Overview'of'Solid-Phase'Peptide'Synthesis'(SPPS)'and'Secondary'Structure'Determination'by'FTIR'

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1 verviewofsolid-phasepeptidesynthesis(spps)andsecondarystructuredeterminationbyftir Introduction Proteinsareubiquitousinlivingorganismsandcells,andcanserveavarietyoffunctions.Proteinscanactas enzymes,hormones,antibiotics,receptors,orserveasstructuralsupportsintissuessuchasmuscle,hair,and skin. Due to the high molecular weight and the difficulty in isolating significant quantities of many proteins, scientistshavebeenworkingfordecadestodevelopmethodstosynthesizenaturallyoccurringpeptides(short proteins)orproteinfragmentsinthelaboratoryinordertostudyormimicthestructureandbiologicalactivity offulllengthproteins.anothermotivationtodevelopefficientpeptidesynthesistechniquesisthepotentialof thesemoleculestoserveastherapeuticagents. 1 Morerecently,thenaturalabilityofpeptides/proteinstoselfDassembleintodefinedstructureshasalsobecome atargetforexploitationinavarietyofmaterialsscienceandbiomedicalapplications.fibrilliaraggregatesand hydrogelsformedfrompeptidesandpeptideconjugateshavebeensuccessfullyusedasbiomimeticcellculture scaffolds, 2 drug delivery vehicles, 3 and stimulidresponsive biomaterials. 4,5 Peptides have also been used to controlthemorphologyoflargerpolymers, 6,7 anddirecttheassemblyofinorganicnanoparticlestoformpeptide basedwires 8 andsensors. 9 Asanintroductiontothisrapidlyexpandingfield,thisexperimentwillcovermethods usedtosynthesizeandcharacterizepeptides,aswellasevaluatethesecondarystructureofapeptidefollowing selfdassembly. BasicPeptideStructure Peptidesareformedbysequentialadditionofspecificaminoacids.Theaminoacidsallhavesimilarstructures thatcontainanamineononeendandacarboxylicacidontheother(hencethename aminoacids ),butthey varyintherdgroupattachedtothealphacarbon.toformapeptide,aminoacidsarejoined headdtodtail by couplingtheamineofoneaminoacidwiththecarboxylicacidofanotheraminoacidtoformanamidebond. Thegeneralstructureofapeptidecontainingfouraminoacids(a tetrapeptide )isshowninfigure1.theendof thepeptidecontainingtheamineiscalledthe Dterminus andtheendcontainingthecarboxylicacidiscalled the CDterminus.ProteinsarenaturallysynthesizedstartingattheDterminus,sobyconvention,theaminoacid sequenceofapeptideistypicallylistedfromthedtocdterminus.forexample,ifyourpeptidecontainsarginine, glycine and aspartic acid, the peptide would be referred to as ArgDGlyDAsp or RGD if using the 1Dletter abbreviationforeachresidue.ote:apeptidewiththesequenceargdglydaspistthesameasaspdglydarg. Figure1.Generalstructureofapeptidecontainingfouraminoacids Solid-PhasePeptideSynthesis(SPPS) Inordertoefficientlysynthesizepeptides,atechniqueknownas soliddphasepeptidesynthesis (SPPS)wasfirst developedinthe1960 s. 10 ThekeyfeatureofSPPSisthesequentialattachmentofaminoacidstoamacroscopic solid support matrix (commonly referred to as resins or beads). While a wide variety of solid supports are available,someofthemostcommonaremadefromsmallbeads(~70d400micronsinsize)ofpolystyreneplastic 1

