Overview of Peptide Synthesis finalpeople.reed.edu/~glasfeld/Chem392/lab/Exp2_peptide.pdf ·...

Background Information

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Overview'of'Solid-Phase'Peptide'Synthesis'(SPPS)'and'Secondary'Structure'Determination'by'FTIR'"Introduction'Proteins"are"ubiquitous" in" living"organisms"and"cells," and"can" serve"a"variety"of" functions."Proteins"can"act"as"enzymes,"hormones,"antibiotics," receptors,"or"serve"as"structural" supports" in" tissues"such"as"muscle,"hair,"and"skin." Due" to" the" high"molecular" weight" and" the" difficulty" in" isolating" significant" quantities" of"many" proteins,"scientists"have"been"working"for"decades"to"develop"methods"to"synthesize"naturally"occurring"peptides"(short"proteins)"or"protein"fragments"in"the"laboratory"in"order"to"study"or"mimic"the"structure"and"biological"activity"of"full" length"proteins."Another"motivation"to"develop"efficient"peptide"synthesis"techniques"is"the"potential"of"these"molecules"to"serve"as"therapeutic"agents.1"""More"recently,"the"natural"ability"of"peptides/proteins"to"selfDassemble"into"defined"structures"has"also"become"a"target" for"exploitation" in"a"variety"of"materials"science"and"biomedical"applications."Fibrilliar"aggregates"and"hydrogels"formed"from"peptides"and"peptide"conjugates"have"been"successfully"used"as"biomimetic"cell"culture"scaffolds,2"drug" delivery" vehicles,3"and" stimuliDresponsive" biomaterials.4 ,5"Peptides" have" also" been" used" to"control"the"morphology"of"larger"polymers,6,7"and"direct"the"assembly"of"inorganic"nanoparticles"to"form"peptide"based"wires8"and"sensors.9"As"an"introduction"to"this"rapidly"expanding"field,"this"experiment"will"cover"methods"used"to"synthesize"and"characterize"peptides,"as"well"as"evaluate"the"secondary"structure"of"a"peptide"following"selfDassembly."""Basic'Peptide'Structure'Peptides"are"formed"by"sequential"addition"of"specific"amino"acids."The"amino"acids"all"have"similar"structures"that"contain"an"amine"on"one"end"and"a"carboxylic"acid"on"the"other"(hence"the"name"‘amino"acids’),"but"they"vary" in" the"RDgroup"attached" to" the"alpha"carbon."To" form"a"peptide,"amino"acids"are" joined" ‘headDtoDtail’"by"coupling"the"amine"of"one"amino"acid"with"the"carboxylic"acid"of"another"amino"acid"to" form"an"amide"bond."The"general"structure"of"a"peptide"containing"four"amino"acids"(a"‘tetrapeptide’)"is"shown"in"Figure"1."The"end"of"the"peptide"containing"the"amine"is"called"the"‘NDterminus’"and"the"end"containing"the"carboxylic"acid"is"called"the"‘CDterminus’."Proteins"are"naturally"synthesized"starting"at"the"NDterminus,"so"by"convention,"the"amino"acid"sequence"of"a"peptide"is"typically"listed"from"the"ND"to"CDterminus."For"example,"if"your"peptide"contains"arginine,"glycine" and" aspartic" acid," the" peptide" would" be" referred" to" as" ArgDGlyDAsp" or" RGD" if" using" the" 1Dletter"abbreviation"for"each"residue."Note:"a"peptide"with"the"sequence"ArgDGlyDAsp"is"NOT"the"same"as"AspDGlyDArg."

"Solid-Phase'Peptide'Synthesis'(SPPS)!'In"order"to"efficiently"synthesize"peptides,"a"technique"known"as"‘solidDphase"peptide"synthesis’"(SPPS)"was"first"developed"in"the"1960’s.10"The"key"feature"of"SPPS"is"the"sequential"attachment"of"amino"acids"to"a"macroscopic"solid" support" matrix" (commonly" referred" to" as" resins" or" beads)." While" a" wide" variety" of" solid" supports" are"available,"some"of"the"most"common"are"made"from"small"beads"(~70D400"microns"in"size)"of"polystyrene"plastic"

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Figure'1."General"structure"of"a"peptide"containing"four"amino"acids"

