Some Novel Methods and Results in the Sulfation of Polysaccharides

9 Some Novel Methods and Results in the Sulfation of

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Polysaccharides K E N N E T H B. G U I S E L E Y Marine Colloids, Inc., Rockland, ME 04841

The methods used in the sulfation of polysaccharides are f a i r l y limited, because the properties of sulfating agents and those of polysaccharides combine to put severe constraints on the synthetic chemist. Thus, in virtually a l l the literature, we find essentially the same techniques - the treatment of a polysaccharide with a Lewis base complex of sulfur trioxide, under anhydrous conditions, usually in the presence of a base such as pyridine. One of the commonest techniques involves the addition of chlorosulfonic acid to pyridine in the cold, followed by introduction of the polysaccharide, and subsequent heating (1-8). The product is usually recovered and purified by alcohol precipitation, dissolution in water, neutralization, dialysis, and alcohol precipitation. The vigor of the sulfating agent is a function of the strength of the Lewis base with which it is combined. Pyridine, being a moderate base, effectively controls the potency of the sulfur trioxide, and the crystalline pyridine-sulfur trioxide complex is readily prepared (9). Triethylamine and trimethylamine form less reactive sulfating reagents (10a), which are so stable, that sulfations in aqueous systems have been reported (4, 11). At the opposite end of the act i v i t y scale are the complexes with dimethylformamide (DMF) and dioxane (10a). These, being much weaker bases, hold the sulfur-trioxide less tightly, permitting reactions at lower temperatures. Virtually a l l polysaccharides have been the object of sulfation reactions, starting with cellulose in 1819 (12). A series of review articles in Industrial and Engineering Chemistry in the 1950's and 1960's, by Gilbert and Jones refer to most of these, and a concise, but thorough, coverage was given the subject in Gilbert's book, "Sulfonation and Related Reactions" (10b). 0-8412-0426-8/78/47-077-148$05.00/0 © 1978 American Chemical Society

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


9.

GuisELEY

Sulfation

of

Polysaccharides

149

Because o f t h e p h y s i o l o g i c a l importance o f h e p a r i n , many attempts have been made over the y e a r s t o produce a s y n t h e t i c h e p a r i n by s u l f a t i o n o f p o l y s a c c h a r i d e s (5-7, 13, 14), and a n t i u l c e r a t i v e m e d i c a t i o n s ( p e p s i n i n h i b i t o r s ) have been s i m i l a r l y p r e p a r e d (1_, 15-17) . D i r e c t s u l f a t i o n o f p o l y s a c c h a r i d e s , as, f o r example, by the p y r i d i n e - c h l o r o s u l f o n i c a c i d method, g e n e r a l l y r e s u l t s i n d e g r a d a t i o n o f t h e polymer, w i t h r e s u l t a n t l o s s o f c o l l o i d a l p r o p e r t i e s . However, employment o f g e n t l e r c o n d i t i o n s o f t e n f a i l s t o e f f e c t enough s u l f a t i o n t o change t h e p r o p e r t i e s o f the p o l y saccharide. Wolfrom and J u l i a n o (_3_) r e c o g n i z e d the need t o r e n d e r p o l y s a c c h a r i d e s more amenable t o s u l f a t i o n through an a c t i v a t i o n p r o c e s s , and Schweiger f u r t h e r e d t h e concept i n a s e r i e s o f p a t e n t s d e s c r i b i n g the s u l f a t i o n o f a l g i n (18), s t a r c h (19), xanthan (20), and c e l l u l o s e (21). In t h e s e c a s e s , t h e a c t i v a t i o n was a c c o m p l i s h e d by t r e a t m e n t o f t h e p o l y s a c c h a r i d e i n a s e r i e s o f s o l v e n t washes which d e h y d r a t e d i t , y e t p r e v e n t e d i t s becoming i n e r t t o s u l f a t i o n , as does oven-drying. The p r i n c i p a l s u b j e c t o f t h i s paper i s a g e n e r a l l y a p p l i c a b l e method o f a c t i v a t i n g p o l y s a c c h a r i d e s , which we d e v e l o p e d a t about the same time. Basically, i t c o n s i s t s o f a two-step p r o c e s s i n v o l v i n g h y d r a t i o n o f t h e gum, f o l l o w e d by d i s t i l l a t i v e s o l v e n t d r y i n g . For i t s h y d r a t i o n , the gum may be t o t a l l y d i s s o l v e d , o r merely s w o l l e n w i t h a l i m i t e d q u a n t i t y o f water. In the d e h y d r a t i o n s t e p , the water and any o t h e r s o l v e n t which may r e a c t i r r e v e r s i b l y w i t h a s u l f a t i n g agent, are removed by d i s t i l l a t i o n i n t h e p r e s e n c e o f a h i g h e r b o i l i n g , water-miscible s o l v e n t . Products of p r e d i c t a b l e degrees o f s u b s t i t u t i o n and h i g h m o l e c u l a r weight can then be p r e p a r e d by c o n v e n t i o n a l methods o f s u l f a tion. Our i n t e r e s t i n s u l f a t i n g p o l y s a c c h a r i d e s was p r i m a r i l y one o f p r o d u c i n g a s y n t h e t i c c a r r a g e e n a n (,22.). Carrageenans a r e g a l a c t a n s u l f a t e s p r e s e n t i n a number o f r e d seaweeds, and which a r e e x t r a c t e d and used f o r t h e i r g e l l i n g , suspending, and v i s c o s i t y - p r o d u c i n g p r o p e r t i e s i n f o o d s , p a r t i c u l a r l y d a i r y p r o d u c t s , and i n such o t h e r d i v e r s e p r o d u c t s as t h e p o p u l a r a i r f r e s h e n e r g e l . A s e m i - s y n t h e t i c gum w i t h p r o p e r t i e s s i m i l a r t o c a r r a g e e n a n c o u l d prove t o be o f g r e a t economic v a l u e i n t h e f a c e o f r i s i n g seaweed c o s t s which put the p r i c e o f carrageenan a t r o u g h l y f o u r times t h a t o f guar and l o c u s t bean gum, and twenty t o t h i r t y times t h a t o f s t a r c h .


