Chemoenzymatic Synthesis Of Homogeneous Hyaluronan Fragments And Their Derivatives

Sugar,also known as carbohydrate,is one of the most widely distributed organic compounds in nature.Glycans and glycoconjugates play indispensable roles in the bioprocesses of all lives.Activated form of corresponding monosaccharides,sugar nucleotides are necessary glycosyl donors of Leloir type glycosyltransferases for the synthesis of complex oligosaccharides or glycoconjugates.Consisting of a monosaccharide and a nucleoside mono-or diphosphate moiety,they are involved in regular metabolism of organisms and the occurrence and development of diseases.At present,a series of strategies have been developed to synthesize sugar nucleotides and their derivatives,such as chemical,enzymatic or chemoenzymatic methods.However,low yield,complicated process and consumption of chemical reagents limit the scaleup of these methods.Commercial sugar nucleotides are expensive,which seriously hinders the biosynthesis of complex glycans and the development of glycobiology.With recent development in bioinformatics,it is possible to mimic the biosynthetic process in vivo and enzymatically produce sugar nucleotides in vitro.The elucidation of de novo synthetic pathways and salvage pathway has made it economically feasible for large-scale production of sugar nucleotides from cheap monosaccharide.However,there are some limitations in current enzymatic synthesis routes,including relatively low substrate concentration,tedious purification process and rather low efficiency.A pratical strategy for large-scale preparation of sugar nucleotides is still urgently needed.To promote applications of environment-friendly enzymatic reactions in the synthesis of glycans and glycoconjugates,we developed a practical and economical strategy for multi-gram scale production of sugar nucleotides in Chapter 2.By optimizing the substrate concentration and co-factors of enzymatic synthesis system,highlyconcentrated multi-enzyme sugar nucleotides cascade synthetic system was explored,and synthetic reaction could be conducted in a test tube with around 40 times improved space-time yield comparing with previously reported 5 mM reaction system.By analyzing the physical and chemical properties of substrates and products,a chromatography-free ion-precipitation purification strategy was also developed.Employing our new highly-concentrated multi-enzyme cascade synthetic system and robust chromatographic-free purification strategy,12 sugar nucleotides and their derivatives were generated from up to 200 mM of cheap and readily available monosaccharides in a 20 mL reaction system.More importantly,up to 2.35 g of product(up to 98%pure by HPLC)could be obtained by selective precipitation.The whole operation could be completed within one week,which greatly improves the efficiency of the preparation of sugar nucleotides and completely solves the bottleneck of enzymatic synthesis of complex oligosaccharides.Subsequently,the synthesized sugar nucleotides were verified as glycosyl donors to generate tumor-associated carbohydrate antigen,Globo H and its trifluoroacetyl modified derivative via in vitro glycosyltransferase-catalyzed stepwise reactions.Compared with sugar nucleotides regeneration system,the use of purified sugar nucleotides could improve the yield of Globo H effectively and simplify the purification process.This strategy could not only realize the large-scale preparation of sugar nucleotides economically and efficiently,but also promote the application and development of sugar nucleotide-dependent glycosyltransferases.Glycosaminoglycans(GAGs)are complex linear hetero-polysaccharides,composed of disaccharide repeating units and modified by different degrees of sulfation and isomerization.GAGs widely exist on the surface of almost all higher animal cells and play an important role in maintaining the structural integrity of cells and tissues.Proteoglycans(PGs)are predominantly dependent on covalently linked GAGs,which mediate an array of biological functions.Hyaluronan(HA)is a released,unbranched,linear polymers composed of repeating disaccharide of 1,4-linked N-acetyl-β-Dglucosamine(β-D-GlcNAc)and 1,3-linked β-D-glucuronic acid(β-D-GlcA)units with molecular weight up to 6 million Daltons.Owing to its excellent water retention,lubrication,viscoelasticity and histocompatibility,it has been widely used in ophthalmic surgery,postoperative adhesion prevention,scald treatment,arthritis treatment,cosmetology and cosmetics.HA hydrogel prepared form chemical crosslinking of its carboxyl and acetyl amino groups among sugar chain was also designed and applied in the fields of drug release and tissue engineering.Besides acting as an important component of cell extracellular matrix,HA is also involved in the occurrence and development of tumors and has been regarded as one of the important markers of tumor prognosis.There are two major methods for commercial production of HA,animal tissue extraction and microbial fermentation.These two methods are still facing problems such as cross-contamination,residual toxins,and difficulty in controlling the length of sugar chains.With the development of molecular biology,in vitro enzymatic synthesis of HA has gradually become a safe and effective alternative method.Based on the sequential one-pot multienzyme(OPME)system,we developed a novel HA in vitro synthetic strategy in Chapter 3 of the thesis.The strategy that combined sugar nucleotide in situ generation and HA chain polymerization could convert cheaper starting monosaccharide into corresponding sugar nucleotides through multi-step enzyme-catalyzed reaction and subsequently acted as glycosyl donor for PmHAS catalyzed HA chain polymerization without further purification.As HA was composed of repeating disaccharide