Sulfated Polysaccharides Metabolized by the Marine Chlorophyceae

13 Sulfated Polysaccharides Metabolized by the Marine Chlorophyceae—A Review

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ELIZABETH

PERCIVAL

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

During the past twenty years my colleagues and I have i n v e s t i g a t e d the polysaccharides o f nine d i f f e r e n t genera o f green s e a weeds. In every case we have been able t o separate a t l e a s t one w a t e r - s o l u b l e p o l y d i s p e r s e heteropolysaccharide s u b s t i t u t e d by h a l f e s t e r s u l f a t e groups. That means not only t h a t each seaweed metabolises molecules c o n t a i n i n g more than a single sugar but t h a t each contains a mixture o f molecules all built up on the same general plan but which differ i n the fine d e t a i l s o f s t r u c t u r e . I should emphasise t h a t the results are only the average o f all the molecules present. Each weed presented its own particular p r o blems, but certain groups o f polysaccharide have emerged. From the Cladophorales and Codiales a s u l f a t e d x y l o g a l a c t o a r a b i n a n , and from the U l v a l e s , A c r o s i p h o n i a l e s and U l o t r i c h a l e s a glucuronoxylorhamnan, sometimes c o n t a i n i n g a small p r o p o r t i o n o f glucose, have been separated. The polysaccharides i n each group appear t o be built up on the same general p l a n , but differ i n the proport i o n s o f the sugars present, and i n the fine d e t a i l s o f s t r u c t u r e . The polysaccharide from the unicellular green a l g a , Acetabulària, a member o f the Dasycladales, resembles the l a t t e r group i n c o n t a i n i n g rhamnose, xylose and g l u c u r o n i c a c i d , but i t s major sugar i s galactose and i t s 4-0-methyl d e r i v a t i v e ( 1 ) . General S t r u c t u r a l

Studies

D e t a i l s o f polysaccharides from the f i r s t group o f seaweeds are given i n Table I. The proportions o f sugars present i n Cladophora and Chaetomorpha polysaccharides d i f f e r i n the s m a l l e r p r o p o r t i o n o f galactose present i n the l a t t e r , and both genera are devoid o f 3,6-anhydrogalactose. Caulerpa polysaccharide appears to be the odd man out i n t h a t i t contains a c o n s i d e r a b l e proport i o n o f mannose residues which d e f i e d f r a c t i o n a t i o n , and t h i s i s true f o r two other species o f Caulerpa we have examined. Nevert h e l e s s a l l four genera c o n t a i n approximately the same p r o p o r t i o n 0-8412-0426-8/78/47-077-203$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society

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


xxxxx xxx xx

xxxxx

xxxxx

xx

L-Arabinose

D-Galactose

D-Xylose

xxxxxx xxx

xxxxx xx

+13°

+46°

+70°

ca.+53°

[a]

D

17.5

17

15

16

+

+

xxx

xx

Caulerpa f i l i f o r m i s

xxxx

Codium f r a g i l e

Sulfate %

3,6-Anhydrogalactose

D-Mannose

Chaetomorpha capillaris

Water-soluble Polysaccharides from

Cladophora rupestris

Constituent Sugars

Table I.


M

H

>

r

c!

M

> Η

> td g Hi ϋ

ο

to ο

13.

Sulfated

PERCivAL

Table I I ,

Water-Soluble

Polysaccharides

Cladophora rupestris

various

Approx. propor. Sugars Gal Ara Xyl

Cladophora sericea*

Scotland Aug. 1969 A p r i l 1970

Gal Ara Xyl

Cladophora laetevirens

Scotland Sept.1969 A p r i l 1970

Gal Ara Xyl

Rhizoclonium r i p a r i urn

Scotland 1969

Gal Ara Xyl

Chaetomorpha melagonium

Scotland 1969

Gal Ara Xyl

Chaetomorpha 1 inum

Scotland 1969 1970

Gal Ara Xyl

Algal


205

Polysaccharides

Key:

Species

Time Place

AgN0 on Papers 3

+++ +++ + ++ ++++ ++ ++ +++++ ++ ++ ++++ ++ ++ ++ +++ + ++ +++ +

GLC Ratio 1.0 1.0 0.4 1.0 1.27 0.80 1.0 1.8 0.9

1.0 1.2 0.45

-

Gal = g a l a c t o s e ; Ara = arabinose; Xyl = xylose

May have been contaminated with C. l a e t e v i r e n s .