CARBOHYDRATE SULFATES

150

Results

and D i s c u s s i o n


The e f f e c t s o f t i m e , temperature, s u l f a t i n g r e a gent, and p r e c i p i t a t i n g s o l v e n t were d e t e r m i n e d on p o l y s a c c h a r i d e s a c t i v a t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method, a f t e r e a r l i e r e x p e r i m e n t s showed t h a t o t h e r methods o f d r y i n g i n h i b i t e d r e a c t i o n (Table 1 ) . E f f e c t o f Temperature. F o r guar and l o c u s t bean gum s u l f a t e d w i t h DMF:S03, t h e e x t e n t o f s u l f a t i o n was q u i t e comparable a t a g i v e n temperature, whereas o t h e r gums v a r i e d somewhat i n t h e i r tendency toward b e i n g sulfated. R e a c t i o n was found t o be q u i t e r a p i d i n i t i a l l y , as i n d i c a t e d i n F i g . 1, w i t h about 80% o f t h e p o s s i b l e s u b s t i t u t i o n f o r a given temperature o c c u r r i n g i n t h e f i r s t hour. U s i n g t h i s knowledge, i t was p o s s i b l e t o o b t a i n a "product w i t h a d e s i r e d DS, merely by employing t h e c a l c u l a t e d amount o f s u l f a t i n g r e a g e n t and c a r r y i n g o u t t h e r e a c t i o n a t a temperature somewhat above t h e temperature which would produce t h a t DS when an e x c e s s o f s u l f a t i n g r e a g e n t was p r e s e n t . An added advantage i n t h i s t e c h n i q u e was t h e f a c t t h a t t h e r e was no r e s i d u a l s u l f a t i n g r e a g e n t l e f t f o l l o w i n g t h e r e action. E f f e c t o f P r e c i p i t a t i n g S o l v e n t . « A f a c t o r which, r a t h e r s u r p r i s i n g l y , a f f e c t e d t h e DS a c h i e v a b l e under a g i v e n s e t o f s u l f a t i o n c o n d i t i o n s , was t h e p a r t i c u l a r s o l v e n t used f o r t h e i n i t i a l p r e c i p i t a t i o n o f t h e gum. T h i s i s i l l u s t r a t e d i n T a b l e 2. With a l l t h r e e gums t e s t e d , methanol was g e n e r a l l y t h e most e f f i c i e n t a t a c t i v a t i n g , whereas DMF v a r i e d t h e most, b e i n g l e a s t e f f i c i e n t w i t h s t a r c h and most w i t h l o c u s t bean gum. I s o p r o p y l a l c o h o l worked w e l l f o r s t a r c h b u t was unimp r e s s i v e w i t h t h e two galactomannans. Acetone was gene r a l l y poor. A p o s s i b l e explanation i s that the order i s , i n general, r e l a t e d t o the dehydrating a b i l i t y of the s o l v e n t , m o d i f i e d by an a f f i n i t y o f each gum f o r a p a r t i c u l a r solvent. In a l l t h e s e c a s e s , t h e gum was a c t i v a t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method and s u l f a t e d a t 25° f o r 24 h o u r s , u s i n g s u f f i c i e n t 1 M DMF:S0 t o t o t a l l y s u l f a t e t h e gum, i . e . , t o produce a DS o f 3.0. P y r i d i n e was p r e s e n t i n an amount e q u a l t o t w i c e t h e number o f moles o f DMF:SC>3. 3

