糖苷键专题.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,本文档所提供的信息仅供参考之用，不能作为科学依据，请勿模仿。文档如有不当之处，请联系本人或网站删除。,概述：基本概念,化学合成糖苷键的基本原理,donor,acceptor,糖基化反应中，,donor中间体,和,promoter,是亲电试剂（electrophile），,acceptor是亲核试剂（nucleophile）,一般而言，糖苷键对酸敏感，对碱不敏感。,Leaving group in glycosyl donor,Activator,Comments,L=,OAc,BF,3,Et,2,O,SnCl,4,TMSOTf,Not for complex oligosaccharides,L=,Br,AgCO,3,AgOTf,Hg(CN),2,HgBr,2,Most commonly used donor,L=,Cl,AgOTf,Hg(CN),2,HgBr,2,More stable than glycosyl bromide,L=,F,SnCl,2,-AgOTf,Can be combined with thioglycosides,L=OC(NH)CCl,3,BF,3,Et,2,O,TMSOTf,Mild reaction conditions,widely used,L=SR,TfOH-NIS,DMTST,IDCP,Can also serve as an acceptor in the absence of thiophilic reagents,Can also serve as an acceptor in the absence of bromine,L=S(=O)R,Tf,2,O,概述：基本概念,化学合成糖苷键的立体选择性,S,N,1 机制：anomeric effect，,a,-anomer as major product。,C-2 is non-participation group(2-OCH,2,Ph,2-N,3,2-deoxy),harsh condition（strong promoter，high temperature）,polar solvent,S,N,2 机制：anchimeric effect（C-2 functional group participation effect）,(-OAc,-NHAc,-I,-SR,-SeR),stable intermidiate,概述：基本概念,化学合成糖苷键的donor和promotor,（I）,三氯乙酰基亚胺酯和酸（BF,3,，TMSOTf）,强碱，利于,b,-TCA donor；弱碱，利于,a,-TCA donor。,概述：基本概念,化学合成糖苷键的donor和promotor,（I）,三氯乙酰基亚胺酯和酸（BF,3,，TMSOTf）（Schmidt）,S,N,2 like reaction;,Stereoselectivity(S,N,1,vs,S,N,2)is subject to solvent,temperature,promotor&acceptors.,BF,3,OEt,2,CH,2,Cl,2,TMSOTf,Et,2,O,概述：基本概念,化学合成糖苷键的donor和promotor,（II）,硫苷和DMTST,NBS,IDCP,M,n+,概述：基本概念,化学合成糖苷键的donor和promotor,（II）,硫苷和DMTST,NBS,IDCP,M,n+,概述：基本概念,化学合成糖苷键的donor和promotor,（II）,硫苷和DMTST,NBS,IDCP,M,n+,概述：基本概念,化学合成糖苷键的donor和promotor,（III）,溴苷和M,n+,（Koenigs-Knorr）,概述：基本概念,化学合成糖苷键的donor和promotor,（IV）,Epoxide donor,概述：基本概念,化学合成糖苷键的保护基策略:常用保护基,缩写，保护和脱保护条件，选择性，稳定性,概述：基本概念,化学合成糖苷键的保护基策略（I）,酰基保护基,Acylations can often be carried out as regioselective reactions to establish selective protection patterns.,In basic condition,acetyl group sometimes can shift.,概述：基本概念,化学合成糖苷键的保护基策略（II）,醚保护基,概述：基本概念,化学合成糖苷键的保护基策略（II）,醚保护基,概述：基本概念,化学合成糖苷键的保护基策略（III）,硅醚保护基,In acidic media,the relative stability is:,TMS(1)TES(64)TBS(20 000)TIPS(700 000)TBDPS(5 000 000),In basic media,the relative stability is:,TMS(1)TES(10-100)TBSTBDPS(20 000)TIPS(100 000),In basic condition,silyl ether sometimes can shift,A:benzylidene acetals,概述：基本概念,化学合成糖苷键的保护基策略（IV）,acetal,概述：基本概念,化学合成糖苷键的保护基策略（IV）,acetal,概述：基本概念,化学合成糖苷键的保护基策略（IV）,acetal,Several free monosaccharides can be converted by isopropylidenation,概述：基本概念,化学合成糖苷键的保护基策略（IV）,acetal,概述：基本概念,化学合成糖苷键:armed&disarmed,概述：基本概念,化学合成糖苷键:armed&disarmed,概述：基本概念,寡糖合成策略,固相合成法,One-pot synthesis,Peter H.Seeberger,Chi-Huey Wong,Samuel Danishefsky,Xuefei Huang,天然糖苷类抗生素,合成稀有糖,Activation-substitution reaction at the,carbohydrate ring,天然糖苷类抗生素,合成稀有糖,Tosylate as leaving group,天然糖苷类抗生素,合成稀有糖,Triflate as leaving group,天然糖苷类抗生素,合成稀有糖,Mesylate as leaving group,Intramolecular substitution,天然糖苷类抗生素,合成稀有糖,2.Deoxyhalo derivatives,Florination