2 that have been chemically modified to attach a linker molecule to the surface of the bead. 11 Each bead has multiple linker molecules on its surface. The number of linker molecules on the surface of a particular batchofbeadsisusuallydesignatedbygivingthemillimolesoflinkerper gram of beads (mmol/g). The chemical structure of the particular resin thatwewilluseinthislabisshowninfigure2(calledwangresin 12 ).The hydroxylgrouphighlightedinblueisthepointofattachment(viaanester linkage)tothecterminalaminoacidinthepeptidechain.therestofthe peptide is then synthesized in a stepdwisefashionbyaddingoneamino acidatatime(seescheme1below).ote:asmentionedabove,proteins arenaturallysynthesizedstartingfromthedterminus,butsppstechniquessynthesizepeptidesstartingfrom thecdterminusforeaseofsynthesisandtominimizeracemizationoftheaminoacids.therefore,tosynthesize thepeptideglydargdasp,youwouldfirstaddasp,thenarg,thenglytotheresin. FmocStrategyinSPPS Sinceeachaminoacidcontainsbothanamineandcarboxylicacidfunctionalgroup,ithasthepotentialtoreact withitself.therefore,inordertosynthesizepeptidescontainingaprecisesequenceofdifferentaminoacids,we mustusecarefulprotectinggroupstrategiessothatwecancontrolwhichendoftheaminoacidcanparticipate inthecouplingreaction.neofthemostcommonlyusedprotectionstrategiesiscalledthe FmocStrategy,in which the aminedend of the amino acids used are first protected with a fluorenylmethoxycarbonyl (Fmoc) group(scheme1). 13,14 Thesederivativesarenowcommerciallyavailablefromavarietyofvendors. TheFmocgroup preventstheaminedend of the amino acid from reacting, so that the coupling is selective between the terminal aminegrouponthesolidphase resin, and the carboxylic acid group on theaminoacidtobeadded.tocontinue the growth of the peptide chain, the Fmoc group can be removed by reaction withastrongbase,suchaspiperidine,as showninscheme2. Scheme1.SynthesisofFmocDprotectedaminoacids. Figure2.Wangresinlinker. Scheme2.MechanismofFmocremovalfromthegrowingpeptide. 2

3 ThegeneralstepscarriedoutinsolidDphasepeptidesynthesisusingtheFmocstrategyareoutlinedinScheme3. Wang resin is commonly sold with one amino acid already attached. Therefore, the resin must first be deprotected byremovingthefmocgrouponthefirstaminoacid(cdterminalaminoacid)usingabasesuchas piperidine.the secondfmocdprotected aminoacidisthenattachedusingacouplingreagenttofacilitatethe reaction (see further discussion of coupling reagents below). The second amino acid is then deprotected by treatmentwithpiperidine,andthenathirdfmocaminoacidcanbecoupled.afterthedesiredpeptidelengthis reached, the peptide undergoes a final deprotection step and can be detached from the solid support using trifluoroaceticacid(tfa).whenthepeptideiscleavedfromthewangresinlinker,thecarboxylicacidterminus willberegenerated. Scheme3:PeptidesynthesisusingtheFmocstrategy. 3

4 ProtectionofReactiveSideChains Severalaminoacidscontainreactivesidechains(D,D,DS,DC)thatmustalsobeprotectedtoprevent sidedreactionsfromoccurring.theprotectinggroupsfortheseaminoacidsmustbechosencarefullysothatthey arecompatiblewiththefmocremovalconditions. 13,14 Whileawidevarietyofoptionsareavailableforallofthe different reactive amino acids, 15 select examples of common protecting groups are given in Figure 3. As discussedabove,thefmocgroupsthatblocktheendofthegrowingpeptidechainareremovedusingabase. Therefore,topreventdegradationduringsynthesis,sideDchainprotectinggroupssuchastertDbutyl(tDBu)ortertD butyloxycarbonyl(boc)canbe employedduetotheirstabilityinbasicconditions.theseparticularprotecting groupsarealsoconvenientwhenusedinconjunctionwithwangresinbeadsastheyareunstableinacid,and canberemovedduringthefinalcleavagestepofthepeptidefromtheresinbeads. Figure3.Selectexamplesofprotectinggroupsforsomeofthereactiveaminoacids. CouplingReagents Inordertogetanefficientreactionbetweenanamineandacarboxylicacidtoformanamidebond,a coupling reagent or activator mustbeused,asillustratedinscheme4.the ofacarboxylicacidisapoorleaving group, making it difficult to directly displace. Therefore, carboxylic acids are typically converted into an activatedester priortoreactioninordertofacilitatedisplacementofthe bythe 2 ontheendofthe growingpeptide. 16 Scheme4.Activationofthecarboxylicacidfacilitatesamidebondformation. 4