E f f e c t o f S u l f a t i n g Reagent. U s i n g DMF as a s o l v e n t , and l o c u s t bean gum as t h e s u b s t r a t e , f o u r s u l f a t i n g r e a g e n t s were t e s t e d f o r t h e i r a c t i v i t y a t 70° (for 4 hours). Here, a g a i n , t h e r e were d i f f e r e n c e s i n DS. G e n e r a l l y , t h e y f o l l o w e d t h e o r d e r p r e d i c t e d from


9.

Sulfation

GuiSELEY

of

Polysaccharides

151

T a b l e 1. E f f e c t o f d r y i n g method on degree o f s u b s t i t u t i o n (DS) and v i s c o s i t y (1%, 2 5 ° , B r o o k f i e l d LVF, 6 rpm) f o r l o c u s t bean gum + 1 mole DMF:SO3/mole anhydro sugar, 24 h r s . , room temperature.


D.S.

25 , cps) 1.0

η

(

Oven D r y i n g , 60°C. ATM.

0. 006

2425

Benzene D i s t i l l a t i o n ,

ATM.

0. 03

3450

Benzene D i s t i l l a t i o n , VAC.

0. 07

1100

DMF D i s t i l l a t i o n , VAC.

0.47

1150

3.0

ρ Sulfation of Guar

2.0

—

D.S. 1.0

70°

0.0

2 3 T I M E , hrs.

Figure 1. Effect of time at different tempera tures in the sulfation of guar with DMF:S0 3

T a b l e 2. E f f e c t o f p r e c i p i t a t i n g s o l v e n t on degree o f s u l f a t i o n o f s t a r c h , guar, and l o c u s t bean gum (DMF:S0 , 25°/24 h r s . ) 3

Starch

Guar

LBG

MeOH

1. 51

DMF

1. 67

1. 37

DMF

0. 70

MeOH

1. 08

Acetone

0. 57

Acetone

0. 65

Py

0. 94

DMF

0. 31

i-PrOH

0. 60

Acetone

0. 53

i-PrOH

0. 47

Me OH

1. 65

i-PrOH


CARBOHYDRATE SULFATES

152

t h e s t r e n g t h o f t h e base used t o complex t h e SO3, w i t h t h e e x c e p t i o n o f t r i e t h y l a m i n e , which produced t h e h i g h e s t DS r a t h e r t h a n t h e second l o w e s t as a n t i c i pated: S u l f a t i n g Reagent Et N:S0 DMF:S0 Py:S0 Me N:S0 3

DS 1.35

3

0.99


3

0.88

3

3

0.

3

39

From a p r a c t i c a l p o i n t o f view, t h e r e i s no advantage i n u s i n g any o f t h e complexes o t h e r t h a n t h a t w i t h DMF, s i n c e i t can be p r e p a r e d and used d i r e c t l y , and i s v e r y efficient. T h i s a s p e c t was i n v e s t i g a t e d no f u r t h e r . A n a l y s i s f o r A c t i v e S u l f a t i n g Reagent. Because o f t h e d e s i r e t o c o n t r o l DS and t o m i n i m i z e polymer degra d a t i o n , i t was n e c e s s a r y t o a n a l y z e f o r t h e c o n c e n t r a t i o n o f b o t h a c t i v e s u l f a t i n g r e a g e n t and h y d r o l y z e d DMF:S0 which may be p r e s e n t . P u b l i s h e d methods f o r such an a n a l y s i s u s u a l l y d i r e c t the r e a d e r t o add an e x c e s s of water and t i t r a t e t h e s u l f u r i c a c i d p r o duced ( 2 3 ) . T h i s method f a i l s t o make t h e n e c e s s a r y d i s t i n c t i o n between t h e a c t i v e and t h e h y d r o l y z e d r e a gent. I f , however, one uses anhydrous methanol i n p l a c e o f water i n a second t i t r a t i o n , i t i s p o s s i b l e t o d e t e r m i n e , by d i f f e r e n c e , b o t h t h e amount o f a c t i v e s u l f a t i n g r e a g e n t and t h a t o f s u l f u r i c a c i d . 3

C a l l i n g the r e a c t i o n w i t h water T i t r a t i o n f u r i c a c i d from a l l s o u r c e s i s d e t e r m i n e d : S0

3

+ H 0

+ H S0

4

(2

meq.

H)

H S0

-> H S 0

4

(2

meq.

