Carbohydrate Sulfates - American Chemical Society


yield starting from 5,6-0-isopropylidene-L^-ascorbic acid. But we ..... 1. 0. 3. 0. 5. I. I. 5. 0. 57. 0. 4. 0. 5. 0. II. I. 1.7. 111. 8. 85. 0. 81. 0...
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1 Monosulfate Esters of L-Ascorbic Acid DONALD W. LILLARD, JR. and PAUL A. SEIB

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Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506

L-Ascorbate 2-Sulfate. In 1972 Halver and his colleagues (1) reported L-ascorbic acid 2-sulfate (AA2S, F i g . 1) was as effective as L-ascorbic acid (AA) in preventing scurvy in rainbow trout and coho salmon. Prior to that time, Ford and Ruoff (2) synthesized AA2S and showed the sulfate ester was more stable than AA to oxidative degradation. Those two reports stimulated the interest of C. W. Deyoe in AA2S because he was engaged in research on formulating catfish feed at Kansas State University. Fish feed is often produced in floating form by extrusion puffing, a process that destroys 70-80% of the L-ascorbic acid in the feed (3). In addition, L-ascorbate 2-sulfate appeared attractive as a potential form of vitamin C since AA2S occurs in Nature; i t is found in the dormant embryo of brine shrimp (4,5), in human urine (6), and in rat l i v e r , spleen, adrenal gland, and urine (7,8). Since there was no commercial supply of AA2S, Prof. Deyoe asked that our group produce a large amount of AA2S. A chronology of events concerning L-ascorbate 2-sulfate is given in Table I. The biological significance of AA2S has not been established. Its antiscorbutic activity appears species-dependent. AA2S is inactive in the guinea pig (14,15), but is active in fish and marginally active in the rhesus monkey (23). It is also p a r t i a l l y active i n insect diets (24). No data are available on man. It has been suggested that AA2S may serve as a storage form of AA, or as a direct sulfate donor in the biosynthesis of s u l fate esters (20,22). Wholebody radioautographs of fish intubated with radioactive L-ascorbate l - 14C or L-ascorbate 2-sulfate-S showed the radioactivity of both compounds was concentrated in the same organs, mainly the skin, fins, lower jaw-plate, kidney, l i v e r , and heart (25). However, in rats and guinea pigs (8), the uptake of AA by organs was different than observed for AA2S. To obtain large amounts of AA2S we f i r s t modified (16) the original procedure of Ford and Ruoff (2) and produced AA2S in 75% yield starting from 5,6-0-isopropylidene-L^-ascorbic acid. But we also found (16) a much better method to prepare AA2S (Figure 2) with the following attractive features; (a) the starting material 35

0-8412-0426-8/78/47-077-001$05.00/0

©

1978 American Chemical Society

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Synthesis of 5,6-0-isopropylidene-L-ascorbate-2-sulfate.

Event

3 5

35

35

1

Demonstration of L - a s c o r b i c a c i d s u l f o t r a n s f e r a s e a c t i v i t y i n r a t l i v e r and "colon homogenates which catalyzed format i o n of AA2S- S from L - a s c o r b i c a c i d and 3 -phosphoadenyls u l f a t e - S or sodium " s u l f a t e - S .

1976

Mohamaram et a l . (22)

I n c o r p o r a t i o n of s u l f a t e from AA2S i n t o c h o i n d r o i t i n s u l f a t e . Discovery of widespread d i s t r i b u t i o n of AA2S s u l f hydrolase.

C a r l s o n et a l . (17), Hatanaka et a l . I s o l a t i o n of AA2S s u l f h y d r o l a s e from a marine organism and and mammalian l i v e r . (18) and Roy, et a l . (19)

1974

1975-6 Hatanaka et a l . (20) and F l u h a r t y et a l . (21)

Seib et a l . (16)

1974

Synthesis of AA2S improved.

AA2S gave no a n t i s c o r b u t i c a c t i v i t y i n guinea p i g s .

(14) and Campeau et

1973- 4 Kuenzig et a l . a l . (15)

structure.

Determination of X-ray c r y s t a l l o g r a p h i c

1972- 3 Bond et a l . (5) and McClelland (13)

Discovery of AA2S i n human u r i n e ; d a i l y e x c r e t i o n reported to be 30-60mg of the dipotassium s a l t . Prevention of scurvy i n coho salmon and rainbow t r o u t by equivalent amount of AA2S or L-ascorbate.

Baker et a l . (6,Π.)

1971

I d e n t i f i c a t i o n of AA2S i n Nature from undeveloped cysts of b r i n e shrimp.

1972-3 Halver et a l . (J., 12)

Mead and Finamore (4)

Chu and Slaunwhite (9) and Mumma (10) în VAJUIO o x i d a t i v e s u l f a t i o n of ft-octanol and androsterone during mild o x i d a t i o n of L-ascorbate 2 - s u l f a t e (AA2S) i n non-aqueous media. ~~

Ford and Ruoff (2)

Reference

1969

1968

1965

Date

Table I. Chronology of L-Ascorbate 2-Sulfate

1.

