OZONE CHEMISTRY AND TECHNOLOGY

Role of Formazan Reaction in Proving Structure of Ozone-Oxidized Carbohydrates LÁSZLÓ MESTER

Downloaded by COLUMBIA UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: January 1, 1959 | doi: 10.1021/ba-1959-0021.ch029

1

Institute of Organic

Chemistry, Technical University, Budapest,

Hungary

1

The formazan reaction is useful for investigating the structure of ozone-oxidized carbohydrates. Ozonizing sugar alcohols in an aqueous solution, aldoses arise during the oxidation process which have been identified by the formazan reaction. Polysaccharides also can be oxidized with ozone a n d then converted into their formazans. Polysaccharides not containing a free primary alcoholic group d o not yield formazan. This finding seems to indicate that the formazan group takes the place of the primary alcoholic group. The new formazans have been successfully transformed to metallic complexes, tetrazolium, a n d thionic acid phenylhydrazide derivatives. The reaction can also be used to establish the structure of the sugar portion of ozone-oxidized glycosides.

W h e n H a r r i e t (2) a t t h e b e g i n n i n g of t h e p r e s e n t c e n t u r y c a r r i e d o u t h i s f u n d a m e n t a l e x p e r i m e n t s w i t h a v i e w t o o z o n i z i n g o r g a n i c c o m p o u n d s , h e also s t u d i e d t h e effect of ozone o n c e r t a i n s u g a r a l c o h o l s . H i s i n i t i a l s t u d i e s s h o w e d t h a t t h e p r i m a r y a l c o h o l i c h y d r o x y l s of s u g a r d e r i v a t i v e s c a n b e o x i d i z e d t o f o r m a l d e h y d e g r o u p s b y u t i l i z i n g ozone. V a r i o u s e x p e r i m e n t s m a d e i n t h i s i n s t i t u t e i n recent y e a r s ( 5, 10-13) s h o w e d t h a t t h e f o r m a z a n r e a c t i o n l e n t i t s e l f r e a d i l y t o studies of t h e s t r u c t u r e of o x i d i z e d s u g a r d e r i v a t i v e s w h e n e v e r free a l d e h y d e g r o u p s were p r e s e n t . Obviously, i t was a useful r e a c t i o n f o r i n v e s t i g a t i o n s i n t o t h e s t r u c t u r e of o z o n e - o x i d i z e d c a r b o h y d r a t e s , a m o n g t h e m the sugar alcohols t h a t h a d been studied b y H a r r i e s . T h e present w o r k showed, i n perfect agreement w i t h H a r r i e s , t h a t o n ozonizing s u g a r a l c o h o l s i n a n a q u e o u s s o l u t i o n , t h e y b e g i n t o o x i d i z e a n d aldoses arise i n t h e course of t h e o x i d a t i o n process. F o r instance, D-mannose was prepared f r o m ozoneo x i d i z e d D - m a n n i t o l ; w i t h t h e a i d of t h e f o r m a z a n r e a c t i o n , D - m a n n o s e w a s t h e n i d e n t i f i e d as D - m a n n o - A ^ i V ' - d i p h e n y l f o r m a z a n (8). 1

Present address, Faculté de Pharmacie, Université de Paris, Paris, France. 195

In OZONE CHEMISTRY AND TECHNOLOGY; Advances in Chemistry; American Chemical Society: Washington, DC, 1959.

196

ADVANCES

Η C/= 0.

CH OH 2

HO-C—H

I

H O - Ç - H —

^

N H

.

H O - Ç - H

N H 2

I

Ο

'N = N * Ç*[ N - N,

[Q- . ]* N

H

N

>•

0

I I

'

H

" Ç -

I H - C - O H

CH OH

CH OH

I

2

>

H

H—Ç—OH

H—C-OH

CH OH

2

SERIES

HO

H—C—OH

•

H - C - O H

CH OH


Q_

H - C - O H

CHEMISTRY

. \>

HO-C—H

l

H-C—OH

,

C/= N - N H — (/

HO—C—H

H O - Ç - H H—C—OH

Η

IN

I

2

2

T o an aqueous solution of mannitol, ozonized for 7 to 14 hours, phenylhydrazine was added. When the solution was kept in the refrigerator overnight, mannose phenylhydrazone separated. O n coupling it in pyridine solution with an ice cold solution of diazotized aniline, D-manno-A^iV'-diphenyl formazan, crystallizing in brownish red needles, was obtained. T o establish optimum conditions for the conversion of mannitol to mannose by 1% ozone, aqueous solutions were ozonized in gas washing bottles provided with shrunken glass niters for 7 and 14 hours, in concentrations of 0.8, 1.6, 3.2, and 4.8 1 0 mole per liter. Under these conditions, the lowest concentration and the longer time of ozonization were found to be favorable for the formation of mannose (Figure 1). - 1

^^^14

hours

7 hours 1 1

0

ι 3

2

1

4

5

Optimum conditions for oxidation of mannitol

ozone

1

Ι Ο " Mole Manitol Figure

Ί.

