Background Information

! 1!

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"

!!

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

Background Information

! 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"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."

"'

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

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

"

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

"

Background Information

! 3!

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."

!"'

Background Information

! 4!

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.""

"

"

Background Information

! 5!

"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.""""

'

'

'

'

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

""

Background Information

! 6!

'

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.""

""""""

Background Information

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. 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.

Background Information

! 8!

'''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

! 9!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!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!!!!

!!!!!!!!!!!

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.!!

!!