Background Information

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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"batch"of"beads"is"usually"designated"by"giving"the"millimoles"of"linker"per"gram" of" beads" (mmol/g)." The" chemical" structure" of" the" particular" resin"that"we"will"use"in"this"lab"is"shown"in"Figure"2"(called"Wang"resin12)."The"hydroxyl"group"highlighted"in"blue"is"the"point"of"attachment"(via"an"ester"linkage)"to"the"C'terminal"amino"acid"in"the"peptide"chain."The"rest"of"the"peptide" is" then" synthesized" in" a" stepDwise" fashion" by" adding" one" amino"acid"at"a"time"(see"Scheme"1"below)."Note:"As"mentioned"above,"proteins"are"naturally"synthesized"starting" from"the"NDterminus,"but"SPPS"techniques"synthesize"peptides"starting" from"the"CDterminus"for"ease"of"synthesis"and"to"minimize"racemization"of"the"amino"acids."Therefore,"to"synthesize"the"peptide"GlyDArgDAsp,"you"would"first"add"Asp,"then"Arg,"then"Gly"to"the"resin."" "" "Fmoc'Strategy'in'SPPS'Since"each"amino"acid"contains"both"an"amine"and"carboxylic"acid"functional"group,"it"has"the"potential"to"react"with"itself."Therefore,"in"order"to"synthesize"peptides"containing"a"precise"sequence"of"different"amino"acids,"we"must"use"careful"protecting"group"strategies"so"that"we"can"control"which"end"of"the"amino"acid"can"participate"in"the"coupling"reaction."One"of"the"most"commonly"used"protection"strategies"is"called"the"‘Fmoc"Strategy’,"in"which" the" amineDend" of" the" amino" acids" used" are" first" ‘protected’" with" a" fluorenylmethoxycarbonyl" (Fmoc)"group"(Scheme"1).13,14"These"derivatives"are"now"commercially"available"from"a"variety"of"vendors."""The" Fmoc" group"prevents" the" amineDend"of" the" amino" acid" from" reacting," so" that"the" coupling" is" selective" between" the"terminal" amine" group"on" the" solid" phase"resin," and" the" carboxylic" acid" group" on"the"amino"acid" to"be"added."To"continue"the" growth" of" the" peptide" chain," the"Fmoc" group" can" be" removed" by" reaction"with"a"strong"base,"such"as"piperidine,"as"shown"in"Scheme"2."

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Figure'2.'Wang"resin"linker."

Scheme'1."Synthesis"of"FmocDprotected"amino"acids.""

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Scheme'2."Mechanism"of"Fmoc"removal"from"the"growing"peptide.""

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Background Information

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The"general"steps"carried"out"in"solidDphase"peptide"synthesis"using"the"Fmoc"strategy"are"outlined"in"Scheme"3."Wang" resin" is" commonly" sold" with" one" amino" acid" already" attached." Therefore," the" resin" must" first" be"‘deprotected’"by"removing"the"Fmoc"group"on"the"first"amino"acid"(CDterminal"amino"acid)"using"a"base"such"as"piperidine." The" second" FmocDprotected" amino" acid" is" then" attached"using" a" coupling" reagent" to" facilitate" the"reaction" (see" further" discussion" of" coupling" reagents" below)." The" second" amino" acid" is" then" deprotected" by"treatment"with"piperidine,"and"then"a"third"Fmoc"amino"acid"can"be"coupled."After"the"desired"peptide"length"is"reached," the" peptide" undergoes" a" final" deprotection" step" and" can" be" detached" from" the" solid" support" using"trifluoroacetic"acid"(TFA)."When"the"peptide"is"cleaved"from"the"Wang"resin"linker,"the"carboxylic"acid"terminus"will"be"regenerated."""

'Scheme'3:"Peptide"synthesis"using"the"Fmoc"strategy."

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Background Information

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Protection'of'Reactive'Side'Chains'Several"amino"acids"contain"reactive"side"chains"(DOH,"DNH,"DSH,"DCOOH)"that"must"also"be"protected"to"prevent"

sideDreactions"from"occurring."The"protecting"groups"for"these"amino"acids"must"be"chosen"carefully"so"that"they"

are"compatible"with"the"Fmoc"removal"conditions.13,14

"While"a"wide"variety"of"options"are"available"for"all"of"the"

different" reactive" amino" acids,15"select" examples" of" common" protecting" groups" are" given" in" Figure" 3." As"

discussed"above,"the"Fmoc"groups"that"block"the"end"of"the"growing"peptide"chain"are"removed"using"a"base."

