NMR Spectroscopy of Ascorbic Acid and Its Derivatives - Advances in

6 N M R Spectroscopy of Ascorbic A c i d and Its Derivatives J. V. PAUKSTELIS—Department of Chemistry, Kansas State University, Manhattan, KS 66506

Downloaded by UNIV OF BATH on October 30, 2014 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch006

D. D. MUELLER—Department of Biochemistry, Kansas State University, Manhattan, KS 66506 P. A. SEIB and D. W. LILLARD, JR. —Department of Grain Science, Kansas State University, Manhattan, KS 66506 1

13

C resonances of L-ascorbic acid (I) at 25.2 MHz were iden tified from proton-coupled spectra, spin-lattice relaxation times, changes in chemical shifts with ionization, and 4-D isotopic substitution. C N M R spectroscopy was used to differentiate 2-O- from 3-O-, and 5-O- from 6-O-substituted derivatives of I. The H NMR spectra of I, 4-D-L-ascorbic acid, 5-D-L-ascorbic acid, D-isoascorbic acid, and 5-D-D-isoascorbic acid were recorded at 600.2 MHz. The proton 13

1

-proton vicinal coupling constants showed the conforma tion of the side-chain of I in water to be the same as that in its crystalline state. Unlike the solid state, however, the con formation did not change when I ionized at OH3. In the proton-coupled C NMR spectrum of I at 25.2 MHz, virtual coupling occurred between H6, H6', and C4. To resolve J spectra must be measured at a field strength exceeding 2.3 T. 13

3

C4H6',

' T h e detailed structure of L-ascorbic acid is important in understanding its biological and chemical properties. The formula of L-ascorbic acid (I) was first deduced in 1933 by Herbert et al. (1), and was later confirmed using x-ray crystallography (2). Hvoslef (3), who also exam ined the structure of sodium l-ascorbate (II), concluded, as others had previously proposed (4,5,6), that the monoanion of I is formed by ionization of OH3, and that the predominant resonance form of the monoanion is the 2,3-enolate form. Data from C NMR studies (7) on II also were in accord with those conclusions. 1 3

1

Current address: Spring and Durum Wheat Quality Research Laboratory, SEA, U.S. Department of Agriculture, North Dakota State University, Fargo, ND 58105. 0065-2393/82/0200-0125$07.75/0 © 1982 American Chemical Society In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

126

ASCORBIC ACID

CH OH I H—C—OH

CHoOH I H — C — O H

2

0

HO

0

OH I


From

OH C

ONa II

the crystal structure data the ring

i n t h e free

acid

(I)

w a s f o u n d t o b e n e a r l y p l a n a r a n d c o n t a i n a s l i g h t d i s t o r t i o n of t h e l a c t o n e atoms o u t of t h e e n e - d i o l p l a n e . I n t h e s i n g l y c h a r g e d a n i o n (II), the situation was reversed, a n d the e n e - d i o l

atoms

were

puckered,

w h e r e a s t h e l a c t o n e g r o u p w a s v i r t u a l l y p l a n a r . S o m e d e r e a l i z a t i o n of e l e c t r o n d e n s i t y o v e r t h e r i n g w a s e v i d e n c e d i n II b y changes i n b o n d lengths a n d angles w h e n I i o n i z e d . I n a d d i t i o n , the p r i m a r y a l c o h o l g r o u p (OH6)

i n II r o t a t e d m o r e t h a n 100° f r o m its p o s i t i o n i n I. T h u s , t h e

conformation about the C 5 - C 6 b o n d changed from that i n I where 0 5 a n d 0 6 a r e n e a r l y a n t i p a r a l l e l t o a gauche o r i e n t a t i o n i n t h e a n i o n (II). C o n f o r m a t i o n a l c h a n g e m a y reflect p r e f e r e n t i a l i n t e r a c t i o n s i n t h e c r y s t a l t h a t a r e n o t necessarily a v a i l a b l e t o t h e m o l e c u l e i n s o l u t i o n . N o i n t r a m o l e c u l a r h y d r o g e n b o n d s w e r e f o u n d i n t h e crystals of I o r II (2, S). The

objectives

of t h i s w o r k

were

t o v e r i f y t h e assignments

of

c a r b o n - 1 3 resonances i n L - a s c o r b i c a c i d ( I ) , t o use t h e c a r b o n - 1 3 c h e m i c a l shifts t o assign positions of s u b s t i t u t i o n i n d e r i v a t i v e s of I, a n d t o d e t e r m i n e t h e c o n f o r m a t i o n a l p r e f e r e n c e of I a n d its s o d i u m salt ( I I ) i n aqueous solution.

C

13

NMR Studies of L-Ascorbic Acid (I) C o m m e n s u r a t e w i t h t h e b i o l o g i c a l a n d c h e m i c a l i m p o r t a n c e of I,

investigators h a v e s t u d i e d t h e N M R p r o p e r t i e s of this m o l e c u l e a n d sev e r a l of its d e r i v a t i v e s (7-10). the

assignments

T h e investigators g e n e r a l l y h a v e a g r e e d t o

originally p u t forth

p r o o f of assignments has b e e n The { H} J

1 3

(7,8).

However,

no

complete

reported.

C N M R s p e c t r u m of L - a s c o r b i c a c i d is s h o w n i n F i g u r e

1 A . T h e most u p f i e l d resonance arose f r o m the p r i m a r y a l c o h o l g r o u p a t C 6 b e c a u s e of its t r i p l e t p a t t e r n i n p r o t o n - c o u p l e d s p e c t r a , a n d b e c a u s e t h e c h e m i c a l shift of 63.1 p p m is t y p i c a l o f p r i m a r y a l c o h o l s i n c a r b o h y d r a t e s (11).

O f the remaining t w o protonated

carbons, w h i c h are

doubles w h e n proton coupled, C 4 w o u l d be expected to occur at lower field t h a n C 5 b e c a u s e of t h e e n e - d i o l g r o u p i n a ^ - p o s i t i o n . T h i s h y p o t h e -

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


160

13

140 8, p.p.m.

120

C2

100

2

J.

OH

—I

80

C4

I—

C5

1

60

C6

13

Figure 1A. Proton-decoupled C NMR spectrum of L-ascorbic acid in H 0, pH 2.0, 33°C. Short-range multiplicities arising from proton coupling are Cl (S), C2 (S), C3 (S), C4 (D), C5 (D), and C6 (T), where S, D, and T refer to singlet, doublet, and triplet, respectively. The direct and long-range H- C coupling constants are summarized in Table VII.

