h r o u g h t h e l a c t o n e a n d e n e - d i o l groups ( 1 . 4 ° vs. 0 . 6 ° ) . T h e s i d e - c h a i n c o n f o r m a t i o n is n e a r l y r e t a i n e d f r o m t h e a c i d , b u t i t is m o r e (by + 3 1 ° )

staggered

by a rotation about C 4 - C 5 .


56

ASCORBIC ACID

W h e r e a s m o s t b o n d l e n g t h s a n d angles agree w e l l w i t h those o f other ascorbate anions ( T a b l e I I ) , t h e h y d r o g e n b o n d i n t e r a c t i o n asso c i a t e d w i t h 0 3 is u n i q u e : i t accepts n o less t h a n f o u r b o n d s r a n g i n g f r o m 2.785 t o 2.993 A . T w o of these a r e f r o m w a t e r m o l e c u l e s .

This unusually

l a r g e n u m b e r of b o n d s e x p l a i n s t h e fact t h a t n o short h y d r o g e n

bonds,

w h i c h a r e u s u a l l y e n c o u n t e r e d i n t h e e n e - d i o l g r o u p , a r e present i n these crystals.


T h e fate of D-isoascorbic a c i d o n o x i d a t i o n has n o t y e t b e e n s t u d i e d b y d i f f r a c t i o n m e t h o d s , b u t N M R results (29) s h o w t h a t i n i n e r t solvents a n a s y m m e t r i c d i m e r is p r e f e r e n t i a l l y f o r m e d .

I n water, however,

a

m i x t u r e of t w o a n o m e r i c p y r a n o s e r i n g s results, a n d t h e s i m p l e , r e v e r s i b l e D-isoascorbic

acid/dehydroisoascorbic

acid

e q u i l i b r i u m is lost.

This

m a y p o s s i b l y b e o n e of t h e factors t h a t reduces t h e b i o l o g i c a l a c t i v i t y of D-isoascorbic a c i d . T h e l a t t e r has a n effect t h a t is o n l y 5% of t h a t of v i t a m i n C (30).

Literature Cited 1. Cox, E. G.; Goodwin, T. H. J. Chem. Soc. 1936, 769. 2. Herbert, R. W.; Hirst, E. L.; Percival, E. G. V.; Reynolds, R. J. W.; Smith, F. J. Chem. Soc. 1933, 1275. 3. Haworth, W. N.; Hirst, E. L.; Tones, J. K. N.; Smith, F. J. Chem. Soc. 1934, 1192. 4. Hvoslef, J. Acta Crystallogr., Sect. B 1972, 28, 916. 5. Hvoslef, J.; Pedersen, B. Acta Chem. Scand., Ser. B 1979, 33, 503. 6. Hvoslef, J. Acta Chem. Scand. 1964, 18, 841. 7. Hvoslef, J. Acta Crystallogr., Sect. B 1968, 24, 23. 8. Ibid., 1431. 9. Sudhakar, V.; Bhat, T. N.; Vijayan, M. Crystallogr., Sect. B 1980, 36, 125. 10. Hvoslef, J. Acta Crystallogr., Sect. B 1969, 25, 2214. 11. Hughes, D. L. J. Chem. Soc., Dalton Trans. 1973, 2209. 12. Hearn, R. A.; Bugg, C. E. Acta Crystallogr., Sect. B 1974, 30, 2705. 13. Hvoslef, J.; Kjellevold, K. E. Acta Crystallogr., Sect. B 1974, 30, 2711. 14. Abrahams, S. C.; Bernstein, J. L.; Bugg, C. E.; Hvoslef, J. Acta Crystal logr., Sect. B 1978, 34, 2981. 15. Sudhakar, V.; Vijayan, M. Acta Crystallogr., Sect. B 1980, 36, 120. 16. Jeffrey, G. A.; Kim, H. S. Carbohydr. Res. 1970, 14, 207. 17. Tolbert, B. M.; Isherwood, D. J.; Atchely, R. W.; Baker, E. M. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1971, 30, 529. 18. McClelland, B. W. Acta Crystallogr., Sect. B 1974, 30, 178. 19. Cutolo, E.; Lorizza, A. Gazz. Chim. Ital. 1961, 91, 964. 20. Lee, C. H.; Seib, P.; Hoseney, R. C.; Liang, Y. T.; Deyoe, C. W. Cereal Chem. 1975, 20, 456. 21. Jernow, J.; Blount, J.; Oliveto, E.; Perotta, A.; Rosen, P.; Toome, V. Tetrahedron, 1979, 35, 1483. 22. Albers, H.; Müller, E.; Dietz, H. Hoppe-Seyler's Z. Physiol. Chem. 1963, 334, 243. 23. Müller-Mulot, W. Hoppe-Seyler's Z. Physiol. Chem. 1970, 351, 50. 24. Hvoslef, J.; Nordenson, S. Acta Crystallogr., Sect. B 1976, 32, 1665. 25. Ibid., 448.


2. HVOSLEF Crystallography of the Ascorbates

57

26. Pedersen, B. Acta Chem. Scand., Ser. B 1980, 34, 429. 27. Azarnia, N.; Berman, H.; Rosenstein, R. D. Acta Crystallogr., Sect. B 1971, 27, 2157. 28. Kanters, J. A.; Roelofsen, G.; Alblas, B. P. Acta Crystallogr., Sect. B 1977, 33, 1906. 29. Hvoslef, J.; Pedersen, B. Carbohydr. Res. 1981, 92, 9. 30. Brubacher, G.; Vuilleumier, J. P. Wiss. Veröff. Dtsch. Ges. Ernähr. 1965, 14, 61.


Received for review January 22, 1981. ACCEPTED April 8, 1981.


Crystallography of the Ascorbates - American Chemical Society

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