5 There are many different coupling reagents that have been developed for this purpose. 16 We will use D (benzotriazold1dyl)d,,, Dtetramethyluronium hexafluorophosphate (BTU),which reacts as shown in the mechanismgiveninscheme5.whilethiscompoundissoldasa uronium salt,itactuallyhastheguanidinium structureshownbelow. 17 Briefly,anFmocDprotectedaminoacidisfirstmixedwithBTUinthepresenceofbase (,Ddiisopropylethylamine, DIPEA) to convert the carboxylic acid to an ester that is activated toward nucleophilicattack.thefreeamineontheendofthegrowingpeptidechaincanthenattackthecarbonyland displacetheactivatorgroup(herehydroxybenzotriazole,bt),forminganamidebond.verthecourseofthis reactiontwobydproductsaregenerated,1,1,3,3dtetramethylureaandbt,whicharesubsequentlywashedout. Scheme5.Activationofthecarboxylicacidtofacilitateamidebondformation. 5

6 CleavageandIsolationofthePeptide Thefinalstepofthesynthesisistocleavethepeptidesfromtheresinbeads.Beforecleavage,anyremaining Fmoc groups are removed. As detailed in Scheme 6, peptides are typically detached from Wang resin using trifluoroaceticacid(tfa),whichregeneratesthecarboxylicacidonthecdterminusofthepeptide.ucleophilic scavengersareoftenaddedtothereactionmixturetopreventfurtherreactionofthebenzylcationproducedon theresin. IfthepeptidehasafreeDterminus,itwillbecomeprotonatedundertheseacidicconditions,andformasalt withtfa.ote:thepeptidewewillsynthesizeisdacylated,thuswillnotformasalt. Scheme6.CleavageofthepeptidefromtheresinusingTFA. AdvantagesandDisadvantagesofSPPS Solidphasereactionshaveadvantagesanddisadvantages. 13 Sincethepeptideisanchoredtoasolidsupportand only has one reactive end, a large excess of reagents at high concentrations can be used to drive coupling reactions to completion. Excess reagents and side products can easily be removed by filtration and washing steps after each coupling step. Disadvantages to this approach are the cost of the solid support, the limited number of linker groups on the surface of the beads, and tedious nature of repetitive stepdwise synthesis (owever,therearecommerciallyavailableinstrumentscalled peptidesynthesizers thatcandotheworkfor you). Typically, only peptides containing less than 30 amino acids are synthesized using this method. Even thoughthereactionconditionshavebeenhighlyoptimizedandarequiteefficient,ifyouget98%ofthecoupled product at each step, after the addition of 30 amino acids only ~55% of your product will have the correct sequence.therefore,longersequencesaremorecommonlyobtainedthroughexpressionbybacterialcellssuch ase.coli. 6

7 Secondary Structure Determination Thus far, we have only discussed the primary structure of peptides and proteins, which refers to the particular sequence of amino acids in the chain. owever, protein function heavily relies on the assembly of the molecule into higher order structures, referred to as secondary, tertiary and quaternary structures. ere we will focus on the secondary structure, which is governed by hydrogen bonding interactions between amide groups in the protein backbone (C=----). Depending on the location and size of the amino acid side chains in the primary structure, different domains within a protein will commonly fold into either an alpha helix (spiral) or beta Figure 4. Illustrations of alpha helix and beta sheet structures. sheet (extended) structure as illustrated in Figure 4. While some proteins will primarily fold into one structure or the other, oftentimes a single protein will have domains of both. Beta sheets can form by association of either parallel or anti-parallel strands, where the strands are either oriented in the same to C direction or in alternating directions, respectively (Figure 5). The close C=---- distances obtained in the anti-parallel beta sheet arrangement typically leads to the strongest hydrogen bonds. Figure 5. ydrogen bonding in parallel vs. anti-parallel beta sheet structures. To determine the 3D structure of proteins, X-ray crystallography and multi-dimensional MR spectroscopy are commonly employed. owever, these techniques are time consuming and require a high level of expertise to interpret the data. ere, we will utilize FTIR spectroscopy to gain some insight into the secondary structure of your peptide. The vibration of the amide C= in the peptide backbone (~ cm -1 ) is particularly sensitive to hydrogen bonds, and can be used to identify the presence of different types of secondary structures. Through a compilation of spectra of many well-characterized proteins, a consensus has emerged regarding peak assignments corresponding to beta-sheets, alpha-helices, random coils, turns, etc. as summarized in Table 1. 18,19 While FTIR analysis of proteins with several different structural domains is quite complex due to overlapping peaks, FTIR can be very useful for simple peptides such as ours. As noted in Table 1, lower C= vibration frequencies are associated with stronger hydrogen bonds. Relevant to your peptide, a prominent shift in the C= vibration from ~1640 cm -1 to ~1625 cm -1 is observed upon transition from a disordered state to a beta sheet structure, 20 due to the strong hydrogen bonds formed in an extended beta conformation. Furthermore, parallel and anti-parallel beta sheet structures can often be distinguished by a weak secondary band around 1645 cm -1 or 1690 cm -1, respectively. 18,19 7

