Background Information

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."

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

"
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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Background Information

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" Figure'2.'Wang"resin"linker."
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" Scheme'1."Synthesis"of"FmocDprotected"amino"acids."
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."

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

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Background Information

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

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"tertD
butyloxycarbonyl" (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."

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

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

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Background Information

"
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.""
"

"
"

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Background Information

'
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."""
'

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

"
""
'"
'"
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.""
"
"
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Background Information

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 Figure 4. Illustrations of alpha helix and beta sheet
structure, different domains within a protein will structures.
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.

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

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

7
Background Information

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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)2
Beta"sheet/"extended" 1621D1640"(strong)"
"""""""""""""Parallel" ~1645"(weak)"
"""""""""""""AntiDparallel" ~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"glycineDalanineD
glycineDalanineDglycineDserine" (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."
"
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Background Information

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
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.

! 9!
Experimental Procedure

!
Peptide!Synthesis!Scheme!
!
!
!
Start with ala-Fmoc Wang resin
O
H
N Step 5: Couple gly-Fmoc
O Fmoc
and remove Fmoc

Step 1: Remove Fmoc O O

H H
N N
O N NH2
O H
O O
NH2
O
Step 6: Couple hexanoic acid

Step 2: Couple gly-Fmoc

O O O
H H
O N N
H O N N
N Fmoc H H
O N O O
H
O
Step 7: Cleave from resin (Day 2)
Step 3: Remove Fmoc
O O O
O H H
H N N
N HO N N
O NH2 H H
O O
O
Step 4: Couple ala-Fmoc
and remove Fmoc
O O
H
N NH2
O N
H
O

!
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!
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!
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!
!

! 1!
Experimental Procedure

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.!
!

! 2!
Experimental Procedure

!
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.!!
!
!
!
!

! 3!
Experimental Procedure

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!
1
H!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.!!
!
!

! 4!
Experimental Procedure

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.!
!
!!

! 5!
Instructor Notes

Instructor)Notes)
!
Equipment)(for)the)entire)experiment))
4!dispensing!pumps!for!solvents!(DCM,!DMF,!20%!piperidine!in!DMF!and!methanol)!
Analytical!balances!(±!0.1!mg)!
Rotary!Evaporator!
Centrifuge!
Vacuum!pump!and!manifold!
Lyophilizer!
Heat!gun!
Water!bath!sonicator!
Hot!plate!
)
)
Glassware)and)consumables)(per)group))
Nitrile!gloves!
weigh!paper!
glass!Pasteur!pipettes!
2!beakers!(250!mL)!for!halogenated!and!nonQhalogenated!waste!
3!glass!shell!vials!(1!dram)!for!the!TLC!solution!(1),!xerogel!solution!(1),!and!NMR!solution!(1)!
4Q!10!mL!beakers!for!the!solvents!and!coupling!agent!mixtures!!
1!spatula!
1!polypropylene!syringe!with!a!frit!(10!mL!Torviq!disposable!reaction!vessel,!www.torviq.com)!
1!round!bottom!flask!(50!mL)!to!evaporate!TFA!
2!plastic!centrifuge!tubes!(15!mL!or!larger)!
1!silica!gel!TLC!plate!(~1x3”)!!
2!capillary!tubes!for!spotting!the!TLC!plate!
TLC!development!chamber!with!lid!
1!NMR!tube!
1!HPLC!autosampler!vial!
2!plastic!conical!Eppendorf!tubes!(1.5!mL)!for!xerogel!formation,!and!for!MS!sample.!
1!scintillation!vial!(20!mL)!and!vial!adapter!(such!as!Chemglass!CGQ1318Q40)!for!evaporation!of!xerogel!solvent!
1!disposable!plastic!syringe!(1!mL)!
1!syringe!filter!(4!mm!diameter,!0.2!µm!pore!size)!
!
!
Instrumentation)
The!specifications!of!the!instrumentation!used!to!collect!the!data!shown!are!given!below.!Modifications!may!be!
needed!to!the!characterization!procedures!if!different!instrumentation!is!used.!!
!
FTIR:! Thermo! Scientific! Nicolet! iS10! FTQIR! Spectrometer! equipped! with! an! attenuated! total! reflectance! (ATR)!
accessory.!
NMR:!Varian!Inova!500!MHz!FTQNMR!Spectrometer!(a!300!MHz!spectrometer!would!also!be!fine)!
HPLC:!Thermo!Scientific!Dionex!Ultimate!3000!with!diode!array!detector.!Column:!Phenomenex!150!x!4.6!mm!
C18!column!with!5!µm!pore!size.!λmax!=!210!nm!for!detection!
Mass.spectrometry:!Applied!Biosystems!API!2000!Triple!Quadrupole!Mass!Spectrometer!running!in!negative!ion!
mode.!!

! 1!
Instructor Notes

Hazards)and)Safety)
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,! and! perform! their! work!
inside! a! fume! hood.! Hazards! associated! with! each! chemical! are! given! in! the! table! below.! In! particular,!
trifluoroacetic!acid!is!very!corrosive,!toxic!and!volatile,!so!special!measures!should!be!taken!to!avoid!exposure!
and! inhalation.! Additional! information! can! be! found! in! the! Material! Safety! Data! Sheet! (MSDS)! database.!
Students! should! be! instructed! to! report! any! incidents! immediately! to! the! instructor! and! to! dispose! of! all!
chemicals!in!appropriate!waste!containers.!
)
Chemicals)(per)group))
The!name,!CAS!number,!and!hazards!associated!with!each!chemical!are!given!in!the!table!below.!The!amounts!
given!are!per!group!(usually!groups!consist!of!2!students),!and!it!is!recommended!to!have!~15%!extra!of!each!on!
hand!in!case!of!spillage/overuse.!

