Conjugation to the chain termini of chitin oligosaccharides

Abstract

Today's polymer science is based largely on the creation of synthetic block copolymer structures. The possibility of creating block copolymer structures from biological polysaccharides with the same physiochemical properties as the synthetic ones is desirable, this would give the altered advantage of biocompatible and biodegradable characteristics as well. One of the limiting factors is the lack of reactivity at the polysaccharide non-reducing end, making only small linear block constructions through the reducing end possible.

The purpose of this master thesis has been to determine if periodate oxidation of chitin can be utilized as a method for construction of block copolymer structures, by activating through the non-reducing end of the oligomer. Chitin oligomers were selected because of the periodate oxidations selectivity towards vicinal diols, which chitin oligomers only have at the non-reducing end. The degradation into chitin oligomers also produces a more reactive reducing end, 2,5 anhydro-D-mannose, which is readily available for further reactions. Two separate strategies were used for this thesis. Strategy one was oxidation of reducing end conjugated oligomers, while strategy two was conjugation of oxidized oligomer.

Chitosan (FA=0.4) was degraded with nitrous acid to obtain the chitin oligomers used, all of them type AnM. The degradation gives a water-soluble low molecular weight (DP<10) fraction and a water insoluble high molecular weight fraction. The water-soluble oligomers were further separated by size exclusion chromatography (SEC) and purified.

For strategy one, the purified oligomers were conjugated to a linker, adipic acid dihydrazide (ADH) or propanediyl bishydroxyiamine dihydrochloride (PDHA), through a reductive amination at the chitin oligomer reducing end. The reaction was proven successful for the formation of activated oligomers of the type AnM-ADH or AnM-PDHA. The reducing agent used was 2-picoline borane. The conjugated oligomers were characterized with 1H-NMR before they were oxidized with periodate, leading to the formation of two reactive aldehydes at the non-reducing end of the oligomers.

Analysis from 1H-NMR illustrated breakdown in the conjugation after oxidation, for both linkers and for different oligomer starting structures. These results suggested that the M-unit of the chitin oligomer carry an instability after conjugation. This presumption was supported by an analysis of ADH in periodate oxidation which showed no significant alterations to the chain structure of the linker.

For strategy two the purified, unmodified oligomers were subjected to periodate oxidation. The characterization of the oxidation products by 1H-NMR revealed the chitin oligomers were intact after the reaction, but are shown to have an oxidation limit, even in excess periodate. Two separate comparison studies were done with the reduction of oxidized oligomer, one with NaBH4 and one with the conjugation to L-tyrosine methyl ester. Both reactions gave successful results. The oxidized chitin oligomer was conjugated to ADH and PDHA in two separate reactions. The reaction products of both reactions were identified by 1H-NMR and MS. The reactions appear to lead to formation of conjugated product at both the reducing and non-reducing end of the oligomer for both linkers. The ADH activated reaction resulted in several products of presumed polymerized ADH, as well as an insoluble fraction probably due to the same internal ADH reaction. The insoluble fraction was characterized in HFIP by 1H-NMR. The PDHA activated reaction gave what appear to be a polymerization of the oligomers.

A brief study of the diffusion rate of commonly used salts for buffers, sodium chloride (NaCl), ammonium acetate (AmAc) and sodium acetate (NaAc) was prepared in two experiments for each salt; stirred and unstirred. The salt solutions of 0.1M were added to dialysis bags and the conductivity inside the bags were measured until all salt had diffused out. The results show that the dialysis rate is strongly dependent on type of salt, with AmAc having the highest rate, then NaAc,so NaCl. With a MWCO of 100-500Da, the time needed for dialysis was found to stretch from 5 days for AmAc to 14 days for NaCl.