LiLLARD AND SEiB

Monosulfate

Esters of

L-Ascorbic

Acid

3

i s L-ascorbic a c i d , (b) the r e a c t i o n solvent i s water, (c) the conversion of AA to AA2S i s 98%, (d) the s u l f a t i o n r e a c t i o n i s r a p i d (< 30 minutes), (e) the s u l f a t i n g agent i s the s u l f u r t r i oxide complex of the l e a s t expensive t e r t i a r y amine ( t r i m e t h y l a mine), (f) the i s o l a t i o n of AA2S i s simple, and (g) a n a l y t i c a l l y pure c r y s t a l s of barium L-ascorbate 2 - s u l f a t e are i s o l a t e d i n 80% yield. Obviously, the chemical synthesis of AA2S presents l i t t l e difficulty (16). L-Ascorbate 6-Sulfate (AA6S). L-Ascorbate 6 - s u l f a t e (AA6S) i s an~intermediate (not i s o l a t e d ) i n t h e production (26) of La s c o r b y l 6-palmitate, a m a t e r i a l that i s used mainly to prevent o x i d a t i v e r a n c i d i t y (27) i n f a t s , o i l s , and dehydrated foods. In a d d i t i o n , L - a s c o r b y l 6-palmitate i s a u s e f u l a d d i t i v e i n breads (28) and cârotenoid c o l o r a n t s (29). L-Ascorbyl 6-palmitate i s synthesized by r e a c t i n g L-ascorbic a c i d TAA) with p a l m i t i c a c i d i n concentrated s u l f u r i c ac"id. The e s t e r i f i c a t i o n of AA i n concentrated s u l f u r i c a c i d i s at f i r s t s u r p r i s i n g , s i n c e L-ascorbic a c i d i s r a p i d l y dehydrated and decarboxylated i n hot a c i d to give almost q u a n t i t a t i v e y i e l d s of f u r f u r a l (30). But the r i n g - s t r u c t u r e of AA i s very s t a b l e i n concentrated s u l f u r i c a c i d at room temperature. We p r e v i o u s l y showed (26) that the absorbance (265nm) of a s o l u t i o n of AA Cx/3xlO" M) i n 95-98% s u l f u r i c a c i d was unchanged over a 46-day p e r i o d . We a l s o found the absorption maximum f o r unionized L-ascorbic a c i d (X 245nm at pH 2) s h i f t e d to longer wavelengtrï ( À 265nm) when AA was d i s s o l v e d i n concentrated s u l f u r i c a c i d , i n d i c a t i n g that a d e l o c a l i z e d c a t i o n had formed i n the s t r o n g l y a c i d i c medium (Figure 3). We f u r t h e r speculated that the primary hyd r o x y l group of AA i s f u l l y s u l f a t e d i n concentrated s u l f u r i c a c i d . Carbon-13 nmr measurements confirmed (26) the formation of the h y d r o x y a l l y l c a t i o n shown i n F i g u r e 3. The ^ C - s p e c t r a of AA i n 99% s u l f u r i c a c i d and of 4-deuterio-L-ascorbic a c i d i n water at pH 2 are shown i n Figure 4. The assignments of the resonance s i g n a l s were made from the r e l a x a t i o n r a t e s of the carbon atoms and from proton-coupled s p e c t r a . The assignments w i l l not be discussed here, s i n c e they are the subject of another paper (31). When AA i s d i s s o l v e d i n 99% s u l f u r i c a c i d , spectrum Β i n Figure 4 shows the r e a c t i o n products a l l have a set of s i x s i g n a l s which c l o s e l y resembles the set of s i g n a l s of AA i n water. Spectrum Β c l e a r l y shows the r e a c t i o n product contains three com­ ponents which are a l l c l o s e l y r e l a t e d to the s t r u c t u r e of AA. Thus, AA does not undergo dehydration and polymerization (32) i n concentrated s u l f u r i c a c i d at room temperature. In t h i s r e p o r t , we describe the i s o l a t i o n and c h a r a c t e r i z a ­ t i o n of the two p r i n c i p a l components formed when AA i s d i s s o l v e d i n concentrated s u l f u r i c a c i d , namely, L-ascorbate 6 - s u l f a t e (AA6S) and L-ascorbate 5 - s u l f a t e (AA5S)T Other references to work on AA6"S" are given i n Table I I ; AA5S has not been reported -

5

m a x

m a x

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

4

CARBOHYDRATE SULFATES

CH-OH HC0H

OSOl

-0

Figure 1. -L-Ascorbate 2-sulfate (AA2S)

CH 0H

CH OH ι
9

H-C-0H

(1 ) a l k a l i , p H 10 7 0 ' , 30 m i n . , N

(CH3) N:SO 3

3

n

(2) c a t i o n e x c h a n g e resin (H ) +

(3) b a r i u m

-o

hydroxide

oso: Figure 2.

Synthesis of L-ascorbate 2-sulfate

CH OS0 H

CHoOH

2

Γ

3

HCOH

HCOH 0^

HO

2

.0^

4H S04 2

Γ

ΌΗ

HO

OH

OH + H3O* + 3HS0 ~ 4

Figure 3.

L-Ascorbic acid in concentrated sulfuric acid

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Figure 4.

