Consequences of chitosan decomposition by nitrous acid: Approach to non-branched oligochitosan oxime

It is well known that chitosan degradation by nitrous acid leads to oligochitosan (oligoCHI^t-ahm) bearing reactive 2,5-anhydromannose (3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-aldehyde) units at the new reducing ends of macromolecules. Standard protocol requires reduction of oligoCHI^t-ahm with NaBH₄ to corresponding oligoCHI^t-hml bearing unreactive hydroxymethyl group instead of reactive aldehyde group. For the first time, HP SEC as well as UV and CD spectroscopy methods have revealed that the reduction leads to an indefinite side modification and the formation of a branched oligoCHI^t-hml with increased molecular weight. Here, it is shown that the branching and modification can be prevented by means of the simple and reproducible reaction of oligoCHI^t-ahm with hydroxylamine that allows preparation of a stable linear oligochitosan oxime, oligoCHI^t-oxm. Cytotoxicity tests show that oligoCHI^t-ahm, oligoCHI^t-hml and oligoCHI^t-oxm are non-toxic at concentration below 2.5 mg/ml, and the cytotoxicity is concentration dependent and decreases in the order oligoCHI^t-ahm > oligoCHI^t-hml > oligoCHI^t-oxm at higher concentrations both before and after long shelf-storage. The elaborated approach and cytotoxicity data give an opportunity to use the non-branched oligoCHI^t-oxm for biomedical applications.