with DAST,(Diethylaminosulfur trifluoride),天然糖苷类抗生素,合成稀有糖,Selective florination,天然糖苷类抗生素,合成稀有糖,Other halogenation,天然糖苷类抗生素,合成稀有糖,3.Deoxygenation of carbohydrates derivatives,Opening of epoxides,天然糖苷类抗生素,合成稀有糖,Reduction of ester,Mechanism:,天然糖苷类抗生素,合成稀有糖,Examples:,Synthesis of methyl 2,4-di-,O,-benzoyl-6-deoxy-3-,O,-(1-imidazolylthiocarbonyl)-,a,-L-galactopyranoside,Synthesis of methyl 2,4-di-,O,-benzoyl-3,6-dideoxy-,a,-L-hexopyranoside,天然糖苷类抗生素,合成稀有糖,4.Epimerizations,Misunobu reaction:,Example:,天然糖苷类抗生素,合成稀有糖,Oxidation-reduction reaction,:,天然糖苷类抗生素,合成稀有糖,合成稀有糖,天然糖苷类抗生素,从非糖原料出发合成糖分子：,Samuel Danishefsky,George A.ODoherty,Peng G.Wang,Peng George Wang,Departments of Biochemistry&Chemistry,The Ohio State University,Carbohydrate Chemistry&Glycobiology,L9 Enzymatic Synthesis of Oligosaccharides and glycopeptides/glycoproteins,Comparison of Organic and Biosynthetic Approaches for Carbohydrate Synthesis,Organic Synthesis,Biosynthetic process,Derivative synthesis,Versatile,Not very flexible,Scale,Usually small scale,Can be Large scale,Regiochemistry,Hard to achieve,Good Stereo-and regioselectivity,Process,Expensive and multistep,Economic and short,Environment problem,Chemical waste,Environment-friendly,Carbohydrate polymer synthesis,Not applicable,Practical approach,Chemical Synthesis of,a,-Gal,High Cost,Multiple protection,deprotection steps,Tedious separation procedures,Synthesis of D-Galactoside Donor,Chemical Synthesis of,a,-Gal,Synthesis of Disaccharide Acceptor,Chemical Synthesis of,a,-Gal,Synthesis of,a,-Gal Trisaccharide,Chemical Synthesis of,a,-Gal,W.Zhang,J.Wang,J.Li,L.Yu,P.G.Wang,J.Carbohydrate Chemistry,199918(8),1009-1017,Enzymatic Synthesis of,a,-Gal,Enzymatic synthesis of oligosaccharides,Glycosyltransferase,Superbeads and superbug technologies,Glycosidase,Glycosynthase,Glycosyltransferase,Advantages:high regio-selectivity,the most practical method,Transfer sugar residues from an activated donor substrate to an acceptor,Donor:A sugar activated by a phosphate,a lipid phosphate,or a,nucleotide-usually a,nucleotide-sugar,Acceptor:a lipid,a protein,DNA,a small molecule acceptor,or a,growing oligosaccharide.,Typically grouped into families based on the type of sugar they transfer,eg.,Galactosyl,transferases,sialyltransferases,Glycosyltransferases are very specific enzymes,with only one particular,transferase being able to catalyze one particular glycosidic linkage,-,One EnzymeOne Linkage,Glycosyltransferase,Sugar Nucleotide,Nine Common Sugar Nucleotides,UDP-Glc,UDP-Gal,UDP-GlcNAc,UDP-GalNAc,UDP-GlcA,UDP-Xyl,GDP-Man,GDP-Fuc,CMP-NeuAc(the only monophosphate),Biosynthesis of Sugar Nucleotides,UDP-Glc,UDP-Gal,UDP-GlcA,UDP-Xyl,UDP-GlcNAc,UDP-GalNAc,GDP-Man and GDP-Fuc,CMP-Neu5Ac,a,1,3GalT Catalyzed Synthesis of,a,-Gal Epitopes and Their Derivatives,Fang J,Chen X,Wang PG,et al.:,J.Org.Chem,.1999,64,4089-4094.,H,O,O,O,H,H,O,H,O,O,U,D,P,H,O,O,O,H,H,O,H,O,O,U,D,P,O,H,O,O,H,H,O,O,O,O,H,H,O,R,1,R,2,H,O,O,H,O,O,H,H,O,H,O,O,H,O,O,H,O,H,O,O,O,O,H,H,O,R,1,R,2,UDP-Gal,4-epimerase,UDP,a,1,3GalT,R,1,R,2,OH,OH,OH,OH,NHAc,b,N,3,b,SPh,b,OAllyl,a,b,c,d,NHAc,OH,e,N,H,2,OH,f,UDP-Gal,100mg/$416,UDP-Glc,5g/$529.5,EC 5.1.3.2,Examples,Synthesis of,a,-Gal Pentasaccharide with,In Situ,Cofactor Regeneration,Fang J,Li J,Chen X,Wang PG,et al:,J.Am.Chem.Soc,.1998,120,6635-6638.,Examples,De Luca,C.;Wong,C.