200

Ul

C-3

150

6 , ρ. ρ .τη.

C-2

100

C-4

C-5

C-6

Fourier transform, carbon-13 spectrum of: (A) 4-deuterio-L-ascorbic acid in deuterium oxide at 30°C, ascorbic acid in concentrated sulfuric acid at30°C

C-l

50 and (B) L-

DSS

δ:

Ci

ο"

S"

ο

Ci

S!"

Co

Ο Ο

M S

Ο

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978. m a x

T o l b e r t (35)

1975

3

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n N , N dimethylformamide (DMF). AA6S was c h a r a c t e r i z e d by p.m.r. and u.v. spectroscopy. Y i e l d not reported

Hatanaka, Ogawa, and Egami (34)

1974

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n DMF. AA6S was p u r i f i e d by DEAE-cellulose chromatography i n 20% yield. The compound had ε 12 χ 10 , X 265nm (pH 7.0).

S u l f a t e d AA with p y r i d i n e - s u l f u r t r i o x i d e complex i n p y r i ­ dine; AA6S i s o l a t e d by DEAE-cellulose chromatography. Y i e l d not r e p o r t e d . Product chromatographically pure.

Allaudeen and Ramakrishnan (33)

1970

'Event

Reference

6-Sulfate (AA6S)

Date

Table I I . Chronology of L-Ascorbate

1.

LiLLARD AND SEIB

Monosulfate

Esters of L-Ascorbic

Acid

7

previously. The i s o l a t i o n of L-ascorbate 6 - s u l f a t e was accomplished as f o l l o w s . A 0.7M s o l u t i o n of L - a s c o r b i c a c i d i n 99% s u l f u r i c a c i d was quenched by dropwise additfion, with s t i r r i n g , of the a c i d i c mixture to e t h y l ether at -65°. A f t e r n e u t r a l i z a t i o n with aqueous barium hydroxide and removal of ether and barium s u l f a t e , i o d i metric t i t r a t i o n showed that 74% of the reducing power i n the s t a r t i n g m a t e r i a l was present i n the aqueous phase. The r e a c t i o n products were separated (35)by ion-exchange column chromatography on DEAE c e l l u l o s e using a gradient of very d i l u t e s u l f u r i c a c i d as e l u t i n g solvent (Figure 5). The percentage of each component i n the r e a c t i o n mixture was determined by i o d i n e t i t r a t i o n and u.v. absorbance at 265nm (Table I I I ) . Component I I I , which accounted f o r 81-85% of the products (u.v. a c t i v i t y ) a p p l i e d to the ion-exchange column, was found to be L-ascorbate 6 - s u l f a t e . The barium s a l t of AA6S was i s o l a t e d as ah amorphous s o l i d i n an o v e r a l l y i e l d of 48% (u.v. on s o l i d s ) . Elemental a n a l y s i s showed the s o l i d contained 81% barium L-ascorbate 6 - s u l f a t e and 19% barium s u l f a t e . No attempt was macTe to f u r t h e r p u r i f y the s o l i d . We found when i s o l a t i n g A A 6 S , the y i e l d of the product can be preserved by using a s l u r r y of barium c a r bonate which had been p r e v i o u s l y de-aerated with n i t r o g e n . Presumably oxygen i s absorbed on the s u r f a c e of the f i n e p a r t i c l e s of barium carbonate, which leads to p a r t i a l o x i d a t i o n of the d e s i r e d product. L-Ascorbate 6 - s u l f a t e was c h a r a c t e r i z e d by u.v. and c.m.r. spectroscopy. The c.m.r. spectrum (Figure 6) shows that, r e l a t i v e to the s h i f t s observed f o r AA, s u l f a t i o n at C-6 s h i f t e d the s i g n a l of C-6 i n water downfield by ^6ppm while the s i g n a l of C-5 moved s l i g h t l y u p f i e l d . Those s h i f t s have been noted by others (36) i n the spectra of sugar s u l f a t e s . The p.m.r. spectrum of AA6S showed that the s i g n a l of the H-6 protons are s h i f t e d downfield by M).5 ppm i n AA6S(ôH-6, H-6 4.14-4.30 at pH 7.0) from those observed f o r L-ascorbate(6 H-6, H-6 3.74 at pH 7). Our method of preparing ana p u r i f y i n g AA6S i s somewhat b e t t e r than those p r e v i o u s l y d e s c r i b e d . When we repeated the method of Allaudeen and Ramakrishnan (33) we found the crude sodium s a l t of L.-ascorbate 6 - s u l f a t e could be i s o l a t e d i n only 26% y i e l d . T o l b e r t (35) obtained AA6S i n 20% y i e l d , which i s probably the same obtained by Hatanaka, et a l . (34). T

1

L-Ascorbate 5 - S u l f a t e . We suspected that Component I I (Figure 5 and Table I I I ) was L-ascorbate 5 - s u l f a t e f o r s e v e r a l reasons: (1) primary hydroxyTs are known to s u l f a t e 3-10 times f a s t e r i n concentrated s u l f u r i c a c i d than are secondary hydroxyls, which explained the small percentage of Component I I ; (2) Component I.I gave one spot on paper chromatography i n the monosulfate r e g i o n , and the spot s t r o n g l y reduced s i l v e r and f e r r i c ions (Table IV); (3) when Component I I was c o l l e c t e d over 6% metaphosphoric a c i d and the a c i d i c s o l u t i o n concentrated at 25° to a s m a l l

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ure 5.

r

10 hr. Time

15 hr.