Therefore,"to"prevent"degradation"during"synthesis,"sideDchain"protecting"groups"such"as"tertDbutyl"(tDBu)"or"tertDbutyloxycarbonyl" (Boc)" can"be"employed"due" to" their" stability" in" basic" conditions." These"particular" protecting"

groups"are"also"convenient"when"used"in"conjunction"with"Wang"resin"beads"as"they"are"unstable" in"acid,"and"

can"be"removed"during"the"final"cleavage"step"of"the"peptide"from"the"resin"beads."

Coupling'Reagents'In"order"to"get"an"efficient"reaction"between"an"amine"and"a"carboxylic"acid"to"form"an"amide"bond,"a"‘coupling"

reagent’"or" ‘activator’"must"be"used,"as" illustrated" in"Scheme"4."The"–OH"of"a"carboxylic"acid" is"a"poor" leaving"

group," making" it" difficult" to" directly" displace." Therefore," carboxylic" acids" are" typically" converted" into" an"

‘activated"ester’"prior"to"reaction"in"order"to"facilitate"displacement"of"the"–OH"by"the"–NH2"on"the"end"of"the"

growing"peptide.16""

''Figure'3."Select"examples"of"protecting"groups"for"some"of"the"reactive"amino"acids."

Scheme'4."Activation"of"the"carboxylic"acid"facilitates"amide"bond"formation.""

"

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Background Information

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"There" are" many" different" coupling" reagents" that" have" been" developed" for" this" purpose.16"We" will" use" OD(benzotriazolD1Dyl)DN,N,N’,N’Dtetramethyluronium" hexafluorophosphate" (HBTU),which" reacts" as" shown" in" the"mechanism"given"in"Scheme"5."While"this"compound"is"sold"as"a"‘uronium’"salt,"it"actually"has"the"guanidinium"structure"shown"below.17"Briefly,"an"FmocDprotected"amino"acid"is"first"mixed"with"HBTU"in"the"presence"of"base"(N,NDdiisopropylethylamine," DIPEA)" to" convert" the" carboxylic" acid" to" an" ester" that" is" ‘activated’" toward"nucleophilic"attack."The"free"amine"on"the"end"of"the"growing"peptide"chain"can"then"attack"the"carbonyl"and"displace"the"activator"group"(here"hydroxybenzotriazole,"HOBt),"forming"an"amide"bond."Over"the"course"of"this"reaction"two"byDproducts"are"generated,"1,1,3,3Dtetramethylurea"and"HOBt,"which"are"subsequently"washed"out.""""

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'

'

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Scheme'5."Activation"of"the"carboxylic"acid"to"facilitate"amide"bond"formation."""

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Background Information

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Cleavage'and'Isolation'of'the'Peptide'The" final" step"of" the"synthesis" is" to"cleave" the"peptides" from"the" resin"beads."Before"cleavage,"any" remaining"Fmoc" groups" are" removed." As" detailed" in" Scheme" 6," peptides" are" typically" detached" from"Wang" resin" using"trifluoroacetic"acid"(TFA),"which"regenerates"the"carboxylic"acid"on"the"CDterminus"of"the"peptide."Nucleophilic"scavengers"are"often"added"to"the"reaction"mixture"to"prevent"further"reaction"of"the"benzyl"cation"produced"on"the"resin."

If" the"peptide"has"a" free"NDterminus," it"will"become"protonated"under"these"acidic"conditions,"and"form"a"salt"with"TFA."Note:"The"peptide"we"will"synthesize"is"NDacylated,"thus"will"not"form"a"salt."""

'

''Advantages'and'Disadvantages'of'SPPS'"Solid"phase"reactions"have"advantages"and"disadvantages.13"Since"the"peptide"is"anchored"to"a"solid"support"and"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"(However," there"are"commercially"available" instruments"called" ‘peptide"synthesizers’" that"can"do"the"work"for"you!)." Typically," only" peptides" containing" less" than" 30" amino" acids" are" synthesized" using" this" method." Even"though"the"reaction"conditions"have"been"highly"optimized"and"are"quite"efficient,"if"you"get"98%"of"the"coupled"product" at" each" step," after" the" addition" of" 30" amino" acids" only" ~55%" of" your" product" will" have" the" correct"sequence."Therefore,"longer"sequences"are"more"commonly"obtained"through"expression"by"bacterial"cells"such"as"E.'coli.""""''''

Scheme'6."Cleavage"of"the"peptide"from"the"resin"using"TFA.""

