Carbohydrate Sulfates - American Chemical Society

14 Sulfated Polysaccharides of the Rhodophyceae— A Review ELIZABETH PERCIVAL

Downloaded by GEORGETOWN UNIV on September 5, 2015 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch014

Bourne Laboratory, Chemistry Department, Royal Holloway College, Egham Hill, Egham, Surrey, TW20 0EX, England

General S t r u c t u r a l Features The major polysaccharides o f the Rhodophyceae a r e galactans which c a r r y varying proportions o f h a l f e s t e r s u l f a t e l i n k e d t o one or more o f the f r e e hydroxyl groups o f the galactose r e s i d u e s . They are readily e x t r a c t e d from the seaweeds by hot water, and research has shown t h a t they all appear t o c o n s i s t o f chains o f a l t e r n a t i n g u n i t s o f 1,3-linked β-galactose and 1 , 4 - l i n k e d α - g a l a c t o s e , t h a t i s a l t e r n a t i n g A and Β u n i t s (Figure 1 ) . How ever, some of the u n i t s may be masked by m o d i f i c a t i o n o r by sub stitution. E x t r a c t s from different seaweeds vary i n the amount of D- and o f L - g a l a c t o s e , i n the extent t o which these residues are modified t o the 3,6-anhydrosugar, by the extent and p o s i t i o n of h a l f e s t e r s u l f a t e and methoxyl groups and by s u b s t i t u t i o n with pyruvic a c i d (Figure 2 ) . I t i s c l e a r t h a t there are a l a r g e number o f ways i n which the e x t r a c t s from the different weeds differ from one another. It is these d i f f e r e n c e s i n fine s t r u c ture which determine the conformation o r shape o f the molecules and, hence, the p h y s i c a l p r o p e r t i e s o f the various e x t r a c t s . The members o f the Rhodophyceae can be d i v i d e d i n t o those genera which s y n t h e s i s e agar-type molecules, t h a t i s a l t e r n a t i n g 1,3-1 inked D-galactose residues and 1 , 4 - l i n k e d L - r e s i d u e s , others that metabolise carrageenan-type molecules i n which all the u n i t s are D-galactose and galactans which show a f f i n i t i e s to both agar and carrageenan (Table I ) . Table I.

Seaweeds M e t a b o l i s i n g Polysaccharides o f :

Agar-type

Carrageenan-type

Mixed-type

Gelidium

Chondrus

Porphyra

Gracilaria

Gigartina

Laurencia

Phyllophora

Eucheuma

Bangia

Pterocladia

Furcellaria

Gloiopeltis

0-8412-0426-8/78/47-077-213$05.00/0 © 1978 American Chemical Society

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


Figure 2.

/

Figure 1.

Β

^Galactose (left) and ^-galactose (right)

Α

Β

(left to right, top to bottom)O-Galactose 6-sulfate, ^-galactose 2,6-disulfate, galactose 4,6-0-1boxylethylidene, a-l,4-linked 3,6-anhydro^-gahctose, and 6-0-methyl-O-galactose

