Sulfated Glycosaminoglycans Obtained by Chemical Modification of

6 Sulfated Glycosaminoglycans Obtained by Chemical Modification of Polysaccharides D E R E K H O R T O N and T A I C H I U S U I

Downloaded by CORNELL UNIV on July 23, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0077.ch006

Department of Chemistry, Ohio State University, Columbus, OH 43210

As part of the general theme of the symposium devoted to sulfated carbohydrates, this report presents some of the work from our laboratory concerned with the natural, sulfated glycosaminoglycan, heparin, both from the standpoint of structural elucidation and more especially from that of synthetic production of similarly constituted, sulfated polysaccharides that have biological properties resembling those of heparin. There is not time to present a detailed historical development of the entire story of the biological role and efforts to establish the chemical structure of heparin, but i t has been known for many years that this biopolymer contains residues of a uronic acid and 2-amino-2-deoxy-D-glucose in approximately equal proportion, and the material is highly sulfated, both at the amino positions and also at certain of the hydroxyl positions (1). The compound is widely distributed in many connective tissues, where i t appears to be produced as an intracellular component in mast cells. It is widely used in therapy as an anticoagulant in cardiovascular disorders resulting from thrombosis, and i t also displays antilipemic (clearing factor) properties. Much current evidence indicates that heparin as used therapeutically is the carbohydrate portion alone of a proteoglycan that is the native tissue component; a long, single, protein chain may be envisaged as being substituted by a large number of lateral chains of the sulfated polysaccharide, attached by a bridge region containing two D-galactose and a D-xylose residue, attached via L-serine to the peptide chain (2). Not only has the total constitution of this native proteoglycan not been elucidated, but controversies still remain concerning the exact constitution of the glycan chains that are detached during the processes used for extraction of heparin on the commercial scale for therapeutic use as an anticoagulant. Details of the exact procedure used in the commercial isolation of heparin from such rich sources as hog gastric mucuosa and beef lung are not readily available, but in general terras, the process involves autolysis of the tissue with concomitant 0-8412-0426-8/78/47-077-095$05.00/0 © 1978 American Chemical Society

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


96

CARBOHYDRATE

SULFATES

degradation of the non-heparin components, f o l l o w e d by a l k a l i n e treatment t o remove as much as p o s s i b l e of the p r o t e i n component, and subsequent p u r i f i c a t i o n by use of c a t i o n i c detergents. The g l y c a n m a t e r i a l thus i s o l a t e d i s of much lower molecular weight than the parent proteoglycan, and the product most widely used i n therapy has a molecular weight i n the region of 12,000, with a d i s t r i b u t i o n of molecular weights above and below t h i s f i g u r e . The molecular weight i s d i f f i c u l t t o estimate a c c u r a t e l y unless c a r e f u l a t t e n t i o n i s p a i d t o the e f f e c t s of the p o l y a n i o n i c c h a r a c t e r of the compound, but the accompanying graph ( F i g u r e l ) from a recent a r t i c l e (3) shows the r e s u l t s of molecular weight determinations on v a r i o u s h e p a r i n p r e p a r a t i o n s by u l t r a c e n t r i f u g a t i o n methods i n comparison with v i s c o s i t y measurements; i t may be seen t h a t the more r e a d i l y a p p l i e d v i s c o s i t y technique may be used at l e a s t as a rough approximation i n estimating the molecular weight of a h e p a r i n sample. The a n t i c o a g u l a n t a c t i v i t y of such heparin preparations i s c r i t i c a l l y dependent on the l e v e l of s u l f a t i o n ; commercial products assayed by a c o n v e n t i o n a l technique u s i n g sheep plasma t y p i c a l l y show a c t i v i t i e s i n the range of 110 t o l 8 0 I n t e r n a t i o n a l U n i t s per m i l l i g r a m f o r m a t e r i a l c o n t a i n i n g approximately 5 s u l f a t e residues per nominal t e t r a s a c c h a r i d e u n i t of the polymer. P a r t i a l d e s u l f a t i o n leads t o lowering of the b i o l o g i c a l a c t i v i t y , without n e c e s s a r i l y causing changes of more-profound s i g n i f i c a n c e i n the polymer; milder i s o l a t i o n techniques l e a d i n g t o products having s t i l l h i g h e r degrees of s u l f a t i o n have d i s p l a y e d a c t i v i t i e s at higher l e v e l s than those encountered normally i n commercial material. E l u c i d a t i o n of the chemical c o n s t i t u t i o n of the heparin chain has proved remarkably d i f f i c u l t ; the high degree of s u l f a t i o n renders the m a t e r i a l i n t r a c t a b l e i n many of the c o n v e n t i o n a l methods f o r p o l y s a c c h a r i d e structure-determination. I t has been e s t a b l i s h e d with l i t t l e doubt that the u r o n i c a c i d component involvesD-glucuronic a c i d and L - i d u r o n i c a c i d , but the exact p r o p o r t i o n s of these, and t h e i r r a t i o i n the n a t i v e proteoglycan, s t i l l remain c o n t r o v e r s i a l . I t i s by no means c e r t a i n t h a t the r a t i o of these u r o n i c a c i d s present i n commercial heparin r e f l e c t s t h a t present i n the o r i g i n a l proteoglycan, as these two u r o n i c a c i d s c o u l d i n t e r c o n v e r t by C-5 e p i m e r i z a t i o n under the a l k a l i n e c o n d i t i o n s used i n the i s o l a t i o n procedure. In the b i o s y n t h e s i s of the polymer chain, i t i s very probable that t h i s C-5 i n v e r s i o n occurs at the polymer l e v e l (U), and i n the n a t i v e s t a t e there may e x i s t an e n t i r e spectrum of glycan chain-compositions ranging from f r a c t i o n s r i c h i n D-glucuronic a c i d on the one hand t o f r a c t i o n s r i c h i n L - i d u r o n i c a c i d on the other. Working on the premise of a l i n e a r , a l t e r n a t i n g chain of amino sugar and u r o n i c a c i d components, work from t h i s l a b o r a t o r y (5.) i n v o l v i n g W and 0 - d e s u l f a t i o n of commercial heparin, f o l l o w e d by c a r b o x y l r e d u c t i o n w i t h diborane and then s e l e c t i v e fragmentation of the r e s u l t a n t , reduced, d e s u l f a t e d heparin l e d ,



