Sequential One-pot Multienzyme Synthesis Of Heterogeneous And Homogeneous Chondroitin And Its Derivatives

Glycosaminoglycans(GAGs),also known as mucopolysaccharides,are negatively charged polysaccharides composed of repeating disaccharide units that exhibit numerous biological roles determined by polysaccharide structures.Owing to important functions in physiological and pathological events,great efforts have been devoted to achieving illustration of structural mechanisms behind diverse biological roles.Based on histocompatibility and biological safety of GAGs,it has been widely used in R&D fields of drug development,clinical medicine and daily cosmetics,etc.Chondroitin sulfate(CS)is one of widely distributed GAGs composed of[-4GlcA?1-3GalNAc?1-]repeating disaccharide unit with various sulfation patterns,leading to structural diversities and functional complexities.CS can be divided into several sub-types according to its diverse sulfation patterns,encoding a variety of biological information and participating in different life processes.Non-sulfated chondroitin(CH)polysaccharide also acts as a structural component of some pathogenic bacteria capsular polysaccharides,probably helping to escape immune response in the infection processes.More importantly,CH has been documented to selectively down-regulate expression level of pro-inflammatory cytokines.It has been well documented that biological activites of CS are corrected with molecular weight and sulfation patterns on sugar chains.High molecular weight CS can be applied to treat inflammation clinically while low molecular weight CS exhibits pro-inflammatory characteristics.In addition to acting as structural component of extracellular matrix,CS also plays an important biological role in the development of infections and tumors,cell signal transduction and immune regulation.Current commercial CS products predominantly isolated from animal tissues,are facing growing safety concerns.Efficient acquisition of CS polysaccharides with well-defined structure is one of the key scientific challenge to comprehensively elaborate biological functions.and develop new utilization.In past 10 years,numerous synthetic strategies have been developed,such as chemical methods,enzyme methods,chemoenzymatic methods,solid-phase synthesis and microbial fermentation,etc.However,the synthesis of CS polymers economically and efficiently still has many challenges such as lengthy steps and complicated purification operations.In this thesis,synthetic methodology was investigate the challenge facing CH polysaccharides preparation.One-pot Multienzyme(OPME)synthetic strategy was optimized and applied to synthesis of CH fragments in vitro by combining sugar nucleotides in situ generation and CH sugar chain polymerization.It could efficiently convert cheap starting monosaccharides into corresponding polysaccharides by enzymatic cascade catalyzed reaction without expensive sugar nucleotides.Further we explored the heterogeneous CH synthetic reaction via hyaluronidase-assisted glycan remodeling process to provide a feasible approach for the generation of homogenous CH fragments.Exogenous CH tetrasaccharide was added to reaction system as the necessary acceptor to trigger PmCS-catalyzed homogenous CH chain elongation in a synchronous manner.In the second chapter,we established a convenient in vitro sequential OPME synthetic strategy that combined in situ sugar nucleotides generation and PmCS-catalyzed CH chain polymerization together.In this strategy,two kinds of monosaccharides,GalNAc and GlcA,were used as cheap starting monosaccharides to convert into corresponding sugar nucleotides UDP-GalNAc and UDP-GlcA,respectively.Once sugar nucleotides were accumulated to comparable yields,PmCS(Chondroitin synthase from Pasteurella multocida)was added to catalyze the polymerization of heterogeneous CH polymers(180 mg,45%)without consumption of exogenous sugar nucleotide donors.Taking advantage of widely substrate promiscuity of prokaryotic enzymes,heterogeneous Azido-CH(167 mg,38%)and TFA-CH(154 mg,34%)derivatives were further synthesized by incorporation of GalNAc derivatives to generate CH derivatives.N-trifluoroacetyl galactosamine(GalNTFA)could be efficiently converted to galactosamine(GalNH2)under alkaline condition,and NH2-CH derivatives(20 mg,91%)were further prepared.Cross-linked CH hydrogel was generated via copper-catalyzed azide-alkyne cycloaddition chemistry reaction.Different from the smooth and sheet-like structure of native CH fragments,cross-linked CH hydrogel exihibted a highly-porous structure and a larger specific surface area.Formed CH hydrogel has broad application prospects in drug sustained release,3D cell culture and long-term arthritis treatment drugs.In the third chapter,sequential OPME glycan remodeling synthetic strategy was explored to generate homogeneous CH polymers to meet the urgent needs for biological studies,CH tetrasaccharide[-4GlcA?1-3GalNAc?1-]2(50 mg,33%)digested from heterogeneous CH fragments was added to reaction system as the necessary acceptor to trigger PmCS-catalyzed homogenous CH chain elongation in a synchronous manner.By controlling theoretical molar ratios of acceptor/donor and reaction conditions,homogeneous CH fragments(36.3?49.2 mg,30?41%)were synthesized with any desired chain length.The result indicated that the low-cost and efficient hyaluronidase-assisted glycan remodeling approach developed in this study was suitable to large scale synthesis of homogeneous CH fragments.Similary,a library of homogeneous Azido-CH(7.6?23.2 mg,6?17%)and TFA-CH(16.5?29 mg,12?21%)derivatives with defined chain length were further generated.The novel synthetic strategy exhibited a well-controlled relationship between the product CH chain length and the molar ratios of reaction substrates.The strategy will guid in vitro synthesis of any desired uniform CH fragments and other GAGs polysaccharides,which can promote the development of glycobiology and structure-activity relationships of complex glycans.