180

Cl

C3

HO

HCOH

9

CH 0H


128

ASCORBIC

sis w a s c o n f i r m e d b y p r e p a r a t i o n of t h e 4-deutero

compound

(Figure

I B ) , w h i c h s h o w e d o n l y a w e a k t r i p l e t c e n t e r e d at 76.7 p p m . assignments agree w i t h those g i v e n b y e a r l i e r w o r k e r s

ACID

These

(7,8).

T h e assignments f o r the three n o n p r o t o n a t e d c a r b o n s i n the d o w n f i e l d p o r t i o n w e r e not as easily d e d u c e d .

T h e l o n g - r a n g e c o u p l i n g patterns of

those resonances w e r e c o m p l e x a n d p r o b a b l y a m b i g u o u s N e v e r t h e l e s s , t h e resonance at 156.4 p p m p r o t o n c o u p l i n g constant ( ~

( F i g u r e 1)

(vida infra).

h a d t h e largest

6 H z ) a n d reasonably c o u l d be assigned

to C 3 . F u r t h e r m o r e , c a r b o n y l carbons are t y p i c a l l y the most d o w n f i e l d resonances

i n compounds

like L-ascorbic acid, a n d lactone

carbonyl


c a r b o n s i n p a r t i c u l a r are k n o w n to f a l l i n the 1 7 0 - 1 8 0 - p p m r a n g e

(II).

T h e r e f o r e , t h e resonance at 174.0 p p m w a s a s s u m e d i n i t i a l l y to c o r r e s p o n d t o C l . T h e p e a k at 118.8 p p m c o u l d b e a s s i g n e d to C 2

by

difference. T o h e l p c o n f i r m t h e p r o p o s e d assignments, the p H d e p e n d e n c e of t h e c h e m i c a l shifts w a s

studied.

A

p H d e p e n d e n c e s t u d y of

I was

r e p o r t e d ( 9 ) , b u t t h e solutions a p p a r e n t l y w e r e not p u r g e d to

remove

a t m o s p h e r i c o x y g e n , a n d u n d e r a l k a l i n e c o n d i t i o n s t h e solutions h a v e c h a n g e d p H d u r i n g t h e r e c o r d i n g s of the spectra. the experiment under an inert atmosphere

We

may

repeated

to a v o i d oxidative

degra

d a t i o n of L - a s c o r b i c a c i d . O u r d a t a ( F i g u r e 2 ) a n d t h a t of B e r g e r

(9)

are i n e x c e l l e n t a g r e e m e n t b e t w e e n p H 2 a n d 7, b u t i n less t h a n satis factory

agreement

between

pH

7

and

11.

The

earlier data

show

c o n s i d e r a b l y s m a l l e r shifts over t h e h i g h e r p H r a n g e . The

s i g n a l at 156.4 p p m

undergoes

a 19.3-ppm

downfield

shift

b e t w e e n p H 2 a n d 6 ( F i g u r e 2 A ) , c o r r e s p o n d i n g to t h e r a n g e o v e r w h i c h O H 3 ionizes ( 1 2 ) .

A l t h o u g h s u c h a shift is m u c h l a r g e r t h a n t h e 4 - 5 - p p m

d o w n f i e l d shift n o r m a l l y o b s e r v e d for i o n i z a t i o n of c a r b o x y l i c a c i d s

(11),

i t a l m o s t c e r t a i n l y arose f r o m C 3 . O v e r the same p H r a n g e , C l w a s d e s h i e l d e d b y 3.9 p p m , w h i c h w a s e n o u g h to p r e v e n t C 3 f r o m m o v i n g d o w n f i e l d of C l ( F i g u r e 2 A ) .

Similarly, C 4 was shifted downfield b y

2.1 p p m ( F i g u r e 2 B ) . O n t h e other h a n d , C 2 m o v e d u p f i e l d b y 4.6 p p m , a n d the s i d e - c h a i n c a r b o n s C 5 a n d C 6 w e r e d e s h i e l d e d b y 0.55 a n d 0.46 p p m , r e s p e c t i v e l y . C o n s e q u e n t l y , i o n i z a t i o n of L - a s c o r b i c a c i d at C 3 also shifts t h e signals of t h e other r i n g carbons m o r e t h a n is o b s e r v e d

for

adjacent

the

carbons

i n simple carboxylic acids.

l e n g t h e n i n g of t h e C 2 - C 3 , C 3 - C 4 , C l - O l ,

T h i s is c a u s e d

by

a n d C 2 - 0 2 bonds i n the

m o n o a n i o n (II) c o m p a r e d w i t h I, as w e l l as t h e s h o r t e n i n g of t h e C 1 - C 2 and C 3 - 0 3 bonds ( 2 , 3 ) .

F u r t h e r m o r e , d e r e a l i z a t i o n of e l e c t r o n d e n

sity t h r o u g h o u t the e n e - d i o l a n d c a r b o n y l g r o u p s p r o b a b l y is m a i n l y responsible

for

the

abnormally

large

downfield

shift

of

C3

ionization.


upon


Cl

C3

13

C2

HO

HCOH

CHgOH

OH

0

C4

C5 C6

h

Figure IB. Proton-decoupled C NMR spectrum of 4-D-L-ascorbic acid in HJD, pH 2.3, 33°C. Deuteration at C4 produced a 0.41-ppm upfield shift in the C4 peak. Shifts are relative to internal Me Si.

B


In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982. 2

2

Figure 2. A . pH dependence of the chemical shifts of iu-ascorbic acid in H 0 for CI, C2, and C3, 33°C. B. pH dependence of the chemical shifts of ^-ascorbic acid in H 0 for C4, C5, and C6, 33°C.


5

• o

s

o o

o

CO

6.

Spectroscopy of Ascorbic Acid

131

B e t w e e n p H 6 a n d 9 n o s u b s t a n t i a l shifts i n t h e c a r b o n

resonances

NMR

PAUKSTELIS E T A L .

of II w e r e n o t e d .

A b o v e p H 10, O H 2 b e g a n to i o n i z e a n d t h e C 2 reso

nance m o v e d downfield r a p i d l y w i t h increasing p H , w h i l e the C 3 a n d C 4 peaks s h i f t e d u p f i e l d , w h i c h r e v e r s e d the t r e n d seen b e t w e e n p H 2 a n d 7. C a r b o n s C l , C 5 , a n d C 6 c o n t i n u e d to s h o w i n c r e a s e d d e s h i e l d i n g at p H v a l u e s a b o v e 10.