HORTON AND usui

Sulfated

Gly cosaminogly cans

Figure 1. Relation between intrinsic viscosity and molecular weight (sedimen tation) from [ ] = J.35 Χ 10~ Μ · in aqueous sodium chloride medium. Data from Ref. 2. v

5

0 9



98

CARBOHYDRATE SULFATES

according t o the h y d r o l y t i c procedure used, t o two disaccharides, as i l l u s t r a t e d i n F i g u r e 2. One of these d i s a c c h a r i d e s , c h a r a c t e r i z e d on a c r y s t a l l i n e b a s i s , had a s t r u c t u r e t h a t i n d i c a t e d an α-ρ-(ΐ-*»*0 linkage between 2-amino-2-deoxy-D-glucose and D-glucuronic a c i d i n the o r i g i n a l polymer. The second d i s a c c h a r i d e "maltosamine", f o r which a reference sample was synthesized from maltose, i n d i c a t e s from i t s s t r u c t u r e the occurence of an a - D - ( l - * 4 ) linkage between g l u c u r o n i c a c i d and 2-amino-2-deoxy-D-glucose i n the o r i g i n a l polymer. This information l e d t o the formulation of a p a r t i a l backbone s t r u c t u r e as shown i n F i g u r e 2, and subsequent unequivocal i d e n t i f i c a t i o n of L - i d u r o n i c a c i d residues i n some preparations of heparin (6) l e d t o the type of g e n e r a l formulation shown i n F i g u r e 3, which d e p i c t s a h y p o t h e t i c a l t e t r a s a c c h a r i d e r e p e a t i n g - u n i t f o r heparin based on the occurence of L - i d u r o n i c a c i d residues and D-glucuronic a c i d residues, complete s u l f a t i o n of the amino groups, complete s u l f a t i o n of the primary hydroxyl groups, and a d d i t i o n a l s u l f a t e residues at one of the secondary p o s i t i o n s , probably p o s i t i o n 2, i n the L - i d u r o n i c a c i d component; the p r e f e r e n t i a l s u s c e p t i b i l i t y of the D-glucuronic a c i d residues toward degradation by periodate provided evidence that the Dg l u c u r o n i c a c i d residues were l a r g e l y non-sulfated at the 273 positions. Since the-time of these formulations, other workers have proposed on the b a s i s of enzymic s t u d i e s with degradation fragments from heparin that some or a l l of the linkages of the g l y c u r o n i c a c i d components have the β-D or α-L c o n f i g u r a t i o n (7), and support f o r these proposals ( F i g u r e h) has been provided from i n s p e c t i o n of n. m.r. spectra of the polymer (8) and a l s o from X-ray d i f f r a c t i o n studies (9) on the o r i e n t e d polymer. The X-ray d i f f r a c t i o n studies do accord with the general idea of f a i r l y short, l i n e a r chains f o r the macromolecule. However, unequivocal s t r u c t u r a l proof by degradation and synthesis of fragments at a l e v e l beyond t h a t of d i s a c c h a r i d e components i s s t i l l l a c k i n g , as i s such evidence obtained from n a t i v e proteoglycan m a t e r i a l that has not been subjected t o an u n s p e c i f i e d l e v e l of p o s s i b l e a l k a l i n e treatment during a commercial i s o l a t i o n process. Our continuing studies on heparin have been d i r e c t e d along two f r o n t s , f i r s t of a l l the systematic s t r u c t u r a l e l u c i d a t i o n of components of the polymer chain at a l e v e l beyond t h a t of the d i s a c c h a r i d e , together w i t h procedures f o r determining uronic a c i d r a t i o s and p o s i t i o n s of s u l f a t i o n i n the chain. In p a r a l l e l i n v e s t i g a t i o n s , we have examined the chemical synthesis of modified polymers whose s t r u c t u r e simulates that of heparin, to a f f o r d p o t e n t i a l replacements f o r the expensive n a t u r a l m a t e r i a l as a t h e r a p e u t i c anticoagulant, and t o provide a product f o r attachment t o the surface of s y n t h e t i c objects f o r implantation i n the c i r c u l a t o r y s y s t e m , with t h e o b j e c t i v e of c o n f e r r i n g a b i o l o g i c a l l y acceptable, nonthrombogenic s u r f a c e - c h a r a c t e r i s t i c f o r such devices. I t i s the l a t t e r aspect of chemical synthesis


