Abstract
Deacetylated hyaluronan (daHA) containing reactive free amino groups is an important intermediate for further modification. Comparing direct and indirect NMR and HPLC to characterize the degree of HA deacetylation (DD), direct NMR approach using area ratio of anomeric CH and CH-NH2 groups was the most precise one.
To describe the substitution pattern, daHA was selectively cleaved by nitrous acid generated in situ or hyaluronan lyase from Streptococcus pneumoniae. The resulting oligomers were identified by LC-ESI-MS.
The experimental distribution of these oligomers was compared with theoretically expected random oligomer distribution. Independently on the starting HA molecular weight and deacetylation conditions, the experimental data differed from the random distribution model and suggested that deacetylation of certain N-acetyl-D-glucosamine had reduced the probability of deacetylation at the neighbouring disaccharide.
This phenomenon was explained by conformational changes of HA caused by intra- and intermolecular interactions between positively charged amino and negatively charged carboxylic groups.