Chemistry of tissues. 4. Methylation studies on hyaluronic acid

Abstract

K hyaluronate was prepared from human umbilical cords and the free acid obtained there from using "Zeo-Karb 215" to remove K. Both were obtained in fibrous form. The acid prepn. gave no satisfactory X-ray diffraction photographs and showed no very highly ordered structure. From an aqueous acid hydrolysate of K hyaluronate gluco-samine HC1 was crystallized and identified by an X-ray powder photograph. The presence of glucosamine and a uronic acid in the hydrolysate was confirmed by paper-strip chromatography. A partially depolymerized hyaluronate prepn. was obtained and methylated with dimethyl sulfate followed by CH3I, to yield a product containing 33% methoxyl. From this, a uronic acid derivative was isolated and identified, after further methylation, as 2,3,4-trimethyl-alpha-methyl-D-glucopyruronoside acid amide. At least 1/3 of the uronic acid residue in the hyaluronic acid molecule is present in a chain, linked glycosidically through its reducing group and one other hydroxyl group and is not present as an end group. No amino sugar derivatives could be identified from the methylated product, the significance of which is discussed. Methylation with Na and CH3I in liquid ammonia (Freudenberg) was unsatisfactory and the method was considered unsuitable for hyaluronic acid.