HORTON AND usui

Sulfated

The

Linkage

C0 H

!

2

OH

:

Glycosaminoglycans Sequence

CH 0H

in

;

2

NHO

Heparin

C0 H

;

2

OH

:

,0 — Desulf ated

CH 0H 2

NH

:

2

Heparin


carboxyl reduction (diborane) Carboxyl — reduced

Heparin

hydrolysis CH OH

CHgOH

2

CH OH 2

OH OH

HO^ta^l

NK, NH^CI

NH3CI

maltosamin e Figure 2.

Disaccharides from carboxyl-reduced heparin

CH OS0 Na 2

3

HNS0 Na 3

CO^Na

CH 0S0 Na 2

OH

3

HNS0 Na

0

3

H, S 0 N a 3

Component R e s i d u e s Figure 3.

Present

i n Heparin

Component residues present in heparin



100


of h e p a r i n - l i k e p o l y s a c c h a r i d e s that c o n s t i t u t e s the p r i n c i p a l theme of t h i s p r e s e n t a t i o n , but the f o l l o w i n g d i s c u s s i o n presents a b r i e f survey of some of the more recent e f f o r t s t o degrade commercial heparin i n t o o l i g o s a c c h a r i d e s above the d i s a c c h a r i d e l e v e l that might be u s e f u l i n s t r u c t u r e - d e t e r m i n a t i o n studies. The next scheme ( F i g u r e 5) i l l u s t r a t e s a degradative method based on the recognized s u s c e p t i b i l i t y of D-glucuronic a c i d residues i n heparin t o a t t a c k by periodate? Exposure of heparin t o 1 molar equivalent of p e r i o d a t e per t e t r a s a c c h a r i d e u n i t gives a product i n which the D-glucuronic a c i d components are s e l e c t i v e l y degraded; subsequent borohydride r e d u c t i o n of t h i s m a t e r i a l , f o l l o w e d by m i l d h y d r o l y s i s w i t h a p o l y s t y r e n e s u l f o n i c a c i d of high molecular weight c o n f i n e d w i t h i n a d i a l y s i s membrane, shows t h a t degradation t o a low molecular weight, t r i s a c c h a r i d e t e t r o n i c a c i d component does take p l a c e by t h i s procedure. The recovery of the degraded, d i a l y z a b l e components i s r e l a t i v e l y low i n p r o p o r t i o n t o the t o t a l product t h a t s t i l l r e t a i n s polymeric c h a r a c t e r (10). T h i s r e s u l t tends t o argue against the idea t h a t the p e r i o d a t e - l a b i l e u r o n i c a c i d residues are u n i f o r m l y d i s t r i b u t e d along the chain. The d i a l y z a t e , a f t e r p u r i f i c a t i o n by p r e p a r a t i v e paper-chromatography, y i e l d s a m a t e r i a l that appears t o be a homogeneous t e t r a s a c c h a r i d e whose c o n s t i t u t i o n may be t e n t a t i v e l y d e p i c t e d as i n F i g u r e 6. I f the p e r i o d a t e - o x i d i z e d , borohydride-reduced heparin i s f u r t h e r subjected t o carboxyl-group r e d u c t i o n , and the product i s then subjected t o a c i d h y d r o l y s i s under c o n d i t i o n s where the h i g h l y a c i d - r e s i s t a n t 2-amino-2-deoxy-D-glucosyl residues would be expected t o remain attached t o t h e i r aglycons, a mixture of products i s obtained from which the aglycons may be detached by deamination w i t h n i t r o u s a c i d . This procedure gives e r y t h r i t o l a r i s i n g from the o r i g i n a l D-glucuronic a c i d r e s i d u e s that had been cleaved by periodate,"and L - t h r e i t o l a r i s i n g from any Li d u r o n i c a c i d r e s i d u e s t h a t had~undergone C - 2 — C - 3 cleavage~by p e r i o d a t e ( F i g u r e 7). Although the observed r a t i o (10) of these two products (2:1) i s s t r o n g l y i n f a v o r of e r y t h r i t o l , the f a c t t h a t some t h r e i t o l i s a l s o formed i n d i c a t e s that some of the Li d u r o n i c a c i d residues i n the o r i g i n a l polymer were not s u l f a t e d at p o s i t i o n s 2 and 3. The q u a n t i t a t i v e r e s u l t s obtained by t h i s sequence of experiments depend t o a l a r g e extent on the method of the treatment of heparin w i t h periodate; there i s no c l e a r - c u t , l i m i t i n g consumption of p e r i o d a t e at the l e v e l of 1 mole per t e t r a s a c c h a r i d e , and the use of an excess of p e r i o d a t e leads to a p r o g r e s s i v e i n c r e a s e i n the t o t a l periodate uptake. The p o s s i b i l i t y that other s u l f a t e d amino sugar polysaccha r i d e s might have anticoagulant p r o p e r t i e s has a t t r a c t e d a good d e a l of a t t e n t i o n . In an e a r l y a p p l i c a t i o n , Wolfrom and Shen Han (11) u t i l i z e d N-deacetylated c h i t i n (chitosan) and s u l f a t e d i t w i t h 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 t o produce an N-sulfated, p a r t i a l l y O - s u l f a t e d product ( F i g u r e 8). This compound had r e l a t i v e l y h i g h a n t i c o a g u l a n t a c t i v i t y , although t h i s was not so