-H.,J.Am.Chem.Soc,.1995,117,5869-5870,One-pot multi-enzyme synthesis of a hyaluronic acid polymer involving glycosyltransferases and sugar nucleotide regeneration systems,E,1,hyaluronic acid synthase;,E,2,UDP-Glc dehydrogenase;,E,3,UDP-Glc pyrophosphorylase;,E,4,UDP-GlcNAc pyrophosphorylase;,E,5,pyruvate kinase;,E,6,lactate dehydrogenase;,E,7,inorganic pyrophosphatase,Examples,Cytels large-scale synthesis of sialyl Lewis X antigen,E1:,a,1,3-fucosyltransferase,E2:pyruvate kinase,E3:GDP-mannose pyrophosphorylase,E4:GDP-4-keto-5-deoxymannose 3,5-epimerase/GDP-4-keto-6-galactose reductase,E5:glucose dehydrogenase,E6:hexokinase,E7:phosphomannomutase,E8:,a,2,3-sialyltransferase,Examples,Neoses large-scale synthesis of sialyated lactose using a CMP-sialic acid synthetase/,a,2,3-sialyltransferse fusion enzyme with in situ regeneration of CMP-NeuAc,E1:CMP-NeuAc synthetase/,a,2,3-sialyltransferase fusion enzyme,E2:myokinase,E3:pyruvate kinase,E4:GlcNAc2-epimerase,E5:sialic acid aldolase,Examples,Kyowa Hakkos technology for large-scale production of UDP-Gal and globotriose utilizing metabolically engineered bacterial cells,Ppa:pyrophosphatase,GalU:glucose-1-phosphate uridylyltransferase,GalT:galactose-1-phosphate uridylyltransferase,GalK:galactokinase,LgtC:,a,1,4-galactosyltransferase,Examples,Examples,Synthesis of tumor-associated antigen,Globo-H hexasaccharide,Step,Enzyme(s),Product,Yield(%),1,LgtC,GalE,Gb3-OBn,90,2,LgtD-WbgU,Gb4-OBn,78,3,LgtD,Gb5-OBn,85,4,WbsJ,Globo-H-OBn,95,Su D.,Eguchi H.,Yi W.,Li L.,Wang PG.and Xia C.,Org.Lett.,2008,in press,Superbug and Superbead technologies for the synthesis of,a,-Gal,Bacteria,N,u,t,r,i,e,n,t,s,S,t,a,r,t,i,n,g,M,a,t,e,r,i,a,l,s,B,i,o,-,s,y,n,t,h,e,t,i,c,p,a,t,h,w,a,y,e,n,g,i,n,e,e,r,i,n,g,a,-Gal,Synthesis of oligosaccharides in biological system,OH,HO,S1,OH,HO,S2,+,O,OH,HO,S1,S2,+H,2,O,OH,O,Nucleotide,Donor,Acceptor,Glycosyltransferase,HO,S1,Sugar-Nucleotide,Biosynthesis,&Regeneration,HO,S1,O,OH,OH,HO,S2,HO,S1,S2,Nucleotide,*,Glycosylation in Eukaryotes,Sugar,Nucleotide,transport,transferase,UDP,UTP,UDP,UMP,UDP,UMP,Gal,P,Gal,UDP,Gal,Gal,Acceptor,Acceptor,Cytosol Golgi,Biosynthetic Pathway for UDP-Gal Regeneration and,a,-Gal Production,ATP,ADP,G,a,l,K,:,g,a,l,a,c,t,o,k,i,n,a,s,e,GalUT:galactose-1-phosphate uridylytransferase,G,a,l,U,:,g,l,u,c,o,s,e,-,1,-,p,h,o,s,p,h,a,t,e,u,r,i,d,y,l,y,l,t,r,a,n,s,f,e,r,a,s,e,P,y,k,F,:,p,y,r,u,v,a,t,e,k,i,n,a,s,e,a,1,3,G,a,l,T,:,a,1,3,-,g,a,l,a,c,t,o,s,y,l,t,r,a,n,s,f,e,r,a,s,e,a,1,3GalT,Gal,a,1,3Lac,L,a,c,t,o,s,e,UDP-Gal,UDP,PEP,Pyruvate,Gal-1-P,Glc-1-P,UDP-Glc,UTP,PykF,GalU,GalK,GalUT,Galactose,PPi,UDP-Gal