Towards block polysaccharides: Terminal activation of chitin and chitosan oligosaccharides by dioxyamines and dihydrazides and the preparation of block structures
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Polysaccharides are highly abundant and due to the large variation in chemical compositions, they possess a range of intrinsic properties, biological functions, and industrial applications. In the context of block copolymers, polysaccharide-containing structures are attracting increasing interest since they also serve as more sustainable alternatives to copolymers exclusively composed of synthetic polymers. Block polysaccharides represent a new class of engineered block polymers, exclusively composed of terminally linked oligo- or polysaccharides. Terminal coupling of blocks will, in contrast to the traditional lateral substitution, retain the intrinsic polysaccharide properties, as it does not perturbate the chain structure. Such block polysaccharides can be relevant for a wide range of applications in e.g. the biomedical and (bio)material fields. Chitin is the second most abundant polysaccharide found in nature after cellulose and is the major structural component of the exoskeleton of crustaceans and insects. Chitin is a water-insoluble high molecular weight unbranched homopolysaccharide composed of β-1,4-linked N-acetylglucosamine (GlcNAc, A) residues, whereas its de-N-acetylated derivative, chitosan, has high water-solubility at acidic pH due to the positively charged amino groups (pKa approx. 6.5) of the glucosamine (GlcN, D) residues. Chitin and chitosan are particularly interesting in the context of block polysaccharides due to their high abundance, biocompatibility, biodegradability, self-assembling properties (chitin) and positive charge (chitosan). In the work of this thesis the terminal activation of chitin and chitosan oligomers by a dioxyamine (O,O’-1,3-propanediylbishydroxylamine, PDHA) and a dihydrazide (adipic acid dihydrazide, ADH) using reductive amination with α-picoline borane (PB) as the reductant was studied in detail. In Paper I and Paper II the chemistry and kinetics of the reducing end activation of chitooligosaccharides (CHOS) were investigated. A simple pseudo first order model was introduced to obtain kinetic data which enable simulation of reactions under different conditions as a tool to develop preparative protocols. Activated CHOS were also purified and thoroughly characterised. In contrast to other “click” reagents, oxyamines and hydrazides can react directly with the reducing end aldehyde of carbohydrates without an intermediate reaction step. PDHA and ADH can therefore serve as linkers between two polysaccharide blocks for the preparation of AB-type antiparallel block structures. The attachment of a second block to the free end of the linkers was therefore studied in detail in Paper II. Preparation of parallel block polysaccharides or more complex block structures (e.g. ABC-type) additionally require functionalisation of the non-reducing end (NRE). Only a few and highly polysaccharide specific NRE functionalisation methods are available. Chitin can, however, be selectively oxidised by periodate to obtain a dialdehyde in the NRE residue. The aldehydes can subsequently be activated by PDHA or ADH which enable conjugation of the reducing end of other polysaccharides to the NRE of chitin. Periodate oxidation and subsequent activation chitin oligomers and the preparation of water-soluble chitinbased block polymers by end-coupling of chitin oligomers were studied in Paper III. DnXA oligomers (X = D or A), prepared by enzymatic degradation of chitosan using chitinase B, and AnM or DnM oligomers (M = 2,5-anhydro-D-mannose), prepared by nitrous acid depolymerisation of chitosan, were used in this study. First, activation (amination) of the reducing end by PDHA and ADH was studied. DnXA oligomers were shown to have very low reactivity towards the linkers as compared to glucose (Glc) terminated oligomers (obtained from dextran or β-1,3-glucan), but a notably higher reactivity compared to CHOS terminating in D residues. The yields and type of conjugates (Schiff bases and/or N-glycosides) formed with DnXA were highly linker dependent, whereas the kinetics was highly pH dependent (in the pH range 3.0-5.0). The best compromise between yield and rates was obtained at pH 4.0. AnM and DnM oligomers were in contrast highly reactive towards PDHA and ADH at all pH values and, due to the pending aldehyde of the M residue, only Schiff bases (oximes and hydrazones, respectively) were formed in high yields. The kinetics was also independent of the fraction of acetylated residues (FA) of the oligomers. PB was introduced as the reducing agent for the reductive amination reactions. This reducing agent has lower HSE concerns compared to sodium cyanoborohydride (NaCNBH3) and has proven efficient for reductive