Amount)needed)
Chemical)Name) CAS)number) per)group) Hazards)
N,NQdimethylformamide!(DMF)!
[68Q12Q2]! 150!mL! Toxic,!flammable,!reproductive!toxin!
(Peptide!Synthesis!Grade)!
[110Q89Q4]! Highly!flammable,!toxic,!caustic,!
20%!(v/v)!Piperidine!in!DMF! 40!mL!
[68Q12Q2]! reproductive!toxin!
Dichloromethane! [75Q09Q2]) 35!mL! Toxic,!carcinogen!
Methanol!(HPLC!grade)! [67Q56Q1]! 5!mL!+!0.5!mL! Toxic,!flammable!
Extremely!flammable,!toxic;!May!form!
Anhydrous!diethyl!ether!) [60Q29Q7]! 40!mL!
explosive!peroxides!
Extremely!flammable,!carcinogen,!
Tetrahydrofuran! [109Q99Q9]! 0.5!mL!
irritant;!May!form!explosive!peroxides!
FmocQAlaQWang!resin!(100Q200!
N/A! 300!mg! N/A!
mesh,!0.72!mmol/gram)!!
25%!(v/v)!!N,NQ
[7087Q68Q5]! Highly!flammable,!caustic,!reproductive!
diisopropylethylamine!(DIPEA)! 8!mL!
[68Q12Q2]! toxin!
in!DMF!!
OQ(BenzotriazolQ1Qyl)QN,N,N′,N′Q
tetramethyluronium! [94790Q37Q1]! 1.4!g! Irritant,!toxic!
hexafluorophosphate!(HBTU)!
FmocQGlyQOH! [29022Q11Q5]! 0.6!g! None!
FmocQAlaQOH! [35661Q36Q3]! 0.3!g! None!
Hexanoic!acid! [142Q62Q1]) 0.1!g! Corrosive,!toxic!!
Trifluoroacetic!Acid!(95%)! [76Q05Q1]! 14!mL! Highly!corrosive,!acidic,!toxic,!volatile!
[67Q66Q3]!
6:1:2!chloroform:!glacial!acetic! Flammable,!carcinogen,!toxic,!acidic,!
[64Q19Q7]! 10!mL!
acid:!methanol! corrosive!
[67Q56Q1]!
[7722Q64Q7]! one!batch!for!
KMnO4!TLC!stain!(KMnO4!K2CO3!
[584Q08Q7]! whole!class!(see! corrosive,!toxic,!oxidant!
NaOH,!water)!
[1310Q73Q2]! below)!
Dimethyl!sulfoxideQd6! [2206Q27Q1]! 1!mL! Flammable!
1:1!nanopure!water:! [75Q05Q8]!
1!mL! Corrosive,!acidic,!toxic,!flammable!
acetonitrile!with!0.1%!TFA! [76Q05Q1]!
)
)

! 2!
Instructor Notes

)
Special)Instructions)
!
Day$One$
!
Allow!all!chemicals!and!resin!beads!to!warm!to!room!temperature!prior!to!use.!!
!
To!prevent!spillage!of!the!expensive!resin!beads,!we!recommend!preQloading!the!resin!into!the!fritted!syringe!
reaction!vessels!for!the!students.!!
!
PreQmix!the!following!solutions!(see!table!above!for!amounts):!
20%!(v/v)!piperdine!in!DMF!
25%!(v/v)!DIPEA!in!DMF!
!
All!other!reagents!and!solvents!were!used!directly!from!the!manufacturers!bottle.!We!recommend!the!use!of!a!
new!bottle!of!DMF.!Any!old/stored!DMF!should!be!sparged!with!dry!argon!or!nitrogen!prior!to!use!to!remove!
any!dimethyl!amine!that!may!form!upon!storage.!Vacuum!distillation!may!be!necessary!if!it!is!quite!impure.!!
!
Provide!halogenated!organic!and!nonQhalogenated!organic!waste!containers.!!
!
Remind$students$to$follow$the$steps$carefully!$
!
Day$Two$
!
Allow!resin!beads!to!warm!to!room!temperature!prior!to!use.!
)
In! addition! to! halogenated! organic! and! nonQhalogenated! organic! waste! containers,! provide! a! special! waste!
container!for!any!TFA!rinses.!Warn!students!that!gas!is!often!produced!when!acetone!is! added!to!a!container!
that!held!TFA,!and!any!glassware!that!has!been!in!contact!with!TFA!should!be!rinsed!carefully!in!a!fume!hood.!!
!
Cleavage. of. the. peptide:! Students! should! be! warned! to! be! careful! when! swirling! the! syringe! containing! TFA!
during!the!cleavage!step.!Occasionally!TFA!collects!in!the!tip!of!the!syringe,!and!may!drip!out!from!the!syringe!
when! swirled.! To! minimize! this! problem,! students! should! pull! back! on! the! plunger! to! pull! any! TFA! that! has!
collected! in! the! nozzle! back! into! the! barrel! prior! to! swirling.! In! addition,! students! should! always! point! the!
syringes!away!from!eyes,!hands,!and!their!lab!partner.!
!!
Isolation.of.the.peptide:!Occasionally!little!or!no!precipitate!is!observed!when!the!ether!is!added!to!the!sample.!
This! happens! either! if! the! students! did! not! remove! enough! of! the! TFA,! or! have! not! synthesized! their! peptide!
correctly.!Students!should!reQevaporate!to!dryness,!and!try!the!precipitation!again.!!!
When!centrifuging!the!peptide!in!ether,!it!often!forms!a!gel!in!the!bottom!of!the!tube.!If!this!happens,!students!
can!gently!invert!the!tube!to!decant!the!ether!supernatant,!and!then!resuspend!the!gel!in!more!ether!for!the!
second!wash.!
$
Lyophilization.of.the.peptides:!Open!the!student!sample!tubes!containing!the!peptides!(may!be!a!gel)!for!a!few!
hours!to!evaporate!any!residual!ether.!Add!3!mL!of!nanopure!water!to!each!tube,!and!sonicate!briefly!in!a!water!
bath!until!dissolved.!Freeze!the!solutions!in!liquid!nitrogen!and!lyophilize!for!at!least!48!hours!(<5!mTorr).!If!a!
lyophilizer!is!not!available,!a!manifold!and!vacuum!pump!may!suffice.!$
!