Superbead,GalU,GalT,GalK,PyKF,Recombinant,E.coli,stratins overexpressing,GalK,GalT,GalU or PykF,1)Fermentation,2)Lysis,3)Add to Ni,2+,resin,Chen X,Fang J,Wang PG,et al.:,J.Am.Chem.Soc,.2001,123,2081-2082.,Beads with:,GalK,GalT,GalU,PykF,Peristaltic pump,for circulation,Reservoir with:,a,1,3GalT,LacOBn 9.6 mM,ATP 0.96 mM,PEP 19.2 mM,UDP 0.96 mM,Glc-1-P 0.96 mM,Gal 12 mM,MgCl,2,10 mM,MnCl,2,10 mM,KCl 100 mM,HEPES 100 mM,pH 7.5,Liu Z,Zhang J,Chen X,Wang PG:,ChemBioChem,2002,3,348-355.,Production of UDP-Gal with Superbeads.,Synthesis of oligosaccharides with UDP-Gal Superbead,a,Gram scale synthesis,others are 100 mg scales,a,-Gal synthesis using Superbug,a,1,3GalT,Gal,a,1,3Gal,b,1,4Glc,Gal,b,1,4Glc,UDP-Gal,UDP,PEP,Pyruvate,Gal-1-P,Glc-1-P,UDP-Glc,UTP,PykF,GalU,GalK,GalT,Galactose,Lactose,Glycolytic,pathway,Glucose,ATP,ADP,Glucose,Pyruvate,PykF,PEP,Glycolytic pathway,PPi,NM522,First Generation of,a,-Gal Superbug,Clone and express individual enzymes in the biosynthetic pathway of,a,-Gal,Construction an artificial biosynthetic gene cluster and transfer into,E.coli,host cell.,Large scale production of,a,-Gal oligosaccharides using fermented and permeated cells.,E,.,c,o,l,i,N,M,5,2,2,p,L,D,R,2,0,-,a,K,T,U,F,a,-,G,a,l,7.2 g Gal,1.1 g ATP,8.4 g Lac,3.6 g Glc,Total volume:1 L,0.3 g UDP-Glc,7.2 g,Chen X,Liu Z,Zhang W,Fang J,Andreana P,Wang PG:,ChemBioChem,.2002,3,47-53.,Large Scale Production of,a,-Gal Trisaccharide,Second Generation of,a,-Gal Superbug,PpK:polyphosphate kinase,Third Generation of,a,-Gal Superbug,Chen X,Zhang J,Kowal P,Andreana P,Wang PG:,J.Am.Chem.Soc,.2001,123,8866-8867.,SusA:sucrose synthase,GalE:UDP-Gal 4-epimerase,Application of Glycosyltransferases-Summary,Pros:,Excellent yield with complete regio-and stereoselectivity.,No protecting groups needed.,Sialyltransferases allow facile sialylation.(This is the hardest glycosylation to carry out by chemical methods.),Reactions can be carried out in water.,Cons:,Most requisite enzymes are not readily available,and those that are available are expensive.,Regio/stereoselectivity means that substrate scope is limited and a unique enzyme is needed for almost every reaction.,Nucleotide sugar donors are very expensive and/or unstable.,Scale is often limited by enzyme availability/volumetric productivity.(A large amount of enzyme produces only a small amount of sugar by weight.),Based on AA sequence similarity,Glycosidase are classified into,107,distinct families.,(,www.cazy.org/index.html,),Currently,37472,known or putative glycosidase in CAZy,433,different glycosidase crystal structures from,76,different families have been reported,Glycosidase and Transglycosidase,In vivo function:hydrolysis of glycosidic bonds,Glycosidase and Transglycosidase,Advantages:available,stable,organic