Perhaps the tendency

t o w a r d increased

derealiza

t i o n seen w i t h t h e first i o n i z a t i o n w a s r e v e r s e d s o m e w h a t w h e n C 2 i o n i z e d . T h e c o n t i n u e d d o w n f i e l d shift of C l , h o w e v e r , w o u l d n o t fit t h a t explanation. F u r t h e r c o n f i r m a t i o n of t h e assignments of t h e n o n p r o t o n a t e d


bons was obtained from measurement (Ti).

R e l a x a t i o n t i m e s f o r t h e carbons

car

of s p i n - l a t t i c e r e l a x a t i o n t i m e s of I d e t e r m i n e d i n d e u t e r i u m

o x i d e s o l u t i o n at 3 3 ° C a n d p H 1.6 are s u m m a r i z e d i n T a b l e I. r e l a x a t i o n times of C 4 , C 5 , a n d C 6 are t y p i c a l of p r o t o n a t e d

The

carbon

atoms i n a n i s o t r o p i c a l l y t u m b l i n g m o l e c u l e of this size, b u t t h e r e l a x a t i o n t i m e s of n o n p r o t o n a t e d carbons are u n u s u a l l y l o n g . t r e n d a m o n g t h e r e l a x a t i o n t i m e s of the n o n p r o t o n a t e d

However,

the

carbons w a s of

increasing time w i t h increasing distance from H 4 . T h i s trend w o u l d be expected

if the

assignments

c o n t r i b u t i o n to t h e

were

correct

and

H 4 made

u s u a l l y inefficient relaxations of t h e n o n p r o t o n a t e d will

be

discussed

elsewhere

(13).

A g a i n the

NMR Studies of Derivatives of L-Ascorbic have used

1 3

major

The

un

c a r b o n atoms i n I

assignments

p r o t o n a t e d carbons agree w i t h those r e p o r t e d e a r l i e r

Researchers

the

d i p o l a r r e l a x a t i o n of t h e r i n g c a r b o n s .

for

non

(7,8).

Acid.

C N M R s p e c t r o s c o p y to assign s t r u c t u r e

to several ester a n d ether d e r i v a t i v e s of I.

T h a t m e t h o d is p a r t i c u l a r l y

u s e f u l to differentiate 2 - 0 - a n d 3 - O - s u b s t i t u t e d d e r i v a t i v e s of I because i o n i z a t i o n of the O H 3 i n d u c e s t h e l a r g e d o w n f i e l d shift of C 3 , as p r e v i ously discussed. T h u s , 2 - O - m e t h y l - L - a s c o r b i c a c i d , b u t not 3 - O - m e t h y l - L a s c o r b i c a c i d , s h o w e d a 1 6 - p p m c h a n g e i n the c h e m i c a l shift of C 3 w h e n t h e p H values of t h e i r solutions w e r e c h a n g e d f r o m 2 to 7 ( T a b l e A t p H 7, the c h e m i c a l shifts of the C 3 carbons i n L - a s c o r b a t e (II)

its 2-sulfate a n d 2-phosphate esters w e r e s i m i l a r i n m a g n i t u d e ( T a b l e

Table I.

0

N o t e : I n D2O

33°C. a

II).

Spin-Lattice Relaxation Times for the Carbon Atoms of L-Ascorbic A c i d 1

Carbon Atom NT^sec)

II).

and i n

132 under N2,

2

3

98

38

4 1.60

p H 1.6 (meter reading i n D2O

5 1.44

6 2.02

u s i n g a glass electrode),

N, n u m b e r of d i r e c t l y bonded protons, if n o t zero.


132

ASCORBIC ACID

Table I I . Chemical Shifts for Carbons i n L-Ascorbic A c i d a n d Several Derivatives 0

Carbon Atom

Derivative of L-Ascorbic Acid

1

3

2

4

5

6

69.9

63.1

pH 2.0-2.1" Unsubsituted (this w o r k )

174.0

2-0-Methyl*


04) 3-0-Methyl* (14, 10)

118.8

156.4

173.7 (-0.3)"

122.5 (3.7)

162.1 (5.7)

77.0 (•- 0 . 1 )

69.7 (-0.2)

62.8 (•- 0 . 3 )

174.1 (0.1)

119.2 (0.4)

155.9 (-0.5)

76.9 (•- 0 . 2 )

69.8 (-0.1)

( -0.3)

77.1

62.8

pH 6.5-7.0" Unsubsituted (this w o r k )

178.0

114.1

176.2

79.2

70.6

2-O-Methyl"

179.4 (1.4)"

119.3 (5.2)

178.1 (2.1)

79.3 (0.1)

70.4 (-0.2)

(-0.3)

174.8 (-3.2)

120.4 (6.3)

155.0 (-21.2)

76.8 ( -2.4)

70.0 (-0.6)

(-0.6)

181.1 (3.1)

111.6 (-2.5)

176.7 (0.5)

79.9 0.7)

70.7 (0.1)

63.5 (0.1)

177.4 (-0.6)

113.2' (-0.9)

177.0 (0.8)

78.7 ( -0.5)

70.1 (-0.5)

( -0.7)

U4) 3-O-Methyl* (14,10)

2-Sulfate (10,15) 2-Phosphate (10,15)

63.6 63.3 63.0

62.9

° C h e m i c a l shifts (5 f r o m M e 4 S i ) . p H meter reading i n D2O using a glass electrode. S i g n a l of O M e at 6 1 2 a n d 61.6 p p m , p H 2 a n d 7, respectively. Difference between c h e m i c a l shift of parent c o m p o u n d ( I o r I I ) a n d d e r i v a t i v e . Signal of O M e at 6 0 5 a n d 60.1 p p m at p H 2 a n d 7, respectively. ' D o u b l e t w i t h s p l i t t i n g 7.3 H z ( C - P c o u p l i n g ) . h e

d

e

1 3

The monoanion

3 1

( I I ) i n v o l v e s O H 3 ( 3 ) ; therefore, 2 - s u b s t i t u t i o n of t h e

p h o s p h a t e a n d sulfate groups is i n d i c a t e d . The

1 3

C N M R s p e c t r u m of 3 - O - m e t h y l - L - a s c o r b i c a c i d a n d t h a t of i t s

parent compound

( I ) were almost identical at p H 2 ( T a b l e I I ) . T h e

3 - m e t h y l d e r i v a t i v e is a v i n y l ether, a n d i t c o u l d h a v e b e e n h y d r o l y z e d at p H 2 d u r i n g r e c o r d i n g of t h e s p e c t r u m .