! 3!
Instructor Notes

Day$Three$
!
PreQmix!the!following!solutions!(see!table!above!for!amounts):!
TLC!running!solvent:!6:1:2!chloroform:!glacial!acetic!acid:!methanol!(in!this!solvent!system,!the!peptide!typically!
has!an!Rf!of!~0.7).!!
TLC! reference:! 5! mg! of! the! peptide! product! (if! available)! dissolved! in! 1! mL! of! a! 3:1! chloroform:methanol!
solution.!!
TLC!potassium!permanganate!stain:!Dissolve!1.5!g!KMnO4,!10!g!K2CO3! and!1.25!mL!10%!NaOH!in!200!mL!water.!
Store!in!a!wide!mouth!glass!jar.!
Solvent!for!MS!samples:!!HPLC!grade!methanol!!
Solvent!for!HPLC!samples:!1:1!nanopure!water:!acetonitrile!with!0.1%!(v/v)!TFA!
$
Xerogel.formation:!We!have!found!that!the!gels!are!difficult!to!work!with!if!formed!in!a!glass!vial.!Therefore,!we!
recommend!transferring!the!peptide!solution!into!a!conical!1.5!mL!Eppendorf!tube!prior!to!gelation.!If!students!
do!not!obtain!a!gel!after!~15!minutes,!have!them!start!over.!Oftentimes!students!do!not!weigh!out!the!peptide!
carefully,!and!the!resulting!solution!is!too!dilute!to!form!a!robust!gel.!Also,!on!occasion,!students!have!observed!
gelation! during! the! heating! process.! If! this! happens,! students! should! immediately! transfer! the! sample! to! a!
plastic!Eppendorf!tube,!and!allow!the!sample!to!cool.!! !
!
The!evaporation!procedure!will!vary!based!on!the!instrumentation!available!at!your!
institution.!The!procedure!we!use!is!as!follows:!!
Once! all! of! the! peptides! have! formed! a! gel,! we! put! all! of! the! tubes! in! a! foam!
holder,!and!place!them!together!in!a!bell!jar!typically!used!for!lyophilization!(see!
photo).!The!jar!is!then!connected!to!a!high!vacuum!pump!(mTorr),!and!carefully!
evacuated.! If! evacuated! too! rapidly,! the! gels! may! burst! out! of! the! tubes.! In! this!
setQup,!it!typically!takes!~20!minutes!to!evaporate!all!of!the!THF.!!
!
Alternate!methods!could!include:!!
QCut! the! lid! off! of! the! plastic! Eppendorf! tube! containing! the! gel,! and! place! the!
tube!into!a!20!mL!scintillation!vial!(2!tubes!can!fit!in!one!vial).!An!adapter!will!be!
needed!to!connect!the!vial!to!a!manifold!/!high!vacuum!system.!!
QPlace!the!tubes!in!a!microcentrifuge!rack,!and!place!in!a!vacuum!desiccator!(may!
take!longer!than!20!mintues!to!dry).!
QIf!a!IR!solutionQcell!is!available,!you!may!not!need!to!remove!the!THF!at!all.!!
!
When! dried,! the! gel! will! collapse! in! the! tube.! Some! samples! have! a! webQlike! structure,! while! others! have! a!
powdery!consistency.!The!macroscale!structure!does!not!seem!to!have!any!bearing!on!the!FTIR!results.!!
!
Ranges!observed!in!the!FTIR!vibration!values:!
Lyophilized!peptide!(before!assembly):!amide!C=O!from!1637!to!1643!cmQ1,!COOH!C=O!from!1723!to!1727!cmQ1!!
Peptide!xerogel!(after!assembly):!amide!C=O!from!1622!to!1625!cmQ1,!amide!C=O!shoulder!from!1690!to!1693!
cmQ1,!COOH!C=O!from!1721!to!1725!cmQ1!
.
.
NMR:!Samples!occasionally!have!a!large!water!peak!that!can!occlude!one!of!the!peaks!in!the!peptide.!For!these!
samples,!we!recommend!using!a!water!suppression!program.!!
!

