Solid-state NMR of N-acylureas derived from the reaction of hyaluronic acid with isotopically-labeled carbodiimides

Article abstract of DOI:10.1021/ja00041a010

Hyaluronic acid (HA) is a naturally-occurring linear polysaccharide consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues. Reaction of the carboxyl group of the glucuronate residues with l-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) in the presence of primary amines yielded only the N-acylurea adducts rather than the expected amide coupling products. To determine the nature of this linkage unambiguously and to deduce the primary structure of the N-acylurea products, ¹³C- and ¹⁵N-labeled l-ethyl-3-(3-(dimethylamino)propyl)carbodiimide were synthesized. The isotopically-labeled carbodiimides were coupled to the carboxyl group of HA (molecular size ca. 2 000 000 Da) in water at pH = 4.75. The modified polysaccharides were then isolated, purified, and examined by cross polarization and magic angle spinning (CP-MAS) solid-state ¹³C and ¹⁵N NMR. The chemical shifts and states of protonation of the nitrogens confirmed the presence of two isomeric N-acylureas in unequal amounts and ruled out the presence of any unrearranged O-acylurea product.