However, hydrolysis d i d not

o c c u r since t h e m e t h y l s i g n a l w a s o b s e r v e d a t 60.2 p p m a n d n o m e t h a n o l s i g n a l w a s f o u n d at 48 p p m . B e c a u s e t h e s i g n a l of H 4 m o v e s u p f i e l d b y a p p r o x i m a t e l y 0.5 p p m w h e n O H 3 i o n i z e s , * H N M R s p e c t r o s c o p y also c a n b e u s e d to differ entiate b e t w e e n 2 - 0 - a n d 3 - O - s u b s t i t u t e d d e r i v a t i v e s of I . T h e u p f i e l d shifts f o r H 4 i n s e v e r a l 2 - O - s u b s t i t u t e d d e r i v a t i v e s of I a r e g i v e n i n T a b l e


6.

NMR


133


Table III. Proton Magnetic Resonances of L-Ascorbic A c i d and Several Derivatives Derivative of L-Ascorbic

Chemical Shift

0

pH

Acid

F r e e a c i d (16) 3-0-Methyl

c

(14)


2 - O - M e t h y l " (14) 2 - S u l f a t e (16) 2 - P h o s p h a t e (16) 2,2'-Phosphoric diester (16)

H4

AH4*

(h)

H5

H6, H6'

2 7

4.97 4.50

0.47

4.09 4.02

3.76 3.74

2 7

4.91 4.90

0.01

4.02 4.01

3.71 3.71

2 7

4.90 4.49

0.41

4.01 4.00

3.76 3.71

1 7

5.02 4.57

0.45

4.20 4.05

3.76 3.73

1 7

5.00 4.60

0.40

4.16 4.05

3.74 3.70

1 12

5.00 4.50

0.50

4.15 4.02

3.78 3.68

—

° D e t e r m i n e d i n D2O at 60 or 100 M H z . C h e m i c a l shifts i n p p m from internal D S S . C h e m i c a l shifts of H 5 a n d H 6 are reported as the center of the recorded peaks. A H 4 is the upfield shift of H 4 o n changing the p H of the m e d i u m . Signal of O M e was 4.18 p p m at p H 2 a n d 7. A t p H 10, the spectrum showed degradation of the 3-methyl ether. Signal of O M e was 3.69 p p m at p H 2 a n d 3.62 p p m at p H 7. 6

c

d

III.

T h e H 4 s i g n a l of the 3 - m e t h y l ether f a i l e d to s h i f t to h i g h e r

field

at p H 7. D a t a f r o m U V s p e c t r o s c o p y are h e l p f u l i n d i s t i n g u i s h i n g 2- a n d 3-derivatives of L - a s c o r b i c a c i d . T h e i o n i z a t i o n of O H 3 is a c c o m p a n i e d b y a b a t h o c h r o m i c shift of a p p r o x i m a t e l y 20 n m ( T a b l e I V ) . Workers have used

1 3

C N M R s p e c t r o s c o p y to a s s i g n structures to

t h e 5- a n d 6-sulfate esters of L - a s c o r b i c a c i d a n d t o t h e 4 Z a n d 4 E isomers of 2-sulfo-2,3,4,6-tetrahydroxyhexa-2,4-dienoate-8-lactone

(Table

V ) . S u l f o n a t i o n at C 6 (C5) of L - a s c o r b i c a c i d s h i f t e d t h e s i g n a l of C 6 (C5) d o w n f i e l d b y 7 - 8 p p m ; the s i g n a l ( s ) of the adjacent c a r b o n ( s ) slightly upfield (18).

moved

T h o s e shifts w e r e n o t e d p r e v i o u s l y b y others

(19)

i n s u g a r sulfates. W h e n I w a s d i s s o l v e d i n c o n c e n t r a t e d s u l f u r i c a c i d - d , 2

1 3

C N M R shows a p p r o x i m a t e l y 9 0 % m o n o s u l f o n a t i o n at C 6

(18).

T h e 2-sulfate ester of 4Z-2,3,4,6-tetrahydroxyhexa-2,4-dienoate-8-lactone (4,5-dehydroascorbate

2-sulfate) w a s c h a r a c t e r i z e d l a r g e l y b y

1 3

C

N M R a n d H N M R . T h e d a t a i n T a b l e V s h o w t h a t the signals of C 4 a n d X

C 5 w e r e s h i f t e d d o w n f i e l d 70 a n d 38 p p m , r e s p e c t i v e l y , c o m p a r e d to t h e i r p o s i t i o n s i n t h e s p e c t r u m of L - a s c o r b a t e at p H 7. I n a d d i t i o n , t h e reso nances of C l a n d C 3 m o v e d u p f i e l d a p p r o x i m a t e l y 5 p p m u p o n i n t r o d u c t i o n of t h e 4,5-double b o n d .


134

ASCORBIC ACID

Table I V .

U V Spectral Properties of L-Ascorbic A c i d and Several Derivatives Acid (pH, 2.0)

Derivative of L-Ascorbic Acid

Base (pH 10.0) (•mM

hmax

*mM

F r e e a c i d (17)

243

10

265

16.5

2-0-Methyl

(U)

239

8.5

260

12.3

3-0-Methyl

(17)

244

9

244

9

233

9.6

257

14.5

258

15

237

8

238

8

263

8

232

11

255

16.3

255

16.3

238

9

258

11.5

264

16.0

236

17.3

258

21.6

259

30.2

2 - 0 - (Phenylphosphono)


Neutral (pH 7.0)

3-0-(Phenylphosphono)

a

a

2 - S u l f a t e (16) 2-Phosphate

(16)

2 , 2 ' - P h o s p h o r i c diester (16) B

— — —

5,6-Isopropylidene acetal; data from Bond et al. (17). T e n t a t i v e l y , the Z - c o n f i g u r a t i o n w a s assigned to t h e 4,5-ene

i n the derivative p r e p a r e d f r o m L-ascorbic acid.

bond

T h i s assignment

was

b a s e d o n t h e s t e r e o c h e m i c a l a r g u m e n t t h a t i n t h e t r a n s i t i o n state of t h e elimination reaction, H 4 a n d 0 5

assume

an antiparallel orientation.