8 Table 1. Consensus amide C= vibrations of proteins in various conformations as measured with FTIR spectroscopy. 18D20 Secondary2Structure2 Vibration2(cm 1 )2 Betasheet/extended Parallel AntiDparallel 1621D1640(strong) ~1645(weak) ~1690(weak) Alphahelix 1651D1662 Randomcoil/Disordered 1638D1655 Turns 1663D1696 Labverview ThepeptidethatyouwillsynthesizeinthislaboratoryexerciseismodeledaftertherepetitiveglycineDalanineD glycinedalaninedglycinedserine(gagags)motiffoundinsilkfibroinproducedbybombyxmorisilkworms. 21 The GAGAGSdomainsinsilkselfDassembleintohighlycrystalline,antiDparallelbetasheets,whichareresponsiblefor the characteristic strength of silk fibers. You will synthesize a peptide mimic of silk containing a short GAGA sequence with an attached alkyl tail to increase solubility and aid in characterization. nce synthesized, directionsareprovidedtoinduceselfdassemblyofthepeptideinanorganicsolvent,resultingintheformationof an organogel (gel in an organic solvent, as opposed to a hydrogel which forms in water). Following solvent evaporation,yourtaskwillbetodeducethesecondarystructureofyourpeptidexerogel(gelwiththesolvent removed)usingftirspectroscopy. 8

9 References (1)a)Bray,B.L.LargeDscalemanufactureofpeptidetherapeuticsbychemicalsynthesis.at.Rev.DrugDiscov. 2003,2,587D593.b)Robinson,J.A. ProteinepitopemimeticsasantiDinfectives.Curr.pin.Chem.Biol.2011, 15,379D86.c)Schall,.;Page,.;Macri,C.;Chaloin,.;Briand,J.P.;Muller,S.J.PeptideDbasedapproaches totreatlupusandotherautoimmunediseases.autoimmun.2012,39,143d153. (2)Matson,J.B.;Stupp,S.I.SelfDassemblingpeptidescaffoldsforregenerativemedicine.Chem.Comm.2011,48 (1), (3) Branco,M.C.;Schneider,J.P.SelfDassemblingmaterialsfortherapeuticdelivery. Acta Biomater. 2009, 5, 817D831. (4)Zhang,S.FabricationofnovelbiomaterialsthroughmolecularselfDassembly.at.Biotechnol.2003,21, (5) Mart, R. J.; sborne, R. D.; Stevens, M. M.; Ulijn, R. V. PeptideDbasedstimuliDresponsivebiomaterials.Soft Matter,2006,2,822D835. (6)Frauenrath,;Jahnke,E.AGeneralConceptforthePreparationofierarchicallyStructuredπDConjugated Polymers.Chem.Eur.J.2008,14,2942D2955. (7)Shu,J.Y.;Panganiban,B.;Xu,T.PeptideDpolymerconjugates:fromfundamentalsciencetoapplication.Annu. Rev.Phys.Chem.2013,64,631D657. (8)Reches,M.;Gazit,E.CastingMetalanowiresWithinDiscreteSelfDAssembledPeptideanotubes.Science 2003,300, (9) Lakshmanan, A.; Zhang, S.; auser, C. A. E. Short selfdassembling peptides as building blocks for modern nanodevices.trendsbiotechnol.2012,30,155d165. (10)Merrifield,R.B.SolidPhasePeptideSynthesis.I.TheSynthesisofaTetrapeptide.J.Am.Chem.Soc.1963,85, (11)SigmaDAldrichChemFilesVol.3,o.4.ResinsforSolidPhasePeptideSynthesis. (12) Wang, S.S. pdalkoxybenzyl alcohol resin and pdalkoxybenzyloxycarbonylhydrazide resin for solid phase synthesisofprotectedpeptidefragments.j.am.chem.soc.1973,95,1328d1333 (13)a)Fields,G.B.;oble,R.L.Solidphasepeptidesynthesisutilizing9Dfluorenylmethoxycarbonylaminoacids. Int.J.Pept.ProteinRes.1990,35,161D214.b)Chan,W.C.;WhiteP.D.FmocSolidPhasePeptideSynthesis:A PracticalApproach;xfordUniversityPress,ewYork,2000. (14)Carpino,L.A.;an,G.Y.The9Dfluorenylmethoxycarbonylaminoprotectinggroup.J.rg.Chem.1972,37, 3404D3409. (15)IsidroDLlobet,A.;Alvarez,M.;Albericio,F.AminoAcidDProtectingGroups.Chem.Rev.2009,109,2455D2504. (16)ElDFaham,A.;Albericio,F.Peptidecouplingreagents,morethanalettersoup.Chem.Rev.2011,111,6557D (17) Carpino, L.; Imazumi,.; ElDFaham, A.; Ferrer, F.; Zhang, C.; Lee, Y.; Foxman, B.; enklei, P.; anay, C.; Mügge,C.;Wenschuh,.;Klose,J.;Beyermann,M.;Bienert,M.TheUronium/GuanidiniumPeptideCoupling Reagents:FinallytheTrueUroniumSalts.Angew.Chem.Int.Ed.2002,41,441D445. (18) Byler, D.M.; Susi,. Examination of the Secondary Structure of Proteins by Deconvolved FTIR Spectra. Biopolymers1986,25,469D487. (19) Miyazawa,T.;Blout,E.R. TheInfraredSpectraofPolypeptidesinVariousConformations:AmideIandII Bands.J.Am.Chem.Soc.1960,83,712D719. (20)u,X.;Kaplan,D.;Cebe,P.DeterminingBetaDSheetCrystallinityinFibrousProteinsbyThermalAnalysisand InfraredSpectroscopy.Macromolecules2006,39,6161D6170. (21)Zhou,C.Z.;Confalonieri,F.;Jacquet,M.;Perasso,R.;Li,Z.G.;Janin,J.Silkfibroin:structuralimplicationsofa remarkableaminoacidsequence.protein2001,44,119d122. 9