amination reactions with carbohydrates. Unreacted AnM and DnM were rapidly reduced by both PB and NaCNBH3 and the reductive amination of these oligomers therefore needs to be performed in two consecutive steps. The reduction of AnM- or DnM-based conjugates formed with PDHA and ADH was fast compared to conjugates formed with the other oligomers and clearly fastest at low pH (3.0). Moreover, hydrazones were faster reduced than oximes. Unreacted DnXA oligomers and oligomers terminating in Glc residues were in contrast essentially stable towards the reducing agents. Reductive amination of these oligomers can therefore be performed as conventional one pot. The one pot reductive amination reactions with DnXA oligomers were however, limited by extremely slow kinetics, especially with ADH. Hence, the kinetics of these reactions needs to be improved by high concentrations of linkers and PB, higher temperatures and longer reaction times. The reduction of DnXA-based conjugates by NaCNBH3 yielded a different and unidentified type of conjugates, and this reducing agent is therefore not applicable for these oligomers. The preparation of chitin- and chitosan-based diblock structures was demonstrated by attaching a second oligomer block to the free end of the linkers. An increased reactivity for the free end of both linkers was observed after activation of the first block, which facilitates the use of PDHA and ADH for the preparation of block polysaccharides. To exploit the particularly high reactivity of the M residues for the preparation of diblock structures, AnM and DnM oligomers can be used as the second block. However, to prevent the rapid reduction of unreacted oligomers, an excess of the activated block is recommended to obtain high amination yields prior to reduction. Due to the low reactivity observed for CHOS terminating in A or D residues, such oligomers (e.g. DnXA) should on the other hand be used as the first block for the preparation of block structures. To improve the kinetics of the diblock formation, an excess of the activated block is recommended, also for these preparative protocols. However, this approach requires subsequent purification of the diblocks, and even though gel filtration chromatography was proven useful for the shorter blocks used in this study, other purification methods need to be considered. A pH dependent degradation of the M residue was observed for the DnM oligomers during isolation and purification, but activation of the oligomers prior to isolation preserved the M residue. De-N-acetylation of AnM-based diblocks was also shown to be an alternative approach for the preparation of chitosan-based diblock structures. The low water-solubility of chitin limits its applications. The preparation of water-soluble chitin-based diblocks was therefore attempted by conjugating water-insoluble AnM oligomers to ADH activated dextran which is highly water-soluble. The results were inconclusive but suggested that the high reactivity of the M residue promotes conjugation to the free end of ADH without the dextran block being able to increase the water-solubility of chitin block. However, very recent (unpublished) results suggest that water-insoluble AnM can react with PDHA in DMAc/LiCl. Hence, this solvent can serve as an alternative for the preparation of chitin-based diblock structures with longer water-insoluble chitin blocks. The vicinal diol in the NRE residue of AnM oligomers can be selectively oxidised by periodate to form a dialdehyde. The subsequent activation of oxidised AnM by PDHA and ADH revealed a high reactivity of both these aldehydes towards the linkers and the reactivity was even higher than for the pending aldehyde of the M residue. Such oxidised and activated AnM oligomers can serve as precursors for more complex block polysaccharides (e.g. ABA- or ABC-type). By reacting oxidised AnM oligomers with a substoichiometric amount of PDHA, a discrete distribution of ‘polymerised’ oligomers was formed. These chitin-based block polysaccharides were, in contrast to chitins of the same length, water-soluble.
Består avPaper 1: Mo, Ingrid Vikøren; Feng, Yiming; Dalheim, Marianne Øksnes; Solberg, Amalie; Aachmann, Finn Lillelund; Schatz, Christophe; Christensen, Bjørn E.. Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides. Carbohydrate Polymers 2020 ;Volum 232. s. 1-10
Paper 2: Mo, Ingrid Vikøren; Dalheim, Marianne Øksnes; Aachmann, Finn Lillelund; Schatz, Christophe; Christensen, Bjørn E.. 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides. Biomacromolecules 2020 ;Volum 21.(7) s. 2884-2895
Paper 3: Mo, Ingrid Vikøren; Schatz, Christophe; Christensen, Bjørn E. (2021). Functionalisation of the non-reducing end of chitin: A new approach to form complex block polysaccharides and soluble chitin-based block polymers. Carbohydrate Polymers; Volume 267 ©2021. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/