! 4!
Instructor Notes

HPLC:!The!peptide!was!eluted!using!a!mobile!phase!gradient!of!MeCN!in!water!containing!0.1%!TFA!at!a!flow!
rate!of!1!mL/min!and!detected!at!210!nm.!The!gradient!increased!from!30%!to!90%!MeCN!from!T!=!0!to!11!min,!
then!was!held!at!90%!MeCN!for!2!min,!and!finally!was!held!at!40%!MeCN!for!2!min.!!
!
!
!
!
Suggestions)for)Adaptation)to)an)InquiryCBased)Experiment)
!
The!experiment!described!is!also!amenable!to!inquiryQbased!approaches,!as!the!alkyl!end!group!of!the!peptide!
can!easily!be!varied.!In!one!version!of!the!lab,!we!divided!our!class!into!three!groups,!each!containing!four!pairs!
of!students.!Each!group!synthesized!a!GAGA!peptide!with!a!different!alkyl!tail!length!(6,!8!and!10!carbons).!All!
peptides!were!successfully!prepared,!and!differences!in!solubility!were!noted.!While!further!characterization!is!
needed,! in! our! initial! observations! the! octylQGAGA! behaved! similarly! to! the! hexylQGAGA! peptide,! but! we! have!
been!thus!far!unsuccessful!at!forming!gels!with!the!decylQGAGA!peptide!due!to!its!increased!solubility!in!THF.!
Further!iterations!that!we!have!envisioned!include!using!branched!or!aromatic!tails!to!discern!the!point!at!which!
the!endQgroup!disrupts!the!ability!of!the!peptide!to!selfQassemble.!!

! 5!
Pre-lab Report
Name: _______________________________

Experiment: Solid-Phase Peptide Synthesis

1. Draw the complete chemical structures (including the full structure of the Fmoc group) of
the following amino acid derivatives and other reagents that we will use in this lab. Give the
molecular weights where indicated.

1
Pre-lab Report
!
2.!You!will!start!your!synthesis!with!Wang!resin!containing!one!FmocDprotected!alanine!already!
attached.!The!structure!is!shown!below.!!!
O
H
N
O Fmoc
CH3
O

!
a) If!the!resin!has!0.72!mmol/g!of!this!linker!on!the!surface!of!the!beads,!and!you!use!300!
mg!of!resin,!how!many!mmol!of!linker!do!you!have?!Show!your!work.!
!
!
!
!
!
!
b) To!ensure!complete!coupling!of!the!second!amino!acid!to!the!resin,!you!will!add!4!molar!
equivalents! of! FmocDGlyDOH! relative! to! the! number! of! moles! of! linker! in! your! first!
coupling!step.!How!many!grams!is!this?!Show!your!work.!
!
!
!
!
!
c) Using! the! mmol! of! linker! calculated! above,! what! is! the! theoretical! yield! of! the! final!
peptide?!Show!your!work.!
!
!
!
!
!
!
3. A! student! synthesized! a! peptide! starting! with! Wang! resin! that! already! had! one! glycine!
coupled! to! the! beads.! He! then! added! FmocDPheDOH! followed! by! FmocDValDOH! (using!
appropriate! deprotection! steps! in! between).! Draw! the! complete! structure! of! the! peptide!
AFTER!it!is!cleaved!from!the!resin!using!TFA.!Draw!the!peptide!in!standard!ND!to!CDterminus!
form,!and!with!proper!stereochemistry.!!
!

2
Pre-lab Report
! ! ! ! ! !!!!!!!!! !!!!!!!!!!!Name:!________KEY__________________!
! ! ! ! ! !!!!!!!!
Experiment:+Solid0Phase+Peptide+Synthesis! !
!
1. Draw!the!complete!chemical!structures!(including!the!full!structure!of!the!Fmoc!group)!of!
the!following!amino!acid!derivatives!and!other!reagents!that!we!will!use!in!this!lab.!Give!the!
molecular!weights!where!indicated.!
hexanoic acid

OH

Molecular Weight = 116

Density = 0.93 g/mL

FMOC-Ala-OH FMOC-Gly-OH

O O
NH O NH O
HO HO
O O

Molecular Weight: 311.33 Molecular Weight: 297.31

Piperidine Diisopropylethylamine (DIPEA)

H
N
N

O-Benzotriazole-N,N,N’,N’-tetramethyl-
Dimethylformamide uronium-hexafluoro-phosphate (HBTU)

O
O N
N
H N N
PF 6
(H 3C) 2N N(CH 3) 2
Molecular Weight: 379.25
*Aldrich catalog shows the uronium not
the guanidinum structure shown here. !
!
1
Pre-lab Report
!
2.!You!will!start!your!synthesis!with!Wang!resin!containing!one!FmocFprotected!alanine!already!
attached.!The!structure!is!shown!below.!!!
O
H
N
O Fmoc
CH3
O

!
a) If!the!resin!has!0.72!mmol/g!of!this!linker!on!the!surface!of!the!beads,!and!you!use!300!
mg!of!resin,!how!many!mmol!of!linker!do!you!have?!Show!your!work.!
!
!
0.72 mmol/g * 0.300 g = 0.216 mmol linker
!
!
b) To!ensure!complete!coupling!of!the!second!amino!acid!to!the!resin,!you!will!add!4!molar!
equivalents! of! FmocFGlyFOH! relative! to! the! number! of! moles! of! linker! in! your! first!
coupling!step.!How!many!grams!is!this?!Show!your!work.!
!
!
0.000216 mol * 4 * 297 g/mol = 0.257 g
!
!
c) Using! the! mmol! of! linker! calculated! above,! what! is! the! theoretical! yield! of! the! final!
peptide?!Show!your!work.!
!
Molecular!weight!of!the!final!peptide!=!372.4!g/mol!

0.216 x 10-3 mol linker * 372.4 g/mol = 0.080 grams or 80 milligrams!

!
!
3. A! student! synthesized! a! peptide! starting! with! Wang! resin! that! already! had! one! glycine!
coupled! to! the! beads.! He! then! added! FmocFPheFOH! followed! by! FmocFValFOH! (using!
appropriate! deprotection! steps! in! between).! Draw! the! complete! structure! of! the! peptide!
AFTER!it!is!cleaved!from!the!resin!using!TFA.!Draw!the!peptide!in!standard!NF!to!CFterminus!
form,!and!with!proper!stereochemistry.!!