10 Experimental Procedure PeptideSynthesisScheme Start with ala-fmoc Wang resin Fmoc Step 5: Couple gly-fmoc and remove Fmoc Step 1: Remove Fmoc 2 Step 6: Couple hexanoic acid 2 Step 2: Couple gly-fmoc Step 3: Remove Fmoc 2 Fmoc Step 4: Couple ala-fmoc and remove Fmoc Step 7: Cleave from resin (Day 2) 2 1

11 Experimental Procedure Solid1PhasePeptideSynthesis(SPPS)Procedure Thereactionvesselyouwillbeusingisshownontheleft.Itconsistsofastandardsyringebarrel,with afritinthebottom.yourinstructorwillpreloadtheresinintothebarrelofthesyringe. Standard washing procedure(useeverytimetheproceduresaysto washtheresin ): Toaddsolventtothesyringe,simplyimmerseopenendintothesolvent,andpullupontheplunger. Turnthesyringeupsidedown(plungersidedown)andswirlgentlyfor1minute. Expelthesolventintoawastecontainerbygentlypushingdownontheplunger.Takecarenotto squishthebeadsalwaysleaveacushionofairbetweenthebeadsandtheplunger. azards Mostofthesolventsandchemicalsusedinthislabaretoxic,sopreventativemeasuresshouldbetakentoavoid exposure.allstudentsshouldwearsafetyglasses,glovesandlabcoatsatalltimes,transportchemicalsinclosed vessels with secondary containment, and perform their work inside a fume hood. In particular, trifluoroacetic acidisverycorrosive,toxicandvolatile,sospecialmeasuresshouldbetakentoavoidexposureandinhalation. Tetrahydrofuran,diethylether,andpiperidinearehighlyflammableandshouldbekeptawayfromheatsources. AdditionalinformationcanbefoundintheMaterialSafetyDataSheet(MSDS)database.Reportanyspillsor incidentsimmediatelytotheinstructor.whendone,disposeofallchemicalsinappropriatewastecontainers. TAKEYURTIMEADFLLWTEDIRECTISCAREFULLY Dayne Stepne:PreparingtheResinandRemovingFmoc a) Youwillbegivenasyringeloadedwith300mgoftheWangresinthatalreadyhasoneFmocprotected alanineattached(resinhas0.72mmolofthelinkerpergramofbead) b) Washtheresin3timeswith5mLofdichloromethane(DCM).Washtheresin3moretimeswith5mLof dimethylformamide(dmf).thesewashingscausetheresintoswell. c) Add5mLof20%(v/v)piperidineinDMFandsoakfor5minutes,drain,thenwashagainwith5mLof 20%piperidineinDMF.ThisremovestheFmocprotectinggroup. d) Washtheresin3moretimeswithDMFalone(5mLeachtime)toremovethepiperidinereagent. StepTwo:GlycineCouplingProcedure e) Inaclean,dry10mLbeakercombinethefollowing:(donotcombineuntilyouarereadytouseit) 0.26g(0.86mmol)ofFmocglycine 0.33 g (0.86 mmol) (benzotriazol1yl),,,tetramethyluronium hexafluorophosphate (BTU) 1.8mLof25%diisopropylethylamine(DIPEA)inDMF f) Mixthoroughlywithaglasspipetteuntilcompletelydissolved(BTUwillactivatethecarboxylicacid), thenimmediatelydrawthissolutionintothesyringebarrelcontainingtheresin.letthissolutionsitfor 30minuteswithoccasionalswirling.Placethesyringeuprightinalargebeakertopreventleakage. g) Drainthereactionsolution,andthenwashtheresin3timeswith5mLofDMF. 2

