Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process

Abstract

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii. Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (approximately 50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.