Figure 1.

Structural features in Cladophora rupestris (left); after reduction and desulfation (right)



17.5

16

[»]„ -87°

n

-47°

11 17-22 -90°

16

21

-74°

-

17

X

probably contaminated with s t a r c h - t y p e p o l y s a c c h a r i d e [ a ] + 1 5 0 ° .

-31°*

7.8

18

19

Sulphate %

X

XX*

D-Glucuronic A c i d %

xxxx

wormskioldii

xxx

xxx

Urospora penicilliformis

xxxx

XX

D-Glucose

XXX

xxx

XXXXX

Ulva lactuca

D-Xylose

XXXXX

Enteromorpha compressa

xxx

Acrosiphonia central i s

Water-Soluble Polysaccharides from

L-Rhamnose

Constituent Sugars

Table IV.


*1

M

to ο

13.

PERCivAL

Sulfated

Polysaccharides

207

of h a l f e s t e r s u l f a t e and a l l have a p o s i t i v e s p e c i f i c r o t a t i o n . Examination of the c o n s t i t u e n t s of a number o f d i f f e r e n t seaweeds belonging to the Cladophorales shows (Table II) a wide v a r i a t i o n i n the proportions of the sugars present ( 6 ) . Table III gives the linkages present i n Cladophora r u p e s t r i s polysaccharide and Figure 1 some of the s t r u c t u r a l features


Table I I I .

Linkages present i n Cladophora r u p e s t r i s

1.3- and 1,6-1 inked g a l a c t o s e ; 1 , 4 - l i n k e d x y l o s e ; 1,4- and t r i p l y l i n k e d arabinose; that have been determined (7) f o r t h i s p o l y s a c c h a r i d e . Although t e n t a t i v e evidence of s i m i l a r linkages i n other genera of t h i s group have been obtained these have not been f i r m l y e s t a b l i s h e d . In c o n t r a s t to the arabinans the glucuronoxylorhamnans a l l have negative s p e c i f i c r o t a t i o n s (Table IV). Furthermore, the proportions o f the sugars i n the d i f f e r e n t species are very s i m i l a r e s p e c i a l l y , i f i t i s remembered t h a t these f i g u r e s are only approximate and are the average o f a l l the molecules present. They are determined on hydrolysates of the p o l y s a c c h a r i d e s , and the a l d o b i o u r o n i c a c i d , 4-0-3-D-glucuronosyl-L-rhamnose, i s i n v a r i a b l y present and not included i n these p r o p o r t i o n s . While t h i s prevents accurate determination of the proportions o f the c o n s t i tuents i t s occurrence i n the hydrolysates from a l l the species examined i s evidence of the o v e r a l l s i m i l a r i t y of t h e i r p o l y s a c c h a r i d e s . A s t r i k i n g d i f f e r e n c e i s the lower p r o p o r t i o n of s u l f a t e i n Acrosiphonia and Urospora w o r m s k i o l d i i . A l l the sugars i n the polysaccharides i n t h i s group are l i n k e d i n the same way (Table V ) . Table V.