Component III

20 hr.

Separation of reaction products of L-ascorbic acid in 99% sulfuric acid. Products were chromât ο graphed on a column of DEAE-cellulose (35).

5 hr.

Component I I

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

6.0

5.0

1.7

1.7

I

II

III

IV

pH

T o t a l Recovery

1464

1118

570

351

Total e f f l u e n t , ml.

89.5

0.2

85.0

4.0

0.3

uv

Percent of

87.0

< 0.5

81.0

5.0

0.5

. _ a mixture Iodine titration

acid.

Percentage determined by comparison of absorbance or i o d i n e t i t e r of a peak to the t o t a l absorbance or iodine t i t e r of the mixture applied to the column.

Effluent

Component

by d i s s o l v i n g L - a s c o r b i c a c i d i n 99% s u l f u r i c

Table I I I . Ion-exchange column chromatography of r e a c t i o n products formed

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

C-T

\

j|

C-3

J -

Figure 6.

150

13

C-4

Λ

V

/

l!,

[

ί

M

C-5 C-6

50

pH 6 . 5

pH 6 . 7

L-Ascorbic acid 5-sulfate

L-Ascorbic acid 6-sulfate

2.0

7J

pH 10/2

pH

pH

L-Ascorbic acid 2 - s u l f a t e

Comparison of the C chemical shifts of L-ascorbic acid and its derivatives

100 6 , p.p.m.

1

C-2

ο

Hi

ο a

> w

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

d

See

Sample

ASC

=

o

b

not

m

i

i

t

y

2

or

The

H-5

is

179.37

116.06

176..14

80.51 69.81

77.64 77.43

under

the

HOD

peak

at

4.5-4.8.

2,2-dimethyl-2-silapentane-5-sulfonate.

266

apparently

(p.m.r.)

signal

acid.

internal

L-ascorbic

D 0.

to

(c.m.r.)

with

relative

external

exchanged

l

from

Experimental.

Downfield

C R

b

244

1 7 5 .,72

71.50

61.41

4.53

4.68

white

113.90

+

177.65

0.5

6-sulfate

267

white

+

0.3

245

4.57

65.12

81.31 72.28

182.,34

113.01

178.08

255

H-4

232

C-6

red

C-5

-

C-4

4.50

C-3

p.m.r .

65.33

C-2

b

80.96 72.26

C-l

pH 7

δ at

i n Water,

c.m.r.,

1 7 7 . ,87

7

2

H 0

Acid

L-Ascorbate

of

Esters

115.70

pH

in

Monosulfate

179.71

λ

the

265

2

,

of

244

pH

u.v.

Properties

white

5-sulfate

Spray

Spray

0.5

Chloride

Nitrate

2-sulfate

Ferric

+

Alcoholic

Silver

a

IV,

Neutral

1.0

ASC°

C h r o m a t o :n ; r a p h y

Unsubstituted

Derivative

R

Paper

Table

9

δ at

3.86

3.73

3.74

H-6'

H-6,

D 0,

4.30

14- 4.14- 4.30

4.

_d

4.05

4.02

H-5

in

6.7

6.5

7.0

7.1

pH

pH

7

b

Ci*

1

IT

Co

ο ο

w

I—I

Η

α

>

CARBOHYDRATE SULFATES

12

volume, the r e s u l t i n g mixture contained only L - a s c o r b i c a c i d as determined by paper chromatography; and (4) tîïe 5- and 6-hydroxyls of AA would be expected to undergo very l i t t l e d i s u l f a t i o n s i n c e i t i s known (37) that v i c i n a l d i o l s do not form d i n i t r a t e s upon r e a c t i o n with the e l e c t r o p h i l i c nitronium i o n . Presumably, r e a c t i o n of d i o l s with s u l f u r t r i o x i d e i n concentrated s u l f u r i c a c i d would g i v e the same r e s u l t . Attempts to i s o l a t e Component I I from the column e f f l u e n t were u n s u c c e s s f u l , because as we discovered l a t e r , s u l f a t e i s e l i m i n a t e d from AA5S i n the presence of barium i o n . In f a c t , Component I was thought to be the 4,5-unsaturated product formed by the e l i m i n a t i o n r e a c t i o n . The u.v. p r o p e r t i e s of Component I support the extended resonance system of three conjugated double bonds. Compared to L - a s c o r b i c a c i d , the a b s o r p t i o n maximum of Component I was s h i f t e d 15-20nm to longer wavelength ( X 260nm and 285nm at pH 2 and 7, r e s p e c t i v e l y , νΟΛΛίιΛ À 245nm and 265nm f o r AA at the same pH). In a d d i t i o n , Component I was very unstable above pH 7. We t h e r e f o r e decided to prepare AA5S by an unequivocal route, which i s shown i n F i g u r e 7. Reaction of L - a s c o r b i c a c i d i n p y r i dine with two e q u i v a l e n t s of t- b u t y l d i m e t h y l s i l y l c h l o r i d e f o l lowed by s u l f a t i o n with p y r i d i n e - s u l f u r t r i o x i d e gave 2 6-0-bZ&(t-butyldimethylsilyl. )-L-ascorbate 5 - s u l f a t e (AA5S). H y d r o l y t i c removal of the 2,6-blockThg group with 80% a c e t i c a c i d followed by ion-exchange chromatography gave the d e s i r e d AA5S. m a x