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Background Information

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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. However, protein function heavily relies on the assembly of the molecule into higher order structures, referred to as secondary, tertiary and quaternary structures. Here we will focus on the secondary structure, which is governed by hydrogen bonding interactions between amide groups in the protein backbone (C=O---H-N). 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 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 N to C direction or in alternating directions, respectively (Figure 5). The close C=O---H-N distances obtained in the anti-parallel beta sheet arrangement typically leads to the strongest hydrogen bonds.

To determine the 3D structure of proteins, X-ray crystallography and multi-dimensional NMR spectroscopy are commonly employed. However, these techniques are time consuming and require a high level of expertise to interpret the data. Here, we will utilize FTIR spectroscopy to gain some insight into the secondary structure of your peptide. The vibration of the amide C=O in the peptide backbone (~1600-1700 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=O vibration frequencies are associated with stronger hydrogen bonds. Relevant to your peptide, a prominent shift in the C=O 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

Figure 4. Illustrations of alpha helix and beta sheet structures.

Figure 5. Hydrogen bonding in parallel vs. anti-parallel beta sheet structures.

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'''Table' 1." Consensus" amide" C=O" vibrations" of" proteins" in" various" conformations" as" measured" with" FTIR"spectroscopy.18D20""Secondary2Structure2 Vibration2(cm'1)2Beta"sheet/"extended""""""""""""""Parallel""""""""""""""AntiDparallel"

1621D1640"(strong)"~1645"(weak)"~1690"(weak)"

Alpha"helix" 1651D1662"

Random"coil/"Disordered" 1638D1655"

Turns" 1663D1696""""Lab'Overview''"The"peptide"that"you"will"synthesize" in"this" laboratory"exercise" is"modeled"after"the"repetitive"glycineDalanineDglycineDalanineDglycineDserine" (GAGAGS)"motif" found" in" silk" fibroin"produced"by"Bombyx'mori" silkworms.21"The"GAGAGS"domains"in"silk"selfDassemble"into"highly"crystalline,"antiDparallel"beta"sheets,"which"are"responsible"for"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." Once" synthesized,"directions"are"provided"to"induce"selfDassembly"of"the"peptide"in"an"organic"solvent,"resulting"in"the"formation"of"an" organogel" (gel" in" an" organic" solvent," as" opposed" to" a" hydrogel" which" forms" in" water)." Following" solvent"evaporation,"your"task"will"be"to"deduce"the"secondary"structure"of"your"peptide"xerogel"(gel"with"the"solvent"removed)"using"FTIR"spectroscopy."""""""""""""""""""""""""

Background Information

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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!References''(1)"a)"Bray,"B.L."LargeDscale"manufacture"of"peptide"therapeutics"by"chemical"synthesis."Nat.'Rev.'Drug'Discov."

2003,"2,"587D593."b)"Robinson,"J.A."“Protein"epitope"mimetics"as"antiDinfectives."Curr.'Opin.'Chem.'Biol."2011,"15,"379D86."c)"Schall,"N.;"Page,"N.;"Macri,"C.;"Chaloin,"O.;"Briand,"J.P.;"Muller,"S."J."PeptideDbased"approaches"to"treat"lupus"and"other"autoimmune"diseases."Autoimmun."2012,"39,"143D153.""

(2)"Matson,"J."B.;"Stupp,"S."I."SelfDassembling"peptide"scaffolds"for"regenerative"medicine."Chem.'Comm.'2011,'48'(1),"26–33."

(3)" Branco,"M." C.;" Schneider," J." P." SelfDassembling"materials" for" therapeutic" delivery."Acta' Biomater."2009,"5,"817D831."

(4)"Zhang,"S."Fabrication"of"novel"biomaterials"through"molecular"selfDassembly."Nat.'Biotechnol."2003,"21,"1171–1178."

(5)"Mart,"R." J.;"Osborne,"R."D.;" Stevens,"M."M.;"Ulijn,"R."V."PeptideDbased"stimuliDresponsive"biomaterials."Soft'Matter,"2006,"2,"822D835."

(6)"Frauenrath,"H;" Jahnke,"E."A"General"Concept" for" the"Preparation"of"Hierarchically"Structured"πDConjugated"Polymers."Chem.'Eur.'J."2008,"14,"2942D2955."

(7)"Shu,"J.Y.;"Panganiban,"B.;"Xu,"T."PeptideDpolymer"conjugates:"from"fundamental"science"to"application."Annu.'Rev.'Phys.'Chem."2013,"64,"631D657."

