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[doc] Synthesis of Cell Adhesive Motif RGD Tripeptide by a Novel Chemical Method and Its Purification

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[doc] Synthesis of Cell Adhesive Motif RGD Tripeptide by a Novel Chemical Method and Its Purification

    Synthesis of Cell Adhesive Motif RGD

    Tripeptide by a Novel Chemical Method

    and Its Purification

Availableonlineatwww.sciencedirect.com

    ;@

    ;CHEM.RES.CHINESEU.2006,22(5),612616

    ;SynthesisofCellAdhesiveMotifRGDTripeptidebyaNovel ;ChemicalMethodandItsPurification

    ;WANGHua,ZHAOMi.feng,MACheng.yun,JINGWei,HUANGYi.bing, ;HOURui-zhen,ZENGHong-bin,XULi’’andZHANGXue-zhong’

    ;1.KeyLaboratoryofMolecularEnzymologyandEngineering,University,Changchun130021,

    ;PR.China:

    ;2.China-JapanUnionHospitalofnUnivers,Changchun130033,R.China;

    ;3.FirstHospita2ofJilinUniversity,Changchun130021,R.China ;ReceivedSept.19,2005

    ;-I1lecelladhesivemotifRGDtripeptidewassynthesizedbyusinganovelchemicalmethod.First,Cly-Asp(GD)

    ;wassynthesizedintwostepsincludingtheehloroacetylationoffreeL-asparticacidandtheammonolysisofthechl0r0-

    ;aeetylatedL-aspartieacid.TheyieldofehloroacetylatedL-asparticacidwas83.O%.Fortheammonolysisofchl0r0.

    ;acetylatedL-asparticacid,theyieldoftheammonolyzedproductwaft,92.3%.Second,thecouplingbetweenAand

    ;Gly-AspwascarriedoutbyusingtheNCAmethod.Thenla~mulnyieldofRGDwasabout50%at0oCandpH=

    ;9.5.nlepreparedRGDtripeptidewasconfmnedbyusingaminoacidcomponentanalysisandmflssspectrographic

    ;analysis.

    ;KeywordsRGDtripeptide;Chemicalsynthesis;Purification ;ArticlelDmo5-9o4o(2oo6)-05-612-05

    ;Introduction

    ;Arg-Gly-Asp(RGD),acharacteristictripede

    ;sequencefoundwithinfibronectinandotherrelatedad- ;hesionmoleculesinextracellularmatrices(ECM),has ;attractedmuchattentionbecauseithasbeenprovedto

    ;bearecognitionsiteforcellularadhesion,spreading, ;andmotilityofcells[?21.RGDandsomesynthetic

    ;RGD-containingpeptidesascompetitiveandreversible ;inhibitorsforthebindingofadhesiveproteinshavebeen ;usedtostudytheadhesiveinteractionsbetweencells

    ;andastheagentstosuppresstumormetastasisand

;plateletaggregationt3.

    ;Recentreseal~hstudieshave

    ;alsoprovedthatRGDcoulddirectlyenterintotumor ;cellsandinduceapoptosisofcancercellsbyactivating ;certainmembersofthecaspasefamilyincluding ;caspase-8andcaspase-9,andoflatecaspase-3,im- ;plyingunexpectedintracellularactionsoftheRGDmo- ;tif[8--10].

    ;Duringthepastfewyears,thesynthesesofRGD ;tripeptideandRGD-containingshortpeptidesbychemi- ;calandenzymaticmethodsandrecentlybyacombina- ;tionofthesetwomethodshavebeenreported[一引.

    ;However.theuseofconventionalchemicalmethodsfor ;thesynthesisofpeptidesincludingtheliquidphase ;methodand,especially,thesolidphasemethodhas ;beenrestrictedtoacertainextentbecauseofthehigh ;costofinstrumentationandreagents(protectiveamino ;acidsandcouplingreagents).

    ;Theenzymaticpeptidesynthesishasmanyimpor- ;tantbenefits.Inorganicmedia,anumberofhydropho- ;bicsmallpeptideshavebeensynthesizedinhighyields ;fromproteases.asreportedpreviously[B’.How.

