Towards Polysaccharide Origami: Preparation of Chitooligosaccharide-Dihydrazide Hybrids
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The recent development of DNA origami shows that biopolymers can form a variety of predetermined nanostructures of considerable sophistication. A corresponding development of polysaccharide origami based on a modular strategy is proposed to include carbohydrates from natural and renewable sources for use in biomaterials. Chitin is claimed to be the second most abundant naturally occurring polysaccharide on earth after cellulose. Chitin and its N-deacetylated derivative, chitosan, have a number of potential applications due to their physicochemical properties. Oligosaccharides obtained from these polymers may thus be applicable in the development of polysaccharide origami to form a new type of polysaccharide based biomaterials. The aim of this master thesis was to establish a library of di-substituted hybrids, composed of chitin oligomers (fully N-acetylated oligomers with a 2,5-anhydro-D-mannose (M)-unit at the reducing end of the type AnM) conjugated to adipic acid dihydrazide (ADH) by reductive amination, of the type AnM-ADH-MAn. Chitosan (FA = 0.4 and 0.48) was degraded by an excess of nitrous acid to obtain the chitin oligomers of the type AnM. The water soluble low molecular weight oligomers obtained by this degradation procedure were separated by size exclusion chromatography (SEC) and purified. The degree of polymerisation (DP) and the chemical structure of the oligomers were determined from the time of elution in the SEC-separation, in addition to proton nuclear magnetic resonance (1H-NMR)-spectroscopy and liquid chromatography mass spectrometry (LC-MS). A protocol for the preparation of di-substituted hybrids by reductive amination was developed and optimised for chitin oligomers of DP = 3, trimers (A2M) in a reaction mixture of MeOH and AcOH. A solubility test of the reducing agent, 2-picoline borane (pic-BH3) in the reaction mixture was conducted prior to the initial reductive amination. A reaction mixture of 50 mg/mL oligomers and 85 vol % MeOH showed complete dissolution of pic-BH3. Purified trimers were conjugated to ADH by reductive amination in a reaction mixture of 85 vol % MeOH and AcOH added to a final concentration of 10 mM. Di-substituted hybrids with trimers were confirmed to have been formed in the reaction by 1H-NMR and LC-MS analysis. Formation of by-products and a background reduction of the trimers were additionally observed in the reaction. The yield of the di-substituted hybrids was increased by increasing the AcOH-concentration in the reaction mixture and by adding pic-BH3 at a later stage of the reaction. By-products formed in the reaction were proposed to be a result of impurities in the starting material and conversion/degradation of the di-substituted hybrids after formation. Acid hydrolysis of the di-substituted hybrids was suggested in the reaction mixtures with high AcOH-concentrations (≥100 mM). The kinetics of the di-substituted hybrid formation was studied and a nearly complete formation of di-substituted hybrids was observed after a reaction time of 6 hours. The formation of stable di-substituted hybrids without the addition of the reducing agent was not obtained under the conditions used. Preparation of di-substituted hybrids with chitin oligomers of DP = 6, hexamers (A5M) was achieved by applying the reaction conditions optimised for the preparation of di-substituted hybrids with trimers. The hexamers showed low solubility in the reaction mixture with high MeOH-concentration (85 vol %) but complete dissolution of neither the oligomer nor pic-BH3 was necessary to obtain di-substituted hybrids. Precipitation of products in the buffer used for the SEC-separation, and the low yield of di-substituted hybrids with hexamers obtained by upscaled production, proposed low solubility of di-substituted hybrids of longer chain length. The solubility of the hybrids was observed to reflect the solubility of the fully N-acetylated oligomers of corresponding chain length. Formation of by-products and background reduction of the starting material suggested sub-optimal reaction conditions for the di-substituted hybrid formation. However, formation of di-substituted hybrids of the type AnM-ADH-MAn with trimers and hexamers was successfully accomplished during the course of this master thesis. The developed procedure may be applied to establish a library of di-substituted hybrids with oligomers obtained from chitosan, and the hybrids may further be screened for intrinsic self-assembling properties for potential application in the development of polysaccharide origami .