Other sugar alcohols, such as D-sorbitol and D-dulcitol, give an aldose phenylhydrazone which is hard to isolate and convert to the formazan. Therefore, the aldose (D-glucose from D-sorbitol) was transformed into glucosazone, which was identified with the aid of the formazan reaction as D-glucosazone formazan (4).

y—

H

JC = =..N - N. H. - / - \ H

ρ c=o

ÇH OH 2

H - C - O H

H O - Ç - H

H - C - O H

H-C—OH

H—C-OH I

H - C - O H l

CH OH 2

Ç = N - N H - ^ sQ-NH-NHa >-

H - C - O H I

CH OH 2

N = r f O "H

J

I Ν Ν ^

Ç=N-NH

H O - Ç - H H - Ç - O H

c

\_j " ΝΛΛ

y=y

I

H - C - O H

H O - Ç - H

ft.

H O - Ç - H +

^QHN=N] >-

H - Ç - O H H-Ç—OH I

CH OH 2

CH 0H 2


\Q>

M E S T E R - F O R M A Z A N REACTION

197

S u c r o s e l i k e w i s e y i e l d s g l u c o s a z o n e i d e n t i f i a b l e as g l u c o s a z o n e f o r m a z a n . T h i s i n d i c a t e s t h a t i n sucrose i t is t h e h y d r o x y l g r o u p o n t h e first c a r b o n a t o m of D - f r u c t o s e w h i c h oxidizes to a n aldehyde group. A s has been observed b y several authors {1,7, 14), i n t h e course of t h e f o r m a t i o n of osazone t h e d i s a c c h a r i d e b o n d (2, 5) b r e a k s u p a n d g l u c o s a z o n e f o r m s f r o m b o t h c o m p o n e n t s of t h e d i s a c c h a r i d e u p o n t h e a d d i t i o n of phenylhydrazine.


F r o m ozone-oxidized sugar alcohols n o f o r m a z a n derivatives have y e t been isolated i n w h i c h t h e p r i m a r y a l c o h o l i c h y d r o x y l s , p r e s u m a b l y p r e s e n t a t b o t h ends of t h e molecule, have oxidized to aldehydes. H o w e v e r , bisformazans, conspicuous for their c a p a c i t y to crystallize a n d their insolubility i n water, have been successfully prepared b y o t h e r m e a n s (IS). P o l y s a c c h a r i d e s , s u c h as cellulose a n d s t a r c h , c a n be o x i d i z e d w i t h ozone a n d t h e n c o n v e r t e d t o t h e i r f o r m a z a n s . A cellulose w a d o r a s a m p l e of s t a r c h p o w d e r , finely divided or suspended i n water, was placed i n a gas-washing bottle w i t h a s h r u n k e n glass filter a n d o z o n i z e d a t r o o m t e m p e r a t u r e f o r 8 t o 18 h o u r s , c a r e f u l l y w a s h e d , a n d r e a c t e d i n acetic a c i d w i t h p h e n y l h y d r a z i n e . T h i s r e s u l t e d i n v i v i d l y y e l l o w p h e n y l h y d r a z o n e s of t h e o x i d i z e d p o l y s a c c h a r i d e s . B y t h e r e a c t i o n w i t h a n i c e - c o l d s o l u t i o n of d i a z o t i z e d a n i l i n e i n p y r i d i n e a n d e t h y l a l c o h o l , t h e b r i g h t r e d f o r m a z a n s o f t h e o z o n e - o x i d i z e d p o l y s a c c h a r i d e s were o b t a i n e d . P o l y s a c c h a r i d e s n o t c o n t a i n i n g a free p r i m a r y a l c o h o l i c g r o u p — e . g . , x y l a n — h a v e f a i l e d t o y i e l d f o r m a z a n u p o n o x i d a t i o n w i t h ozone. T h i s finding a n d t h e o b s e r v a t i o n s m a d e i n t h e p r e s e n t w o r k i n c o n n e c t i o n w i t h o z o n e - o x i d i z e d s u g a r a l c o h o l s seem t o s u p p o r t t h e suggestion t h a t t h e f o r m a z a n g r o u p t a k e s t h e p l a c e of t h e p r i m a r y a l c o h o l i c group.

I t h a s l o n g b e e n k n o w n t h a t o x i d a t i o n of s i m p l e f o r m a z a n s gives t e t r a z o l i u m c o m p o u n d s w h i c h , e v e n u n d e r b i o l o g i c a l c o n d i t i o n s , a r e e a s i l y r e d u c e d once m o r e t o r e d formazans. F o r instance, triphenyltetrazolium chloride, prepared b y oxidizing t r i p h e n y l f o r m a z a n , is a n a p p r o v e d b i o l o g i c a l i n d i c a t o r . L i k e t h e s u g a r f o r m a z a n s (9), t h e f o r m a z a n s p r e p a r e d f r o m o z o n e - o x i d i z e d p o l y s a c c h a r i d e s r e a d i l y o x i d i z e w i t h i V - b r o m o s u c c i n i m i d e t o t h e c o r r e s p o n d i n g colorless tetrazolium compounds.