O
O O
F3C O H
H3N N
N OH
H
O

Peptides with a free N-terminus will form a salt with TFA

2
Post-lab Report
!!!!!!!!!!!!Name:!_______________________________!
! ! ! ! !!!!!!!!
Solid&Phase,Peptide,Synthesis! !
!
Lab$Results$
!
1. Draw!the!peptide!that!you!synthesized!in!standard!N8!to!C8terminus!form,!and!with!proper!
stereochemistry.!Calculate!its!molecular!weight.!!
!
!
!
!
!
!
!
2. How!many!milligrams!of!peptide!did!you!obtain?!___________!!
!
3. What!was!your!%!yield!for!the!reaction?!Calculate!the!theoretical!yield!based!on!the!moles!
of!linker!on!the!beads!that!you!started!with.!Show$your$work.!!
!
!
!
!
!
!
!
!
!
!
!
4. Attach!the! 1H!NMR!spectra!of!the!peptide!to!this!report,!and!assign!all!of!the!peaks!to!your!
peptide!structure!(be!specific!).!!
!
5. Draw! the! TLC! plate! of! your! peptide,! and! give! Rf! values! for! each! spot.! Based! on! the! TLC!
results!alone,!was!your!peptide!pure?!Explain.!!
!
!
!
!
!
!
!
!
!
!
!
!
1
Post-lab Report
!
6.! ! Attach! a! copy! of! your! HPLC! chromatogram,! and! your! mass! spectral! data.! Label! the! peaks!
belong! to! your! peptide,! and! if! possible,! identify! any! other! peaks! observed.! Based! on! this!
data,!was!your!peptide!pure?!Explain.!!
!
!
!
!
!
!
!
!
7. Attach! a! copy! of! the! FTIR! spectra! of! your! peptide! before$ and$ after$ the! self8assembly!
procedure.!Identify$all$of$the$major$vibrations.!What!secondary!structure!did!your!peptide!
form?!Use!your!data!and!the!observations!you!made!in!lab!to!support!your!answer.!!
!
!
!
!
!
!
!
!
Questions$
!
Q1.!Explain!the!purpose!of!the!following!steps!in!the!synthesis!procedure:!!
!
a. Why!are!peptides!typically!synthesized!using!a!solid!support!resin?!
!
!
!!
!
b. Why!was!the!resin!first!rinsed!with!DCM?!
!
!
!
!
c. What!is!the!purpose!of!adding!DIPEA!to!the!coupling!agent!solution?!
!
!
!
!
!
d. What!is!the!purpose!of!adding!cold!ether!at!the!end?!!
!
!
!
2
Post-lab Report
!
Q2.!Draw!the!structure!of!lysine!and!glutamic!acid.!What!special!precautions!must!you!take!if!
you!want!to!incorporate!either!of!these!amino!acids!into!your!peptide?!Be!specific!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Q3.! !a.!Draw!the!tetrapeptide!CHEM.!(N8!to!C8terminus,!proper!stereochemistry)!!
!
!
!
!
!
!
!
!
b.!If!you!wanted!to!synthesize!CHEM!using!SPPS!techniques,!which!amino!acid!would!you!
attach!to!the!resin!first?!Briefly!explain!why.!!
!
!
!
!
!
Q4.! PyBOP! (benzotriazol818yl8oxytripyrrolidinophosphonium! hexafluorophosphate)! is! another!
coupling!reagent!used!in!peptide!synthesis.!!
!
a. !Draw!the!structure!of!PyBOP:!!
!
!
!
!
!
!
!
!
!
3
Post-lab Report
!
!
b. Draw!the!complete!mechanism!for!the!following!reaction,!which!uses!PyBOP!as!the!
coupling!agent.!Be!sure!to!show!all!intermediates,!and!use!arrows!to!show!the!flow!
of!electrons!at!each!step.!What!are!the!two!by8products!of!this!reaction?!Draw!them!
in!the!boxes!provided.!
!
O O
H 1) PyBOP, DIPEA H
N H3C N
HO Fmoc N Fmoc
2) CH3-NH2 H
R R

By-product #1 By-product #2

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

4
Post-lab Report
!!!!!!!!!!!!Name:!_________KEY_______________!
! ! ! ! !!!!!!!!
Solid&Phase,Peptide,Synthesis! !
!
Lab$Results$
!
1. Draw!the!peptide!that!you!synthesized!in!standard!N;!to!C;terminus!form,!and!with!proper!
stereochemistry.!Calculate!its!molecular!weight.!!
!
O O O
H H
N N
N N OH
H H
O O

Molecular Weight: 372.42 !

!
!
2. How!many!milligrams!of!peptide!did!you!obtain?!___________!(40!mg!is!typical)!!
!
!
3. What!was!your!%!yield!for!the!reaction?!Calculate!the!theoretical!yield!based!on!the!moles!
of!linker!on!the!beads!that!you!started!with.!Show$your$work.!!
!

Theoretical yield = 80 mg (see pre-lab for calculations)!

!
!
!
4. Attach!the! 1H!NMR!spectra!of!the!peptide!to!this!report,!and!assign!all!of!the!peaks!to!your!
peptide!structure!(be!specific!).!!
!
See!student!data!provided.!(Students!will!likely!need!extra!help!assigning!these!peaks.)!
!
!
5. Draw! the! TLC! plate! of! your! peptide,! and! give! Rf! values! for! each! spot.! Based! on! the! TLC!
results!alone,!was!your!peptide!pure?!Explain.!!
!
Typical!Rf!for!the!peptide!is!~0.7!
!
!
6.! ! Attach! a! copy! of! your! HPLC! chromatogram,! and! your! mass! spectral! data.! Label! the! peaks!
belong! to! your! peptide,! and! if! possible,! identify! any! other! peaks! observed.! Based! on! this!
data,!was!your!peptide!pure?!Explain.!!
!
Will!vary!based!on!conditions!used.!
!
!