Α



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215

In every case, the polysaccharides c o n t a i n a f a m i l y o f molecules which d i f f e r i n the f i n e d e t a i l s o f s t r u c t u r e d e t a i l e d above. It has been p o s s i b l e t o separate the extremes of the s t r u c t u r e s i n some i n s t a n c e s . The separation o f agarose (Figure 3 ) , a n o n - s u l f a t e d g e l l i n g f r a c t i o n from agar, where the chains c o n s i s t o f a l t e r n a t i n g 1,3-1 inked D-galactose residues and 1 , 4 - l i n k e d 3, 6-anhydro-L-galactose, l e a v i n g a g a r o p e c t i n , a mixture of v a r i o u s l y s u l f a t e d molecules, behind i s one example. The p r e c i p i t a t i o n by potassium ions o f kappa-carrageenan (Figure 4) where the 1,3l i n k e d u n i t s c a r r y s u l f a t e groups a t C-4, a l s o a separation o f a g e l l i n g f r a c t i o n from a whole e x t r a c t of Chondrus c r i s p u s o r G i g a r t i n a species i s another example o f t h i s f r a c t i o n a t i o n . Again, i n carrageenan the m a t e r i a l l e f t i n s o l u t i o n , the s o - c a l l e d "lambda"-carrageenan, a n o n - g e l l i n g f r a c t i o n , i s a mixture o f v a r i o u s l y s u b s t i t u t e d molecules with a low 3,6-anhydro content. In recent f u r t h e r f r a c t i o n a t i o n work, Dr. Rees (1) suggests t h a t the term lambda-carrageenan should be r e s t r i c t e d to one f r a c t i o n of t h i s m a t e r i a l (Table I I ) . Methods f o r the Determination of the S i t e of S u l f a t e Groups I t i s important to know the p o s i t i o n o f the s u l f a t e groups on the i n d i v i d u a l galactose residues and t h i s has been determined by (1) removal o f the s u l f a t e by a l k a l i , (2) methylation o f the p o l y s a c c h a r i d e , (3) p a r t i a l a c i d h y d r o l y s i s o f the p o l y s a c c h a r i d e , and (4) i n f r a r e d spectroscopy. 1. A c t i o n o f a l k a l i . - E a r l y studies on model s u l f a t e d monosaccharides revealed t h a t s u l f a t e could be removed on treatment w i t h a l k a l i i f there was an adjacent trans f r e e hydroxyl group on the sugar moiety ( 2 ) , then cleavage occurred w i t h Walden i n v e r s i o n . I f there i s no f r e e trans hydroxyl groups, then the s u l f a t e i s s t a b l e t o a l k a l i . At the same t i m e , any s u l f a t e l i n k e d to C-6 o f the sugar residue w i l l be cleaved by a l k a l i i f the hydroxyl group on C-3 i s f r e e . Walden i n v e r s i o n does not occur i n these circumstances, but a 3,6-anhydro-ring i s formed (Figure 5 ) . t i n s ' l a t e r s t a t e o f a f f a i r s occurs f r e q u e n t l y i n these g a l a c t a n s . This was o f commercial importance during the l a s t war when the supply o f agar from Japan was c u t o f f , and carrageenan, which i s i s o l a t e d from Chondrus c r i s p u s and G i g a r t i n a s p e c i e s , was the source i n Canada and Great B r i t a i n o f m a t e r i a l to r e p l a c e agar. Carrageenan has a lower g e l l i n g strength than agar, but i t was found t h a t treatment with a l k a l i increased the gel s t r e n g t h . Research l a t e r showed t h a t the r e a c t i o n j u s t o u t l i n e d had taken p l a c e , and i t has s i n c e been shown t h a t a l l p o l y saccharides w i t h a high 3,6-anhydrogalactose content give strong gels. I t i s common p r a c t i c e i n Japan a t the present time to increase the gel strength o f e x t r a c t s o f G r a c i l a r i a s p e c i e s , a somewhat low gel strength agar, i n t h i s way. In the p l a n t an enzyme brings about t h i s change so t h a t the seaweed i s able to r e g u l a t e the p r o p o r t i o n o f galactose 6 - s u l f a t e and 3,6-anhydro-



A UNITS

D-Galactose 2-sulphate

3

- A^

UNITS

Β

A

_3 UL

nu

mu

NAME

lambda

D-Galactose 2 , 6 - d i s u l p h a t e

3,6-Anhydro-D-galactose 2-sulphate theta

xi


kappa 3,6-Anhydro-D-galactose 3,6-Anhydro-D-galactose 2-sulphate i o t a

D-Galactose 2 , 6 - d i s u l p h a t e

A

_3 1A.


Repeating Units of Carrageenans


Table II.


H W

r >

1

cl

>

ο Κ

W

>

1

to h-

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Figure 3.

of the

Agarose R=H

Figure 4.

Rhodophyceae

217

or OMe

κ-Carrageenan

+ Na S0 +H 0 2

Figure 5.