    ;everthesynthesisofchargedaminoacidresidue-con- ;tainingpeptidesgenerallyresultsinaratherlowyield ;becauseofthelowsolubilityofhydrophilicaminoacids ;inorganicsolvents.TheRGDtripeptidecontainstwo ;chargedresidues(ArgandAsp)andoneneutralresi- ;due(Gly).Onemethodthatiscurrentlyavailableto ;overcomethedisadvantageofthelowsolubilityofhy- ;drophilicaminoacidsubstrateinorganicsolventsisthe ;useofreversemicelleasthereactionmedia,asrepor- ;tedpreviously[.

    ;However,thisreactionsystem

    ;makestheseparationandpurificationofpeptideprod- ;uctsdifficultbecauseofthepresenceofsurfactantmol- ;SupportedbytheSpecialResearchGrantfromtheStateAdministrationofTra

    ditionalChineseMedicineofChina(No.

    ;2004ZDZX003).

    ;Towhomcorrespondenceshouldbeaddressed.E-mail:xuli@jlu.edu.en;zha

    ngxz@mail.jlu.edu.en

    ;

    ;No.5WANGHuaeto1.613

    ;ecules.

    ;Recently,anovelchemicalmethodforthesynthe

;sisofRGDprecursordipeptideGlyAsp(GD)hasbeen

    ;developed,whichisnoticeablydifferentfromthecon

    ;ventionalliquidphaseandsolidphasechemical ;methods.Itissimple,feasible,andefficient,witha ;lowcostonascaleofgrams.

    ;Inthisarticle,thesynthesisoffreeRGDisdealt ;th.First.GlyAspwassynthesizedintwostepsin

    ;cludingchloroacetylationofL?-asparticacidandam?-

    asparticacid.Second, ;monolysisofthechloroacetylL

    ;thecouplingbetweenRandGDwascarriedoutby ;usingtheNCAmethod.Inthismethod,freeRGD ;tripeptideCallbesynthesizedsimplyfromfreeGly,Asp ;withoutprotection/deprotection,andZArg.

    ;Experimental

    ;1Materials

    ;LAsparticacidwaspurchasedfromGLBiochem ;(Shanghai,China).Chloroacetylchloridewaspur- ;chasedfromBeijingHengyeZhongyuanChemicalCo.. ;ZArgOHwaspurchasedfromSigma(St.Louis,MO, ;USA).TrifluoroaceticacidwasfromMerck(Darms

    ;tadt,Germany).AcetonitrilewasofHPLCgrade.All ;otherorganicsolventswereofanalyticalgrade.Sepha

    ;dexG10waspurchasedfromPharmacia.Silicagel ;GF254forTLCwaspurchasedfromQingdaoOceania ;Chemical(Qingdso,China).

    ;2ChemicalSynthesisofGD

    ;TheGlyAsp(GD)dipeptidewassynthesizedin ;twosteps:chloroacetylationofLasparticacidandam

    ;monolysisofthechloroacetylatedLasparticacid.

    ;2.ChloroacetylationofL-AsparticAcid ;LAsparticacid(12.0g)wasdissolvedin20mL ;of27%(massfraction)NaOHfollowedbytheaddition ;0f20mLofethylacetatethcontinuousstirring. ;Chloroacetylchloride(7.5mL)and27%(massfrac

    ;tion)NaOHweresimultaneouslyaddedtotheabove

    ;mentionedsolutionwithcontinuousstirringinanice

    ;saltbath.rrhepHvalueofthereactionwascontroHed ;inarangeofll12.

    ;Afterthecompletionofthereaction,thereaction ;mixturewasstirredforanother30min,andthepHval

    ;ueofthereactionmixturewasadjustedto2.0withcon

    ;centratedhydrochloricacid.Thereactionproductwas ;extractedwith50mLofethylacetate.Thisstepwasre

    ;peatedtwice.AwhiteproductofchloroacetylatedLas

;particacidwasobtainedaftertheremovalofethylace

    ;tateunderreducedpressure.

    ;2.2AmmonolysisofChloroacetylatedL-AsparticAcid ;rrheabovementionedproduct(3.0g)wasdis

    ;solvedin30mLofNH4OH(massfraction28%),wid1 ;constantstirringinanicesaltbath.After36h,the ;NHgaswasremovedunderreducedpressure.There

    ;suhingsticky,lightyeHowproductwasdissolvedinab

    ;soluteethano1.withthoroughshaking.rrhesupematant ;wasthendecanted.Ethanolwasaddedagaintothesys

    ;ternundercontinuousstirring.GDdipeptidewasob

    ;tainedasawhitesolidproductafterrotaryvaporization ;underreducedpressure.

