Combating vancomycin-resistant bacteria with catalytic antibodies : the synthesis of a transition-state analog PDF
Preview Combating vancomycin-resistant bacteria with catalytic antibodies : the synthesis of a transition-state analog
COMBATINGVANCOMYCIN-RESISTANTBACTERIAWITHCATALYTIC ANTIBODIES: THESYNTHESISOFATRANSITION-STATEANALOG By KAVITHAVEDHA-PETERS ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2002 Tomybelovedhusbandandfriend,Matt ACKNOWLEDGMENTS FirstandforemostIwouldliketothankmyadvisor,Dr.JonStewart,forallofhis guidance,encouragement,andpatienceduringthecourseofmystudies. Hehastaught meatremendousamount,andIhaveenjoyedworkinginhislab. Iwouldalsoliketo expressmyappreciationtoallofthepastandpresentStewartgroupmembersfortheir helpfuldiscussionsandfriendship. SoniaRodriguez,JenniferTonzello,CarlosMartinez andArisPolyzosprovidedaverysupportiveandopenenvironmentformewhenIfirst joinedthegroupandwasverynervousandintimidated;forthisandtheirfriendshipIwill beforevergrateful. MydeepestgratitudegoestoCarlosforallofhishelpwithmy projectandforhispatiencewithmydailybarrageofquestionsduringmyfirstthreeyears inthelab. IwonaKaluznahaskeptmeentertainedthepasttwoyearswiththeinteresting thingsthatshedoes,butshehasalsobecomeagoodfriendwhoalwayslistenstome, whichItrulyappreciate. BrianKyte,whosedaily"gestures"willbemisseddearly, deservesacknowledgementfortheideaforthelabelingexperimentfortheMitsunobu reactionproduct. AdamWaltonhasbeenaconstantsourceoffactsaboutCaliforniaand IhaveenjoyedhearingabouthisquestsinthelabtomakeDr.Stewartdoajigonthe roof. BrentFeskehasactuallymademeputthewords"dude"and"whoadie"inmy workingvocabularyandIwishhimluckwiththecontinuationofthevancomycin- resistanceproject. in IamverygratefultotheHorenstein,Hudlicky,Scott,andWrightgroups,wholet meborrowmanychemicalsandequipmentovertheyearsthathelpedmewithmy project. Idon'tthinkIwouldhavemadeittothispointwithoutthecontinualloveand supportofmyparents. Theyalwaysencouragedmycuriosityasachildandhavetaught metheimportanceofaneducation. Finally,myhusbandMatthasbeenanintegralpartofmygraduatecareeron manydifferentlevels. Asacolleaguehehasprovidedaconstantsourceofdiscussionfor myresearchideasandproblems,andwouldoftengivemeveryhelpfulsuggestionsfor mychemistry. IhavealsofoundMatt'senthusiasmandloveofscienceinspiringand appreciatethemanynewthingsIhavelearnedfromourconversationsonhisnewbook oftheweek(eventhoughhethoughtIwasneverlistening). Butaboveall,IthankMatt forbeingthemostwonderfulhusbandandfriendintheworld. Hisendlessloveand supporthelpedmakethisPh.D.possible. IV TABLEOFCONTENTS page ACKNOWLEDGMENTS iii SYMBOLSANDABREVIATIONS vii ABSTRACT xi CHAPTERS BACKGROUNDANDINTRODUCTION 1 1 ABriefIntroductiontoVancomycin 1 BacterialCellWallBiosynthesis 2 Vancomycin-ResistantBacteria 5 StrategiestoFightVancomycin-ResistantBacteria 7 IntroductiontoResearchGoal 13 CatalyticAntibodies 14 IntroductiontoSyntheticGoal 18 MethodsfortheSynthesisofPhosphonateEsters 19 SynthesisofUnsymmetricalPhosphonateDiesters 19 SynthesisofPhosphonateMonoesters 25 Solid-PhasePeptideSynthesis 26 SegmentCondensation 30 2 MODELSTUDYOFTHEMITSUNOBUREACTIONANDSIDE-CHAIN DEPROTECTIONSTEPSFORTHETRANSITION-STATEANALOG SYNTHESIS 32 MitsunobuReactionwithaMonomethylPhosphonateEster 33 Oxygen-18LabelingExperiment 36 ChiralGCExperiment 41 Side-ChainDeprotectionSteps 43 PhosphonateDiesterDemethylationwithBromotrimethylsilane 43 SaponificationofCarboxylateEster 43 3 SYNTHESISOFTHETRANSITION-STATEANALOG 46 Solid-PhaseSyntheticApproachtotheTransition-StateAnalog 47 ProtectionofL-LacticAcid 48 CouplingtotheWangResin 49 ConvergentApproachtotheTransition-StateAnalog 52 Solid-PhasePeptideSynthesisoftheTetrapeptideFragment 53 SynthesisofthePhosphonateEsterFragment 58 ModelStudiesoftheSegmentCondensationStep 59 CouplingoftheTetrapeptideandPhosphonateFragments 61 DeprotectionofthePhosphonateMethylEsterandCarboxylateEsters 62 Conclusion 67 4 CONCLUSIONSANDFUTUREWORK 68 5 EXPERIMENTAL 71 GeneralMethods 71 ExperimentalProcedures 72 REFERENCES 91 BIOGRAPHICALSKETCH 99 VI SYMBOLSANDABREVIATIONS Ac acetyl ATP adenosinetriphosphate Bn benzyl Boc terr-butoxycarbonyl BOP (l//-benzotriazole-1-yloxy)tris(dimethylamino)pl hexafluorophosphate BSA bovineserumalbumin Bu butyl Cbz benzoxycarbonyl DBU l,8-diazabicyclo[5.4.0]undec-7-ene DCC dicyclohexylcarbodiimide DIAD diisopropylazodicarboxylate DIC diisopropylcarbodiimide DIEA diisopropylethylamine DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline ESI electrosprayionization Et ethyl FAB fastatombombardment vn Fmoc 9-fluorenylmethoxycarbonyl GC gaschromatography HBTU 0-(l//-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HMPA hexamethylphosphoramide HOBt 1-hydroxybenzotriazole HPLC highperformanceliquidchromatography IBCF isobutylchlorotbrmate IR infrared KLH keyholelimpethemocyanin Lac lactate Me methyl MS massspectrometry MSRA methicillin-resistantStaphylococcusaureus NAD+ nicotinamideadeninedinucleotide,oxidizedform NADH nicotinamideadeninedinucleotide,reducedform NADP^ nicotinamideadeninedinucleotide,phosphate,oxidizedform NAG N-acetylglucosamine NAM N-acetylmuramicacid NMM N-methylmorpholine NMP N-methylpyrrolidinone NPEOC 4-nitrophenethyloxycarbonyl PAM phenylacetamidomethyl PCR polymerasechainreaction viu Ph phenyl Pr propyl PyBOP (\H-1-benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate SPPS solid-phasepeptidesynthesis TBAF tetrabutylammoniumfluoride TBDMS terf-butyldimethylsilyl TFA trifluoroaceticacid THF tetrahydrofuran TMS trimethylsilyl TSA transition-stateanalog VRE vancomycin-resistantenterococci AminoAcidCodes alanine Ala A arginine Arg R asparagine Asn N asparticacid Asp D cysteine Cys C glutamine Gin Q glutamicacid Glu E glycine Gly G histidine His H isoleucine lie I leucine Leu L IX methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T W tryptophan Trp tyrosine Tyr Y valine Val V
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