N末端的生物素标记

• Wash0.1mmolresinwithDMF.
• Dissolve0.244g(+)-biotin(1mmol,MW244.3)in5mLDMF-DMSO(1:1)solution.Alittlewarmingisnecessary.
• Add2.1mL0.45MHBTU/HOBtsolutionand0.3mLDIEAtothesolutionpreparedinstep2.
• Addtheactivatedbiotinsolutiontotheresinandletstirovernight.
• Checkresintomakesurecouplingiscompleteasevidencedbynegativeninhydrintest(colorless).
• WashresinwithDMF-DMSO(1:1)(2x)toremoveexcess(+)-biotin.
• WashresinwithDMF(2x)andDCM(2x).
• Lettheresindrybeforeproceedingtocleavage.

在2-氯TRT树脂上挂第一个FMOC氨基酸的方法

• Weigh10g2-chlorotritylchlorideresin(15mmol)inareactionvessel,washwithDMF(2x),swelltheresinin50mLDMFfor10min,drainvessel.
• Weigh10mmolFmoc-aminoacidinatesttube,dissolveFmoc-aminoacidin40mLDMF,transferthesolutionintothereactionvesselabove,add8.7mLDIEA(50mmol),swirlmixturefor30minatroomtemperature.
• Add5mLmethanolintothereactionvesselandswirlfor5min.
• DrainandwashwithDMF(5x).
• Checksubstitution.
• Add50mL20%piperidinetoremovetheFmocgroup.Swirlmixturefor30min.
• WashwithDMF(5x),DCM(2x),putresinontissuepaperoverafoampadandletdryatroomtemperatureovernightunderthehood.Covertheresinwithanotherpieceoftissuepaper,presslightlytobreakaggregates.
• Weighloadedresin.
• Packinappropriatecontainer.

检测树脂上面是否挂上第一个FMOC氨基酸的方法

• Weighduplicatesamplesof5to10mgloadedresininanEppendorftube,add1.00mL20%piperidine/DMF,shakefor20min,centrifugedowntheresin.
• Transfer100µLoftheabovesolutionintoatubecontaining10mLDMF,mixwell.
• Pipette2mLDMFintoeachofthetwocells(referencecellandsamplecell),setspectrophotometertozero.Emptythesamplecell,transfer2mLofthesolutionfromstep2intothesamplecell,checkabsorbance.
• Subs=101(A)/7.8(w)
  A=absorbance
  w=mgofresin
• Checkabsorbancethreetimesat301nm,calculateaveragesubstitution.

FMOC方法合成多肽操作步骤(0.25mmol)

• WashresinwithDMF(4x)andthendraincompletely.
• Addapproximately10mL20%piperidine/DMFtoresin.Shakeforoneminanddrain.
• Addanother10mL20%piperidine/DMF.Shakefor30min.
• DrainreactionvesselandwashresinwithDMF(4x).Makesurethereisnopiperidineremaining.Checkbeadsusingninhydrintest,beadsshouldbeblue.
• CouplingStep-Preparethefollowingsolution:
  1mmolFmoc-aminoacid
  2.1mL0.45MHBTU/HOBT(1mmol)
  348µLDIEA(2mmol)
  Addabovesolutiontotheresinandshakeforaminimumof30min.Thiscouplingstepcanbelongerifdesired.
• DrainreactionvesselandwashresinwithDMF(4x).
• PerformNinhydrintest:
  • Ifnegative(colorless),proceedtostep2andcontinuesynthesis.
  • Ifpositive(blue),returntostep5andre-couplethesameFmoc-aminoacid.Increasethecouplingtimeifnecessary.

合成含有磷酸化酪氨酸多肽的方法
Reagent:N-a-Fmoc-O-phosphotyrosine

• For0.1mmolor0.25mmolsynthesis,use0.483gFmoc-Tyr(PO3H2)-OH(1mmol,MW483.4).ForABIsynthesizers,packFmoc-Tyr(PO3H2)-OHinacartridge.
• ThecycleprogramforcouplingFmoc-Tyr(PO3H2)-OHisthesameasforotherFmoc-aminoacidsexceptforthecouplingtime(seestep3).(Note:ABIsynthesizersuseHBTU/HOBTastheactivatingreagent.)
• ThecouplingtimeforFmoc-Tyr(PO3H2)-OHneedstobeincreased.ForABImodel430Apeptidesynthesizer,insertseveralsteps(i.e.,vortexon,wait990sec,vortexoff,toincreasethecouplingtime).ForABImodel431Apeptidesynthesizer,addadditional"I"s.Overnightcouplingmaybenecessaryforsomesequences.
• AfterthecouplingstepforFmoc-Tyr(PO3H2)-OH,performninhydrintesttoensurecompletecoupling.Negative(colorless)ninhydrintestindicatescompletecoupling,whileapositive(blue)ninhydrintestindicatesincompletecoupling.
• Increasethecouplingtimeoftheaminoacidresiduesafterthephosphotyrosineorperformdoublecoupling.(Note:Thecouplingofaminoacidsafterthephosphotyrosinecanbedifficult.)
• Thereisalimitonthenumberofaminoacidresiduesthatcanbecoupledafterthehosphotyrosine.Sincethephosphogroupisunprotected,sidereactionsarelikelytoccur.(Note:Peptideshavebeensuccessfullycoupledwithsequencescontainingupotenadditionalaminoacidsfollowingthephosphotyrosineresidue.)