12 Experimental Procedure StepThree:RemovingFmoc h) Repeatsteps(c)and(d)abovetoremovetheFmocgroup. StepFour:AlanineCoupling/FmocRemoval i) Repeatsteps(e)through(h),substituting0.27gofFmocalaninefortheFmocglycineinpart(e). StepFive:GlycineCoupling/FmocRemoval j) Repeatsteps(e)through(h). StepSix:AlkylChainCoupling k) Inaclean,dry10mLbeakercombine: 0.10mL(0.86mmol)hexanoicacid(liquid) 0.33g(0.86mmol)BTU 1.8mLof25%DIPEAinDMF l) Mixthoroughlywithapipetteuntilcompletelydissolved,thenimmediatelydrawthissolutionintothe syringebarrelcontainingtheresin.letthissolutionsitfor30minuteswithoccasionalswirling. m) Drainthereactionsolution,thenwashtheresinoncewith5mLmethanol,threetimeswith5mLDMF, andthreetimeswith5mldcm. ResinStorage Expel any residual solvent, label your syringe with your name,andgive to the instructor to store under refrigerationuntilthefollowinglabperiod. DayTwo StepSeven:PeptideCleavage **Trifluoroacetic-acid-(TFA)-is-a-volatile,-corrosive-acid.-Take-precautions-to-prevent-breathing-the-vapors,-and- be-careful-not-to-spill-any-on-your-skin.-- a) Draw5mLDCMintothesyringe.Letthebeadssoakfor15minuteswithoccasionalswirling(beadstend tofloatindcm).drain. b) Add5mLof95%TFAtothebeads.Thiswillcleavethepeptidefromtheresin.Letthissolutionsitin contactwiththebeadsfor1hour, swirling occasionally.ccasionally TFA collects in the tip of the syringe,andmaydripoutfromthesyringewhenswirled.tominimizedrips,pullbackontheplungerto pull any TFA that has collected in the nozzle back into the barrel prior to swirling. Place the syringe uprightinalargebeakertopreventleakage. c) d) Toensurecompleterecoveryofthepeptidefromthebeads,washtheresintwomoretimeswith4mL of95%tfaandaddeachofthewashestotheroundbottomflask. 3