1
Post-lab Report
!
7. Attach! a! copy! of! the! FTIR! spectra! of! your! peptide! before$ and$ after$ the! self;assembly!
procedure.!Identify$all$of$the$major$vibrations.!What!secondary!structure!did!your!peptide!
form?!Use!your!data!and!the!observations!you!made!in!lab!to!support!your!answer.!!
!
See! student! data! provided.! Most! should! see! the! C=O! peak! shift! from! 1637! cm;1! in! the!
lyophilized! peptide! to! 1622! cm;1! with! a! shoulder! at! 1691! cm;1! in! the! xerogel,! which! is!
consistent!with!an!anti;parallel!beta!sheet!structure.!!
!
!
Questions$
!
Q1.!Explain!the!purpose!of!the!following!steps!in!the!synthesis!procedure:!!
!
a. Why!are!peptides!typically!synthesized!using!a!solid!support!resin?!
!
Ease!of!purification,!block!one!reactive!end.!
!
b. Why!was!the!resin!first!rinsed!with!DCM?!
!
Swell!the!beads!
!
c. What!is!the!purpose!of!adding!DIPEA!to!the!coupling!agent!solution?!
!
The!DIPEA!deprotonates!the!carboxylic!acid.!!
!
d. What!is!the!purpose!of!adding!cold!ether!at!the!end?!!
!
The!peptide!is!not!soluble!in!ether,!so!it!precipitates!and!can!be!easily!isolated.!!
!
!
!
!
Q2.!Draw!the!structure!of!lysine!and!glutamic!acid.!What!special!precautions!must!you!take!if!
you!want!to!incorporate!either!of!these!amino!acids!into!your!peptide?!Be!specific!!
!
Need! to! protect! the! amine/acid! side! chains! so!
they! do! not! interfere! with! the! coupling! between!
amino! acids.! A! typical! protecting! group! for! lysine!
is!tert;butyloxycarbonyl!(Boc)!and!tert;butyl!(t;Bu)!
for!glutamic!acid.!Both!of!these!protecting!groups!
are!stable!in!base!during!the!Fmoc!removal!steps,!
but!can!be!easily!cleaved!in!acid.!!
!
!
!
!
2
Post-lab Report
!
Q3.! !a.!Draw!the!tetrapeptide!CHEM.!(N;!to!C;terminus,!proper!stereochemistry)!!
!
!
N S
NH

O O
H
H2N N OH
N N
H H
O O
SH

O ! OH
!
!
b.!If!you!wanted!to!synthesize!CHEM!using!SPPS!techniques,!which!amino!acid!would!you!
attach!to!the!resin!first?!Briefly!explain!why.!!
!
methionine: always synthesize from C to N terminus
!
!
Q4.! PyBOP! (benzotriazol;1;yl;oxytripyrrolidinophosphonium! hexafluorophosphate)! is! another!
coupling!reagent!used!in!peptide!synthesis.!!
!
a. !Draw!the!structure!of!PyBOP:!!
!
!
!
!
!
!
b. Draw!the!complete!mechanism!for!the!following!reaction,!which!uses!PyBOP!as!the!
coupling!agent.!Be!sure!to!show!all!intermediates,!and!use!arrows!to!show!the!flow!
of!electrons!at!each!step.!What!are!the!two!by;products!produced?!
!
O O
H 1) PyBOP, DIPEA H
N H3C N
HO Fmoc N Fmoc
2) CH3-NH2 H
R R

By-product #1 By-product #2

O N
N
P
N N N
HO
N

3
Post-lab Report

N
O
O P
H N N
N
N N
HO R N
N

R O

O N
N
P
R O O N N
N N
N N
R O P
O O
N N

N
N Activated amino acid

by-product #1

O N N
O N N

N
N
R O
R O
N H
CH3-NH2 H3C
H

O
O
N N N
N
CH3
R N CH3
N N
R N HO
H O H
H

by-product #2 !

4
Student'Characteriza/on'Data'
All) data) shown) is) from) lyophilized) pepKdes) synthesized) by) students.) Samples) that) had) only)
one) spot) on) the) TLC) plate) were) chosen) for) further) analysis.) No) further) purificaKon) was)
performed.))

Chemical)Formula:)C16H28N4O6)
Exact)Mass:)372.20)

HPLC'Chromatogram' •  The)pepKde)was)dissolved)in)a)soluKon)
containing)50%)acetonitrile)(MeCN))and)50%)
deionized)water)with)0.1%)TFA)(~1)mg/mL)),)and)
pepKde) then)filtered)through)a)0.22)μm)syringe)filter.)
•  Thermo)ScienKfic)Dionex)UlKmate)3000)with)
diode)array)detector.)Column:)Phenomenex)150)
x)4.6)mm)C18)column)with)5)µm)pore)size))
solvent) •  The)pepKde)was)eluted)using)a)mobile)phase)
gradient)of)MeCN)in)water)containing)0.1%)TFA)
at)a)flow)rate)of)1)mL/min.)The)gradient)
increased)from)30%)to)90%)MeCN)from)T=0)to)
11)minutes,)then)was)held)at)90%)MeCN)for)2)
min,)then)held)at)40%)MeCN)for)2)min.))

