| As important biological macromolecules in organisms,carbohydrates play essential roles in many important physiological and pathological processes.Because of the micro-heterogeneity of carbohydrates in nature,it is extremely challenging to obtain homogeneous and structurally well-defined carbohydrates from natural source.To tackle these difficulties,various strategies have been developed for the synthesis of oligosaccharides.Chemical synthesis of oligosaccharides requires multiple tedious protection-deprotection manipulations;as an alternative or complementary approach to chemical synthesis,enzymatic glycosylation has many advantages,such as high stereo-and regioselectively,without the need of protecting group manipulation on acceptor and donor,mild reaction conditions.Despite these advantages,the reported enzymatic synthesis approaches still suffer some difficulties,including the source of enzyme,complex reaction system,labor-intensive and time-consuming purification steps.To resolve the aforementioned problems,this study mainly focused on the development of two efficient,convenient and general liquid-phase enzymatic synthesis/solid-phase "capture-release" separation and purification strategies for the rapid synthesis and purification of various oligosaccharides.Both strategies have been successfully applied for the preparation of various biologically important complex oligosaccharides.The first method is based on a highly efficient and convenient thiol-tag assisted liquid-phase enzymatic synthesis and solid-phase "capture-release" purification strategy.For this strategy,a thiol-tagged glycosyl acceptor was extended by liquid-phase enzymatic synthesis.Then,the thiol tag facilitates solid-phase workup by reversible immobilization of the glycosylation products onto the Thiopropyl Sepharose 6B resin via a disulfide bond.The products were released from the resin by eluting with a solution of reducing agent of dithiothreitol(DTT).This part mainly contains the following contents:(1)Chemical synthesis of two thiol-tagged lactoside receptorsStarting from peracetylated lactose,two thiol-tagged lactoside receptors ?-1 and ?-2 were prepared by chemical synthesis.(2)Study on the thiol-tag assisted "capture-release" purification strategyWe firstly evaluated the compatibility of the Sepharose-immobilized thiol-tagged lactoside acceptors ?-1 and ?-2 using 5 different glycosyltransferases.Unsurprisingly,the receptors were not recognized or only showed low transformation rates(<50%)for the Thiopropyl Sepharose 6B resin immobilized acceptors.In contrast,both non-immobilized acceptors ?-1 and ?-2 were well recognized by all 5 tested glycosyltransferases under the liquid-phase enzymatic synthesis.The resulted products with a thiol tag could be quickly and efficiently immobilized to the Thiopropyl Sepharose 6B resin and purified by the solid-phase"capture-release" strategy.(3)Thiol-tag assisted "capture-release" strategy for the enzymatic synthesis of oligosaccharidesThe thiol-tagged lactosides ?-1 and ?-2 were extended by liquid-phase enzymatic synthesis and purified by solid-phase "capture-release".By sequential glycosylation using 10 enzyme modules for introducing Gal,GalNAc,Fuc,GlcNAc and sialic acid residues for chain elongation,various oligosaccharides including human ABO blood group antigens,ganglioside GM1,poly-LacNAc glycans and its sialyl and fucosyl derivatives were rapidly obtained.(4)One-step removal of the anomeric thiol tagsThe anomeric thiol groups in ?-10 and ?-25 were rapidly removed by treatment with N-bromosuccinimide in a mixture solvent to yield free oligosaccharides.The second method is based on fluorous-tag assisted liquid-phase enzymatic synthesis and solid-phase "capture-release" purification strategy.Firstly,Fluorous-tagged lactoside receptors were extended by liquid-phase enzymatic synthesis.The fluorous-tagged glycosylation products were selectively captured by fluorous silica gel.After removal the non-fluorinated impurities such as sugar nucleotides and salts,the products released from the resin could be directly applied for the next round enzymatic glycosylation.This part mainly contains the following contents:(1)Chemical synthesis of fluorous-tag bearing lactoside receptorsTo obtain fluorous tags with good solubility in water,two single-chain fluorous tags ?-1,?-2 and a double-chain fluorous tag ?-3 were chemically synthesized.The chemical glycosylation of fluorous tags ?-1,?-2 and ?-3 with glycosyl donor ?-26,single-chain fluorous-tagged lactoside ?-16,?-17 and double-chain fluorous-tagged ?-18 were obtained.(2)Study on the fluorous-tag assisted "capture-release" purification strategyTo study the influence of fluorine content under fluorous-tags assisted capture-release purification step,three kinds of fluorous tags were evaluated for the enzymatic synthesis of oligosaccharides.Unsurprisingly,when the receptors ?-16 and ?-17 were extended to pentasaccharide(?-16 as the receptor)or hexasaccharide(?-17 as the receptor)using liquid-phase enzymatic synthesis,the non-fluorinated impurities and fluorous-tagged products could not be achieved separation by solid-phase purification strategy.Fortunately,?-18 receptors were extended the oligosaccharides chain,non-fluorinated impurities and products could be efficiently separated.(3)Fluorous-tag assisted enzymatic synthesis of complex oligosaccharidesThe ?-18 receptor was extended by liquid-phase enzymatic synthesis/fluorous-tags assisted solid-phase "capture-release" purification strategy.By sequential glycosylation with 15 enzyme modules for introducing Gal,GalNAc,Fuc,GlcNAc and sialic acid residues respectively,a series of complex oligosaccharides were obtained,such as ABH blood group antigen and its derivatives,ganglioside GM1 and its derivatives,poly-LacNAc,Lewis antigen and its derivatives,Globo H antigen and its derivatives.(4)Removal and regeneration of fluorous tagsEfficient removal of the fluorous tags was realized under the common catalytic hydrogenation condition to obtain free glycans.The benzyl-type fluorous tags might be regenerated in two steps including simple oxidation and reduction reaction.The recycling and reuse of fluorous tags could cut the cost and provide an atom economical synthetic approach.The novelties of this study are listed below:1.In this study,two novel strategies based on liquid-phase enzymatic synthesis and solid-phase purification were developed.These strategies are compatible with various glycosyltransferases,which showed high efficiency of enzymatic synthesis and solid-phase purification.2.The sequential liquid-phase glycosylation coupled with solid-phase purification provides easy access to various complex oligosaccharides such as poly-LacNAc,human ABH blood group antigen,Lewis antigen and its sialic acid derivatives,GM1 and its derivatives,Globo H and its derivatives.3.The anomeric thiol tag can be easily removed in one simple step chemical reaction to afford free oligosaccharides.The fluorous tags could be regenerated in two steps and the regenerated fluorous tags can be reused,which greatly cut the cost and provides an atom economical synthetic approach.4.The thiol-tagged oligosaccharides could be valuable tools for the synthesis of glycopeptides through sulfide formation,which can enhance the stability of glycopeptides. |
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