Linkages Present i n Glucuronoxylorhamnans

1,3- and 1 , 4 - l i n k e d and end group rhamnose; 1,3- and 1.4- l i n k e d x y l o s e , 1,3-1 inked glucose (when p r e s e n t ) ; 1,4linked glucuronic acid However, the proportions o f the d i f f e r e n t types o f linkages appear to vary i n the d i f f e r e n t s p e c i e s . S t r u c t u r a l features found i n the polysaccharide from Ulva l a c t u c a (13) a r e : GA(Ti4jR; GA(l*3)Xy; GA(l->4)Xy; G(l+3)Xy 2 S GA(l*4)R(l+3)GA(l*3)Xy; R(l*4)Xy(l*3)G/GA. GA = D-glucopyranuronic a c i d ; R = L-rhamnopyranose^O Xy = D-xylopyranose; G = D-glucopyranose; S = S-0-


208

CARBOHYDRATE SULFATES


The S i t e of Sulphate Groups These polysaccharides e x i s t as mucilages i n the p l a n t and the presence of s u l f a t e groups must play a v i t a l r o l e i n t h e i r c o n f o r mation and p h y s i c a l p r o p e r t i e s . I t i s important, t h e r e f o r e , to determine the s i t e of these groups. This has been achieved i n a v a r i e t y of ways, depending upon the p a r t i c u l a r p o l y s a c c h a r i d e . 1. Cleavage by A l k a l i . A l k a l i w i l l cleave s u l f a t e groups from a polysaccharide i f there i s a f r e e adjacent hydroxyl group trans to the s u l f a t e , t h i s occurs v i a an epoxide r i n g and Walden i n v e r s i o n of the hydroxyl c a r r y i n g the s u l f a t e group. Cleavage of the epoxide r i n g can occur i n two ways and two sugars u s u a l l y r e s u l t . Cleavage a l s o occurs w i t h s u l f a t e on C-6 of the hexose provided the hydroxyl on C-3 i s f r e e . In t h i s event removal of the s u l f a t e r e s u l t s i n the formation of the 3,6-anhydride of the p a r t i c u l a r hexose. In both the U l v a l e s and the Cladophorales t h i s method of r e moval of s u l f a t e has been p a r t i c u l a r l y u s e f u l . The s u l f a t e con t e n t i n the rhamnan from U. l a c t u c a was reduced from 14.1 to 12.5% by the a c t i o n of a l k a l i , and the hydrolysate of the r e c o v e r ed polysaccharide was found to contain arabinose (14). This could only have a r i s e n from s u l f a t e d xylose (Figure 2 ) . I t could have come from xylose 2- or 3 - s u l f a t e . However, treatment of the p o l y saccharide with sodium methoxide gave 3-0-methylarabinose which could only have been derived from xylose 2 - s u l f a t e . S i m i l a r l y the treatment of the x y l o g a l a c t o a r a b i n a n from Cladophora r u p e s t r i s w i t h sodium methoxide gave mainly 2-0-methyl-L-xylose and l i t t l e 3-0-methyl-L-arabinose (3) i n d i c a t i n g t h a t the s u l f a t e was i n i t i a l l y l i n k e d to C-3 of arabinose. P a r t i a l h y d r o l y s i s s t u d i e s con firmed t h i s r e s u l t . The removal of s u l f a t e w i t h a l k a l i and the formation of 3,6anhydrogalactose was demonstrated i n the s u l f a t e d polysaccharide from Codium f r a g i l e (4). The s u l f a t e decreased from approximately 13% to 97o and the 3,6-anhydrogalactose content increased from 1% to 3% on t h i s treatment (Figure 3). 2. The S i t e of S u l f a t e from Periodate Oxidation S t u d i e s . Treatment of the rhamnan from U. l a c t u c a with dry methanolic hydrogen c h l o r i d e reduced the s u l f a t e to 5% and periodate o x i d a t i o n of the i n i t i a l and the d e s u l f a t e d polysaccharide at 2° i n buffered s o l u t i o n stopped a f t e r 70 hours. I t was found t h a t the d e s u l f a t e d polysaccharide had reduced twice as much periodate as the i n i t i a l polysaccharide (14). P a r a l l e l o x i d a t i o n experiments were c a r r i e d out at room temperature. Again, the d e s u l f a t e d polysaccharide reduced approximately twice as much periodate as the i n i t i a l p o l y saccharide. From t h i s i t can be deduced, t h a t removal of s u l f a t e groups l e d to the formation of a d d i t i o n a l α-glycol groups. I t i s known t h a t , i n buffered s o l u t i o n at low temperature, v i c i n a l c i s hydroxyl groups i n sugars are s e l e c t i v e l y o x i d i s e d by p e r i o d a t e . We were a b l e , t h e r e f o r e , to conclude t h a t d e s u l f a t i o n f u r n i s h e d c i s r a t h e r than trans g l y c o l groupings. The only sugar present i n