U s i n g t h e same a r g u m e n t t h e E - c o n f i g u r a t i o n w a s a s s i g n e d to t h e i s o m e r p r e p a r e d f r o m D - i s o a s c o r b i c a c i d (III).

The

1 3

C data i n Table V

show

t h a t b o t h isomers h a v e b e e n i s o l a t e d . T h e scheme u s e d to p r e p a r e t h e

CH OH 2

H O — C — H

III 4 , 5 - d e h y d r o d e r i v a t i v e s is s h o w n i n S c h e m e 1. O t h e r 4 , 5 - d e h y d r o d e r i v a tives of I are k n o w n

(20).

T h e s t r u c t u r e of t h e 4 , 5 - d e h y d r o c o m p o u n d s w a s v e r i f i e d u s i n g

X

H

N M R s p e c t r o s c o p y ; t h e s p e c t r u m of t h e Z - i s o m e r at p H 6.2 is s h o w n i n F i g u r e 3. T h e s i g n a l of H 5 w a s a t r i p l e t c e n t e r e d at 5.59 p p m w i t h J 5 , H 6 H

=

7.3 H z , a n d H 6 w a s a d o u b l e t at 4.34 p p m . F o r t h e E - i s o m e r at p H



1 3

6

8

Prepared starting from L-ascorbic acid. Prepared starting from D-isoascorbic acid.

172.6 (-5.4)

(E) - 4 , 5 - D e h y d r o - 2 - s u l f ate (this w o r k ) "

179.4 (1.4)

6 - S u l f a t e (18)

172.7 (-5.3)

177.6 (-0.4)

5 - S u l f a t e (18)

(Z) - 4 , 5 - D e h y d r o - 2 - s u l f ate (this w o r k ) *

178.0

Cl

13

C2

116.0 (1.9)

114.2 (0.1)

116.1 (2.0)

113.9 (-0.2)

114.1

2

170.2 (-6.0)

171.2 (-5.0)

176.1 (-0.1)

175.7 (-0.5)

176.2

CS

Chemical Shifts in H 0 at pH 6.5-7.0)

C N M R and U V D a t a

C (S-Values

V.

S o d i u m salt (this work)

L - A s c o r b i c Acid

Table

148.4 (69.2)

149.4 (70.2)

80.5 (1.3)

77.6 (-1.6)

79.2

C4

13

108.6 (38.0)

108.2 (37.6)

69.8 (-0.8)

77.4 (6.8)

70.6

C5

2

Monosulfate

Esters

58.4 (-5.2)

57.7 (-5.4)

71.5 (7.9)

61.4 (-2.2)

63.6

C6

C Chemical Shifts (S-Values in H 0 at pH 6.5-7.0)

for Several


260

260

244

245

244

pH2

247

244

266

267

265

H7 P

2

UV in H 0)

ASCORBIC ACID


136



6.

NMR


137


Figure 3. 100-MHz H NMR spectrum of 4Z-2-sulfo-2,3,4,6-tetrahydroxyhexa-2,4-dieonate-$-lactone (4,5-dehydroascorbate 2-sulfate) in D 0, pH (meter reading) 6.2, 33°C. Shifts are from internal Me Si. 2

2

h

5.9, H 5 w a s a t r i p l e t at 5.80 p p m w i t h

/ 5,H6 H

=

6.6 H z , a n d H 6 w a s a

d o u b l e t at 4.59 p p m . A s m e n t i o n e d p r e v i o u s l y , i o n i z a t i o n of O H 3 i n L - a s c o r b i c a c i d is a c c o m p a n i e d b y a shift i n A opposite

is t r u e i n t h e case

m a x

of

to l o n g e r w a v e l e n g t h s .

( T a b l e V ) . A t p H 0.5-1.0 those d e r i v a t i v e s gave A at p H 7 A

m a x

H o w e v e r , the

the 4,5-dehydro-2-sulfate m a x

derivatives

of 2 6 0 - 2 6 2 n m , b u t

w a s 2 4 4 - 2 4 7 n m . A p p a r e n t l y most of the c h a r g e i n t h e a n i o n

is o n O l ( S c h e m e 2 ) , w h e r e a s most of the c h a r g e o n t h e ascorbate m o n o a n i o n ( I I ) is o n 0 3

Scheme 2.

(3).

Ionization of 4,5-dehydroascorbate-2-sulfate.


138

ASCORBIC

Tolbert a n d W a r d have reviewed the

1 3

C N M R data on

ACID

dehydro-

L-ascorbic a c i d i n chapter 5 i n this book.

H NMR Spectroscopy and the Conformations of L-Ascorbic Acid

1

and D-isoascorbic Acid in Aqueous Solution I n 1977

C N M R s p e c t r o s c o p y w a s u s e d to a s s i g n a

1 3

to t h e s i d e - c h a i n of L - a s c o r b i c

acid (I)

i n water

conformation That

(21).

report

s t i m u l a t e d us to m e a s u r e t h e 6 0 0 - M H z * H N M R s p e c t r a of I a n d D - i s o


a s c o r b i c a c i d ( i n ) a n d t h e i r 4- a n d 5-deutero d e r i v a t i v e s . T h e 6 0 0 - M H z * H N M R s p e c t r a w e r e a n a l y z e d , a n d t h e v i c i n a l c o u p l i n g constants w e r e u s e d to p r e d i c t p r e f e r r e d

conformations.

T h e * H N M R s p e c t r u m of L - a s c o r b i c a c i d at l o w observed

by

many

100 M H z g a v e H6,

3

groups

/H4,H5

a n d was p u b l i s h e d

(22).

fields

has

The

were

H o w e v e r , t h e 1 0 0 - M H z s p e c t r u m of I c a n b e s i m u l a t e d

b y m a c h i n e c o m p u t a t i o n w i t h a h i g h d e g r e e of c o r r e l a t i o n w h e n 3

at

of 1.8 H z , b u t the c o u p l i n g constants b e t w e e n H 5 ,

a n d H 6 ' c o u l d not b e o b t a i n e d b y i n s p e c t i o n b e c a u s e t h e y

strongly coupled. and

been

results

3

/ 5,H6 H

/ H 5 , H G ' are a s s u m e d to b e 6.6 H z , w h i c h is a t y p i c a l v a l u e i n ethane

d e r i v a t i v e s (23).