H)

2

2

Titration S0

3

4

2

2

3

3

H S0 2

4

3

-> H S 0 2

4

+

( 1 meq.

H+)

(2

H)

meq.

species:

+

Therefore, A =

2 X meq.

S0

3

+ meq.

H S0

4

Β =

meq.

S0

3

+ meq.

H S0

4

meq.

S0

3

A-B

=

sul

+

Β d i s t i n g u i s h e s between t h e two

+ CH OH -> C H O S 0 H

A,

2

2

and,


9.

Sulfation

GuiSELEY

2B = 2 X meq. A = 2B-A

=

of

Polysaccharides

153

H S0

4

+ 2 X meq.

SO

meq.

H S0

4

+ 2 X meq.

SO

meq.

H S0

2

2

2

4

To i l l u s t r a t e t h i s , 10 ml. o f DMF:S0 r e a g e n t added to 50 ml. o f water r e q u i r e d 22.65 ml. o f 1 Ν NaOH. A n o t h e r 10-ml. p o r t i o n was added t o 10 ml. o f anhydrous DMF p l u s 2.5 ml. o f anhydrous methanol and 5 ml. anhydrous pyridine. The m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t , then 50 ml. o f water was added, and the s o l u t i o n t i t r a t e d t o a p h e n o l p h t h a l e i n e n d p o i n t w i t h 1 Ν NaOH: 11.90 ml. was required.


3

A-B 2B-A

= 22.65 - 11.90

= 10.75

= 23.80 - 22.65 =

1.15

meq. meq.

S0

3

H S0 2

4

E f f e c t of R e s i d u a l Water A f t e r A c t i v a t i o n . This a n a l y t i c a l scheme was put t o use t o d e t e r m i n e how much r e s i d u a l water was p r e s e n t i n an a c t i v a t e d gum, by c a r r y i n g out t h e a n a l y s i s on the r e a c t i o n m i x t u r e a f t e r t h e r e a c t i o n was complete. Guar gum was a c t i v a t e d by t h e d i s s o l u t i o n / r e p r e c i p i t a t i o n method, f o u r samples b e i n g p r e p a r e d i d e n t i c a l l y except t h a t each was taken t o a d i f f e r e n t s t a t e of d r y n e s s w i t h the DMF. S u l f a t i o n was c a r r i e d out w i t h two moles o f DMF:S0 per mole o f anhydrohexose, and two moles o f p y r i d i n e f o r each mole o f DMF:S0 , employing c o n d i t i o n s o f time and temperature t h a t would n o r m a l l y produce a DS o f about 1.25. When t h e r e a c t i o n was complete, the p r o d u c t was s e p a r a t e d and worked up, and the r e a c t i o n m i x t u r e ana l y z e d f o r a c t i v e DMF:S0 and s u l f u r i c a c i d . The v i s c o s i t y o f the p r o d u c t and i t s DS were d e t e r m i n e d and compared w i t h the l e v e l o f s u l f u r i c a c i d found. These r e s u l t s are shown i n T a b l e 3. I t i s quite c l e a r that d i f f e r e n c e s i n v i s c o s i t y a r e not due t o v a r i a t i o n s i n DS, s i n c e t h o s e are v i r t u a l l y i d e n t i c a l , but i t does appear s i g n i f i c a n t t h a t even i n the p r e s e n c e o f an ex cess of p y r i d i n e , degradation occurs i n p r o p o r t i o n to the amount of s u l f u r i c a c i d produced. In view o f the f a c t t h a t the s u l f a t e e s t e r o f the polysaccharide s h o u l d be as a c i d i c as the s u l f u r i c a c i d (pyridinium i o n b e i n g the c o u n t e r i o n i n b o t h c a s e s ) , a p o s s i b l e explanation i s that oxidative degradation i s occurring, r a t h e r than h y d r o l y t i c c h a i n c l e a v a g e . (The t i t r a t a b l e a c i d i t y would then be e x p l a i n e d i n terms o f H S 0 plus unreduced H S 0 ) . 3

3

3

2

2

3

4

Other A c t i v a t i o n Methods.