(8)"Reches,"M.;"Gazit,"E."Casting"Metal"Nanowires"Within"Discrete"SelfDAssembled"Peptide"Nanotubes."Science"2003,"300,"625–627."

(9)" Lakshmanan," A.;" Zhang," S.;" Hauser," C." A." E." Short" selfDassembling" peptides" as" building" blocks" for"modern"nanodevices."Trends'Biotechnol."2012,"30,"155D165.!

(10)"Merrifield,"R.B."Solid"Phase"Peptide"Synthesis."I."The"Synthesis"of"a"Tetrapeptide."J.'Am.'Chem.'Soc."1963,"85,"2149–2154."

(11)""SigmaDAldrich"ChemFiles"Vol."3,"No."4."Resins"for"Solid"Phase"Peptide"Synthesis."(12)" Wang," S.S." pDAlkoxybenzyl" alcohol" resin" and" pDalkoxybenzyloxycarbonylhydrazide" resin" for" solid" phase"

synthesis"of"protected"peptide"fragments."J.'Am.'Chem.'Soc."1973,"95,"1328D1333"(13)"a)"Fields,"G.B.;"Noble,"R.L."Solid"phase"peptide"synthesis"utilizing"9Dfluorenylmethoxycarbonyl"amino"acids."

Int.'J.'Pept.'Protein'Res.'1990,"35,"161D214."b)"Chan,"W.C.;"White"P.D."Fmoc'Solid'Phase'Peptide'Synthesis:'A'Practical'Approach;'Oxford"University"Press,"New"York,"2000."

(14)" "Carpino,"L.A.;"Han,"G.Y."The"9Dfluorenylmethoxycarbonyl"amino"protecting"group."J.'Org.'Chem."1972,"37,"3404D3409."

(15)"IsidroDLlobet,"A.;"Alvarez,"M.;"Albericio,"F."Amino"AcidDProtecting"Groups."Chem.'Rev."2009,"109,"2455D2504."!(16)"ElDFaham,"A.;"Albericio,"F."Peptide"coupling"reagents,"more"than"a"letter"soup."Chem.'Rev."2011,"111,"6557D

6602."!(17)" Carpino," L.;" Imazumi," H.;" ElDFaham," A.;" Ferrer," F.;" Zhang," C.;" Lee," Y.;" Foxman," B.;" Henklei," P.;" Hanay," C.;"

Mügge,"C.;"Wenschuh,"H.;"Klose,"J.;"Beyermann,"M.;"Bienert,"M."The"Uronium/Guanidinium"Peptide"Coupling"Reagents:"Finally"the"True"Uronium"Salts."Angew.'Chem.'Int.'Ed.""2002,"41,"441D445.""

(18)" Byler," D.M.;" Susi," H." Examination" of" the" Secondary" Structure" of" Proteins" by" Deconvolved" FTIR" Spectra."Biopolymers"1986,"25,"469D487."

(19)"Miyazawa," T.;" Blout," E." R." The" Infrared" Spectra" of" Polypeptides" in"Various"Conformations:"Amide" I" and" II"Bands."J.'Am.'Chem.'Soc."1960,"83,"712D719."

(20)"Hu,"X.;"Kaplan,"D.;"Cebe,"P."Determining"BetaDSheet"Crystallinity"in"Fibrous"Proteins"by"Thermal"Analysis"and"Infrared"Spectroscopy."Macromolecules"2006,"39,"6161D6170."

(21)"Zhou,"C.Z.;"Confalonieri,"F.;"Jacquet,"M.;"Perasso,"R.;"Li,"Z.G.;"Janin,"J."Silk"fibroin:"structural"implications"of"a"remarkable"amino"acid"sequence."Protein"2001,"44,"119D122.

Experimental Procedure

! 1!

!Peptide!Synthesis!Scheme!!!!

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OHN

O

ONH

NH2O

OHN

O

ONH2

Step 3: Remove Fmoc

Step 6: Couple hexanoic acid

Step 7: Cleave from resin (Day 2)

Step 4: Couple ala-Fmocand remove Fmoc

Start with ala-Fmoc Wang resin

OHN

O

ONH

HN

O

ONH2

OHN

O

ONH

HN

O

ONH

HOHN

O

ONH

HN

O

ONH

O

OHN

O

ONHFmoc

Step 1: Remove Fmoc

O

OHN

O

Fmoc

Step 2: Couple gly-Fmoc

ONH2

O

Step 5: Couple gly-Fmocand remove Fmoc

Experimental Procedure

! 2!