B u t i f H 6 a n d H 6 ' h a v e different c h e m i c a l shifts a n d

different c o u p l i n g constants, t h a t is, computer

3

/ 5,H6 H

simulation w i t h the assumed

does n o t e q u a l

3

/H5,H6',

equivalent coupling

the

constants

w o u l d s t i l l a r r i v e at a n a p p a r e n t l y c o r r e c t s o l u t i o n b e c a u s e at 100 M H z it is v e r y difficult to e x p e r i m e n t a l l y m e a s u r e a l l t h e s p e c t r a l l i n e s .

To

d e t e c t the difference b e t w e e n t h e t w o c o u p l i n g constants i n q u e s t i o n , t h e spectrometer w o u l d h a v e to r e c o r d s p e c t r a l lines t h a t are 0 . 1 %

of t h e

m o s t intense lines. T h a t s e n s i t i v i t y w o u l d r e q u i r e a signal-to-noise r a t i o i n excess of 2 0 0 0 : 1 , w h i c h is not r e a d i l y o b t a i n a b l e at 100 M H z . high-field

spectra

of

L-ascorbic

acid have

been

published,

No

although

some p r e l i m i n a r y s p e c t r a at 360 M H z w e r e m a d e a v a i l a b l e to us

(24).

W e h a v e e x a m i n e d L - a s c o r b i c a c i d a n d its 4 - D a n d 5 - D d e r i v a t i v e s at 600.2 M H z at the C a r n e g i e - M e l l o n N M R F a c i l i t y for

Biomedical

Studies. W e also o b t a i n e d d a t a o n D-isoascorbic a c i d a n d its 5 - D d e r i v a t i v e . T h e s i m u l a t e d a n d o b s e r v e d s p e c t r a for H 5 , H 6 , a n d H 6 ' of I are s h o w n at p H 2 a n d 7 i n F i g u r e s 4 a n d 5. T a b l e V I presents the c o u p l i n g constants o b t a i n e d f r o m c o m p u t e r s i m u l a t i o n of t h e s p i n system u s i n g a standard iterative (25)].

fitting

program

[provided

The N M R data were recorded

by

Nicolet

Corporation

using correlation spectroscopy,

w h i c h gave a p o i n t r e s o l u t i o n of a b o u t 0.1 H z . T h e least-squares fits g a v e differences b e t w e e n o b s e r v e d a n d c o m p u t e d l i n e positions t h a t a v e r a g e d 0.04 H z . C o n s e q u e n t l y , t h e J v a l u e s are a c c u r a t e to at least ± 0 . 1 H z .



6.

PAUKSTELIS

ET

139

NMR Spectroscopy of Ascorbic Acid

AL.

pH 2.0 (SIMULATED)

I A

pH 2.0

1

1

1

I

I

440

I

I

1

I

1

-540

1 -640

Hz, upfield from HOD Figure 4. A. Observed 600.2-MHz *H NMR spectrum for H5, H6, and H6' of L-ascorbic acid in D O, pH (meter reading) 2.1. B. Computer simulated spectrum giving the calculated H- H coupling constants listed in Table VI. x

1

1


1

140

ASCORBIC

ACID


pH 7.25 (SIMULATED)

I

I

-460

I

I

I

I

I

•

1

-560 Hz, upfield from HOD

1

-660

Figure 5. A . Observed 600.2-MHz *H NMR spectrum for H5 and H6 H6' of L-ascorbic acid in D 0, pH (meter reading) 7.25. B. Computer simulated spectrum giving the calculated H- H coupling constants listed in Table VI. 9

2

1

1


6.


141


Table V I . Proton—Proton Coupling Constants and A8e,6' for L-Ascorbic and D-Isoascorbic Acids Compound

pH

L-Ascorbic 4-D-L-Ascorbic 5-D-L-Ascorbic

3

5-Z)-D-Isoascorbic

J 6,«'

1.83

5.81

7.34

-11.55"

7.25

1.93

5.65

7.63

-11.60

2.04

5.76

7.36

-11.55

6.29

7.09

5.74

7.65

-11.73

14.42

-11.61

6.37

—

14.63

2.35

D-Isoascorbic

2

2.06'

6.48 14.77

—

-11.58

NR*

NR

NR

NR

7.09

2.96

3.24

7.93

-11.99

6.19

-11.85

12.62

—

—

—

-11.95

6.25

7.20


J

2.45 7.76

NR

NR

Note: Data computed from spectra obtained at 6002 MHz in D2O. Probable error in J ~ 0.10 Hz. pH meter readings in D2O using a glass electrode. Assumed to be negative in the simulation, and all vicinal coupling constants assumed to be positive. Not recorded. 0

6

0

T h e i n f o r m a t i o n i n T a b l e V I m a y b e s u m m a r i z e d as f o l l o w s :

3

JHS,H6

d i d n o t e q u a l / 5 , H 6 ' f o r e i t h e r i s o m e r ; n o changes i n g e m i n a l o r v i c i n a l 3

H

c o u p l i n g constants o c c u r r e d b e t w e e n p H 2 a n d 7 f o r L - a s c o r b i c a c i d , except for a slight change i n / 5 , H6', w h i c h w a s barely outside the error 3

H

l i m i t s ; a t p H 2 t h e difference i n c h e m i c a l shifts b e t w e e n H 6 a n d H 6 ' f o r L - a s c o r b i c a c i d w a s 6.4 H z b u t a t p H 7 i t w a s 14.6 H z ; a n d t h e d i f f e r ence i n c h e m i c a l shifts b e t w e e n H 6 a n d H 6 ' w a s r e v e r s e d f o r D-isoascor b i c , t h a t i s , t h e difference w a s 12.6 H z at p H 2 a n d 6.2 H z a t p H 7. T h e fact that J , H 6 3

5 H

d i d not equal / 5 , H 6 ' indicated that i t might 3

H

b e p o s s i b l e t o d e t e r m i n e t h e c o n f o r m a t i o n of t h e s i d e - c h a i n i n L - a s c o r b i c a c i d . F u r t h e r m o r e , t h e N M R d a t a g a v e n o e v i d e n c e of c o n f o r m a t i o n c h a n g e b e t w e e n p H 2 a n d 7, i n contrast w i t h x - r a y d a t a ( 3 ) . T h e c o n f o r m a t i o n p r e v i o u s l y a s s i g n e d (21) t o t h e C 5 - C 6 b o n d of I i n w a t e r is n o t consistent w i t h t h e 6 0 0 - M H z

J

H N M R data.