m a x

9

We found Component I I was indeed i d e n t i c a l to AA5S. The two m a t e r i a l s co-migrated on paper chromatograms and on column chromatograms packed with 0-(diethylaminoethyl)-cellulose. The s t r u c t u r e of AA5S was r e a d i l y confirmed by u.v., c.m.r., and p.m.r. The carbon-13 spectrum (Figure 6, Table IV) showed that C-5 s h i f t e d downfield by ^6 ppm while C-4 and C-6 s h i f t e d s l i g h t l y upfield. In the p.m.r. spectrum the s i g n a l s of H-4 and H-6 were s i m i l a r to those of L - a s c o r b i c a c i d , whereas the s i g n a l of H-5 was apparently s h i f t e d downfield by 0.5-0.8 p.p.m. under the HOD peak (Table IV). The i s o l a t i o n of barium L-ascorbate 5 - s u l f a t e i n pure s o l i d form i s d i f f i c u l t because of the tendency of the molecule to undergo e l i m i n a t i o n of the s u l f a t e group. In one attempt, a s o l i d was i s o l a t e d that contained mostly AA5S as shown by c.m.r. s p e c t r o scopy. When an aqueous s o l u t i o n of the barium s a l t of AA5S was allowed to stand at room temperature, barium s u l f a t e p r e c i p i t a t e d i n i n c r e a s i n g amounts with time. Experimental General. A l l evaporations were done under reduced pressure below 50°. M e l t i n g p o i n t s were determined with a Thomas-Hoover apparatus. O p t i c a l r o t a t i o n s were obtained using a Swiss-made Kern p o l a r i m e t e r . L-Ascorbic a c i d was obtained from ICN

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

LiLLARD AND SEiB

Monosulfate

CH OH

Acid

CH OR

2

H-C-OH

Esters of L-Ascorbic

CH OH

2

(l)pyridine +

H-Ç-OSO"

RCI

2

80%

acetic

H-C-OSO

acid, 30° 2hr.

(2)pyridine: SO

CH

3

R= C h U - C - S i 0

r CH,

Figure 7.

Synthesis of L-ascorbate 5-sulfate

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

14

CARBOHYDRATE SULFATES

Pharmaceutical, Inc., C l e v e l a n d , Ohio. Concentrated s u l f u r i c a c i d (99%, 37.0±0.15N) was obtained from F i s h e r S c i e n t i f i c , F a i r l a w n , NJ. T h i n - l a y e r chromatography was performed on microscope p l a t e s coated with s i l i c a g e l G (Brinkman Instruments, Inc., Westburg, New York). Compounds were heated by spraying with 50% aqueous s u l f u r i c a c i d followed by c h a r r i n g on a hot p l a t e . Descending paper chromatography was done u s i n g e t h y l acetate, a c e t i c a c i d , and water (6:3:2, V:V). Components were l o c a t e d using one of three d i p reagents. R&agznt A. Strongly reducing components were detected by d i p p i n g a chromatogram i n a s o l u t i o n of 0.7% (by weight) of s i l v e r n i t r a t e i n a mixture of acetone, concen­ t r a t e d ammonium hydroxide, and water (200:1:1). Reagent B. Mod­ erate to weakly reducing components were detected by d i p p i n g i n Reagent A followed by d i p p i n g i n a l c o h o l i c sodium Hydroxide (39). Rtag&nt C. Components with e n o l i c hydroxyls were detected by d i p p i n g i n a 1% f e r r i c c h l o r i d e i n 95% methanol (40). The r e l a ­ t i v e m o b i l i t i e s and c o l o r r e a c t i o n s of L - a s c o r b i c a c i d and i t s monosulfate e s t e r s are given i n Table IV. U.v. s p e c t r a were obtained i n aqueous s o l u t i o n s using a Beckman Model DB-G spectrophotometer. Nuclear magnetic resonance s p e c t r a were recorded with a V a r i a n Model XL-100 spectrometer. For carbon-13 s p e c t r a , the n.m.r. instrument was coupled to a N i c o l e t 1084 pulse F o u r i e r transform system. Chemical s h i f t s are given i n ppm downfield from e x t e r n a l (^C) or i n t e r n a l (^H) sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). L-Ascorbate 6-Sulfate (AA6S). To o b t a i n good y i e l d s of AA6S, molecular oxygen should be e l i m i n a t e d from reagents and s o l v e n t s by b o i l i n g and/or purging with n i t r o g e n . ( P r e - p u r i f i e d grade, Matheson, Ε., Rutherford, New J e r s e y ) . L-Ascorbic a c i d (3g) was d i s s o l v e d with s t i r r i n g at 25° i n 99% s u l f u r i c a c i d (lOmL), and the s o l u t i o n held 4 h at 25°. The v i s c o u s r e a c t i o n mixture was added dropwise with r a p i d s t i r r i n g to e t h y l ether (300mL) a t -65°, which caused the s u l f a t e e s t e r s to p r e c i p i t a t e as a v i s c o u s gum. The ether was maintained a t -65° by o c c a s i o n a l , d i r e c t a d d i t i o n of s o l i d carbon d i o x i d e . While the ether mixture was s t i l l at -65°, aqueous saturated barium hydroxide was added (200mL) and the r e s u l t i n g mixture was allowed to warm to - 5 ° . The ether l a y e r was drawn o f f and washed twice with water (2 χ 150mL). The combined aqueous l a y e r s were kept a t 0-5° and n e u t r a l i z e d by a d d i t i o n of s o l i d barium hydroxide. Barium s u l f a t e was removed by f i l t r a t i o n , and the f i l t r a t e made to volume (lOOOmL). An a l ­ iquot (lO.OmL) of the r e a c t i o n mixture reduced 3.65mL of 0.1N i o d i n e s o l u t i o n , whereas an a l i q u o t (5.0mL) of a known amount (1.015g) of L - a s c o r b i c a c i d i n 6% aqueous metaphosphoric a c i d (100.OmL) recTuced 11.6mL of 0.1N i o d i n e . The t i t e r s showed the aqueous e x t r a c t contained 74% of the e n e - d i o l groups put i n t o the concentrated s u l f u r i c a c i d . The remaining f i l t r a t e (990mL) was evaporated to a small