6.

HORTON A N D usui

Sulfated


101

η


Figure 4.

Heparin: a suggested tetrasaccharide repeating-unit

HEPARIN

1) i o " 4

2) BH ~ 4

POLYALCOHOL pH 2 . 1 dialysis

^58° MIXTURE Sephadex G-15

void-volume fraction

(70%)

with polystyrenesulfonic Figure 5.

tetrasaccharide fraction (18*)

lower fragments (-10*)

acid

Degradative scheme for heparin based on selective periodate cleavage of Ό-glucuronic acid residues


102


1) i o 2) BH4 4


3)

H ,S0

Figure 6.

H +

3

Tetrasaccharide fragment from periodate-oxidized, borohydride-reduced heparin

1)

0.25 mol

I0

4

2) B H ~ OXIDIZED

,

REDUCED

HEPARIN

I ) CMC 2) B H "

3)

CH OH Q

CH 0H

J~°\

+

CH 0H

o

CH 0H

o

/ ~ \

,θΝ_^5\ίΙ/

H

' ° H2

HoMYLi H C

N H

2

H

I CHÔH

o

/ Α

'

Η2θΗ

Ho\_/UcH 1. HNOg

/ ~ \

Α ί « ν

Ηθ\_/1—OS!-/

(20°)

2. B H 6LUCIT0L +

2 , 5 - ANHYDROMANNITOL + T H R E I T O L

+

ERYTHRITOL

+

IDITOL

Figure 7. Alditol components from heparin after sequential periodate oxidation, borohydride reduction, carboxyl reduction, acid hydrolysis, nitrous acid deamination, and borohydride reduction



6.

HORTON AND usui

Sulfated

Glycosaminoglycans

103

high as that of heparin i t s e l f . Part of the reason f o r t h i s d i f f e r e n c e may probably be t r a c e d t o the s t r u c t u r a l d i s s i m i l a r i t y between the c h i t o s a n backbone and the a l t e r n a t i n g backbone of heparin i t s e l f , but another important f a c t o r i s undoubtedly the f a c t t h a t the s u l f a t e d c h i t o s a n had a molecular weight some 20 t o hO times higher than the range considered optimal f o r high a c t i v i t y i n heparin. The m a t e r i a l showed acute t o x i c i t y i n the mouse very s i m i l a r t o t h a t of heparin, although there i s evidence f o r a disadvantageous, delayed t o x i c i t y with t h i s m a t e r i a l . In subsequent s t u d i e s by W h i s t l e r and Kosick (12), a s i m i l a r approach was used, but i n a d d i t i o n , the procedure i n c l u d e d an o x i d a t i o n step by use of d i n i t r o g e n t e t r a o x i d e or oxygen—platinum t o introduce some c a r b o x y l groups i n t o the polymer ( F i g u r e 9). This m a t e r i a l l i k e w i s e showed a c t i v i t y of the same order of magnitude as that obtained by Wolfrom and Shen Han ( l l ) , and i t was shovm that s u l f a t e d and o x i d i z e d products were somewhat more a c t i v e than m a t e r i a l s that were s u l f a t e d only; i n a l l instances the a c t i v i t y i n c r e a s e d with i n c r e a s i n g l e v e l s of s u l f a t i o n , and products of the highest a c t i v i t y were obtained when the sequence was allowed t o give s u f f i c i e n t degradation t o shorten the polymer chain t o molecular-weight l e v e l s (osmometry) of the same order of magnitude as t h a t of heparin. In view of the somewhat n o n s p e c i f i c and degradative c h a r a c t e r i s t i c of d i n i t r o g e n t e t r a o x i d e as an oxidant, an e f f o r t was made i n our l a b o r a t o r y ( F i g u r e 10) t o achieve o x i d a t i o n of c h i t o s a n at C-β with a high degree of s p e c i f i c i t y (15). To accomplish t h i s o b j e c t i v e , c h i t o s a n was converted i n t o i t s p e r c h l o r a t e s a l t , f o l l o w i n g the hypothesis that the s t r o n g l y c a t i o n i c group at C-2 should p r o t e c t the hydroxyl group at C-3 against o x i d a t i o n and allow s p e c i f i c and complete o x i d a t i o n at C-6. The l a t t e r step was indeed achieved by use of chromium t r i o x i d e , and a product was obtained t h a t was completely carboxylated; i t presumably e x i s t s as an inner s a l t . S u l f a t i o n of the l a t t e r product gave the f u l l y N-sulfated, C-6 carboxylated c h i t o s a n analog. The p r o p e r t i e s of t h i s product are d i s p l a y e d i n F i g u r e 11, where i t may be observed that the product does have anticoagulant p r o p e r t i e s , although the procedure used l e d i n l a r g e measure t o r e t e n t i o n of the high-polymeric nature of the o r i g i n a l polysaccharide, and so the molecular-weight range of the product thus obtained l a y above t h a t considered optimal f o r h e p a r i n - l i k e activity. C o n t r o l l e d degradation p r i o r t o s u l f a t i o n of t h i s product would o f f e r promise as a p o t e n t i a l method f o r obtaining a s y n t h e t i c h e p a r i n o i d of higher a c t i v i t y . The former approaches, based as they are upon the c h i t o s a n s t r u c t u r e , do s u f f e r from the fundamental drawback t h a t the polymer chain m a n i f e s t l y d i f f e r s from t h a t i n heparin i t s e l f . In view of t h i s disadvantage, we i n i t i a t e d a new s e r i e s of i n v e s t i g a t i o n s based on an abundant ( l - * * h ) - l i n k e d a-D-glucan, namely amylose. In the i n i t i a l s e r i e s of s t u d i e s (15), amylose was converted by o x i d a t i o n of i t s 6 - t r i t y l ether with methyl