solvent compatible,low cost,Disadvantages:low yields(competing product hydrolysis),unpredictable regioselectivity,Useful catalysts for in vitro formation of glycosidic bonds,Thermodynamic control:,increasing substrate concentrations,decreasing product concentrations by absorption,elevating reaction temperatures,adding water-miscible organic co-solvents,Kinetic control:using activated glycosyl donors and exogenous,nucleophiles,Synthetic Application of Glycosidases,While glycosidases normally cleave glycosidic linkages,they can be coerced to“run backwards”(to some extent)in the presence of an appropriate glycosyl donor.,The synthesis of 2 Tn-antigen epitopes is illustrative:,Application of Glycosidases,Application of Glycosidases,Synthesis of glycoprotein using endo-glycosidase,Li B.,Zeng Y.,Hauser S.,Song H.,and Wang L.,J.Am.Chem.Soc.,2005,9692-93,Endo-A hydrolyze the,b,1,4 linkage in the core,N,N-,diacetylchitobiose moiety of,N,-glycoprotein to release the,N,-glycan.,Application of Glycosidases-Summary,Pros:,Many glycosidases available(esp.in comparison to glycosyltransferases).,Glycosidases are less expensive and more stable than transferases.,Specificity is generally relaxed rel.to transferases,allowing broader substrate scope.,Generally good regio-and stereoselectivity.,No protecting groups.,Cons:,Enzymatic glycosylations still less scalable that chemical reactions.,Volumetric productivity still low.,Yields much lower than with transferases-glycosidase activity(which degrades the product)competes with glycosylation.,Glycosynthase,Specifically mutated glycosidases that efficiently synthesize oligosaccharides but do not hydrolyse them.,Modification of the catalytic nucleophile into a non-nucleophile residue,inactivates the enzyme.However,reactivation of the mutant by an external nucleophile,and analysis of the reaction products enable identification of the residue of interest.,Under these conditions,several retaining glycosidases act as inverting enzymes,which can use a glycosyl fluoride with the opposite anomeric configuration to that of the normal substrate,Reactivated-glycosidase,Invertingting-glycosynthase,Application of Glycosynthases,XynB2(E335G)is a nucleophiline-deficient mutant of,b,-xylosidase from,G.stearothermophilus,.,XT6B2(E265G)is a mutant strain of extracellular xylanase from,G.stearothermophilus,XT6,Synthesis of oligosaccharide using Glycosynthase,xylooligosaccharides,Ben-David A.,Bravman T.,Balazs Y.,Czjzek M.,Schomburg D.,Shoham G.,Shoham Y.,ChemBioChem,2007 2145-51,Application of Glycosynthases,Glycosynthase-mediated synthesis of glycosphingolipids,Vaughan M.,Johnson K.,DeFrees S.,Tang X.,Warren RAJ.and Withers S.,J.Am.Chem.Soc.,2006,6300-6301,Wild type reaction,b)Mutant strain(glycosynthase)reaction,*EGC II:endoglyceramidase II from,Rhodococcus,strain M-777,X=Ser,Ala,and Gly,lyso,-GM3,Chemo-enzymatic synthesis of iGb3,+,Example,