ESI9Mass'Spectrum''
[MWH]W) •  Applied)Biosystems)API)2000)Triple)
Quadrupole)Mass)Spectrometer))
•  NegaKve)mode)
•  PepKde)was)dissolved)in)HPLC)grade)
methanol,)and)directly)injected.)
ATR9FTIR'spectra'before'and'aCer'assembly''
•  Thermo)ScienKfic)Nicolet)iS10)FTIR)
equipped)with)an)ATR)accessory)
•  )Approx.)5)mg)samples)
•  )Ambient)environment)
•  )32)scans) Lyophilized)PepKde)
1726)
Typical)ranges)observed)in)the)FTIR)
vibraKon)values:)
Lyophilized)pepKde)(before)assembly))
amide)C=O):))1637)to)1643)cmW1)) Xerogel))
COOH)C=O):)1723)to)1727)cmW1)) 1637)
1721)
)
PepKde)xerogel)(aier)assembly))) 1691)
amide)C=O):)1622)to)1625)cmW1))
amide)C=O)shoulder):)1690)to)1693)cmW1)
COOH)C=O):)1721)to)1725)cmW1)

1622)

Lyophilized)PepKde)

PepKde)Xerogel))

4000) 3500) 3000) 2500) 2000) 1500) 1000)

Wavenumbers'(cm91)'
 O
o
o
6t'e9 t t
eL'022,

CL'AW'
,g'vgvl
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LO

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o)o)o)o)o)@@@
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@@l-f-Nl- (c, (o
l.* @
a \f N o
(c) @,(c) (() @ (o $ c{o
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ecuenlLusueJl%
 _90'66/

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9Z'990 t
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9V'6VVl
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te'2181
6Z'0e62 9V'9962
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ecuelllLusueJl%
 1H'NMR9'Student'Sample'#1'

• )Varian)Inova)500)MHz)NMR) C) H)
• )~10)mg/mL)in)DMSOWd6) D) I) L) N)
• )8)scans)
A) F)
K) M) O)
B) E) G) J)

iii)
i)
E) C) J)H) BG)

MN) O)
K) L)
impurity)

ii)
D)I)

AF)
iii)

ii)

DMSO)

i)

impurity)
 1.272
1.257
1.240
1.222
1.208
0.860
0.846

1.493
1.478
1.464

2.106
2.090
0.832

2.120

8.223
7.999

8.131
8.014
3.703

8.117
8.048
3.691

4.219
4.166

9 8 7 6 5 4 3 2 1 ppm

PULSE SEQUENCE: PRESAT OBSERVE H1, 499.7522374 DATA PROCESSING Presat Watersupression
Relax. delay 2.000 sec FT size 32768
Pulse 90.0 degrees Total time 1 minute
Acq. time 2.048 sec Solvent: dmso
Width 8000.0 Hz Ambient temperature
8 repetitions Operator: winpiah
File: PRESAT_01
Plotname: PRESAT_01_plot03 INOVA-500 "inovanmr.chem.wwu.edu"
 1.272
1.257
1.240
1.222
1.208
0.846

2.106
0.860
0.832

2.090

2.120
1.464

1.493
1.478

2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 ppm

0.68
2.18
2.15
3.00

10.49

PULSE SEQUENCE: PRESAT OBSERVE H1, 499.7522374 DATA PROCESSING Presat Watersupression
Relax. delay 2.000 sec FT size 32768
Pulse 90.0 degrees Total time 1 minute
Acq. time 2.048 sec Solvent: dmso
Width 8000.0 Hz Ambient temperature
8 repetitions Operator: winpiah
File: PRESAT_01
Plotname: PRESAT_01_plot02 INOVA-500 "inovanmr.chem.wwu.edu"
 8.131
8.223
7.999
3.703

8.117
8.048
8.014
3.691

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm

4.58
2.09
1.69

PULSE SEQUENCE: PRESAT OBSERVE H1, 499.7522374 DATA PROCESSING Presat Watersupression
Relax. delay 2.000 sec FT size 32768
Pulse 90.0 degrees Total time 1 minute
Acq. time 2.048 sec Solvent: dmso
Width 8000.0 Hz Ambient temperature
8 repetitions Operator: winpiah
File: PRESAT_01
Plotname: PRESAT_01_plot01 INOVA-500 "inovanmr.chem.wwu.edu"
 1H'NMR9'Student'sample'#2'with'water'suppression'

• )Varian)Inova)500)MHz)NMR)
• )~10)mg/mL)in)DMSOWd6)
• )8)scans)
• )Water)suppression)program:)
PULSE)SEQUENCE:)PRESAT)
)Relax.)delay)2.000)sec)
)Pulse)90.0)degrees)
C) H)
)Acq.)Kme)2.048)sec) D) I) L) N)
A) F)
)Width)8000.0)Hz)
K) M) O)
B) E) G) J)

iii)

1.272
1.257
1.240
1.222
1.208

0.846
8.131
8.223

7.999
8.117
8.048
8.014

3.703
BG)
2.106

0.860

0.832
3.691
2.090
2.120

1.464
1.493
1.478
O)
MN)
K) L)

2.8
E)2.6
C) J) H)2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6

i) ii) iii)
0.68

2.18

2.15

10.49

3.00
1.272
1.257
1.240
1.222
1.208
0.860
0.846
PULSE SEQUENCE: PRESAT
Relax. delay 2.000 sec
OBSERVE H1, 499.7522374 AF)
DATA PROCESSING
FT size 32768
DI) Presat Watersupressi

