Process for the sulfation of uronic acid-containing...

Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives

Reexamination Certificate

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C536S122000, C536S123000, C536S123100, C536S124000

Reexamination Certificate

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06388060

ABSTRACT:

BACKGROUND OF THE INVENTION
Polysaccharides containing uronic acid residues are found in virtually every species in Nature. These polymers include plant polysaccharides such as pectin, alginic acid, and many gums and mucilages; animal polysaccharides such as the glycosaminoglycans; and bacterial polysaccharides such as a variety of capsular materials. In such polysaccharides, the uronic acid residues may account for 10 to 100 percent of the total monosaccharide residues in the polymer. Uronic acid types that are found in these polymers include D-glucuronic, D-galacturonic, D-mannuronic, L-iduronic, and L-guluronic acids.
A number of animal uronic acid-containing polysaccharides such as heparins, chondroitin sulfates, and dermatan sulfates occur naturally with sulfate substituents in various positions on both their uronic acid and their hexosamine residues. Interestingly, the presence of sulfate groups on these uronic acid-containing polymers leads to species that have various biological and therapeutic activities. For example, heparin exhibits important anticoagulant and antithrombotic activities, whereas the bacterial
E. coli
K5 polysaccharide, which has a structure like that of heparin but which is not sulfated, lacks these activities. Similarly, chondroitin sulfate and dermatan sulfates have activities similar to those of heparin, whereas their structurally-analogous
E. coli
K4 polysaccharide, which is not sulfated, lacks these activities. In the heparin case, it has been demonstrated that both the uronic acid carboxyl groups and the sulfates are essential for the anticoagulant activity. Consequently, sulfation of unsulfated uronic acid-containing polysaccharides may convert such polymers to biologically active species. Similarly, it may be possible to generate substances with unique activities by further sulfating polymers that occur naturally as partially sulfated polysaccharides.
Two general approaches have been used extensively to sulfate polysaccharides. These include (a) the use of amine conjugates of sulfur trioxide as the sulfating agent (Gilbert (1962) Chem. Rev. 62: 550-589; Nagasawa, et al. (1986)Carbohyd. Res. 158: 183-190; Casu, et al. (1994) Carbohyd. Res. 263: 271-284), and (b) the use of a mixture of sulfuric acid and chlorosulfonic acid as the sulfating agent (Naggi, et al. (1987) Biochem. Pharmacol. 36: 1895-1900). We report here a new method for sulfation of uronic acid-containing polysaccharides. This method involves the generation of a sulfating reagent by activation of sulfate ions with carbodiimides. Such active sulfates react with the hydroxyl groups of polysaccharides, generating products with degrees of sulfation that can be controlled by modulation of the reaction conditions. The sulfuric acid/carbodiimide method described here offers a number of advantages over sulfation methods that have been reported in the scientific and the patent literature. As one example the sulfuric acid/chlorosulfonic acid reagent causes depolymerization of the polysaccharide as the sulfation proceeds so that the molecular weight of the final product is difficult to control; the carbodiimide/sulfuric acid method does not cause any depolymerization.
Previous reports have described the use of sulfuric acid and a carbodiimide to sulfate monosaccharides (Mumma, et al (1970) Carbohyd. Res. 14: 119-122; Takano, R., T. Ueda, et aL (1992) Biosci. Biotech. Biochem. 56: 1413-1416) and neutral polysaccharides (Takano, R., S. Yoshikawa, et al. (1996) J. Carbohyd. Chem. 15: 449-457). In these reports it was shown that C6 of the monosaccharides was sulfated much more rapidly than the secondary hydroxyls, but a high level of sulfation of these carbohydrates, i.e. >1 sulfate per monosaccharide residue, could not be achieved. Furthermore, this method was not applied to uronic acid-containing polysaccharides, where the use of the carbodiimide-activated sulfate reagent presents some special problems. First of all, carbodiimides activate the carboxyl groups of the uronic acid-containing polysaccharides, rendering them reactive with a variety of nucleophiles that might be present in the reaction mixture (amines, alcohols, or hydrides (Hoiberg and Mumma (1969) J. Am. Chem. Soc. 91: 4273-4278; Danishefsky, and Siskovic (1971) Carbohyd. Res. 16: 199-205; Taylor and Conrad (1972) Biochemistry 11: 1383-1388), yielding undesired products. Thus, for example, carbodiimide-activated uronic acid residues may react with hydroxyl groups on adjacent chains of the polysaccharide in the reaction mixture to form ester cross-links, thus increasing the molecular weight of the products. Also, the sulfate groups that are already present on the substrates to be sulfated may be activated by the carbodiimide, rendering them quite strong electrophiles and allowing sulfate cross-linking of the polymer chains. Furthermore, during the sulfation reaction, the formation of mixed anhydrides of the sulfuric acid and carboxylic acids may occur. In addition to yielding undesired products, these side reactions consume the sulfation reagent, lowering the concentration of the reagent and thus the extent of sulfation of the desired product. Thus, for the sulfation reaction, it is important to prevent side reactions so that the polymer is altered only by the addition of sulfates to the alcoholic hydroxyl groups. The method described here uses conditions that have been developed to minimize the side reactions and to control the degree of sulfation, yielding, maximally, greater that two sulfate residues per monosaccharide.
SUMMARY OF THE INVENTION
The present invention provides news methods for the preparation of sulfated (or oversulfated or hypersulfated) uronic acid-containing polysaccharides, including glycosaminoglycans. In such methods, tertiary or quaternary alkyl amine salts of uronic acid-containing polysaccharides are dissolved in an aprotic solvent, e.g. dimethylformamide (DMF), and an N,N-carbodiimide and sulfuric acid are added. The sulfuric acid is activated by the carbodiimide and the free hydroxyl groups of the heparinoid react readily with these activated anions. The methods described herein work efficiently for uronic acid-containing polysaccharides, and are superior to previously used carbodiimide/sulfuric acid methods described for monosaccharides and neutral polysaccharides, wherein the extent of sulfation was limited to <1 sulfate/monosaccharide. With the previously reported, more limited reaction conditions, the sulfation occurred primarily at C6 positions of the monosaccharide residues, i.e., positions that are already extensively sulfated in many of the naturally-occurring sulfated polysaccharides, including chondroitin 6-sulfate and heparinoids. The present invention provides methods whereby the conditions employed minimize the modifications of the carboxyl groups while maximizing the degree of sulfation. In the methods of the present invention, the carbodiimide or sulfate derivatives of the carboxylic acids formed during the reaction are converted back to the original carboxylic acids. Furthermore, the polysaccharides do not become degraded or cross-linked during the reaction.
More particularly, the present invention provides a process for the preparation of sulfated uronic acid-containing polysaccharides, the process comprising: (1) converting the uronic acid-containing polysaccharide into an amine salt, and (2) 0-sulfating the amine salt of the uronic acid-containing polysaccharide with carbodiimide and sulfuric acid. The first step, wherein the alkali metal salt of the uronic acid-containing polysaccharide is converted to an amine salt, can be done by either ion exchange chromatography or by the addition of a solution of the tertiary or quaternary amine to an about 2% to about 10% solution of the uronic acid-containing polysaccharide.
In one embodiment, the present invention relates to methods for the preparation of oversulfated heparinoids, the methods generally comprising: (1) conversion of the heparinoids into solvent-soluble salts; (2) 0-sulfation of the heparinoids with carbodiimide and

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