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 OH 2 3 OH 4 OHequatorial OH axial 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 promotor acceptors BF3OEt2CH2Cl2 TMSOTf Et2O 概述 基本概念 化学合成糖苷键的donor和promotor II 硫苷和DMTST NBS IDCP Mn 概述 基本概念 化学合成糖苷键的donor和promotor II 硫苷和DMTST NBS IDCP Mn 概述 基本概念 化学合成糖苷键的donor和promotor II 硫苷和DMTST NBS IDCP Mn 概述 基本概念 化学合成糖苷键的donor和promotor III 溴苷和Mn Koenigs Knorr 概述 基本概念 化学合成糖苷键的donor和promotor IV Epoxidedonor 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 Acylationscanoftenbecarriedoutasregioselectivereactionstoestablishselectiveprotectionpatterns Inbasiccondition acetylgroupsometimescanshift 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 Inacidicmedia therelativestabilityis TMS 1 TES 64 TBS 20000 TIPS 700000 TBDPS 5000000 Inbasicmedia therelativestabilityis TMS 1 TES 10 100 TBS TBDPS 20000 TIPS 100000 Inbasiccondition silylethersometimescanshift A benzylideneacetals 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 Severalfreemonosaccharidescanbeconvertedbyisopropylidenation 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 potsynthesisPeterH SeebergerChi HueyWongSamuelDanishefskyXuefeiHuang 天然糖苷类抗生素 合成稀有糖 Activation substitutionreactionatthecarbohydratering 天然糖苷类抗生素 合成稀有糖 Tosylateasleavinggroup 天然糖苷类抗生素 合成稀有糖 Triflateasleavinggroup 天然糖苷类抗生素 合成稀有糖 Mesylateasleavinggroup Intramolecularsubstitution 天然糖苷类抗生素 合成稀有糖 2 Deoxyhaloderivatives FlorinationwithDAST Diethylaminosulfurtrifluoride 天然糖苷类抗生素 合成稀有糖 Selectiveflorination 天然糖苷类抗生素 合成稀有糖 Otherhalogenation 天然糖苷类抗生素 合成稀有糖 3 Deoxygenationofcarbohydratesderivatives Openingofepoxides 天然糖苷类抗生素 合成稀有糖 Reductionofester Mechanism 天然糖苷类抗生素 合成稀有糖 Examples Synthesisofmethyl2 4 di O benzoyl 6 deoxy 3 O 1 imidazolylthiocarbonyl a L galactopyranoside Synthesisofmethyl2 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 Misunobureaction Example 天然糖苷类抗生素 合成稀有糖 Oxidation reductionreaction 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 SamuelDanishefsky GeorgeA O Doherty PengG Wang PengGeorgeWangDepartmentsofBiochemistry ChemistryTheOhioStateUniversity CarbohydrateChemistry Glycobiology L9EnzymaticSynthesisofOligosaccharidesandglycopeptides glycoproteins ComparisonofOrganicandBiosyntheticApproachesforCarbohydrateSynthesis ChemicalSynthesisofa Gal HighCostMultipleprotection deprotectionstepsTediousseparationprocedures SynthesisofD GalactosideDonor ChemicalSynthesisofa Gal SynthesisofDisaccharideAcceptor ChemicalSynthesisofa Gal Synthesisofa GalTrisaccharide ChemicalSynthesisofa Gal W Zhang J Wang J Li L Yu P G Wang J CarbohydrateChemistry 199918 8 1009 1017 EnzymaticSynthesisofa Gal Enzymaticsynthesisofoligosaccharides GlycosyltransferaseSuperbeadsandsuperbugtechnologiesGlycosidaseGlycosynthase Glycosyltransferase Advantages highregio selectivity themostpracticalmethod TransfersugarresiduesfromanactivateddonorsubstratetoanacceptorDonor Asugaractivatedbyaphosphate alipidphosphate oranucleotide usuallyanucleotide sugarAcceptor alipid aprotein DNA asmallmoleculeacceptor oragrowingoligosaccharide Typicallygroupedintofamiliesbasedonthetypeofsugartheytransfereg Galactosyltransferases sialyltransferases Glycosyltransferasesareveryspecificenzymes withonlyoneparticulartransferasebeingabletocatalyzeoneparticularglycosidiclinkage OneEnzyme OneLinkage Glycosyltransferase SugarNucleotide NineCommonSugarNucleotides UDP Glc UDP GalUDP GlcNAc UDP GalNAcUDP GlcA UDP XylGDP Man GDP FucCMP NeuAc theonlymonophosphate BiosynthesisofSugarNucleotides UDP Glc UDP Gal UDP GlcA UDP Xyl UDP GlcNAc UDP GalNAc GDP ManandGDP Fuc CMP Neu5Ac a1 3GalTCatalyzedSynthesisofa GalEpitopesandTheirDerivatives FangJ ChenX WangPG etal J Org Chem 1999 64 4089 4094 Examples Synthesisofa GalPentasaccharidewithInSituCofactorRegeneration FangJ LiJ ChenX WangPG etal J Am Chem Soc 1998 120 6635 6638 Examples DeLuca C Wong 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 Cytel slarge 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 Neose slarge scalesynthesisofsialyatedlactoseusingaCMP sialicacidsynthetase a2 3 sialyltransfersefusionenzymewithinsituregenerationofCMP NeuAc E1 CMP NeuAcsynthetase a2 3 sialyltransferasefusionenzymeE2 myokinaseE3 pyruvatekinaseE4 GlcNAc2 epimeraseE5 sialicacidaldolase Examples KyowaHakko stechnologyforlarge 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 http www cazy org 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 Specificallymutatedglycosidasesthatefficientlysynthesi