13.

PERCivAL

Sulfated

209

Polysaccharides

the polysaccharide i n which c i s - g l y c o l groupings occur i s L-rhamnose at p o s i t i o n 2 and 3. I t may be presumed t h e r e f o r e t h a t the s u l f a t e groups are l i n k e d e i t h e r t o C-2 or C-3 o f rhamnose. I n f r a r e d a n a l y s i s o f the polysaccharide gave a strong peak a t 850 cm"" , c h a r a c t e r i s t i c o f a x i a l s u l f a t e i n galactose or glucose(15). If t h i s i s a p p l i c a b l e to L-rhamnose, the m a j o r i t y o f the s u l f a t e i n t h i s polysaccharide i s l i n k e d to C-2 o f the L-rhamnose i n i t s most s t a b l e 1C conformation. In s i m i l a r periodate o x i d a t i o n experiments on Enteromorpha compressa p o l y s a c c h a r i d e , the r e l a t i v e proportions o f the uncleaved sugars i n the i n i t i a l polysaccharide and i n the d e s u l f a t e d polysaccharide a f t e r o x i d a t i o n are given i n Table V I .


1

Table VI.

Enteromorpha S u l f a t e d Polysaccharide Percentage Uncleaved: Glucose:Xylose :Rhamnose

I n i t i a l Polysaccharide

26

16

58

Desulfated Polysaccharide

40

16

44

The amount o f rhamnose and, to a l e s s e r e x t e n t , o f xylose was reduced i n the d e s u l f a t e d p o l y s a c c h a r i d e . From these r e s u l t s i t was c a l c u l a t e d t h a t about 22% o f the rhamnose i n the i n i t i a l p o l y saccharide was cleaved by periodate and about 40% i n the d e s u l f a t e d p o l y s a c c h a r i d e , t h a t i s an increase o f 18% o f f r e e hydroxyl groups i n the rhamnose residues proving t h a t a high p r o p o r t i o n of the s u l f a t e groups are attached to t h i s sugar r e s i d u e . Infrared a n a l y s i s o f the i n i t i a l polysaccharide gave a peak a t 850 c m " which i n galactose i n d i c a t e s a x i a l s u l f a t e . A g a i n , i f t h i s i s a p p l i c a b l e to L-rhamnose which i s i n i t s s t a b l e 1C conformation then p o s i t i o n 2 has an a x i a l h y d r o x y l , and t h i s would t h e r e f o r e , as i n the Ulva p o l y s a c c h a r i d e , be the s i t e of s u l f a t e . 3. The S i t e of S u l f a t e from Methylation R e s u l t s . Methylation before and a f t e r d e s u l f a t i o n and comparison of the two sets of methylated sugars i n the r e s p e c t i v e hydrolysates has not proved a very s a t i s f a c t o r y method f o r determining the s i t e o f the e s t e r s u l f a t e i n these p o l y s a c c h a r i d e s . T h e i r high s u l f a t e content makes complete methylation impossible and so the e f f e c t of the removal o f the s u l f a t e groups cannot be p r o p e r l y assessed. However, with the glucuronoxylorhamnan from Urospora p e n i c i l l i f o r m i s i t was found (11) t h a t the hydrolysate from the methylated d e s u l f a t e d polysaccharide contained considerably more 2 , 4 - d i - 0 - m e t h y l rhamnose than the hydrolysate from the i n i t i a l methylated p o l y saccharide w h i l e the p r o p o r t i o n of both the monomethyl and f r e e rhamnose i n the l a t t e r were reduced to t r a c e amounts. From these r e s u l t s i t was p o s s i b l e to deduce t h a t the 1,3-1 inked rhamnose was monosulfated at C-4 and a l s o d i s u l f a t e d a t C-2 and C-4. The 1