4

2

CARBOHYDRATE SULFATES


218

galactose i n any p o l y s a c c h a r i d e . 2. M e t h y l a t i o n . - Methylation has a l s o been used to d e t e r mine the p o s i t i o n o f s u l f a t e groups. As long ago as 1 9 4 7 , E. G. V. P e r c i v a l and h i s colleague {3)_ found t h a t 2 , 6 - d i - 0 methylgalactose was the major methylated sugar i n the hydrolysate a f t e r methylation of carrageenan from G i g a r t i n a s t e l l a t a . This i n d i c a t e d t h a t C-3 and C-4 were i n v o l v e d i n l i n k a g e e i t h e r to other residues or to s u l f a t e . These authors had p r e v i o u s l y shown t h a t the s u l f a t e was s t a b l e t o a l k a l i and, t h e r e f o r e , i t was not l i n k e d t o C - 3 , and they were able to deduce t h a t the galactose residues were mainly 1,3-1 inked w i t h s u l f a t e on C-4. Confirmat i o n of t h i s conclusion was obtained i n 1966 Ç 4 ) . by the i s o l a t i o n and c h a r a c t e r i s a t i o n of 0-a-3,6-anhydrogalactopyranosyl ( 1 - 3 ) galactose 4 - s u l f a t e from a p a r t i a l enzymic h y d r o l y s a t e . Dolan and Rees (5) used methylation s t u d i e s i n a d i f f e r e n t way t o provide evidence o f the p o s i t i o n o f s u l f a t e groups i n lambda -carrageenans. These authors found t h a t f o u r treatments with 1.5%-methanolic hydrogen c h l o r i d e a t 3 5 ° f o r 48 hours reduced the s u l f a t e content from 31 to 2%, and they recovered the d e s u l f a t e d polysaccharide i n 60% y i e l d . M e t h y l a t i o n o f the o r i g i n a l polysaccharide and the d e s u l f a t e d m a t e r i a l and comparison of the methyl sugars i n the hydrolysates o f the two products allowed assignment of the s u l f a t e groups. From these r e s u l t s , i t was f o l l o w e d that 4?% o f the 1 , 4 - l i n k e d residues are s u l f a t e d a t C-2 and C-6 and a high p r o p o r t i o n of the 1,3-1 inked residues a l s o c a r r y s u l f a t e a t C-2 and, i n c o n t r a s t to kappa-carrageenan, only a small p r o p o r t i o n o f these residues are s u l f a t e d a t C-4. 3. P a r t i a l A c i d H y d r o l y s i s . - A c i d h y d r o l y s i s of these p o l y saccharides r a r e l y gives any s u l f a t e d fragments, the g l y c o s i d i c l i n k s and the s u l f a t e groups having s i m i l a r l a b i l i t y . Painter ( 6 ) , however, found t h a t a u t o h y d r o l y s i s o f the f r e e a c i d form a v a r i e t y o f carrageenans i n a d i a l y s i s sac surrounded by d i s t i l l e d water c o n t a i n i n g a suspension of barium carbonate gave a mixture of galactose and galactose monosulfates. From t h i s mixture he was able t o separate and c h a r a c t e r i s e galactose 2 - , 4 - , and 6-monos u l f a t e s , again confirming the deductions from m e t h y l a t i o n . 4. I n f r a r e d Spectroscopy. - This has proved a useful d i a g n o s t i c t o o l i n the a l l o c a t i o n of the s i t e of s u l f a t e groups. Study on model galactose s u l f a t e s (7)_ has shown t h a t peaks a t 820 cm are c h a r a c t e r i s t i c of s u l f a t e at a primary a l c o h o l i c group, t h a t i s a t C - 6 ; a t 830 c m " o f e q u a t o r i a l s u l f a t e , t h a t i s a t C-2 and a t 850 o f a x i a l s u l f a t e , t h a t i s a t C-4. M

M

α

1

Types o f Carrageenans By means o f these and s i m i l a r s t u d i e s , two major groups o f carrageenans have been recognised ( 8 ) . In the f i r s t , the 1 , 3 Ifnked u n i t s are s u l f a t e d i n the 4 - p o s i t i o n , w h i l e i n the second, the s u l f a t e i s i n p o s i t i o n 2 (Table I I ) . This f i r s t group i s subdivided according t o the nature of the 1 , 4 - l i n k e d u n i t s .