CHpOH

ÇH OS0 -Na +

, CHN (A)

CIS0 H

5

3

J 2

5

3

( heterogeneous) -

\P

or HCONMe ' S 0 (B) 2

3

( homogeneous) Method A'. D. P. 1280 Anticoagulant activity 56 Iu/mg LD Wolfrom

5 0

( mouse, IV) 380mg/kg

and Shen Han (1959) Method Β '.

D. P. 530


Anticoagulant activity 50 Iu/mg LD50 Figure 8.

CH (OH)OS0 H

2

2

f~

HCONMegS0 / HCONMe

N0 2

NH2

4

7

t

or Pt/0

vST /

2

(CO^MCHgOSOjH)

3

°\T^

3

ONlL/

775mg/kg

Sulfation of chitosan

CH OH

X

(mouse, IV)

\ £

2

3

NHSO^

Whistler

and Kosi k , 1971

Figure 9.

Sulfation and oxidation of chitosan

CHOH 2

-o

HCI0

4

NH CI0

NHAc

+

3

4

Chitosan

Chit i η C0 H 2

Cr0

I) CHN

3

5

5

3" NaOH v

NH+CI0

w

5.8 x l O

Figure 10.

3

NHS0 Na

+

3

Disodium ( l - 4 ) - 2 - d e o x y - 2 sulf oamino-/3-D-glucopyranuronan

d.s. 1.0 by carboxyl M

4

5

M , u

4.3 χ I0

5

Specific C-6 oxidation and ^-sulfation of chitosan



6.