A l t e r n a t i v e methods o f



154

CARBOHYDRATE

SULFATES

h y d r a t i n g the gums were i n v e s t i g a t e d i n t h e i n t e r e s t of economy - t h e d i s s o l v i n g and r e i s o l a t i o n o f a gum i s v e r y c o s t l y i n view o f t h e energy r e q u i r e m e n t s f o r s o l v e n t r e c o v e r y . In one, t h e gum was b r i e f l y soaked i n aqueous i s o p r o p y l a l c o h o l , then the m i x t u r e was b o i l e d r a p i d l y t o evaporate the a l c o h o l . The r e s u l t i n g wet, s w o l l e n powder was mixed w i t h DMF, broken up, and a l l o w e d t o s t a n d o v e r n i g h t . The water and DMF were r e moved by vacuum d i s t i l l a t i o n as i n t h e p r e c i p i t a t i o n method, and the r e s u l t i n g a c t i v a t e d gum s u l f a t e d . For a g i v e n s u l f a t i o n c o n d i t i o n , the DS and m o l e c u l a r weight (as i n d i c a t e d by v i s c o s i t y ) were dependent on the c o n c e n t r a t i o n o f a l c o h o l i n t h e o r i g i n a l m i x t u r e (Table 4 ) . A t low t i t e r s o f a l c o h o l , low v i s c o s i t i e s suggested incomplete dehydration with r e s u l t i n g degradation. At intermediate t i t e r s , v i s c o s i t i e s increased markedly, a c t u a l l y e x c e e d i n g t h e v i s c o s i t y and DS o f the t y p i c a l DS 1.2 5 guar s u l f a t e s d e s c r i b e d above. F i n a l l y , a t the h i g h e r t i t e r s , t h e DS a g a i n f e l l , p r o b ably a r e s u l t of incomplete a c t i v a t i o n . A v a r i a t i o n o f t h i s p r o c e s s was t h e use o f aqueous DMF t o h y d r a t e t h e gum. T h i s d i d not work w e l l when the o r i g i n a l m i x t u r e p l a c e d on t h e gum was d i s t i l l e d o f f i n vacuo, but i f t h e m i x t u r e were h e a t e d , and the gum t h e n f i l t e r e d o f f and d e h y d r a t e d w i t h f r e s h DMF, s u l f a t i o n w i t h o u t e x c e s s i v e d e g r a d a t i o n was p o s s i b l e . For example, h e a t i n g guar w i t h 3 0% aqueous DMF, filtering, d e h y d r a t i n g , and s u l f a t i n g under c o n d i t i o n s t h a t gave a DS o f 1.25 and a v i s c o s i t y o f about 2000 cps w i t h p r e c i p i t a t e d guar, a DS o f 1.09 and a v i s c o s i t y of 4240 cps were o b t a i n e d , a g a i n somewhat b e t t e r than by t h e p r e c i p i t a t i o n method. A v e r y unexpected r e s u l t came o u t o f a m o d i f i c a t i o n of t h e s e methods: guar was mixed w i t h 60% aqueous i s o p r o p y l a l c o h o l and r e f l u x e d 15 m i n u t e s , then f i l t e r e d o f f and d e h y d r a t e d and s u l f a t e d as d e s c r i b e d above. The p r o d u c t had a DS o f o n l y 0.16, but a v i s c o s i t y o f 29,000 cps a t 1%. (The u n t r e a t e d guar had a v i s c o s i t y of 6600 cps measured by t h e same procedure.) A repeat of t h e experiment r e s u l t e d i n a p r o d u c t w i t h a DS o f 0.20 and a 1% v i s c o s i t y o f 26,000 cps. E f f e c t o f Bases. Because t h e p r i m a r y g o a l o f t h e work was t o make a s y n t h e t i c c a r r a g e e n a n , and s i n c e c a r r a g e e n a n i s used so e x t e n s i v e l y i n f o o d s , an attempt was made t o r e p l a c e p y r i d i n e i n t h e r e a c t i o n m i x t u r e w i t h a l e s s n o x i o u s base. The d i f f i c u l t y was t o f i n d one which would not be so a l k a l i n e as t o r e a c t p r e f e r e n t i a l l y w i t h the DMF:SC>3, n o r so weak as t o f a i l t o p i c k up p r o t o n s as t h e y were l i b e r a t e d (the p o s s i b i l i t y


9.

Sulfation

GuiSELEY

of

Polysaccharides

155


Table 3. E f f e c t of s u l f u r i c acid i n reaction mixture on v i s c o s i t y of sulfated guar. (8.1 g. guar •> 13.1 g. Na guar SO4) . Meq. H S0 2

4

From D M F i S O o (92 ml. 1.106 M)

From H 0 i n Guar 2

Total

9. 9

10. 0

19.9

2375

1.27

9. 9

14.3

24.2

2175

1.27

9.8

26.8

36. 6

1725

1.22

9. 9

38. 5

48.4

1525

1.24

ηJf (cps) 0

DS

T a b l e 4. E f f e c t o f a l c o h o l t i t e r on DS and v i s c o s i t y o f guar gum h y d r a t e d by a l c o h o l b o i l - o f f (8.1 g. guar + 100 ml. aqueous i s o p r o p y l a l c o h o l ) .