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1.

LiLLARD AND SEiB

Monosulfate

Esters of L-Ascorbic

Acid

15

volume, and made to 25.0mL with water. An a l i q u o t (lO.OmL) was subjected to ion-exchange chromatography on a column (2.6 χ 40cm) of (die thy lamino e t h y l ) - c e l l u l o s e (Whatman DE-52, H. Reeve Angel, Inc., C l i f t o n , New J e r s e y ) , which had been p r e v i o u s l y converted to the hydrogen s u l f a t e form by washing with 0.1M s u l f u r i c a c i d . The column was developed at a flow r a t e of 45ml h~* using a l i n e a r g r a d i e n t (0 -> 0.18M of s u l f u r i c a c i d . The absorbance of the e f f l u e n t was monitored at 254nm, and the components (Figure 5) were c o l l e c t e d i n a v o l u m e t r i c f l a s k over 6% metaphosphoric a c i d . The amount of each component was determined by i t s ab­ sorbance (245nm) and by i o d i m e t r i c t i t r a t i o n (Table I I I ) . Component I I I , which accounted f o r 81% of the r e a c t i o n pro­ ducts, was found to give a s i n g l e spot (paper chromatography, AA 0.59), which co-migrated with a sample of L-ascorbate 6 - s u l ­ f a t e prepared as described by Allaudeen and Ramakrishnan (33). The spot r a p i d l y reduced s i l v e r and f e r r i c i o n s . Both samples had u.v. p r o p e r t i e s i d e n t i c a l to those of L - a s c o r b i c a c i d ( À 245nm at pH 2 and X 265nm at pH 7). ~ In a separate experiment Component I I I was i s o l a t e d as i t s barium s a l t a f t e r s e p a r a t i o n of the products obtained from a r e a c t i o n s t a r t i n g with 2.85g of L - a s c o r b i c a c i d . The column (2.6 χ 40cm) was developed as p r e v i o u s l y d e s c r i b e d , except a t o t a l of 400mL of 0.18M s u l f u r i c a c i d was used i n the l i n e a r , gradient e l u t i o n step, followed by 1800mL of 0.18M s u l f u r i c a c i d . Compo­ nent I I I was c o l l e c t e d i n a slowly s t i r r e d mixture of water (50ml) and barium carbonate that had p r e v i o u s l y been purged with nitrogen. A f t e r removal of barium s u l f a t e , the f i l t r a t e was concentrated to dryness to give barium L.-ascorbate 6 - s u l f a t e (5.0g) as an amor­ phous s o l i d which was 75"% barium AA6S as determined by u.v. analy­ sis . Anal. C a l c u l a t e d f o r 0.73 moles CftHôOgSBa +0.27 moles BaSO^ C, 13.44; H, 1.13; 0, 34.35; Ba, 41.50. Found: C, 13.33; H, 1.13; 0, 35.56; Ba, 47.54. The p.m.r. spectrum of barium AA6S at pH 7 (Table IV) agreed with that reported by Hatanaka, et a l . (34). R

m a x

m a x

L-Ascorbate 5 - S u l f a t e . To a p y r i d i n e (lOmL) s o l u t i o n of La s c o r b i c a c i d (1.5g) cooled i n an i c e bath was added 2.1 equival e n t s of ^ - b u t y l d i m e t h y l s i l y l c h l o r i d e (3.0g) ( A l d r i c h Chemical Company, Milwaukee, Wisconsin). A f t e r 3 hr s t i r r i n g at 25°, t . l . c . (benzene, e t h y l acetate 9/1, V/V) showed the r e a c t i o n mixture contained one p r i n c i p a l (R 0.7) and one minor component (Rf 0.8). P y r i d i n e - s u l f u r t r i o x i d e (1.35g) was added, and the s u l f a t i o n r e a c t i o n allowed to proceed overnight at 25°. A f t e r s u l f a t i o n t . l . c . (chloroform, a c e t i c a c i d 3/2, V/V) showed one major spot at Rf 0.7. f