198

A D V A N C E S IN

CHEMISTRY SERIES

A s i n t h e f o r m a z a n s of t h e s i m p l e sugars (15), t h o s e of t h e o z o n e - o x i d i z e d p o l y s a c c h a r i d e s u n d e r g o r e d u c t i v e d i s i n t e g r a t i o n b y t h e a c t i o n of h y d r o g e n s u l f i d e . T h u s , t h e f o r m a z a n o f o z o n e - o x i d i z e d s t a r c h gives t h e c o r r e s p o n d i n g t h i o n i c a c i d p h e n y l hydrazide.

A g a i n , s i m i l a r t o t h e s i m p l e f o r m a z a n s (6), t h o s e of t h e o z o n e - o x i d i z e d p o l y s a c c h a r i d e s r e a d i l y f o r m c o m p l e x salts w i t h h e a v y m e t a l s . T h e p r o b a b l e s t r u c t u r e of a c o p p e r c o m p l e x o b t a i n e d f r o m t h e f o r m a z a n of o z o n e - o x i d i z e d cellulose is :

00

4

ο

ο

P a r t i c u l a r l y fine colors a r e d i s p l a y e d b y t h e m e t a l c o m p l e x e s of f o r m a z a n s d e r i v e d f r o m viscose r a y o n . T h e c o p p e r , c o b a l t , a n d u r a n i u m cellulose c o m p l e x e s r e p r e s e n t a n e w t y p e of t h e s o - c a l l e d c h e m i c a l l y c o l o r e d t h r e a d s . U n d e r t h e e l e c t r o n m i c r o s c o p e t h e u r a n i u m c o m p l e x e s of t h e f o r m a z a n s of o z o n i z e d p o l y s a c c h a r i d e s d i s p l a y a d i s t i n c t s h a d i n g effect, t h e p o l y s a c c h a r i d e s s h o w i n g a deep b l a c k c o l o r . O n t h i s basis i t is t o be h o p e d t h a t a n e w e l e c t r o n m i c r o s c o p i c m e t h o d w i l l s o o n be e l a b o r a t e d (6).


MESTER-FORMAZAN

199

REACTION

T h e f o r m a z a n r e a c t i o n c a n also b e u s e d t o e s t a b l i s h t h e s t r u c t u r e of t h e s u g a r p o r t i o n of o z o n e - o x i d i z e d glycosides. Some earlier investigations i n this laboratory w i t h glycosides c o n t a i n i n g a r o m a t i c o r c y c l i c aglycones s h o w t h a t t h e l a t t e r i s a t t a c k e d d u r i n g o z o n i z a t i o n , w h i l e a c o n s i d e r a b l e p a r t of t h e s u g a r p o r t i o n r e m a i n s u n c h a n g e d a n d c a n b e i s o l a t e d , e v e n i f i t is d i s a c c h a r i d e (3). F o r instance, f r o m «-phenylmannoside t r e a t e d w i t h ozone i t w a s possible t o isolate D - m a n n o - i V , i V ' - d i p h e n y l f o r m a z a n , and f r o m ozonized quercitrin, L - r h a m n o - A ^ i V ' - d i p h e n y l formazan.


OH

I n these cases t h e o x i d a t i o n of t h e s u g a r p o r t i o n i s o b v i o u s l y p r e c e d e d b y t h e d i s i n t e g r a t i o n of t h e a g l y c o n . E x p e r i m e n t s t o disclose t h e t h e o r e t i c a l a n d p r a c t i c a l v a l u e of o z o n e - o x i d i z e d c a r b o hydrates a n d the nitrogenous derivatives prepared f r o m t h e m are being continued. Acknowledgment T h e a u t h o r wishes t o express h i s t h a n k s t o A d a m M a j o r f o r h i s assistance i n t h e e x p e r i m e n t a l p a r t of t h i s w o r k a n d t o I l o n a B a t t a f o r m a k i n g t h e m i c r o a n a l y s e s . Literature

Cited

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Freudenberg, K., Knauber, H., Cramer, F., Chem. Ber. 84, 144 (1951). Harries, C., Langheld, Κ., Z. physiol. Chem., Hoppe-Seyler's 51, 373 (1907). Mester, L., "Glycosidok ozonidos bontàsa," Dissertation, Budapest, 1942. Mester, L., J. Am. Chem. Soc. 77, 4301 (1955). Ibid., p. 5452. Mester, L., J. Polymer. Sci., in press. Mester, L., Magyar Kém. Folyóirat 50, 134 (1944). Mester, L., Major, Α., J. Chem. Soc. 1956, 1403. Mester, L., Messmer, Α., Ibid., 1957, 3208. Mester, L., Moczar, E., Chem. & Ind. 1956, 823.

(11) (12)

Ibid., Ibid.,

1957, 761. p. 764.

(13) Mester, L., Moczar, E., J. Chem. Soc. 1956, 3228. (14) Rabaté, J., Bull. soc. chim. 7, 565 (1940). (15) Zemplén, G., Mester, L., Messmer, Α., Chem. RECEIVED for review June 6, 1957.

Ber. 86, 697 (1953).

Accepted June 19, 1957.


OZONE CHEMISTRY AND TECHNOLOGY

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