% i-PrOH

DS

η

10

0.70

840

20

0.33

605

30

1.25

850

40

1.26

1450

50

1.46

3400

60

0.88

5700

70

0.21

7300

1 < Q

(cps


CARBOHYDRATE


156

SULFATES

o f o x i d a t i v e d e g r a d a t i o n had n o t been c o n s i d e r e d a t the time). A l t h o u g h none was found t h a t worked as w e l l as p y r i d i n e ( l i k e l y because o f t h e h e t e r o g e n e i t y o f t h e system) , ones t h a t showed promise were sodium and pot a s s i u m a c e t a t e s and d i s o d i u m phosphate. In T a b l e 5 are g i v e n some r e s u l t s . In t h e s e i n s t a n c e s , t h e aqueous DMF method was used except f o r t h e example w i t h p o t a s s i u m a c e t a t e ; t h i s was c a r r i e d o u t w i t h f r e s h l y p r e c i p i t a t e d guar. C o n t r o l o f DS. An i n t e r e s t i n g s i d e l i g h t t o o u r work was d e m o n s t r a t i n g t h e apparent i d e n t i t y o f funoran and s u l f a t e d a g a r o s e . Funoran i s a seaweed p o l y s a c c h a r i d e used i n g r e a t q u a n t i t i e s i n t h e F a r E a s t . Ext r a c t e d from G l o i o p e l t i s f u r c a t a and r e l a t e d s p e c i e s , i t f i n d s use- as a t h i c k e n e r o r g l u e . I t was i d e n t i f i e d as a g a l a c t a n s u l f a t e i n t h e e a r l y 1 9 0 0 ' s , and t h e p r e s e n c e o f 3 , 6 - a n h y d r o - L - g a l a c t o s e was e s t a b l i s h e d i n 1956 by H i r a s e , A r a k i , and I t o ( 2 4 ) . T h i s meant t h a t i n s t e a d o f b e i n g a c a r r a g e e n a n , i t was more l i k e a s u l f a t e d agarose. When agarose was s u l f a t e d t o t h e same e x t e n t as f u n o r a n , and t h e i r i n f r a r e d s p e c t r a compared ( F i g . 2), they were found t o be e s s e n t i a l l y i d e n t i c a l as proposed by S t a n c i o f f and S t a n l e y i n 1968 (2_5) · A d d i t i o n a l p r o o f o f f u n o r a n s s t r u c t u r e was g i v e n by Penman and Rees i n 1 9 7 3 (26J . 1

Conclusion - Properties o f the Products. S u l f a t e d p o l y s a c c h a r i d e s p r e p a r e d i n t h i s work were found t o have one f e a t u r e i n common - t h e y were n o n - g e l l i n g . As such, they were most l i k e lambda-carrageenan, and when t e s t e d i n a v a r i e t y o f a p p l i c a t i o n s , demonstrated an a b i l i t y t o r e p l a c e lambda-, b u t n o t kappa- o r i o t a carrageenan. Of a l l t h e p r o d u c t s made, t h e most comm e r c i a l l y p r o m i s i n g was a guar s u l f a t e h a v i n g a DS o f 2 . 1 9 - i t c o u l d be used t o s t a b i l i z e a c o l d - m i x e d m i l k shake a t o n e - f i f t h t h e l e v e l o f a c a r r a g e e n a n used f o r t h a t purpose. S e v e r a l o f t h e p r o d u c t s made s a t i s f a c t o r y c h o c o l a t e syrups which, when mixed w i t h c o l d m i l k , would t h i c k e n i t and p r e v e n t s e t t l i n g o f t h e cocoa, as i s c h a r a c t e r i s t i c o f lambda-carrageenan. A few o f t h e p r o d u c t s were a l s o s a t i s f a c t o r y as t o o t h p a s t e b i n d e r s a n o t h e r f u n c t i o n n o r m a l l y performed by l_ambda-carrageenan. A number o f f a c t o r s combined t o l e s s e n t h e commerc i a l i n t e r e s t i n t h e s e s e m i - s y n t h e t i c gums, t h e p r i n c i p l e ones b e i n g t h e i n c r e a s e d c o n c e r n over t h e s a f e t y o f f o o d a d d i t i v e s (and t h e r e q u i r i n g o f e x t e n s i v e and c o s t l y animal t e s t i n g ) , and t h e a v a i l a b i l i t y o f l a r g e r q u a n t i t i e s o f seaweeds through t h e s u c c e s s f u l d e v e l o p -


Sulfation

GuisELEY

of

Polysaccharides

T a b l e 5. Replacement o f p y r i d i n e by s a l t s o f weak acids. (Guar gum heated w i t h aqueous DMF, f i l t e r e d d e h y d r a t e d , and s u l f a t e d w i t h excess DMFrSO^).