HORTON AND usui

Sulfated


105

s u l f o x i d e — a c e t i c anhydride t o give a product l a r g e l y o x i d i z e d t o the 2-keto d e r i v a t i v e at c e r t a i n of the residues ( F i g u r e 12). Conditions a f f o r d i n g a degree of s u b s t i t u t i o n of approximately 0. 5 were selected. This product was s u c c e s s i v e l y oximated and then reduced with l i t h i u m aluminum hydride t o give, a f t e r t r i t y l a t i o n , an aminated amylose i n which the amino groups had been l a r g e l y i n c o r p o r a t e d at the 2 - p o s i t i o n i n the D-gluco c o n f i g u r a t i o n , as demonstrated by a c i d h y d r o l y s i s . "The o x i d a t i o n procedure used a l s o introduced (methylthiο)methyl ether groups at 0-3, at l e a s t i n c e r t a i n of the residues. T r i f l u a r o a c e t y l a t i o n of the f r e e amino groups and the r e s i d u a l hydroxyl groups i n the r e d u c t i o n product, followed by d e t r i t y l a t i o n , exposed the h y d r o x y l groups at C-6, and these were subjected t o o x i d a t i o n by the a c t i o n of oxygen—platinum. In view o f the f a c t that t h i s o x i d a t i o n proceeds much more r a p i d l y i n D-glucose residues than i n those of 2-amino-2-deoxy-D-glucose, i t was hoped that the procedure would l e a d t o a polymer i n which residues of 2-amino-2-deoxy-D-glucose would be l a r g e l y unoxidized at the C-6 p o s i t i o n , whereas those of the non-aminated, D-glucose residues would be o x i d i z e d t o the corresponding D-glucuronic a c i d residues. Removal of r e s i d u a l p r o t e c t i n g groups, followed by s u l f a t i o n of t h i s polymer, gave a compound c o n t a i n i n g the r e q u i s i t e f u n c t i o n a l groups f o r a h e p a r i n l i k e s t r u c t u r e , although, as shown i n F i g u r e 12, the anticoagulant a c t i v i t y of t h i s m a t e r i a l was low. This f a c t o r may a r i s e from the f a c t t h a t the product was of lower molecular weight than the values considered optimal f o r h e p a r i n - l i k e a c t i v i t y . This degradation occurred p r i n c i p a l l y at the stage where r a t h e r severe c o n d i t i o n s were r e q u i r e d t o remove the 0-(methylthiο)methyl groups t h a t had become introduced during the o x i d a t i o n step with dimethyl s u l f o x i d e — a c e t i c anhydride. In view of the drawbacks of the use of t h i s p a r t i c u l a r oxidant, Dr. Usui performed a new s e r i e s of experiments i n which 6 - 0 - t r i t y l a m y l o s e was o x i d i z e d by mixtures of N,N-dicyclohexylcarbodiamide (DCC) and dimethyl s u l f o x i d e , i n v a r i o u s proportions. A range of o x i d i z e d products was obtained, as shown i n F i g u r e 13, and these were f r e e of s u l f u r . Reduction of these polymers by borohydride, followed by a n a l y s i s of the sugar composition of the products by means of the a l d i t o l acetates, revealed t h a t o x i d a t i o n had taken place s e l e c t i v e l y at the 2 - p o s i t i o n at low l e v e l s of oxidation. At higher l e v e l s of oxidation, the 3 p o s i t i o n s were a l s o a f f e c t e d , as shown by the nature of the a l d i t o l s produced i n the a n a l y t i c a l sequence of r e a c t i o n s . Oximation of the 6 - 0 - t r i t y l a r a y l o s e s that had been o x i d i z e d t o a f a i r l y low degree of s u b s t i t u t i o n gave the corresponding oximes, whose n i t r o g e n content corresponded t o the l e v e l of o x i d a t i o n determined p r e v i o u s l y by r e d u c t i o n of the o x i d i z e d products w i t h sodium borodeuteride, f o l l o w e d by a n a l y s i s f o r the deuteriumc o n t a i n i n g sugar component ( F i g u r e 1*0. The oximated product was i n turn a c e t y l a t e d t o a c y l a t e the f r e e hydroxyl groups i n the polymer chain and a l s o the h y d r o x y l group of the oxime f u n c t i o n .



106

LD

(intraperitoneal , mouse), 237 mg/kg

5 0

Anticoagulant


Leukemia

activity ,

L - 1210 i£ vitro 50%

Figure

26 I.U./mg

at

inhibition,

0.2 μ,Μ ( 100 >xg/ml)

11. Biological properties of C-6 oxi dized, N-sulfation chitosan

CHgOTr 2

2

2. H 0 N H , C H N 2

— 0

5

5

OH

5 5 H

2

(NH ) (OH) 2

· 3»°5 5 5.CIS0 H,

4 N

δ 0

Η

3

( NHCOCFa) (NH ) 2

HORTON Figure 12.

—0

(NOH) ( HOH)

2

(CF C0) 0 C

tetrahydrofuran

/(OH) ÔCHgSMefl

1. 0 , Pt 2. H + 3. OH"

CHoOTr 3

-0

Li AIHL

1. M e S 0 , A c 0

and

J U S T , 1973

(NHCOCF) (OH)

Ν

— 0

C5H5N

(NHSO^Na+î (OH) Anticoagulant a c t i v i t y , l7IU/mg

Conversion of amylose into a 6-carboxyl, 2-sulfoamino analog


HORTON AND usui

Sulfated

CH OH

ChUOTr

9

A


.

DCC , C H N , C F C O ^ 5

5

3

Me S0 , 1 . 4 - dioxane 2 o

—Ο

OH Amy I ose (Super I ose)

D.S.

of

products


controlled

by

proportions

of

DCC

3

and

—0

CF C0 H

0'

2

x

max

OH 5.8/wn

Suif u r Figure 13.

free

Alternative oxidation procedure for 6-O-trityhmylose

ANALYTICAL

A.

METHOD

Oximation ( Ν - content)

8. NaBH^

reduction

Det r i tylat ion 1

Η

and

l 3

C n.m.r

C. Reduction Hyd roly si s G. I.c. — m. s of a l d i t o l acetates

Figure 14.

Procedures for analysis of oxidized tritylamyloses

6-0-



CH 0Tr

}H OT -0

CHoOTr

2

-Q

1

NH«OH k

2

-ο

AcoO

r

OH

— ο By DCC method

NOH

NOAc

D.S. 0.35 by ox i me Sulfur - f ree

\ηοχ · . · >"" 5

6

5

7

1. H"


1

THF

2. NaOMe

—ο NH

NH

C

D.S. 0.35 by N H Figure 15.