units,to assembly HA chain in vitro,the first issue was to generate equal amounts of both sugar nucleotides in a one-pot system.In order to improve the synthesis efficiency,reaction system was optimized to realize the synthesis of UDP-GlcNAc and UDP-GlcA with the same conversion rates.This strategy provided an economic and efficient method for the large-scale preparation of HA,with more than 70%yield.Owing to the substrate flexibility of prokaryotic enzymes,the OPME strategy was further explored to synthesize partially labeled HA derivatives.HA hydrogels were prepared using Azido-alkyne click reaction with intra-and intermolecular cross-linking.Scanning electron microscopy(SEM)showed that the surface of cross-linked HA was loose and porous,which was quite different from the smooth and uniform sheet structure of natural HA polymer.The preparation of HA hydrogel provides potential applications for drug delivery and 3D cell culture.Despite its simple chemical structure,nature HA has a rather broad molecular weight distribution.In tumor microenvironment,HA with different lengths exhibit conflicting biological functions.High molecular weight HA has inhibitory antiangiogenic and anti-inflammatory properties.Whereas,low molecular weight HA,especially HA oligosaccharides,appears to be an endogenous "danger signal" that promotes proliferation and inflammation.Access to structurally well-defined homogeneous HA oligosaccharides and polysaccharides is a prerequisite for the systematic elucidation of the structure-function relationship of HA and its biological information.PmHAS from Pasteurella multocida,is the only known type Ⅱ hyaluronan synthase to date.It is a bifunctional enzyme with two catalytic domains,the N-terminal GlcNAc transferase(GN-T)and the C-terminal GlcA transferase(GA-T).The bifunctional activity of PmHAS was resolved by site directed mutagenesis to obtain two mutant enzymes(GN-T and GA-T)with a single catalytic activity respectively.In our previous studies,there are initial rate-limiting steps existing in PmHAS-catalyzed nascent HA polymerization,thus leading to heterogeneous characteristics of formed HA chain.To generate well-defined HA fragments,chapters 4 and 5 of this thesis were focused on developing chemoenzymatic synthetic methodologies to produce a series of chemically well-defined HA oligosaccharides,HA hybrid oligosaccharides and their derivatives for evaluation of their biological function.In Chapter 4,we developed a chemoenzymatic remodeling synthesis strategy to generate HA oligosaccharide fragments.By using conveniently available exogenous HA tetrasaccharide as starting glycosyl acceptor,a "one-cycle-one-disaccharide-unit"strategy for structure-defined synthesis of HA oligosaccharide fragments was established by alternating addition of GN-T and GA-T.Owing to repeating disaccharide units of HA chain,the synthesis route was further divided into odd-numbered and evennumbered oligosaccharides synthesis to achieve the efficient preparation of HA oligosaccharides.A series of photoactive fluorescent HA probes with well-defined chemical structures have been synthesized by derivatization of HA using bioorthogonal reactions including biotin-avidin and azido-alkyne to introduce photoactive and fluorescent groups.A metabolic assay of HA oligosaccharide fragments was developed on human non-small cell lung cancer cell line A549.The results demonstrated that the length of HA chain could affect the metabolism efficiency of exogenous glycans.Both HA oligo-and polysaccharides photoactive probes could enter cells and the fluorescent probe prepared from HA dodecasaccharide had the highest internalization efficiency.This provides a toolbox for the use of HA fluorescent probes in the fields of selective targeting,multi-analyte detection and imaging.The HA photoactive probe could further be applied as a ’bait’ in a pull-down experiments,to unearth HA binding proteins and receptors by proteomics.Chondroitin sulfate(CS)is one of the most abundant GAGs in mammals.The structure of unsulfated chondroitin(Chondroitin,CH)is similar to that of HA.CS is involved in key cellular regulatory processes.The CS side chains of chondroitin sulfate proteoglycans are involved in various interactions in extracellular matrix and have important biological implications in malignancies.CH is also a constituent of the capsular polysaccharides of some pathogenic bacteria.Chondroitin synthetase(PmCS),from Pasteurella multocida,is a bifunctional enzyme which is 90%similar with PmHAS at the nucleotide and amino acid levels.It was found that both recombinant PmHAS and PmCS can recognize oligosaccharide fragments such as HA tetrasaccharide and CS trisaccharide as acceptors for glycan extension with varied efficiencies.In Chapter 5,we established a modular enzymatic synthesis strategy to generate HA-CH chimeric oligosaccharides.Based on the structural similarity of HA and CH,parallel reactions were carried out to investigate substrate specificity of PmHAS,GNT,GA-T and PmCS.The resulst showed that PmCS can recognize UDP-GalNAc as glycosyl donor to transfer GalNAc residues to the nonreducing end of HA tetrasaccharide better than GN-T or PmHAS.Meanwhile,PmCS,GA-T and PmHAS exhibited comparable GlcA transferase activities towards GalNAc and GlcNAc residue in the nonreducing end as glycosyl acceptors.Modular synthesis of HA-CH chimeric oligosaccharides could be achieved by synthetic reaction catalyzed by PmCS/GN-T and GA-T in an alterantive manner.The synthetic efficiency was improved since one disaccharide unit could be elongated in one synthetic cycle without any purification.As a result,a series of structurally well-defined HA-CH chimeric fragments from 6 to 16 saccharides were generated,aiming to reveal structure-fuctional relationships of HA polysaccharides.