糖苷键.ppt

概述：基本概念,化学合成糖苷键的三个考虑因素,1）如何活化糖基分子,donor,acceptor,2）如何实现立体选择性：aorb,3）如何实现连接选择性选择性地保护acceptor中的羟基利用羟基活性的差别6-OH2,3-OH4-OHequatorial-OHaxial-OH,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。,概述：基本概念,化学合成糖苷键的立体选择性,SN1机制：anomericeffect，a-anomerasmajorproduct。C-2isnon-participationgroup(2-OCH2Ph,2-N3,2-deoxy)harshcondition（strongpromoter，hightemperature）polarsolventSN2机制：anchimericeffect（C-2functionalgroupparticipationeffect）(-OAc,-NHAc,-I,-SR,-SeR)stableintermidiate,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（BF3，TMSOTf）,强碱，利于b-TCAdonor；弱碱，利于a-TCAdonor。,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（BF3，TMSOTf）（Schmidt）,SN2likereaction;Stereoselectivity(SN1vsSN2)issubjecttosolvent,temperature,promotorWong,C.-H.J.Am.Chem.Soc.1995,117,5869-5870,One-potmulti-enzymesynthesisofahyaluronicacidpolymerinvolvingglycosyltransferasesandsugarnucleotideregenerationsystems,E1,hyaluronicacidsynthase;E2,UDP-Glcdehydrogenase;E3,UDP-Glcpyrophosphorylase;E4,UDP-GlcNAcpyrophosphorylase;E5,pyruvatekinase;E6,lactatedehydrogenase;E7,inorganicpyrophosphatase,Examples,Cytelslarge-scalesynthesisofsialylLewisXantigen,E1:a1,3-fucosyltransferaseE2:pyruvatekinaseE3:GDP-mannosepyrophosphorylaseE4:GDP-4-keto-5-deoxymannose3,5-epimerase/GDP-4-keto-6-galactosereductaseE5:glucosedehydrogenaseE6:hexokinaseE7:phosphomannomutaseE8:a2,3-sialyltransferase,Examples,Neoseslarge-scalesynthesisofsialyatedlactoseusingaCMP-sialicacidsynthetase/a2,3-sialyltransfersefusionenzymewithinsituregenerationofCMP-NeuAc,E1:CMP-NeuAcsynthetase/a2,3-sialyltransferasefusionenzymeE2:myokinaseE3:pyruvatekinaseE4:GlcNAc2-epimeraseE5:sialicacidaldolase,Examples,KyowaHakkostechnologyforlarge-scaleproductionofUDP-Galandglobotrioseutilizingmetabolicallyengineeredbacterialcells,Ppa:pyrophosphataseGalU:glucose-1-phosphateuridylyltransferaseGalT:galactose-1-phosphateuridylyltransferaseGalK:galactokinaseLgtC:a1,4-galactosyltransferase,Examples,Examples,Synthesisoftumor-associatedantigen,Globo-Hhexasaccharide,SuD.,EguchiH.,YiW.,LiL.,WangPG.andXiaC.Org.Lett.2008,inpress,SuperbugandSuperbeadtechnologiesforthesynthesisofa-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,a-Gal,Synthesisofoligosaccharidesinbiologicalsystem,OH,HO,S1,OH,HO,S2,+,O,OH,HO,S1,S2,+H2O,OH,O,Nucleotide,HO,S1,Sugar-NucleotideBiosynthesis&Regeneration,HO,S1,O,OH,OH,HO,S2,HO,S1,S2,*,GlycosylationinEukaryotes,SugarNucleotidetransport,transferase,UDP,UTP,UDP,UMP,UDP,UMP,Gal,P,Gal,UDP,Gal,Gal,Acceptor,Acceptor,CytosolGolgi,BiosyntheticPathwayforUDP-GalRegenerationanda-GalProduction,ATP,ADP,a1,3GalT,Gala1,3Lac,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-GalSuperbead,GalU,GalT,GalK,PyKF,RecombinantE.colistratinsoverexpressingGalK,GalT,GalUorPykF,ChenX,FangJ,WangPG,etal.:J.Am.Chem.Soc.2001,123,2081-2082.,Beadswith:GalKGalTGalUPykF,Peristalticpumpforcirculation,Reservoirwith:a1,3GalTLacOBn9.6mMATP0.96mMPEP19.2mMUDP0.96mMGlc-1-P0.96mMGal12mMMgCl210mMMnCl210mMKCl100mMHEPES100mMpH7.5,LiuZ,ZhangJ,ChenX,WangPG:ChemBioChem2002,3,348-355.,ProductionofUDP-GalwithSuperbeads.,SynthesisofoligosaccharideswithUDP-GalSuperbead,aGramscalesynthesis,othersare100mgscales,a-GalsynthesisusingSuperbug,a1,3GalT,Gala1,3Galb1,4Glc,Galb1,4Glc,UDP-Gal,UDP,PEP,Pyruvate,Gal-1-P,Glc-1-P,UDP-Glc,UTP,PykF,GalU,GalK,GalT,Galactose,Lactose,Glycolyticpathway,Glucose,ATP,ADP,Glucose,Pyruvate,PykF,PEP,Glycolyticpathway,PPi,NM522,FirstGenerationofa-GalSuperbug,Cloneandexpressindividualenzymesinthebiosyntheticpathwayofa-GalConstructionanartificialbiosyntheticgeneclusterandtransferintoE.colihostcell.Largescaleproductionofa-Galoligosaccharidesusingfermentedandpermeatedcells.,ChenX,LiuZ,ZhangW,FangJ,AndreanaP,WangPG:ChemBioChem.2002,3,47-53.,LargeScaleProductionofa-GalTrisaccharide,SecondGenerationofa-GalSuperbug,PpK:polyphosphatekinase,ThirdGenerationofa-GalSuperbug,ChenX,ZhangJ,KowalP,AndreanaP,WangPG:J.Am.Chem.Soc.2001,123,8866-8867.,SusA:sucrosesynthaseGalE:UDP-Gal4-epimerase,ApplicationofGlycosyltransferases-SummaryPros:Excellentyieldwithcompleteregio-andstereoselectivity.Noprotectinggroupsneeded.Sialyltransferasesallowfacilesialylation.(Thisisthehardestglycosylationtocarryoutbychemicalmethods.)Reactionscanbecarriedoutinwater.Cons:Mostrequisiteenzymesarenotreadilyavailable,andthosethatareavailableareexpensive.Regio/stereoselectivitymeansthatsubstratescopeislimitedandauniqueenzymeisneededforalmosteveryreaction.Nucleotidesugardonorsareveryexpensiveand/orunstable.Scaleisoftenlimitedbyenzymeavailability/volumetricproductivity.(Alargeamountofenzymeproducesonlyasmallamountofsugarbyweight.),BasedonAAsequencesimilarity,Glycosidaseareclassifiedinto107distinctfamilies.(/index.html),Currently37472knownorputativeglycosidaseinCAZy,433differentglycosidasecrystalstructuresfrom76differentfamilieshavebeenreported,GlycosidaseandTransglycosidase,Invivofunction:hydrolysisofglycosidicbonds,GlycosidaseandTransglycosidase,Advantages:available,stable,organicsolventcompatible,lowcostDisadvantages:lowyields(competingproducthydrolysis),unpredictableregioselectivity,UsefulcatalystsforinvitroformationofglycosidicbondsThermodynamiccontrol:increasingsubstrateconcentrationsdecreasingproductconcentrationsbyabsorptionelevatingreactiontemperaturesaddingwater-miscibleorganicco-solventsKineticcontrol:usingactivatedglycosyldonorsandexogenousnucleophiles,SyntheticApplicationofGlycosidasesWhileglycosidasesnormallycleaveglycosidiclinkages,theycanbecoercedto“runbackwards”(tosomeextent)inthepresenceofanappropriateglycosyldonor.,Thesynthesisof2Tn-antigenepitopesisillustrative:,ApplicationofGlycosidases,ApplicationofGlycosidases,Synthesisofglycoproteinusingendo-glycosidase,LiB.,ZengY.,HauserS.,SongH.,andWangL.J.Am.Chem.Soc.,2005,9692-93,Endo-Ahydrolyzetheb1,4linkageinthecoreN,N-diacetylchitobiosemoietyofN-glycoproteintoreleasetheN-glycan.,ApplicationofGlycosidases-SummaryPros:Manyglycosidasesavailable(esp.incomparisontoglycosyltransferases).Glycosidasesarelessexpensiveandmorestablethantransferases.Specificityisgenerallyrelaxedrel.totransferases,allowingbroadersubstratescope.Generallygoodregio-andstereoselectivity.Noprotectinggroups.Cons:Enzymaticglycosylationsstilllessscalablethatchemicalreactions.Volumetricproductivitystilllow.Yieldsmuchlowerthanwithtransferases-glycosidaseactivity(whichdegradestheproduct)competeswithglycosylation.,Glycosynthase,Specificallymutatedglycosidasesthatefficientlysynthesizeoligosaccharidesbutdonothydrolysethem.,Modificationofthecatalyticnucleophileintoanon-nucleophileresidueinactivatestheenzyme.However,reactivationofthemutantbyanexternalnucleophile,andanalysisofthereactionproductsenableidentificationoftheresidueofinterest.,Undertheseconditions,severalretainingglycosidasesactasinvertingenzymes,whichcanuseaglycosylfluoridewiththeoppositeanomericconfigurationtothatofthenormalsubst