Preparation of 2-amino-2-deoxyamylose of d.s. 0.35

Properties Ninhydrin:

NH

( aid i toi

acetates

from

Soluble

H 0 ,Me SO

Glc

74%

acid

GlcN

23%

2

0.35)

Analysis positive

Jodine: blue complex in

dilute

Aminated amylose (D.S.

2

Aminated amylose

2

I.r,

:

2

2

Ή n.m.r

base

no NHAc,

δ 5.34 ( H - l of GlcN),

by DCC route

5.25 ( H - l

of GlcN)

hydrolyzate)

ManN

3%

AJIN-3 0 % GlcN-3

0%

Mol. wt. : 4 , 0 0 0 - 20,000 ISephadex ID Figure 16.

G - 2 5 , G-75)

7xlO" M (L-I2I0 cells) 6

5 0

Characterization and properties of aminated amylose


Sulfated

HORTON AND USUI


CH OSO~Na

CHUOH

+

2

CHgOSO^Na +

so? Me SO 2

dialysis (Na D.S.


[ex]

salt)

0.35 + 168 ( H 0) 2

NHSO~Na D.S. 0.35 by

sulfoamino

D.S.

sulfate

1.8

by

Toluidine Blue test: positive Ninhydrin test: negative All primary OH sulfated [ C nmr] Mol. wt. 3,000 - 20,000 Anticoagulant activity* 45 IU/mg ,3

Figure 17.

OSiMe Figure 18.

Sulfation of aminated amylose

3

OSiMe

3

OH

Preparation of 6-O-acetylamylose


+


λ

and Ε η. m. r. a n a l y s i s

1

- Superlose 39 +55^.

- Superlose 1^997^.

trace

U$

k

o f reduced, d e t r i t y l a t e d product.

trace

1$

3$

- By gel-permeation chromatography o f reduced, d e t r i t y l a t e d product.

C

99$

20$

1:0.25

1 3

95$

35$

2:0. 5

- Supported by

93$

80$

h:l

2^

21$

h%

75$

R e s u l t s of g. 1. c. a n a l y s i s — Man All Glc

D.S. by oximation

70$

2

2

5:1

3

DCC/CF C0 H per mol.

3

Analysis of D C C — C F C 0 H - O x i d i z e d 6-O-Tritylamylose

1*

Expt. no.

Table I.


+ +

~ 20,000 ~ 20,000 35,000

Iodine

8,000

Mol. vrt. —


6.

HORTON AND usui

Sulfated


111

Reduction of the l a t t e r product by diborane proved t o be a much more s a t i s f a c t o r y method f o r i n t r o d u c i n g the amino group than the sequence p r e v i o u s l y used t h a t i n v o l v e d d i r e c t r e d u c t i o n of the oxime with l i t h i u m aluminum hydride. The sequence (Figure 15) gave, a f t e r d e t r i t y l a t i o n , an aminated amylose whose degree of s u b s t i t u t i o n corresponded t o the o r i g i n a l oxime content of the precursor, and the p r o p e r t i e s of the keto products at d i f f e r e n t degrees of s u b s t i t u t i o n and as obtained by d i f f e r e n t means of p r e p a r a t i o n are shown i n Table I. A c i d h y d r o l y s i s of the aminated amylose showed that the net amination had been achieved with high r e g i o - and stereoselectivity. At a d. s. l e v e l of 0.35, e s s e n t i a l l y a l l of the amino groups had become i n c o r p o r a t e d at p o s i t i o n 2 i n the D-gluco c o n f i g u r a t i o n ; no s i g n i f i c a n t amino sugar components a r i s i n g from o x i d a t i o n at C-3 were detected i n the product at t h i s degree of s u b s t i t u t i o n . D e t r i t y l a t i o n of the product was r e a d i l y achieved by use of methanolic hydrogen c h l o r i d e followed by treatment with sodium methoxide i n methanol to remove the r e s i d u a l 0 - a c e t y l groups. The product (Figure 16) was r e a d i l y s o l u b l e i n water, i n d i l u t e a c i d , and a l s o i n d i l u t e a l k a l i , s t i l l shewed the i o d i n e - s t a i n i n g a b i l i t y c h a r a c t e r i s t i c of amylose, but a l s o showed the n i n h y d r i n r e a c t i o n c h a r a c t e r i s t i c of the presence of amino groups; the i n f r a r e d spectrum, showing absence of carbonyl absorption, e s t a b l i s h e d t h a t no migration of a c e t y l groups from oxygen t o n i t r o g e n had occurred during the s a p o n i f i c a t i o n step. F i g u r e 17 shows the r e s u l t s of s u l f a t i o n of t h i s aminated amylose by use of dimethyl s u l f o x i d e — s u l f u r t r i o x i d e . This product, obtained i n 7^$ y i e l d a f t e r d i a l y s i s , was s o l u b l e i n water, gave a negative n i n h y d r i n t e s t f o r f r e e amino groups, and a p o s i t i v e T o l u i d i n e Blue t e s t f o r sulfoamino groups. Its anticoagulant a c t i v i t y , as assayed with sheep plasma, was I n t e r n a t i o n a l U n i t s p e r m i l l i g r a m , namely about hOô of t h a t of commercial heparin. The C n.m. r. spectrum of the product showed that the primary a l c o h o l groups were e s s e n t i a l l y completely s u l f a t e d , because the CH resonance normally occurring near 6 l . 5 p. p.m. was s h i f t e d downfield by about 6 p. p. m. by the s u b s t i t u e n t e f f e c t of the s u l f a t e groups. The product was q u i t e p o l y d i s p e r s e , and i t s molecular-weight range (3,000-20,000) i n d i c a t e d that some measure of degradation had occurred during the t o t a l sequence u t i l i z e d . Approximately one h a l f of the m a t e r i a l i n t h i s product had a molecular weight below the range considered optimum f o r heparin a c t i v i t y . Continuing work i n our l a b o r a t o r y i s concerned with the f r a c t i o n a t i o n of the m a t e r i a l j u s t described and assay of the anticoagulant a c t i v i t y of products containing t h e i r major component i n the 10 t o 12,000 molecular weight range, with v a r i o u s l e v e l s of amination at the C-2 p o s i t i o n . In a f u r t h e r development of p o t e n t i a l u t i l i t y ( F i g u r e 18) a procedure has been e s t a b l i s h e d (15) f o r s p e c i f i c 6 - s u b s t i t u t i o n of amylose by an a c e t y l group; l 3