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These may be present as galactose 6 - s u l f a t e (mu-carrageenan) o r galactose 2 , 6 - d i s u l f a t e (nu-carrageenan) or as the corresponding 3,6-anhydrides i n kappa- and i o t a - c a r r a g e e n a n . In nature the 3,6-anhydrides o f kappa- and iota-carrageenan are formed by enzymatic e l i m i n a t i o n o f the 6 - s u l f a t e from the mu and nu forms, but the conversion i s not always complete. In some seaweeds, these carrageenan types can be i s o l a t e d i n almost pure form, w h i l e i n others they e x i s t as copolymers. In the second group, the 1 , 4 - l i n k e d u n i t s are s u l f a t e d i n the 2 - p o s i t i o n . In lambda-carrageenan, the 6 - p o s i t i o n i s a l s o s u l f a t e d w h i l e i n theta-carrageenan, i t i s n o t . P h y s i c a l P r o p e r t i e s and Conformational E f f e c t s Many o f these e x t r a c t s have the a b i l i t y t o form r e v e r s i b l e gels which l i q u i f y when heated and s e t when c o l d . Some give s t i f f gels i n d i l u t e s o l u t i o n , t h a t i s give gels which r e t a i n a d e f i n i t e shape even though they c o n s i s t of 99.9% water and others have no g e l l i n g p r o p e r t i e s a t a l l . These p r o p e r t i e s depend l a r g e l y on the presence o r absence o f s u l f a t e groups i n c e r t a i n p o s i t i o n s on the sugar r e s i d u e s , and the theory o f change from random c o i l t o double h e l i x conformation i n the s o l gel t r a n s f o r mation, developed by Dr. D. A. Rees and h i s colleagues ( 1 ) , has gone a long way t o e x p l a i n t h i s . X-ray d i f f r a c t i o n s t u d i e s o f o r i e n t a t e d f i b e r s o f kappa- and modified iota-carrageenan i n d i cated two p a r a l l e l 3 - f o l d h e l i c a l c h a i n s , each t w i s t e d around the other and having a r i s e of 26A° per t u r n . The two chains move past each other from r e l a t i v e p o s i t i o n s t h a t are p e r f e c t l y general to form a s p e c i a l arrangement t h a t i s e x a c t l y staggered w i t h i d e n t i c a l groups moved t o h a l f the i n i t i a l spacing (Figure 6 ) . Only the presence o f the 3,6-anhydro-ring allows the sugar r i n g to be so c o n s t r a i n e d as to have three e q u a t o r i a l C-H bonds, an arrangement t h a t increases the f l e x i b i l i t y o f the chain and allows winding and unwinding of the double h e l i x . The conformation i s such t h a t hydrogen bonding takes place between the 0-2 and 0-6 o f galactose residues i n d i f f e r e n t stands o f the same double h e l i x . Thus, every u n s u b s t i t u t e d hydroxyl group i n iota-carrageènan i s engaged i n hydrogen bonding, making the conformation very s t a b l e . On the other hand, a s u l f a t e group on the 0-2 of the 1,31 inked galactose residue as i n lambda-carrageenan w i l l i n h i b i t double h e l i x formation. Even a f t e r a l k a l i treatment t o convert the 1 , 4 - l i n k e d residues i n t o 3,6-anhydrogalactose, t h i s c a r r a geenan w i l l not g e l . Rees by means o f o p t i c a l r o t a t i o n s t u d i e s ( 9 ) , confirmed t h a t when the molecules are i n s o l u t i o n they e x i s t i n random c o i l formation and when the s o l u t i o n i s c o o l e d , the chains l i n k by t h i s double h e l i x formation to give a three dimens i o n a l framework (Figure 7 ) , the i n t e r s t i c e s o f which are occupied by water. Iota-carrageenan i s a copolymer o f D-galactose 4 - s u l f a t e and 3,6-anhydro-D-galactose 2 - s u l f a t e w i t h masking by r e placement o f 1/10th o f the anhydride by D-galactose 2 , 6 - d i s u l f a t e



220


Figure 6.