The r e a c t i o n mixture was evaporated to remove p y r i d i n e , and water (20mL) was added twice and the mixture re-evaporated to a t h i c k syrup. The syrup was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (16mL), water (4mL) added, and the r e s u l t i n g mixture was held at 25°. A f t e r 12 hours t . l . c . (chloroform, a c e t i c a c i d 3/2, V/V)

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

CARBOHYDRATE SULFATES

16

showed the r e a c t i o n mixture contained one p r i n c i p a l product at R.£ 0.2. Evaporation of the h y d r o l y s i s r e a c t i o n mixture to a small volume (lOmL) removed water, a c e t i c a c i d , and ^ - b u t y l d i m e t h y l s i lanol. Aqueous barium hydroxide was added to pH 7.0, and the mix­ ture was concentrated to ^25mL and a p p l i e d to a DEAE-cellulose (H , SO^") column. Developing the column with a gradient of s u l ­ f u r i c a c i d (0 ->• 0.18M) eluted a m a t e r i a l whose m o b i l i t y was the same as that of Component I I (Figure 5). The column e f f l u e n t was c o l l e c t e d over barium carbonate as p r e v i o u s l y described to give 2.03 g of an amorphous s o l i d . The s o l i d barium s a l t of L - a s c o r b i c 5 - s u l f u r i c a c i d had i d e n t i c a l paper and chromatographic p r o p e r t i e s as those of Component I I . The s o l i d product d i d not give an ac­ ceptable elemental a n a l y s i s . The u.v., p.m.r., and c.m.r. s i g n a l s of AA5S are given i n Table IV. +

L-Ascorbic A c i d 4-d. The t i t l e compound was prepared by a mod­ i f i c a t i o n of the procedures of .Tolbert (41) and Brenner (42). LAscorbic a c i d (0.05 moles) was d i s s o l v e d and evaporated from de^ aerated deuterium oxide (lOmL) three times. To the dry m a t e r i a l was added 4M sodium methoxide i n methanol-cl (50mL), and the mix­ ture was r e f l u x e d 24 hours. The cooled a l c o h o l i c s o l u t i o n was added to water (lOOmL) c o n t a i n i n g 45g of s t r o n g l y a c i d i c , c a t i o n exchange r e s i n ( H ) . The r e s i n was removed, the f i l t r a t e f r e e z e d r i e d , and the residue d i s s o l v e d i n warm a c e t o n i t r i l e . Cooling gave white c r y s t a l s (2.8g, 32%) which had m.p. and a mixed m.p. of 192-3° with authentic L - a s c o r b i c a c i d . +

Ac knowledgment s The authors thank D. Mueller measurements.

and

J . P a u k s t e l i s f o r c.m.r.

Abstract Methods to prepare L-ascorbate 2-,5-, and 6 - s u l f a t e s are given. The compounds wer e c h a r a c t e r i z e d by 1 H - a n d Cnuclear magnetic resonance spectroscopy and by u l t r a v i o l e t spectroscopy. The b i o l o g i c a l p r o p e r t i e s of L-ascorbate 2 - s u l f a t e are reviewed. 1 3

Literature Cited 1. Halver, J . E . , Johnson, C. L., Smith, R. R., Tolbert, Β. M . , and Baker, F. M. Fed. Proc. Fed. Amer. Soc. Exp. B i o l . , (1972) Abstract No. 2764. 2. Ford, E. A. and Ruoff, P. M. Chem. Commun. (1965) 628. 3. Shenouda, M., M. S. Thesis, Kansas State University (1976); Quadri, S. F . , Liang, Y. T . , Seib, P. Α . , Deyoe, C. W., and Hoseney, R. C. J . Food Sci. (1975) 40 837. 4. Mead, C. G. and Finamore, F. J . Biochemistry (1969) 8 2652.

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

1.