T h e v a l u e s of / 5 , H 6 a n d / 5 , H 6 ' o b s e r v e d f o r I , w h i c h w e r e 5.7 a n d 3

3

H

H

7.5 H z , r e s p e c t i v e l y , d i d n o t a p p e a r a t first g l a n c e t o b e consistent w i t h t h e t h e o r e t i c a l v a l u e s of a p p r o x i m a t e l y 3 H z a n d 10 H z n o r m a l l y o b served

for gauche

a n d antiparallel conformations

of v i c i n a l

protons.

H o w e v e r , e l e c t r o n e g a t i v e substituents m o d i f y t h e m a g n i t u d e o f v i c i n a l c o u p l i n g constants ( 2 6 ) . I n c y c l i c c o m p o u n d s , s u c h as steroids (27) o r 4 - f - b u t y l c y c l o h e x a n o l s ( 2 8 ) , c o u p l i n g v a l u e s o f 5.5 ±

1 H z vs. 2.5-3.2 H z

a r e p o s s i b l e f o r p r o t o n s s e p a r a t e d b y i d e n t i c a l d i h e d r a l angles of 6 0 ° .


142

ASCORBIC ACID

T h e difference i n m a g n i t u d e is a t t r i b u t e d to the o r i e n t a t i o n of a c o u p l e d p r o t o n w i t h respect to a n a d j a c e n t O H g r o u p . T h e o r i e n t a t i o n effect w a s s h o w n b y B o o t h ( 2 9 ) to b e m a x i m a l ( s m a l l e s t / )

w h e n an electronega

t i v e s u b s t i t u e n t is a n t i p a r a l l e l to e a c h of t h e c o u p l e d p r o t o n s . If the e l e c t r o n e g a t i v i t y effect is t a k e n i n t o a c c o u n t , the c o u p l i n g constant of 1.83 H z for A

3

/ 4,H5 H

observed

i n I m a y be assigned to r o t a m e r

( F i g u r e 6 ) w h e r e H 4 is a n t i p a r a l l e l to 0 5 a n d H 5 is a n t i p a r a l l e l to

04.

R a p i d rotation (averaging) a r o u n d the C 4 - C 5 b o n d i n I w o u l d be

e x p e c t e d to m a k e / 5 , H 4 l a r g e r , n o t s m a l l e r , t h a n t h e o b s e r v e d v a l u e . 3

H

T h u s , the v a l u e of 1.83 H z is q u i t e r e a s o n a b l e f o r t h e c o n f o r m a t i o n s h o w n


i n F i g u r e 6 A , w h i c h is i d e n t i c a l w i t h t h e o r i g i n a l a s s i g n m e n t of

the

side-chain i n L-ascorbic acid (21). T h e i n c r e a s e d m a g n i t u d e of (HI)

compared

3

(2.94 H z ) f o r D-isoascorbic a c i d

/ 4,H5 H

to 1.8 H z i n I p r o v i d e s

L - a s c o r b i c a c i d . T h e v a l u e of 2.94 H z for

3

support for rotamer 9 A i n / 4,H5 H

also suggests a gauche

o r i e n t a t i o n of H 4 a n d H 5 i n I I I ( r o t a m e r s B a n d C , F i g u r e 7 ) .

Those

rotamers c o n t a i n o n l y one p r o t o n a n t i p a r a l l e l to a n o x y g e n , a n d / H 4 , H S 3

w o u l d b e e x p e c t e d to b e greater i n m a g n i t u d e i n I I I t h a n i t is i n I . T h e three s t a g g e r e d rotamers a r o u n d t h e C 5 - C 6 b o n d of L - a s c o r b i c a c i d are s h o w n i n F i g u r e 8. C o m p o u n d I at p H 2 ( T a b l e V I ) g a v e t h e c o u p l i n g constants / 5 , H 6 , 3

H

—11.55

H z , respectively.

3

/H5,H6', and

Rotamer

2

J 6,H6'

9C

H

e q u a l to 5.81,

can be

7.34,

r u l e d out

c o u p l i n g constants are u n e q u a l f o r H 5 - H 6 a n d H 5 - H 6 ' .

and

since

the

Rotamer 9B

a p p e a r s to fit t h e N M R d a t a better t h a n r o t a m e r 9 A . B o t h rotamers h a v e a p a i r of a n t i p a r a l l e l protons t h a t c a n b e a s s i g n e d to t h e c o u p l i n g c o n stant 7.34 H z , b u t the other c o u p l i n g constant of 5.81 H z is b e t t e r a s s i g n e d to the gauche c o u p l i n g i n r o t a m e r 9 B , w h e r e n e i t h e r of t h e p r o tons is a n t i p a r a l l e l to a n a d j a c e n t o x y g e n . T h e o b s e r v e d v a l u e of 5.81 H z is just s l i g h t l y greater t h a n t h e r a n g e ( 4 . 5 - 5 . 5 H z ) p r o p o s e d ( 2 9 ) for this t y p e of c o u p l i n g . A p p l i c a t i o n of t h e same a r g u m e n t s to the C 6 - C 5 b o n d I I I ( F i g u r e 9 ) shows r o t a m e r 1 0 A to b e t h e most p r o b a b l e . T h e c a l c u l a t e d c o u p l i n g constants f o r I I I at p H 7.09 w e r e 3.24, 7.93, a n d - 1 1 . 9 9 H z for 3

/ H 5 / H 6 % a n d / 6 , H 6 ' , respectively.

and

3

H

T h e c o u p l i n g constants for

H 6 - H 6 ' w e r e a l m o s t i d e n t i c a l to those o b s e r v e d f o r I .

3

/ 5,H6, H

H5-H6' T h e only

v a l u e t h a t d i f f e r e d s u b s t a n t i a l l y w a s / 5 , H 6 , w h i c h w a s 3.24 H z . B a s e d 3

H

o n these v a l u e s , r o t a m e r 1 0 B c a n b e e l i m i n a t e d i m m e d i a t e l y .