210

proportions of the d i f f e r e n t methylxyloses and g l u c u r o n i c acids were the same i n the two hydrolysates i n d i c a t i n g t h a t both of these residues are e s s e n t i a l l y devoid o f s u l f a t e . From the Codiolum, the u n i c e l l u l a r stage i n the l i f e h i s t o r y °f Urospora w o r m s k i o l d i i , a s u l f a t e d mannan i n a d d i t i o n to the glucuronoxylorhamnan, was separated (12). This on methylation and h y d r o l y s i s gave 2 , 3 , 4 , 6 - t e t r a - , 2 , 4 , 6 - t r i , 3,6- and 2,3di-O-methylmannoses i n the r e l a t i v e proportions of 1:13:1.5:1. This mannan i s , t h e r e f o r e , 1 , 4 - l i n k e d , s u l f a t e d and/or branched at C-2 and C-6 and has an average chain length of 16. A f t e r d e s u l f a t i o n with 0.08 M-methanolic hydrogen c h l o r i d e , the s u l f a t e content decreased from 6.5 to 0.6%. The d e s u l f a t e d m a t e r i a l was methylated and hydrolysed and the above methylmannoses were obtained i n the approximate r e l a t i v e proportions of 1:13:0.2:1. The f a c t t h a t the 3,6-di-0-methylmannose had almost disappeared i n the d e s u l f a t e d m a t e r i a l and the q u a n t i t y of 2,3di-0-methylmannose remained v i r t u a l l y unchanged provided evidence t h a t the mannan i s s u l f a t e d on C-2 and branched at C-6. 4. The S i t e of S u l f a t e from P a r t i a l H y d r o l y s i s . This has a l s o been useful f o r the determination of the s i t e of the s u l f a t e groups i n the Chlorophyceaean p o l y s a c c h a r i d e s . For example, h y d r o l y s i s of the Codium polysaccharide with N-sulphuric a c i d f o r one hour at 100° l e d to the separation of galactose 4- and 6-monosulfates (4). These were c h a r a c t e r i s e d as f o l l o w s : Each had a DP o f 1. On h y d r o l y s i s they both gave only g a l a c t o s e . The molar r a t i o s of e s t e r s u l f a t e to galactose were 1.15:1.0 f o r the 4 - s u l f a t e and 1.04:1.0 f o r the 6 - s u l f a t e . Methylation gave r e s p e c t i v e l y 2,3,6- and 2 , 3 , 4 - t r i - 0 - m e t h y l g a l a c t o s e s p r o v i d i n g unequivocal proof of the s i t e of e s t e r s u l f a t e groups as galactose 4- and galactose 6 - s u l f a t e . S i m i l a r h y d r o l y s i s o f the x y l o g a l a c t o a r a b i n a n from Cladophora r u p e s t r i s (3) l e d to the separation of arabinose 3 - s u l f a t e and galactose 6 - s u l f a t e . The l a t t e r was c h a r a c t e r i s e d i n the same way as t h i s d e r i v a t i v e from Codium. The former a f t e r methylation gave 2,4- and 2 , 5 - d i - 0 ^ e t h y l a r a b i n o s e s , showing t h a t C-3 i n v o l v e d i n l i n k a g e to s u l f a t e , thus confirming the r e s u l t s a r r i v e d at by a l k a l i treatment of t h i s p o l y s a c c h a r i d e . Biological