Solid1Phase!Peptide!Synthesis!(SPPS)!Procedure!!"The!reaction!vessel!you!will!be!using!is!shown!on!the!left.!It!consists!of!a!standard!syringe!barrel,!with!a!frit!in!the!bottom.!Your!instructor!will!pre"load!the!resin!into!the!barrel!of!the!syringe.!!!Standard!‘washing’!procedure!(use!every!time!the!procedure!says!to!‘wash!the!resin’):!!"To!add!solvent!to!the!syringe,!simply!immerse!open!end!into!the!solvent,!and!pull!up!on!the!plunger.!!"Turn!the!syringe!upside"down!(plunger!side!down)!and!swirl!gently!for!1!minute.!!"Expel! the!solvent! into!a!waste!container!by!gently!pushing!down!on! the!plunger.!Take!care!not! to!squish!the!beads"!always!leave!a!cushion!of!air!between!the!beads!and!the!plunger.!!!

!

Hazards!Most!of!the!solvents!and!chemicals!used!in!this!lab!are!toxic,!so!preventative!measures!should!be!taken!to!avoid!exposure.!All!students!should!wear!safety!glasses,!gloves!and!lab!coats!at!all!times,!transport!chemicals!in!closed!vessels!with!secondary!containment,!and!perform!their!work! inside!a! fume!hood.! In!particular,! trifluoroacetic!acid!is!very!corrosive,!toxic!and!volatile,!so!special!measures!should!be!taken!to!avoid!exposure!and!inhalation.!Tetrahydrofuran,!diethyl!ether,!and!piperidine!are!highly!flammable!and!should!be!kept!away!from!heat!sources.!Additional! information! can!be! found! in! the!Material! Safety!Data! Sheet! (MSDS)!database.!Report! any! spills! or!incidents!immediately!to!the!instructor.!When!done,!dispose!of!all!chemicals!in!appropriate!waste!containers.!

!!

TAKE!YOUR!TIME!AND!FOLLOW!THE!DIRECTIONS!CAREFULLY!!!

Day!One!!Step!One:!Preparing!the!Resin!and!Removing!Fmoc!!

a) You!will!be!given!a!syringe!loaded!with!300!mg!of!the!Wang!resin!that!already!has!one!Fmoc"protected!alanine!attached!(resin!has!0.72!mmol!of!the!linker!per!gram!of!bead)!!

b) Wash!the!resin!3!times!with!5!mL!of!dichloromethane!(DCM).!Wash!the!resin!3!more!times!with!5!mL!of!dimethylformamide!(DMF).!These!washings!cause!the!resin!to!swell.!!

c) Add!5!mL!of!20%!(v/v)!piperidine!in!DMF!and!soak!for!5!minutes,!drain,!then!wash!again!with!5!mL!of!20%!piperidine!in!DMF.!This!removes!the!Fmoc!protecting!group.!!

d) Wash!the!resin!3!more!times!with!DMF!alone!(5!mL!each!time)!to!remove!the!piperidine!reagent.!!

Step!Two:!Glycine!Coupling!Procedure!e) In!a!clean,'dry'10!mL!beaker!combine!the!following:!(do!not!combine!until!you!are!ready!to!use!it)!!

• 0.26!g!(0.86!mmol)!of!Fmoc"glycine!!• 0.33! g! (0.86! mmol)! O"(benzotriazol"1"yl)"N,N,N',N'"tetramethyluronium! hexafluorophosphate!

(HBTU)!• 1.8!mL!of!25%!diisopropylethylamine!(DIPEA)!in!DMF!!

f) Mix!thoroughly!with!a!glass!pipette!until!completely!dissolved!(HBTU!will!activate!the!carboxylic!acid),!then!immediately!draw!this!solution!into!the!syringe!barrel!containing!the!resin.!Let!this!solution!sit!for!30!minutes!with!occasional!swirling.!Place!the!syringe!upright!in!a!large!beaker!to!prevent!leakage.!

g) Drain!the!reaction!solution,!and!then!wash!the!resin!3!times!with!5!mL!of!DMF.!!

Experimental Procedure

! 3!

!Step!Three:!Removing!Fmoc!!

h) Repeat!steps!(c)!and!(d)!above!to!remove!the!Fmoc!group.!!!Step!Four:!Alanine!Coupling/!Fmoc!Removal!!

i) Repeat!steps!(e)!through!(h),!substituting!0.27!g!of!Fmoc"alanine!for!the!Fmoc"glycine!in!part!(e).!!