Pulse 90.0 degrees Total time 1 minute

1.493

Acq. time 2.048 sec Solvent: dmso

1.478

Width 8000.0 Hz Ambient temperature

1.464
2.106

8 repetitions
2.090

Operator: winpiah

0.832
File: PRESAT_01
Plotname: PRESAT_01_plot02 INOVA-500 "inovanmr
2.120
8.223

7.999
8.131

8.014

3.703
8.117
8.048

3.691
4.219
4.166

5.5 5.0
8.0 4.5
7.5 4.0
7.0 ppm
6.5 6.0
2.09

1.69
4.58

i) ii)
DMSO)

PULSE SEQUENCE: PRESAT Presat Watersupression

OBSERVE H1, 499.7522374 DATA PROCESSING
Relax. delay 2.000 sec FT size 32768
9Pulse 90.0 88 degrees 77 66 5
5 4
4 3
3 2Total time
2 11 1 minute
ppm
ppm
Acq. time 2.048 sec Solvent: dmso
4.58 1.69 *) 2.18 10.49
WidthPRESAT
PULSE SEQUENCE: 8000.0 OBSERVE
Hz H1, 499.7522374 Ambient
DATA PROCESSING temperature
2.09 0.68 Presat
2.15Watersupression
3.00
Relax. delay 2.000 sec FT size 32768
8 repetitions
Pulse 90.0 degrees
Operator:
Total time 1 minute
winpiah
Acq. time PULSE
2.048 SEQUENCE:
sec PRESAT OBSERVE H1, 499.7522374 *IntegraKon)is)underesKmated)due)to)interference)by)water)suppression)program)
File: PRESAT_01
DATA PROCESSING Solvent:
Presat dmso
Watersupression
Width 8000.0 Hz delay 2.000 sec
Relax. FT size 32768 Ambient temperature
8 repetitions
Pulse 90.0 degrees INOVA-500 "inovanmr.chem.wwu.edu"
Total time 1 minute Operator: winpiah
Plotname:
Acq. time 2.048PRESAT_01_plot01
sec Solvent:
File: dmso
PRESAT_01
Width 8000.0 Hz Ambient temperature
INOVA-500 "inovanmr.chem.wwu.edu"
Plotname: PRESAT_01_plot03
8 repetitions Operator: winpiah
File: PRESAT_01
 1.281
1.266
1.258
1.256
1.250
1.245
1.228
1.214
0.876
0.862

3.715
3.703
1.493

2.113
0.848

2.099

2.128

2.513
2.509

2.517

8.066
8.198
7.985
7.972
4.187

8 7 6 5 4 3 2 1 ppm

1.00 2.00 4.09 2.07 2.88

1.06 2.09 2.06 10.03

PULSE SEQUENCE OBSERVE H1, 499.7522374 DATA PROCESSING

Relax. delay 1.000 sec FT size 32768
Pulse 45.0 degrees Total time 1 minute Solvent: dmso
Acq. time 2.048 sec Temp. 25.0 C / 298.1 K
Width 8000.0 Hz Operator: albint
8 repetitions File: PROTON_01
INOVA-500 "inovanmr.chem.wwu.edu"
Plotname: PROTON_01_plot03
 1.281
1.266
1.258
1.256
1.250
1.245
1.228
1.214
0.862
0.848

0.876

2.113
2.099

2.128
1.493

2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 ppm

2.06 10.03
2.07 2.88

PULSE SEQUENCE OBSERVE H1, 499.7522374 DATA PROCESSING

Relax. delay 1.000 sec FT size 32768
Pulse 45.0 degrees Total time 1 minute Solvent: dmso
Acq. time 2.048 sec Temp. 25.0 C / 298.1 K
Width 8000.0 Hz Operator: albint
8 repetitions File: PROTON_01
INOVA-500 "inovanmr.chem.wwu.edu"
Plotname: PROTON_01_plot07
 3.715
3.703

4.187

4.254
4.172

4.200

4.240

4.269
4.3 4.2 4.1 4.0 3.9 3.8 3.7 ppm

2.09 10.03
2.06
4.09 2.07
2.88

PULSE SEQUENCE OBSERVE H1, 499.7522374 DATA PROCESSING

Relax. delay 1.000 sec FT size 32768
Pulse 45.0 degrees Total time 1 minute Solvent: dmso
Acq. time 2.048 sec Temp. 25.0 C / 298.1 K
Width 8000.0 Hz Operator: albint
8 repetitions File: PROTON_01
INOVA-500 "inovanmr.chem.wwu.edu"
Plotname: PROTON_01_plot06
 8.198
8.080
8.066
8.006
7.985
7.972

7.994

8.187
8.018

8.210
8.3 8.2 8.1 8.0 7.9 ppm

1.00 2.00 4.09

2.07
2.88
1.06 10.03
2.09
2.06

PULSE SEQUENCE OBSERVE H1, 499.7522374 DATA PROCESSING

Relax. delay 1.000 sec FT size 32768
Pulse 45.0 degrees Total time 1 minute Solvent: dmso
Acq. time 2.048 sec Temp. 25.0 C / 298.1 K
Width 8000.0 Hz Operator: albint
8 repetitions File: PROTON_01
INOVA-500 "inovanmr.chem.wwu.edu"
Plotname: PROTON_01_plot05
13C'NMR'

B) E) J) M) O)
D) G) I)
A) F) K)
• )Varian)Inova)500)MHz)NMR) L) N) P)
• )~10)mg/mL)in)DMSOWd6) C) H)
• )64)scans) iii)
M) O) P)
N)
ii)
BG) L) CH)

EJ)

i) F)K) DI)
A)

DMSO

iii)

i) ii)