13 Experimental Procedure PeptideIsolationProcedure e) RemovetheTFAbyrotaryevaporation.Again,takeprecautionstopreventinhalationoftheTFAvapors. Evaporatecompletelyuntilonlyanoilyresidueremainsonthebottomoftheflask. f) Cooltheflaskcontainingtheresidualpeptideinanicebath,andadd30mLice<coldanhydrousdiethyl ethertoprecipitatethepeptide.youshouldseeawhiteprecipitateintheflask.ifyoudonot,seethe instructor. g) Pipetthepeptide/ethermixture to two plastic centrifuge tubes (do not use glass). Splitthesolution evenlybetweenthetubes.ifalotofwhitesolidremainsintheflask,scrapeitfromthesidesandadd moreetherandtransferthistothecentrifugetubesaswell.(mayneedtodothisinbatches) h) Centrifugefor5minutesat3000rpm.Thewhitepeptidesolidshouldcollectatthebottomofthetube. i) Carefully removeanddiscard the ether with a Pasteur pipet, making sure not to disturb the peptide pellet(mayformagelinthebottomofthetube). j) Add5mLoffreshethertoeachtube,andpipettevigorouslytoresuspendthepeptidepellet(orgel). k) Centrifugefor5minutesat3000rpm. l) CarefullyremovetheetherwithaPasteurpipet,makingsurenottodisturbthepeptidepellet(orgel). m) Labelyourtubes,andsubmittotheinstructorforfreezedrying. DayThree:Characterization Yield Carefullytransferthepeptideproductfrombothcentrifugetubestoclean,taredweighpaperandrecordthe mass (preweighing the centrifuge tubes is usually not accurate enough given the small amount of peptide product).do not wear gloves during this process, as the static from the gloves will cause your peptide to go flyingcalculatethepercentyield.carefullyreturnthepeptidetooneofthetubesforstorage.doyourbestto minimizeairexposureasthepeptidetendstoabsorbmoisturefromtheair(especiallyonhumiddays),andmay collapseintoagooeyball. TLCAnalysis Inacleanglassvial,dissolveasmallflakeofyourpeptideinonedropofmethanol.SpotthissolutionontoaTLC plate,aswellasthereferencesolutionofthedesiredpeptideprovidedbyyourinstructor.developtheplatein thesolventmixtureprovided(6:1:2chloroform:glacialaceticacid:methanol).visualizethespotsontheplateby dippingtheplateinapotassiumpermanganatestain(turnspink)followedbyheatingwithaheatgununtilthe spotsappear(yellow).recordther f valuesforthereferencepeptideandthespot(s)seeninyoursample. MRSpectroscopy neortwogroupsfromeachclasswillbechosentosubmittheirsampleformr,andthespectrumwillbe sharedwiththeotherstudentsintheclass.dissolve~10mgofthesolidpeptide(usuallythesampleinoneof thecentrifugetubeswillsuffice)in0.75mldimethylsulfoxided 6.PlacesolutioninanMRtube,andobtainan 1 MRspectrumofyoursample(withthehelpoftheinstructor). ATR1FTIRSpectroscopy negroupfromeachclasswillbechosentotakeanirspectrumofthefreezedriedpeptide(beforeassembly). Thespectrumwillbesharedwiththerestoftheclass.Allgroupsshouldtakeindividualspectraoftheirxerogels. btainacopyofbothspectratoanalyzeandturninwithyourreport. 4

14 Experimental Procedure PLCAnalysis Dissolve a small portion (~1mg) of your peptide in 1 ml of the solution provided (1:1 nanopure water: acetonitrilecontaining0.1%tfa).drawthesolutionintoadisposable1mlsyringe,attacha0.2µmfiltertothe end, and expel the solution through the filter into the autosampler vial provided. Label with your name, and submittoyourinstructorforplcanalysis. MassSpectrometry InaplasticEppendorftube,dissolveasmallportion(~1mg)ofyourpeptidein0.5mLPLCgrademethanol. Labelthetubewithyourname,andsubmittoyourinstructorforMSanalysis. Self1AssemblyandFTIRAnalysis a) Combine5mgofthepeptidewith0.5mLtetrahydrofuran(TF)inacleanglassshellvial. b) Sonicateinawaterbathfor5minutes. c) eatthevialgentlyonahotplatejustuntilpeptidedissolvesorsolventbeginsboiling(verylightbubbles). TE:donotcapthevialwhileheating d) Removethevialfromheatandquicklytransferthesolutiontoa1.5mLconicalplasticEppendorftube. e) Let the solution slowlycooltoroomtemperature(~10 minutes). Do not disturb the sample during gel formation. f) Whencool,invertthetubetolookforgelformation.Carefullydecantanysolutionthatdidnotgel.Ifthe entiresampleisstillliquid,repeattheprocedure(mayneedtoaddmorepeptide). g) RemovetheTF solventunderhighvacuum seeinstructorforfurtherinstructions.(takesapprox.30 minutes). h) Take anatrftir spectrum of the dried xerogel powder, and compare with the one provided of the freezedriedproductbeforeassembly. 5