Step!Five:!Glycine!Coupling/!Fmoc!Removal!!j) Repeat!steps!(e)!through!(h).!

!Step!Six:!Alkyl!Chain!Coupling!!

k) In!a!clean,'dry!10!mL!beaker!combine:!!!• 0.10!mL!(0.86!mmol)!hexanoic!acid!(liquid)!• 0.33!g!(0.86!mmol)!HBTU!• 1.8!mL!of!25%!DIPEA!in!DMF!!

l) Mix!thoroughly!with!a!pipette!until!completely!dissolved,!then!immediately!draw!this!solution!into!the!syringe!barrel!containing!the!resin.!Let!this!solution!sit!for!30!minutes!with!occasional!swirling.!!

m) Drain!the!reaction!solution,!then!wash!the!resin!once!with!5!mL!methanol,!three!times!with!5!mL!DMF,!and!three!times!with!5!mL!DCM.!!

Resin!Storage!Expel! any! residual! solvent,! label! your! syringe! with! your! name,! and! give! to! the! instructor! to! store! under!refrigeration!until!the!following!lab!period.!!!!Day!Two!!Step!Seven:!Peptide!Cleavage!!**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) Draw!5!mL!DCM!into!the!syringe.!Let!the!beads!soak!for!15!minutes!with!occasional!swirling!(beads!tend!to!float!in!DCM).!Drain.!!

b) Add!5!mL!of!95%!TFA!to!the!beads.!This!will!cleave!the!peptide! from!the!resin.!Let! this!solution!sit! in!contact! with! the! beads! for! 1! hour,! swirling! occasionally.! Occasionally! TFA! collects! in! the! tip! of! the!syringe,!and!may!drip!out!from!the!syringe!when!swirled.!To!minimize!drips,!pull!back!on!the!plunger!to!pull! any! TFA! that! has! collected! in! the! nozzle! back! into! the! barrel! prior! to! swirling.! Place! the! syringe!upright!in!a!large!beaker!to!prevent!leakage.!!

c) Expel!the!TFA!solution!containing!the!peptide!into!a!50"mL!round!bottom!flask.!Do-not-throw-away-this-solution-@-it-contains-your-peptide!!!

d) To!ensure!complete!recovery!of!the!peptide!from!the!beads,!wash!the!resin!two!more!times!with!4!mL!of!95%!TFA!and!add!each!of!the!washes!to!the!round!bottom!flask.!!

!!!!

Experimental Procedure

! 4!

Peptide!Isolation!Procedure!e) Remove!the!TFA!by!rotary!evaporation.!Again,'take'precautions'to'prevent'inhalation'of'the'TFA'vapors.!

Evaporate!completely!until!only!an!oily!residue!remains!on!the!bottom!of!the!flask.!f) Cool!the!flask!containing!the!residual!peptide!in!an!ice!bath,!and!add!30!mL! ice<cold'anhydrous!diethyl!

ether!to!precipitate!the!peptide.!You!should!see!a!white!precipitate!in!the!flask.!If!you!do!not,!see!the!instructor.!

g) Pipet! the! peptide/ether!mixture! to! two!plastic' centrifuge! tubes! (do! not! use! glass).! Split! the! solution!evenly!between!the!tubes.!If!a! lot!of!white!solid!remains!in!the!flask,!scrape!it!from!the!sides!and!add!more!ether!and!transfer!this!to!the!centrifuge!tubes!as!well.!(may!need!to!do!this!in!batches)!!

h) Centrifuge!for!5!minutes!at!3000!rpm.!The!white!peptide!solid!should!collect!at!the!bottom!of!the!tube.!!i) Carefully! remove! and!discard! the! ether!with! a! Pasteur! pipet,!making! sure! not! to! disturb! the! peptide!

pellet!(may!form!a!gel!in!the!bottom!of!the!tube).!!j) Add!5!mL!of!fresh!ether!to!each!tube,!and!pipette!vigorously!to!re"suspend!the!peptide!pellet!(or!gel).!!!k) Centrifuge!for!5!minutes!at!3000!rpm.!!l) Carefully!remove!the!ether!with!a!Pasteur!pipet,!making!sure!not!to!disturb!the!peptide!pellet!(or!gel).!!m) Label!your!tubes,!and!submit!to!the!instructor!for!freeze"drying.!

!!Day!Three:!Characterization!