Of

the

r e m a i n i n g t w o r o t a m e r s , 1 0 A is p r e f e r r e d o v e r 1 0 C b e c a u s e 1 0 A s h o u l d h a v e a s m a l l e r v a l u e of / 5 , HG t h a n r o t a m e r 1 0 C . F u r t h e r m o r e , r o t a m e r 3

H

1 0 C contains n o oxygens a n t i p a r a l l e l to e i t h e r H 5 or H 6 . D i f f e r e n t c o u p l i n g constants b e t w e e n H 5 - H 6 a n d H 5 - H 6 ' c a n o c c u r o n l y i f there is a p r e f e r r e d c o n f o r m a t i o n f o r L - a s c o r b i c a c i d a r o u n d t h e C 5 - C 6 bond.

T h e C 4 - C 5 b o n d of I also exists i n a p r e f e r r e d c o n f o r m a -






ASCORBIC


144


ACID




6.


145

ASCORBIC ACID


146


6.

NMR

PAUKSTELis E T A L .

147


t i o n i n s o l u t i o n , as e v i d e n c e d b y the s m a l l v a l u e for / H 4 , H 5 of 1.83 H z . 3

T h i s v a l u e is too l o w t o r e s u l t f r o m t i m e - a v e r a g i n g , a n d therefore r e quires a preferred conformation.

T h e preferred rotamer 7 A

around

C 4 - C 5 a n d r o t a m e r 9 B a r o u n d the C 5 - C 6 b o n d are essentially t h e same c o n f o r m a t i o n s f o u n d b y H v o s l e f (2)

i n crystalline L-ascorbic acid. Since

no intramolecular hydrogen bonds occur i n the crystal ( 2 ) , none w o u l d b e e x p e c t e d w h e n I is d i s s o l v e d i n a h y d r o g e n - b o n d i n g solvent s u c h as w a t e r . T h u s , o u r r e s u l t is n o t s u r p r i s i n g . W h a t is s u r p r i s i n g is t h a t n o c h a n g e i n the c o n f o r m a t i o n of the s i d e - c h a i n is a p p a r e n t w h e n a s o l u t i o n of I is c h a n g e d f r o m p H 2 to 7.

O n e m i g h t expect a c o n f o r m a t i o n a l


c h a n g e c o r r e s p o n d i n g to t h a t o b s e r v e d i n t h e c r y s t a l l i n e state ( 3 ) . V i c i n a l coupling

constants

however,

in Ή

N M R data do

not

indicate

any

a p p r e c i a b l e c h a n g e i n s i d e - c h a i n c o n f o r m a t i o n u p o n i o n i z a t i o n at O H 3 .

C NMR Spectroscopy and the Conformation of L-Ascorbic Acid

13

T h e p a p e r of O g a w a et a l . (21) 1 3

C

NMR. Hz,

T h o s e w o r k e r s (21)

Ή

r e p o r t e d t h a t / C 4 , H 6 a n d Λ Μ , Η 6 ' e q u a l 2.4 3

3

w h i c h w o u l d fix the c o n f o r m a t i o n a r o u n d t h e C 5 - C 6 b o n d of I as

shown i n rotamer 9A.

T h e y also c o n c l u d e d t h a t r o t a m e r 7 A w a s r e

q u i r e d t o e x p l a i n the o b s e r v e d de

p r o m p t e d us to e x a m i n e t h e use of

N M R s p e c t r o s c o p y to v e r i f y t h e c o n f o r m a t i o n of I d e t e r m i n e d b y

Bie

(30)

suggested

c o u p l i n g constants.

that no

conformation

Spoormaker

was preferred

and

around

the C 5 - C 6 b o n d , but instead that equal rotamer populations explained the o b s e r v e d d a t a . W e recently recorded the proton-coupled Our

Ή-

1 3

0

1 3

C N M R s p e c t r u m of I.

c o u p l i n g constants a n d t h e l i t e r a t u r e v a l u e s are p r e s e n t e d

in Table V I I . The Ή

N M R d a t a at 600 M H z affirmed t h a t r o t a m e r 9 B

is t h e p r e f e r r e d c o n f o r m a t i o n at t h e C 5 - C 6 b o n d . T h e Ή -

1 3

0

coupling

d a t a h a v e l e d to a different c o n f o r m a t i o n a l a s s i g n m e n t t h a n the

Ή - Ή

c o u p l i n g d a t a b e c a u s e v i r t u a l c o u p l i n g is i n v o l v e d i n t h e A B X s p i n s y s t e m formed by H 6 , H 6 ' , a n d C 4 . T h e virtual coupling yields a deceptively s i m p l e s p e c t r u m at 25.2 M H z ; this s p e c t r u m shows too f e w lines a n d y i e l d s (31)

a n average v a l u e of / c 4 , H e a n d / C 4 , H « ' . T h e c o n d i t i o n s f o r 3

3

v i r t u a l c o u p l i n g i n v o l v i n g H 6 a n d H 6 ' are p r e s e n t i n L - a s c o r b i c a c i d . T h e H6

a n d H 6 ' n u c l e i are n e a r l y i s o c h r o n o u s

y e t are n o t m a g n e t i c a l l y

e q u i v a l e n t , as s h o w n i n t h e 600 M H z spectra. f e r e n c e b e t w e e n H 6 a n d H 6 ' at 2.3 Τ

field

T h e c h e m i c a l shift d i f is a b o u t

1.1 H z w i t h

a

c o u p l i n g constant of a b o u t 11 H z . T h u s Δ δ / J e q u a l s a p p r o x i m a t e l y 0.1 and

t h e c o u p l i n g constants c a n n o t b e d e t e r m i n e d b y i n s p e c t i o n (32).

d e t e r m i n e i f v i r t u a l c o u p l i n g is t h e cause of t h e d i s c r e p a n c y , c o u p l e d NMR

To 1 3

C

s p e c t r a n e e d to b e o b t a i n e d at t h e h i g h e s t p o s s i b l e field. A t 9.4 T ,

Δδ/J =

0.4 a n d i f a n e x p e r i m e n t ^ e r e r i u n ^ t j c f c v a t e d p H , s u c h as 7, t h e


148

ASCORBIC

Table V I I .

C a r b o n - P r o t o n Coupling Constants for L-Ascorbic

ACID

Acid


13Q

Proton

Cl

cz

cs

C4

H4

1.8T (2.0)'

1.96 (2.0)

5.70 (5.9)

153.1 (152.8)

H5

NMR Spectroscopy of Ascorbic Acid and Its Derivatives - Advances in

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