Helical structure

Figure 7.



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221

Rhodophyceae

and a much s m a l l e r p r o p o r t i o n by D-galactose 6 - s u l f a t e (Figure 8 ) . The masking residues cause k i n k i n g of the h e l i c a l carrageenan c h a i n . Thus, the physical and b i o l o g i c a l f u n c t i o n o f these r e s i dues i s to serve as h e l i x - b r e a k i n g i n t e r r u p t i o n s t h a t cause each chain to enter i n t o double h e l i c a l a s s o c i a t i o n w i t h more than one partner (Figure 7 ) . This i s necessary i f a 3-dimensional network i s to be formed r a t h e r than a c o l l e c t i o n o f i s o l a t e d chain p a i r s . I t i s thought that i n gel formation the aggregation of groups of double h e l i c e s i n t o p a r a l l e l bundles takes place (Figure 7) as a secondary process and adds to the strength o f the g e l . 4 - 0 s u l f a t e groups i n the 1 , 3 - l i n k e d residues and 2 - 0 - s u l f a t e groups in the 3,6-anhydro residues are s i t u a t e d on the o u t s i d e o f the h e l i c e s and can, t h e r e f o r e , be expected to a f f e c t the degree o f aggregation. Thus, the i n c r e a s i n g s u l f a t e i n the s e r i e s : agarose -> f u r c e l l a r a n -> kappa-carrageenan -> iota-carrageenan c o i n c i d e s with i n c r e a s i n g e l a s t i c i t y and decreasing b r i t t l e n e s s o f the g e l s . E f f e c t o f M e t a l l i c Ions I t i s understandable t h a t the nature o f the metal i o n combined w i t h the s u l f a t e groups w i l l i n f l u e n c e the degree o f aggregation of the h e l i c e s . For example, the sodium s a l t of kappacarrageenan gives l i t t l e or only weak g e l l i n g products; whereas, potassium gives a strong g e l . In c o n t r a s t , the gel strength o f iota-carrageenan with potassium i s comparatively weak, whereas, with calcium i t gives a strong e l a s t i c g e l . Commercially, the manufacturer combines d i f f e r e n t e x t r a c t s to give the a p p r o p r i a t e p r o p e r t i e s r e q u i r e d by the buyer. E x t r a c e l l u l a r Polysaccharides from M i c r o s c o p i c Red Algae Recent s t u d i e s on the h i g h l y viscous e x t r a c e l l u l a r p o l y saccharides i s o l a t e d from c u l t u r e d samples of the u n i c e l l u l a r microscopic red a l g a e , Rhodella maculata ( 1 1 ) , Porphyridium cruentum (12-14) and Porphyridium aerugineum (14) reveal complex s u l f a t e d p o l y s a c c h a r i d e s . Not only g a l a c t o s e , but c o n s i d e r a b l e proportions o f x y l o s e , g l u c u r o n i c a c i d and glucose are c o n s t i tuents o f these mucilages. Both Rhodella and F. aerugineum cont a i n a p p r e c i a b l e amounts o f 3-0-methylxylose (11,14) and 3- and 4-0-methylgalactoses are c o n s t i t u e n t s o f both Porphyridium species (14). In a d d i t i o n , F. aerugineum contains as much as 10% o f 2,4d i - 0 - m e t h y l g a l a c t o s e . 2-0-Methylglucuronic a c i d has a l s o been reported f o r F. cruentum polysaccharide (13). In both Porphyridium species the s u l f a t e groups i n the polysaccharides are l a b i l e t o methylation and periodate o x i d a t i o n c o n d i t i o n s , although i n cruentum mucilage they are somewhat more s t a b l e than i n aerugineum. While i n f r a r e d a n a l y s i s o f Rhodella polysaccharide (15) gave peaks at 1200 c m " , 874 c m " and 950 c m " which disappear on d e s u l f a t i o n , the two Porphyridium polysaccha1

1


1


222


Figure 8.