用Wang树脂和2-氯TRT树脂同时合成多肽
PeptideswhichdifferintheC-terminicanbesimultaneouslysynthesizedinonereactionvesselbyemployingresinsthatpossessdifferentcleavageproperties.Theresinsusedweretheweakacidlabile2-chlorotritylresinsandtheTFAlabileWangresins.Thesuccessofthisapproachwasshownbytheco-synthesisofACTH(4-10)withACTH(4-11)andNeuropeptideY,aC-terminalamidepeptidewithitscorrespondingC-terminalfreeacidanalog.
*HongA.,LeT.,andPhanT.TechniquesinProteinChemistryVI,531-562(1995).
切割程序
StartingResin:Chlorotritylresins
Reagentsfor1gPeptide-Resin:
1mLaceticacid(AcOH)
2mLtrifluoroethanol(TFE)
7mLdichloromethane(DCM)

• Prepareabovemixture.
• Addpeptide-resintothemixtureandletitstiratroomtemperaturefor1h.
• Filterandwashresinwith10mLTFE:DCM(2:8)(2x)toensurethatalloftheproductisrecovered.
• Evaporatethesolventuntilthereislessthan5mLofliquid.
• Addethertoatesttubecontainingabout100mLoftheabovesolution.Checksolubilityofthefullyprotectedpeptideinether.Iftheproductprecipitates,proceedtostep6.Ifnoprecipitateisobserved,proceedtostep7.
• Addcoldethertotheresidualliquidinstep4toprecipitatethefullyprotectedpeptide.Filterthroughafinesinteredfunneltoobtaintheproduct.
• Somefullyprotectedpeptidesaresolubleinether.Inthiscase,addwatertoprecipitatethemout.Filterthroughafinesinteredfunneltoobtaintheproduct.

从Fmoc-Lys(Mtt)氨基酸上脱去Mtt的方法
Reagent:
Fmoc-Lys(Mtt)-OH

• SwellresininDCM.
• Washresinwith3%TFA/DCM(2x)(sincetheresinisswolleninDCM,thisstepofwashingtheresinquicklywith3%TFA/DCMensuresthattheactualconcentrationofTFAis3%).
• Shaketheresinin3%TFAfor10min.
• Repeatstep3.
• WashresinwithDCM(3x),DMF(3x),isopropanol(3x),andDCM(3x).
• Lettheresindryinair.

多肽标记FITC方法
Reagents:
FITC
Fmoc-e-Ahx-OH

• CoupleFmoc-e-Ahx-OHtotheaminoterminalofthepeptide-resinusingstandardcouplingconditions.
• "De-Fmoc"withpiperidineusingthestandard20%piperidineprocedure.
• WashresinwithDMF(3-4x).
• SwellresinwithDCManddrain.
• Preparesolutionof1.1equivalentofFITCinpyridine/DMF/DCM(12:7:5).Usejustenoughsolutiontoformaslurrywiththeresin.Donotusetoomuchsolutionsincetherateofthereactionisproportionatetotheconcentrationofthesolution.
• Addthesolutionpreparedinstep2totheresin.
• Letmixovernight.
• Checkthecompletionofthereactionusingninhydrintest.
• IfthecouplingofFITCtotheaminogroupisnotcomplete,ninhydrintestwillgiveabluecolor.RepeatthecouplingwithFITC(steps5-7)ifnecessary.
• WashresinwithDMF(2x),isopropanol(2x),andDCM(2x).

多肽标记FAM方法
Reagent:
5-carboxyfluorescein(5-FAM)

Usestandardcouplingmethodtocouple5-carboxyfluoresceintotheaminogroupofthepeptide.Forcostsavingpurposes,use2xexcesscomparedtothemmolofresin,insteadofthestandard4xexcessusedforFmoc-aminoacids.For0.1mmolsynthesis,use75mg5-carboxyfluorescein,76mgHBTU,and70mLDIEA.

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