,


Base

S0 n. 4

Conditions

n

DS

25 l e 0

, , (c s) P

Na HP0

4

70°/l h r .

0.59

600

Na HP0

4

70°/2 h r s .

0.50

350

NaOAc

50°/l h r .

0. 63

1150

NaOAc

50°/2 h r s .

0. 66

1100

NaOAc

50°/2 h r s .

0. 60

3580

KOAc

70°/4 h r s .

0.77

1130

2

2

5.0

6Ό

7.0

MICRONS ao

iq.o

π.ο

13.0

,

,16,

A

A = AGAR0SE

\\

G » G L 0 I 0 P E L T I S POLYSACCHARIDE S » S U L F A T E D AGAROSE

I

2000

1

1

1800

.

1

1600

.

1

1400

.

J

Y\J

1

1200

1 1000

1 800

CM -I Figure 2.

IR spectra of agarose, sulfated agarose (25.5% SO4), and funoran, the polysaccharide from Gloiopeltis furcata (24.7% SO4)


CARBOHYDRATE


158

SULFATES

ment o f seaweed farms i n t h e P h i l i p p i n e s ( 2 7 , 28) . In a d d i t i o n , s i n c e t h e major uses o f carrageenan a r e de pendent on i t s g e l l i n g p r o p e r t i e s , f u r t h e r developmen t a l work w i t h t h e s e n o n - g e l l i n g d e r i v a t i v e s was n o t warranted. N o n e t h e l e s s , a c o n t r o l l a b l e means o f p r o d u c i n g p o l y s a c c h a r i d e s u l f a t e s was d e v e l o p e d and be cause o f i t s g e n e r a l a p p l i c a b i l i t y , i t can r e a d i l y be p u t i n t o p r a c t i c e , u t i l i z i n g v i r t u a l l y any p o l y s a c c h a ride available. Experimental Materials. P o l y s a c c h a r i d e s used were o f s t a n d a r d commercial f o o d grade. S u l f u r t r i o x i d e was o b t a i n e d from A l l i e d Chemical Corp. as S u l f a n B. I s o p r o p y l a l c o h o l was t e c h n i c a l grade, 9 9 % . T r i m e t h y l a m i n e - s u l f u r t r i o x i d e was p u r c h a s e d from Hexagon L a b o r a t o r i e s , I n c . , Bronx, N.Y., and used as r e c e i v e d . A l l o t h e r s o l v e n t s and c h e m i c a l s were r e a g e n t grade. DMF:S03 was p r e p a r e d by t h e method o f G a r b r e c h t (23_) . Pyridine-sulfur t r i o x i d e was made by t h e method g i v e n i n I n o r g a n i c Syn t h e s e s (9_) , except t h a t S u l f a n Β was used i n p l a c e o f chlorosulfonic acid. In t h i s way, t h e p r o d u c t was f r e e from c h l o r i d e i o n . T r i e t h y l a m i n e - s u l f u r t r i o x i d e was p r e p a r e d as d e s c r i b e d by W h i s t l e r and Spencer ( 2 9 J · A c t i v a t i o n o f P o l y s a c c h a r i d e s . 1. Dissolution/ Reprecipitation. The p o l y s a c c h a r i d e was d i s p e r s e d i n t o r a p i d l y a g i t a t e d water a t room temperature, g e n e r a l l y a t a l e v e l o f 1%. I f n e c e s s a r y , t h e m i x t u r e was heated t o d i s s o l v e t h e p o l y s a c c h a r i d e . F o r guar, i t was n o t n e c e s s a r y ; l o c u s t bean gum, 85°C; s t a r c h and agarose were b o i l e d . The s o l was t h e n poured, i n a s t e a d y stream, i n t o t w i c e i t s volume o f t h e s o l v e n t s e l e c t e d , w i t h a g i t a t i o n p r o v i d e d by a g l a s s s t i r r i n g r o d . The gum was s e p a r a t e d by s t r a i n i n g i t through a p i e c e o f p o l y e s t e r c l o t h suspended on a s c r e e n o r c o l l a n d e r , and f u r t h e r d r i e d by drawing up t h e c o r n e r s o f t h e c l o t h and s q u e e z i n g o u t e x c e s s l i q u i d . The p o l y s a c c h a r i d e was t h e n shredded by hand i n t o a l a r g e necked s t a n d a r d t a p e r round-bottom f l a s k , and c o v e r e d w i t h * anhydrous DMF t o t h e e x t e n t o f about t e n times t h e i n i t i a l weight o f gum. The f l a s k was t h e n a t t a c h e d t o a r o t a t i n g e v a p o r a t o r and t h e s o l v e n t s removed a t 5 - 1 0 mm p r e s sure. The temperature o f t h e b a t h s u r r o u n d i n g t h e f l a s k was a l l o w e d t o i n c r e a s e t o 8 0 ° . D i s t i l l a t i o n was stopped when no v i s i b l e l i q u i d remained i n t h e f l a s k . 2. Aqueous a l c o h o l b o i l - o f f . The p o l y s a c c h a r i d e was mixed w i t h aqueous i s o p r o p y l a l c o h o l i n t h e r a t i o o f 8 . 1 g. gum t o 100 ml l i q u i d , and t h e m i x t u r e q u i c k l y



9.