    ;3SynthesisofRGD

    ;3.1PreparationofNCA-Arg

    ;ZArgon(10.0gwassuspendedin20omLof

    ;tetrabydrofuran(THF),followedbyarapidadditionof ;50mLofcatalystPBr3toTHF(5oC)(volumeratio: ;1:4undervigorousstirringatroomtemperature.After ;3h.THFthatisfoundfloatingonthesurfacewasde

    ;canted,andtheheavy,oillikeproductwasdeposited

    ;atthebottom.Theoillikeproductwaswashedrepeat

;edlywith20omLofTHF.NCAArgwasobtainedasa

    ;lightyellowtarbyvacuumdrying.

    ;3.2SynthesisofRGD

    ;GDdipeptide(9.0g)wasdissolvedin180mLof ;0.05mol/LBoraxNaOHbufferatpH=10.ThepH ;valueofthereactionsolutionwasadjustedto9.5with ;1mol/LNaOH;subsequently,onedropofn-octanol ;andthesynthesizedNCAArgwereaddedtothereac

    ;tionmixturerapidly.Meanwhile,thepHofthereaction ;systemwaskeptconstant(9.5)byadding1mol/L ;NaOHevery2min.Finally,thepHofthereactionsys

    ;ternwasadjustedto5.0withconcentratedH,SOd.Asa ;result.CO,wasreleasedandfreeRGDwasformed. ;Thewholereactionprocesswascarriedoutwithvigor- ;OUSstirringinanicebath.Ultimately,thecrudeRGD ;productwasobtainedasawhite,stickymaterialby1”o—

    ;taryvaporizationunderreducedpressure. ;4PurificationofthePepfideProducts

    ;GlyAspdipeptidewaspurifiedonaSephadex ;G10column(16inrfl×1000rain)equilibratedand ;elutedwithwateratanelutionrateof1.0mL/min. ;Theelutionprocesswasmonitoredat220nm.Thewa

;terinthecollectedfractionwilt8removedbyroLRryva

    ;pofizationunderreducedpressure.

    ;TheRGDtripeptidesamplewasdissolvedinalit

    ;tiewaterfollowedbytheadditionofacertainamountof ;ethanoltoprecipitateRGD.ThecrudeRGDwassepa- ;ratedandpurifiedonaSephadexG10column

    ;(16inrfl×1000rain)equilibratedandelutedwithwa

    ;teratanelutionrateof1.0mL/rain.Theelution ;processwasmonitoredat220nm.Thecollectedfrac

    ;tionsofRGDwerelyophilized.

    ;

    ;614CHEM.RES.CHINESEUVo1.22

    ;5AnalysisofthePepfideProducts

    ;equMitativeanalysesofthepepfideproductsin

    ;cludingGDdipeptideandRGDtripepfidewerecarried ;outbyusingTLC.TLCwasperformedonapreeoated ;plateofsilicagel,GF254(2.0cm×5.0cm).Amix

    ;tureofnbutanol/aceticacid/waterfthevolumeratio ;was4:1:11wasusedastheliquidphase.

    ;,I’llequantitativeanalysesofthepeptideproducts

    ;werecarriedoutbyusingHPLC(Shimadzumode1) ;withareversephaseCl8column(ZorbaxODSCl8).

;,I’llecalibrationcurveswereconstructedfromthepeak

    ;areasofthepurifiedproductsat220nm.epeakwas ;identifiedbycomparingtheretentiontimeoftheprod

    ;uctwiththatofthestandardcompound.

     ;Analysesoftheaminoacidcomponentsofthepep

    ;tideproductsweremadebyusingaHitachimodel835

    ;50aminoacidautomaticanalyzer.Molecularweightsof ;GDandRGDwereconfirmedbyusingFABmassspec

    ;troscopy.

    ;ResultsandDiscussion

    ;1SynthesisofGDDipeptide

    ;GlyAspwassynthesizedintwosteps.Inthefirst ;step,theehloroaeetylationofLasparticacidwascar-

    ;riedoutunderanalkalinecondition.,I’llepHvalueof

    ;thereactionwasthekeyfactortothesynthesisandhad ;tobemaintainedbetween11and12.Ethylacetatewas ;71fH

    ;Ph-CH20C0CH(CH2)3NHCNH2

    ;A-Arg+C

    ;.

    ;.Hsp

    ;N

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