!Yield!Carefully! transfer! the!peptide!product! from!both! centrifuge!tubes!to!clean,! tared!weigh!paper!and!record!the!mass! (pre"weighing! 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!flying!!Calculate!the!percent!yield.!Carefully!return!the!peptide!to!one!of!the!tubes!for!storage.!Do!your!best!to!minimize!air!exposure!as!the!peptide!tends!to!absorb!moisture!from!the!air!(especially!on!humid!days),!and!may!collapse!into!a!gooey!ball.!!!TLC!Analysis!In!a!clean!glass!vial,!dissolve!a!small!flake!of!your!peptide!in!one!drop!of!methanol.!Spot!this!solution!onto!a!TLC!plate,!as!well!as!the!reference!solution!of!the!desired!peptide!provided!by!your!instructor.!Develop!the!plate!in!the!solvent!mixture!provided!(6:1:2!chloroform:!glacial!acetic!acid:!methanol).!Visualize!the!spots!on!the!plate!by!dipping!the!plate!in!a!potassium!permanganate!stain!(turns!pink)!followed!by!heating!with!a!heat!gun!until!the!spots!appear!(yellow).!Record!the!Rf!values!for!the!reference!peptide!and!the!spot(s)!seen!in!your!sample.!!NMR!Spectroscopy!One!or! two!groups! from!each!class!will!be!chosen! to!submit! their! sample! for!NMR,!and! the!spectrum!will!be!shared!with!the!other!students!in!the!class.!Dissolve!~10!mg!of!the!solid!peptide!(usually!the!sample!in!one!of!the!centrifuge!tubes!will!suffice)!in!0.75!mL!dimethyl!sulfoxide"d6.!Place!solution!in!an!NMR!tube,!and!obtain!an!1H!NMR!spectrum!of!your!sample!(with!the!help!of!the!instructor).!!ATR1FTIR!Spectroscopy!One!group!from!each!class!will!be!chosen!to!take!an!IR!spectrum!of!the!freeze"dried!peptide!(before!assembly).!The!spectrum!will!be!shared!with!the!rest!of!the!class.!All!groups!should!take!individual!spectra!of!their!xerogels.!Obtain!a!copy!of!both!spectra!to!analyze!and!turn!in!with!your!report.!!!!

Experimental Procedure

! 5!

HPLC!Analysis!Dissolve! a! small! portion! (~1! mg)! of! your! peptide! in! 1! mL! of! the! solution! provided! (1:1! nanopure! water:!acetonitrile!containing!0.1%!TFA).!Draw!the!solution!into!a!disposable!1!mL!syringe,!attach!a!0.2!µm!filter!to!the!end,! and!expel! the! solution! through! the! filter! into! the!autosampler! vial!provided.! Label!with!your!name,!and!submit!to!your!instructor!for!HPLC!analysis.!!Mass!Spectrometry!In!a!plastic!Eppendorf! tube,!dissolve!a!small!portion! (~1!mg)!of!your!peptide! in!0.5!mL!HPLC!grade!methanol.!Label!the!tube!with!your!name,!and!submit!to!your!instructor!for!MS!analysis.!!!Self1Assembly!and!FTIR!Analysis!a)! Combine!5!mg!of!the!peptide!with!0.5!mL!tetrahydrofuran!(THF)!in!a!clean!glass!shell!vial.!b)! Sonicate!in!a!water!bath!for!5!minutes.!c)!!! Heat!the!vial!gently!on!a!hot!plate!just!until!peptide!dissolves!or!solvent!begins!boiling!(very!light!bubbles).!

NOTE:!do!not!cap!the!vial!while!heating!!d)! Remove!the!vial!from!heat!and!quickly!transfer!the!solution!to!a!1.5!mL!conical!plastic!Eppendorf!tube.!e)! Let! the! solution! slowly! cool! to! room! temperature! (~10!minutes).! Do! not! disturb! the! sample! during! gel!

formation.!f)! When!cool,!invert!the!tube!to!look!for!gel!formation.!Carefully!decant!any!solution!that!did!not!gel.!If!the!

entire!sample!is!still!liquid,!repeat!the!procedure!(may!need!to!add!more!peptide).!g)! Remove! the!THF! solvent!under!high! vacuum!–! see! instructor! for! further! instructions.! (takes! approx.! 30!

minutes).!h)! Take! an! ATR"FTIR! spectrum! of! the! dried! ‘xerogel’! powder,! and! compare!with! the! one! provided! of! the!

freeze"dried!product!before!assembly.!!

!!