GuiSELEY

Sulfation

of

159

Polysaccharides

brought t o a b o i l i n a s t e a m - j a c k e t e d s t a i n l e s s s t e e l beaker. The a l c o h o l and some o f t h e water were q u i c k l y removed. The r e s u l t i n g damp, s t i c k y gum was mixed with 150 ml. o f DMF, a l l o w e d t o s t a n d o v e r n i g h t , and the vacuum d i s t i l l a t i o n c a r r i e d out as i n 1. 3. Aqueous DMF. The p o l y s a c c h a r i d e was mixed with about 12 p a r t s by weight o f aqueous DMF and h e a t e d i n a b o i l i n g water b a t h f o r 5-10 minutes. The m i x t u r e was s e p a r a t e d by f i l t r a t i o n , and the gum d e h y d r a t e d by v a c uum d i s t i l l a t i o n as i n 1. 4. Aqueous a l c o h o l soak. The p o l y s a c c h a r i d e was mixed w i t h aqueous i s o p r o p y l a l c o h o l and r e f l u x e d f o r 15 minutes o r a l l o w e d t o s t a n d a t room temperature o v e r n i g h t , then s e p a r a t e d by f i l t r a t i o n and d e h y d r a t e d by DMF d i s t i l l a t i o n as i n 1. S u l f a t i o n o f P o l y s a c c h a r i d e s . The a c t i v a t e d p o l y s a c c h a r i d e was s u l f a t e d i n t h e f l a s k i n which i t had been d r i e d down by d i s t i l l a t i o n . An amount o f a p p r o x i mately 1 M DMF:SC>3 ^ded, a c c o r d i n g t o the d e s i r e d r e s u l t or experimental c o n d i t i o n being i n v e s t i g a t e d . For example, f o r 8.1 g. o f s t a r c h , guar, o r l o c u s t bean gum, each o f which has an average o f t h r e e h y d r o x y l groups p e r anhydro sugar, t h e r e i s a t o t a l o f 150 meq. of h y d r o x y l . To p r o v i d e enough s u l f a t i n g agent t o comp l e t e l y s u l f a t e t h e gum t o a DS o f 3.0, 150 ml. o f 1 M DMF:SC>3 would be used; f o r a DS o f 1.0, o n l y 50 ml. o f the r e a g e n t would be added. P y r i d i n e was added a t twice t h e m i l l i e q u i v a l e n t l e v e l o f the s u l f a t i n g reagent. The f l a s k was s t o p p e r e d and p l a c e d on a shaker f o r the des i r e d time. F o r work a t e l e v a t e d t e m p e r a t u r e s , a water b a t h was p o s i t i o n e d beneath the arm o f the shaker and the f l a s k immersed i n i t . When t h e r e a c t i o n time had e l a p s e d , t h e m i x t u r e was q u i c k l y poured through a s i n t e r e d g l a s s f u n n e l on a f i l t e r f l a s k and the gum washed w i t h d r y DMF and/or i s o p r o p y l a l c o h o l t o remove a d h e r i n g u n r e a c t e d s u l f a t ing reagent. I t was then d i s p e r s e d i n water (about 100 p a r t s , based on i n i t i a l w e i g h t ) , and s t i r r e d . D i l u t e (1 N) sodium h y d r o x i d e was added t o m a i n t a i n a pH somewhat over 7 as t h e gum d i s s o l v e d . Usually, i t was h e a t e d toward t h e end o f t h e d i s s o l u t i o n p r o c e s s , then added t o 2 volumes o f i s o p r o p y l a l c o h o l t o r e p r e c i p i t a t e i t . I t was washed 2-3 t i m e s w i t h 85% i s o propyl a l c o h o l s u f f i c i e n t to cover i t , i n order to r e move r e s i d u a l p y r i d i n e . F i n a l l y , i t was squeezed out and d r i e d i n a c i r c u l a t i n g a i r oven a t 60° and ground through a 40-mesh s c r e e n i n a l a b o r a t o r y W i l e y M i l l . w

a

s

a

Some Novel Methods and Results in the Sulfation of Polysaccharides

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