LiLLARD AND sEiB

Monosulfate

Esters of L-Ascorbic

Acid

17

5. Bond, A. D., McClelland, B. W., Einstein, J. R., and Finamore, F. J., Arch. Biochem. Biophys. (1972) 153, 207. 6. Baker, Ε. Μ., Hammer, D. C., March, S. C., Tolbert, Β. Μ., and Canham, J. Ε., Science (1971) 173, 826. 7. Mumma, R. O. and Verlangieri, A. J., Biochem. Biophys. Acta (1972) 273 249. 8. Hornig, D., Ann. Ν. Y. Acad. Sci. (1975) 258, 103; Hornig, D. World Rev. Nutr. Dietetics (1975) 23, 225. 9. Chu, T. M. and Slaunwhite, W. R., Jr., Steroids (1968) 12, 309. 10. Mumma, R. D., Biochem. Biophys. Acta (1968) 165, 571. 11. Baker, Ε. Μ., Kennedy, J. Ε., Tolbert, Β. Μ., and Canham, J. E. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1972). Abstr. No. 2760. 12. Halver, J. Ε., Ashley, L. Μ., Smith, R. R., Tolbert, Β. M., and Baker, Ε. M. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1973) Abstr. No. 4010. 13. McClelland, B. W., Acta Crystallogr. (1974) 30, 178. 14. Kuenzig, W., Avenia, R., and Kamm, J. J., J. Nutr. (1974) 104, 952. 15. Campeau, J. D., March, S. C. and Tolbert, Β. Μ., Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1973) Abstr. No. 4008. 16. Seib, P. Α., Liang, Y. T., Lee, C. H., Hoseney, R. C., and Deyoe, C. W. J. Chem. Soc., Perkin I (1974), 1220. 17. Carlson, Β. Μ., Downing, Μ., Tolbert, Β. M. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1974) 33, 1377. 18. Hatanaka, H., Ogawa, Y., Egami, F., J. Biochem. Tokyo. (1974) 75, 861. 19. Roy, A. B., Biochem. Biophys. Acta (1975) 377, 356. 20. Hatanaka, H. and Egami, F. J. J. Biochem. (1976) 80, 1215. 21. Fluharty, A. L . , Stevens, R. L . , Miller, R. T., Shapiro, S. S., and Kihara, H. Biochem. Biophys. Acta (1976) 429, 508. 22. Mohamram, M., Rucker, R. Β., and Hodges, R. E. Biochem. Biophys. Acta (1976) 437, 305. 23. Brin, M., Machlin, Κ. J., Kuenzig, W., and Garcia, F. Fed. Proc. Fed. Amer. Soc. Exp. Biol. (1975) 34, 884. 24. Kramer, K. J., Hendricks, L. Η., Liang, Y. T., and Seib, P. A. submitted to J. Ag. Food Chem. 25. Halver, J. Ε., Smith, R. R., Tolbert, Β. Μ., and Baker, Ε. Μ., Ann. New York Acad. Sci. (1975) 258, 81. 26. Cousins, R. C., Seib, P. Α., Hoseney, R. C., Deyoe, C. W., Liang, Y. T., and Lillard, D. W., Jr., J. Am. Oil Chem. Soc. (1977) 54, 308. 27. Cort, W. M. J. Am. Oil Chem. Soc. (1974) 51, 321; Food Tech (1975) 29(11) 46. 28. Hoseney, R. C., Seib, P. Α., and Deyoe, C. W., Cereal Chem. (1977) 54, 1062. 29. Klaüi, H. Wiss. Ver. Deutschen Gesell. fur Ernährung. (1963) 9 390.

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CARBOHYDRATE SULFATES

30. Feather, M. S. and Harris, J. F. Adv. in Carbohyd. Res. (1973) 28, 161. 31. Tolbert, B. M., Matwiyoff, Ν. Α., Lillard, D. L., Mueller, D. D., Paukstelis J., and Seib, P. A. in preparation. 32. Liler, Μ., "Reaction Mechanisms in Sulfuric Acid, "Aca­ demic Press, New York, N.Y. (1971); Gillespie, R. S., and Robinson, Ε. Α., "Non-Aqueous Solvents," Waddington, T. C., ed. Academic Press, New York (1965), pp. 117-120. 33. Allaudeen, H. S., and Ramakrishnan, R., Arch. Biochem. and Biophys. (1970) 140, 245. 34. Hatanaka, Η., Ogawa, Υ., and Egami, F., J. Biochem. (1974) 75, 861. 35. Tolbert, Β. Μ., Spears, A. H., Isherwood, D. J., Atchley, R. Η., and Baker, Ε. Μ., Abstracts of the Fed. of Exp. Biol. Soc, (1972) 31 (No. 2), Abstr. No. 2761. Tolbert, Β. Μ., personal communication (1974). 36. Honda, S., Yuku, Η., and Takiura, Κ., Carbohyd. Res. (1963) 28, 150. 37. Mahoney, J. F., and Purves, C. Β., J. Am. Chem. Soc. (1942) 64, 9; Yin, T. P., and Brown, R. K. Can. J. Chem. (1959) 37, 444. 38. Lillard, D. L., "L-Ascorbic Acid in Concentrated Sul­ furic Acid; Improved Synthesis of L-Ascorbic Acid 6-Sulfate," M. S. Thesis, Kansas State University (1977). 39. Trevelyan, W. Ε., Parker, C. P., and Harrison, J. S., Nature, (1950) 166, 144. 40. Vestling, C. S., and Rebstock, M. C., J. Biol. Chem. (1948) 161, 285. 41. Tolbert, Β. M. personal communication (1975) 42. Brenner, G. S., Hinkley, D. F., Perkinds, L. Μ., and Wever, W., J. Org. Chem., (1969) 29, 2389. RECEIVED F e b r u a r y 6, 1 9 7 8 .

Schweiger; Carbohydrate Sulfates ACS Symposium Series; American Chemical Society: Washington, DC, 1978.