Chemoenzymatic Synthesis Of Homogeneous Chondroitin And Its Derivatives

As an important organic compound and one of the three important biological macromolecules,carbohydrate exists in almost all living organisms.It not only acts as structural components,energy donors and signal-transmitting substances in life processes,but also has been widely used in R&D fields of clinical medicine,novel drug development,daily chemicals,etc.Chondroitin sulfate(CS)is a widely distributed and extremely important sulfated glycosaminoglycan composed of[-4GlcA?1-3GalNAc?1]disaccharide repeating units.Owing to its diverse sulfation patterns,CS can be divided into several sub-types,encoding huge biological information and exhibiting dramatic biological functions.Unsulfated CS(abbreved as CH)was also exhibited on the cell surface of several pathogens,such as Pasteurella multocida Type F,Escherichia coli K4 and Caenorhabditis elegans,etc.,presubmed to be important for the infection processes.CS-A with different molecular weights and N-acetylation degree play vital roles in T cell activity of murine splenocytes.N-sulfonated CH polysaccharides exhibit better complement inhibitory activities than CS polysaccharides,and fucosylated chondroitin sulfate(FucCS)has potential anticoagulant and antithrombotic biological activities.Current commercial available CS products that produced by mainly extracted from animal tissues have rather broad molecular weights and diverse sulfation patterns and are facing with growing safety concerns.Recently,several in vitro synthetic strategies have been developed including chemical methods,enzymatic methods,chemical enzyme methods,and solid-phase synthesis.However,it is still a challenge to obtain homogeneous CH polysaccharides to meet the need for biological studies.In the second chapter,we developed a novel homogenous CH synthetic strategy that combined step-wise CH oligosaccharides enzymatic synthesis and one-pot synchronized CH chain polymerization.By using GalNAc?-N3 as starting acceptor,CH oligosaccharides GlcA?1-3GalNAc?-N3(CH2P-N3,32 mg,86%)and GalNAc?1-4GlcA?1-3GalNAc?-N3(CH3P-N3,27 mg,90%)were successfully generated in PmCS-catalyzed step-wise synthetic manner.Subsequently,9 kinds of CH polysaccharides(10.8-22.5 mg with average yields of 58-84%)were formed in PmCS-catalyzed one-pot synthetic system by well-controlling acceptor/donor molar ratios,respectively.Further analysis results showed.that 3 kinds of CH polysaccharides synthesized using CH3?-N3 as acceptor had rather narrow molecular weight distribution and excellent PDI values(ranging from 1.05-1.29),which could be regarded as homogeneous polysaccharides.Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions.The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution.More importantly,owing to substrate promiscuous of PmCS,the strategy was further expanded to synthesize 5 kinds of CH polymers by incorporating sugar nucleotide derivatives UDP-GalNTFA into the synthetic system.Homogeneous CH derivatives 5(10 mg,31%)and 6(7 mg,47%)containing different proportions of[-4GlcA?1-3GalNTFA?1-]repeating unit were obtained by adding different proportions of sugar-nucleotide derivatives UDP-GalNTFA to synthesis system.Zwitterionic polysaccharides,CH derivatives 7(6 mg,64%)and 8(4 mg,73%)containing different repeating units[-4GlcA?1-3GalNH2?1-]were efficiently prepared by treated with NaOH solution.Subsequently,homogeneous N-sulfonated CH polymer derivative(4 mg,67%)was formed by treated with Py·SO3.This universal synthetic strategy can also be applied for the synthesis of homogenous CH derivatives and provide more resources for glycobiology research.In the third chapter,we applied fucosyltransferase(FucT)to in vitro synthesis of fucosylated chondroitin sulfates(FucCS).Substrate specificity of Hp?l,3FucT and Hm?1,2FucT toward CH oligosaccharides acceptors synthesized by step-wise enzymatic method were well studies in parallel reaction system.Hp?1,3FucT exhibited novel catalyze activity that could recognize CH oligosaccharides containing GalNAc?1-4GlcA sequence as the acceptor to generate fucosylated CH oligosaccharide.GalNAc?1-4(Fucal-3)GlcA?l-3GalNAc?-N3(6 mg,46%)were further synthesized by using GalNAc?1-4GlcA?1-3GalNAc?-N3 as acceptor and well characterized by ESI-MS and NMR,respectively.The results demonstrated that Hp?l,3FucT could transfer fucose from GDP-Fuc to 3-O position of GlcA motif in the acceptor to form branched ?1-3 fucosylated modification.The above results proved the possibility for in vitro enzymatic synthesis of FucCS,and the establishment of in vitro enzymatic synthesis methods will provide more structural species of FucCS.