Imp!ications

U n l i k e the g a l a c t a n s , none of these p o l y s a c c h a r i d e s , a f t e r e x t r a c t i o n , gives gels i n aqueous s o l u t i o n , the most t h a t can be s a i d i s t h a t i n some cases the s o l u t i o n s are somewhat v i s c o u s . The l a t e Dr. Haug (16) reported t h a t an aqueous s o l u t i o n of Ulva polysaccharide gave a strong gel i f i t was t r e a t e d w i t h borate and calcium ions at a concentration and a pH found i n seawater. He experimented with a wide v a r i e t y of m e t a l l i c ions found i n the sea, but t h i s was the only combination w i t h which Ulva polysacchar i d e gave a g e l . He p o s t u l a t e d t h a t the C-2 and C-3 in the 1,4l i n k e d rhamnose u n i t s complex with borate and t h a t the Ca ions



13.

Sulfated Polysaccharides

PERCivAL

211

2-0-Me-Darabinose

3-0-Me-D xylose

Figure 2.

OH Figure 3.

1

—

Galactose 6-sulfate (left) and 3,6-anhydrogahctose (right)

Ο'

O H

H O

O

B . o ^ — ^ o . Ca

2 +

CI"

Figure 4.


212


form bridges or act as s t a b i l i s i n g influences (Figure 4 ) . When C-2 o f these rhamnose u n i t s are s u l f a t e d complex formation w i t h borate i s prevented, and i t may be t h a t t h i s i s the way i n which Ulva l a c t u c a r e g u l a t e s the extent of complexing w i t h borate and hence, the s t i f f n e s s of the polysaccharide g e l . C a r r y i n g out s i m i l a r s t u d i e s on the Cladophora r u p e s t r i s p o l y s a c c h a r i d e , i t was found t h a t calcium ions alone produced a strong gel at the pH of seawater (17).


Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Smestad, B e r i t and Percival, E l i z a b e t h , Carbohyd. Res. (1972) 25, 299. F i s h e r , I . S. and Percival, E l i z a b e t h , J. Chem. Soc. (1957) 2666. H i r s t , S i r Edmund, Mackie, W. and Percival, E l i z a b e t h , J. Chem. Soc. (1965) 2958. Love, J. and Percival, E l i z a b e t h , J. Chem. Soc. (1964) 3338. Mackie, I . M. and Percival, E l i z a b e t h , J. Chem. Soc. (1961) 3010. Percival, E l i z a b e t h and Young, Margaret, Phytochemistry (1971) 1 0 , 807. Bourne, E. J., Johnson, F. G. and Percival, E l i z a b e t h , J. Chem. Soc. (C) 1970, 1561. O ' D o n n e l l , J. J. and Percival, E l i z a b e t h , J. Chem. Soc. (1959) 2168. M c K i n n e l l , J. F. and Percival, E l i z a b e t h , J. Chem. Soc. (1962) 3141. M c K i n n e l l , J. F. and Percival, E l i z a b e t h , J. Chem. Soc. (1962) 2082. Bourne, E. J., Megarry, M. L. and Percival, E l i z a b e t h , J. Carbohyd. Nucleosides, Nucleotides (1974) 1, 235. C a r l b e r g , G. and Percival, E l i z a b e t h , Carbohyd. R e s . , (1977) i n the p r e s s . Haq, Q. N. and Percival, E l i z a b e t h i n "Some Contemporary Studies i n Marine Science" (1966) e d i t e d by Harold Barnes, p. 355. George A l l e n and Unwin Ltd., London. Percival, E l i z a b e t h and Wold, J. Κ., J. Chem. Soc. (1963) 5459. Turvey, J. R., Adv. Carbohyd. Chem. (1965) 20, 183. Haug, Α . , Acta Chem. Scand. Β (1976) 30, 562. Percival, E l i z a b e t h , unpublished work.

RECEIVED February 6,

1978.


Sulfated Polysaccharides Metabolized by the Marine Chlorophyceae

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