2


CARBOHYDRATE

112

SULFATES

t h i s 6-0-acetylamylose and analogous primary monoesters of p o l y s a c c h a r i d e s may prove more u s e f u l than the t r i t y l ethers as s t a r t i n g p o i n t s f o r s y n t h e s i s of heparin analogs. Acknowledgments This work was supported by Grant No. HL-11U89 from the N a t i o n a l Heart, Lung, and Blood I n s t i t u t e , N a t i o n a l I n s t i t u t e s of Health, Department of Health, Education, and Welfare, Bethesda, Md. 2001U.


Literature Cited 1 Jeanloz, R. W., in W. Pigman and D. Horton, Eds., "The Carbohydrates", 2nd ed., Vol. IIB, pp. 609—615, Academic Press, New York, 1970; Lindahl, U., MTP Int. Rev. Sci. Ser. Two, Vol. 7, Carbohydr. (1976) 283—312. 2 Jaques, L. Β., Methods Biochem. Anal. (1977) 24, 203—312; Muir, Η., and Hardingham, Τ. Ε., MTP Int. Rev. Sci. Ser. One, Vol. 5 Biochem. Carbohydr. (1975) 153—222. 3 Johnson, E. A. and Mulloy, Β., Carbohydr. Res. (1976) 51, 119— 127. 4 Hőők, Μ., Lindahl, U., Bäckstrőm, G., Malmstrőm, A., and Fransson, L. -Å., J. Biol. Chem. (1974) 249, 3908—3915. 5 Wolfrom, M. L., Vercellotti, J. R., and Horton, D., J. Org. Chem., (1964) 29, 540—550; Wolfrom, M. L., El Khadem, H. S., and Vercellotti, J. R., ibid. (1964) 29, 3284—3286; Wolfrom, M. L., Tomomatsu, Η., and Szarek, W. Α., ibid. (1966) 31, 1173—1178. 6 Wolfrom, M. L., Honda, S., and Wang, P. Υ., Carbohydr. Res. (1969) 10, 259-265. 7 Hovingh, P., and Linker, Α., Biochem. J. (1977) 165, 287—293; but see also Silva, Μ. Ε., Dietrich, C. P., and Nader, Η. Β., Biochim. Biophys. Acta (1976) 437, 129—141. 8 Perlin, A. S., Mackie, D. Μ., and Dietrich, C. P., Carbohydr. Res. (1971) 18, 185—194. 9 Nieduszynski, I. Α., and Atkins, E. D. T., Biochem J. (1973) 135, 729—731. 10 Horton, D., Liav, A., and Toman, R., unpublished data. 11 Wolfrom, M. L., and Shen Han, Τ. -Μ., J. Am. Chem. Soc. (1959) 81, 1764—1766. 12 Whistler, R. L., and Kosik, Μ., Arch. Biochem. Biophys. (1971) 142, 106—110. 13 Horton, D., and Just, Ε. Κ., Carbohydr. Res. (1973) 29, 173— 179. 14 Horton, D., and Just, Ε. Κ., Carbohydr. Res. (1973) 30, 349— 357. 15 Horton, D., and Lehmann, J., Carbohydr. Res. (1978) 61, 553-556. RECEIVED

M a y 8,

1978.


Sulfated Glycosaminoglycans Obtained by Chemical Modification of

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