Highly sulfated derivatives of K5 polysaccharide and their preparation

Abstract

The purification of the E. coli K5 polysaccharide by treatment with isopropyl alcohol and elimination of lipophilic substances is described. The purified product can be used to prepare, after N-deacetylation, new N,O-sulfated polysaccharides with high degree of sulfation.

1 . Pure K5 polysaccharide showing no signals in the region below 1.5 ppm of its:  1 H-NMR spectrum. 
     
     
         2 . The K5 polysaccharide of  claim 1 , wherein nucleic acids are not detectable in the UV spectrum at 260 nm and the protein content is below 0.5%. 
     
     
         3 . The process of  claim 4  wherein (a1) a K5 from fermentation, dissolved at a 5% concentration in 4 M sodium chloride solution at 4° C., is treated with 1 volume of isopropanol; (a2) the salt solution is brought to a 3 M concentration by addition of the calculated amount of sodium chloride; (a3) the solution is left to stand overnight at 4° C.; and (a4) the product thus obtained is isolated by centrifugation and the salts are eliminated by ultrafiltration whereby a K5 polysaccharide free from lipophilic substances is obtained. 
     
     
         4 . A process for the purification of K5 polysaccharide which comprises treating a K5 polysaccharide from fermentation with isopropanol in a 2-5 M aqueous saline solution. 
     
     
         5 . The process of  claim 4  wherein said K5 starting material is dissolved in said saline solution at a concentration of from 0.5 to 10% and said isopropanol is added in 1-3 volumes. 
     
     
         6 . The process of  claim 5  wherein said saline solution is a 2-5 M aqueous sodium chloride solution. 
     
     
         7 . The process of  claim 4  wherein the K5 starting material is dissolved in a 2-5 M sodium chloride solution at a concentration of from 0.5 to 10%, treated with 1-3 volumes of isopropanol at a temperature of 0-8° C. and the thus obtained solution is brought to 2-4 M by further addition of sodium chloride, whereby a K5 polysaccharide free from lipophilic substances is obtained and isolated by filtration or centrifugation.