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r i d e s give the c h a r a c t e r i s t i c peaks f o r galactose s u l f a t e s , t h a t i s broadish bands, a t 820-830 c m " and a sharper band a t 850 c m " (14) i n d i c a t i n g t h a t primary, e q u a t o r i a l and a x i a l s u l f a t e may be present. A l l the s u l f a t e i n F. cruentum polysaccharide i s s t a b l e to a l k a l i whereas i n t h a t from F. aerugineum the s u l f a t e i s decreased from 9% t o 3.7%. I n f r a r e d a n a l y s i s of a s u l f a t e d f r a g ment i s o l a t e d from an autohydrolysate o f the f r e e a c i d form o f F. aerugineum (14) gives a peak a t 830 c m " , i n d i c a t i v e o f equa t o r i a l s u l f a t e . This has been c h a r a c t e r i s e d . 1

1

1


Conclusions Although chemical s t u d i e s have been made on only a m i n o r i t y of known species of red a l g a e , i t appears t h a t the type o f water e x t r a c t a b l e polysaccharides present f o l l o w s the b o t a n i c a l c l a s s i f i c a t i o n i n t o genera. There are d i s t i n c t groups o f polymers which, although having much i n common, d i f f e r i n d e t a i l , the pro p o r t i o n and p o s i t i o n of s u l f a t e groups being an important v a r i able. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Rees, D. A. Adv. Carbohyd. Chem. (1969) 24, 267 and refer ences c i t e d t h e r e i n . Percival, E. G. V. Quart. Rev. (1949) 3, 369. Dewar, Ε. T. and Percival, E. G. V. J. Chem. Soc. (1947) 1622. W e i g l , J., Turvey, J. R. and Yaphe, W. Proc. V t h . I n t . Seaweed Symp. (1965) H a l i f a x , Nova S c o t i a (Edited E. G. Young and J. L. McLachlan) p. 329. Pergamon P r e s s , Oxford. Dolan, T. C. S. and Rees, D. A. J. Chem. Soc. (1965) 3534. P a i n t e r , T. J. Proc. Vth I n t . Seaweed Symp. (1965) H a l i f a x , Nova S c o t i a (Edited E. G. Young and J. L. McLachlan) p. 305. Pergamon P r e s s , Oxford. Turvey, J. R. Adv. Carbohyd. Chem. (1965) 20, 183 and references c i t e d t h e r e i n . S t a n c i o f f , D. J. and Renn, D. W. i n " P h y s i o l o g i c a l E f f e c t s of Food Carbohydrates" ACS Symposium 15, (1974). Edited by A l l e n e Jeanes and John Hodge p. 282. Rees, D. Α . , S c o t t , W. E. and W i l l i a m s o n , F. B. Nature (Lond.) (1970) 227, 390. Anderson, N. S., Dolan, T. C. S. and Rees, D. A. J. Chem. Soc. (C) (1973) 2173. Sheik Fareed, V. and Percival, E. Carbohyd. Res. (1977) 53, 276. Medcalf, D. G., S c o t t , J. R., Brannon, J. H., Hemerick, G. Α . , Cunningham, R. L., Chessen, J. H. and Shah, J., Carbohyd. Res. (1975) 44, 87. Heaney-Kieras, J. and Chapman, D. J. Carbohyd. Res. (1976) 52, 169.


224 14. 15.


Percival, E. and F o y l e , R. unpublished work. Evans, L. V. and Callow, M. E., Percival, E. and Sheik Fareed, V. J. Cell. S c i . (1974) 16, 1. 1978.


RECEIVED F e b r u a r y 6,


Carbohydrate Sulfates - American Chemical Society

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