synthetic strategies in solid-phase peptide synthesis: n ...€¦ · ii synthetic strategies in...
TRANSCRIPT
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SyntheticStrategiesinSolid-PhasePeptideSynthesis:N-TerminusModification
by
MaxRyanWeissman
AthesissubmittedinconformitywiththerequirementsforthedegreeofMasterofScience
DepartmentofChemistryUniversityofToronto
©CopyrightbyMaxRyanWeissman2018
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ii
SyntheticStrategiesinSolid-PhasePeptide
Synthesis:N-TerminusModification
MaxRyanWeissman
MasterofScience
DepartmentofChemistry
UniversityofToronto
2018
AbstractSolid-phasepeptidesynthesis(SPPS)isatechniqueusedinthesynthesisof
peptides.SPPSismainlyusedtosynthesizelinearpeptidesusingnaturalα-amino
acidprecursors.Inthisthesis,differentwaysofmodifyingtheN-terminusof
polymer-supportedpeptideswasinvestigated.TheN-terminuswasconvertedinto
ureaandguanidinefunctionalitieswiththeuseofisocyanatesequivalents.
Alternatively,anothermethodconvertedtheN-terminusintoisocyanatesand
carbodiimides,whichthenreactedwithvariousamines.Inaddition,lesscommon
peptidecouplingreagents,suchasacylchloridesandanhydrides,wereexamined
withrespecttopolymerboundpeptides.Thesereagentswerethencomparedwith
moretypicalones,suchasHATU,onavarietyofsubstrates.
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iii
Acknowledgments
I’dliketothankmysupervisor,Rob,andallthemembersoftheBateygroupfor
theirhelpduringmytimehere.Theyhavealltaughtmemanythingsandguidedme
throughthisproject.Iappreciatealltheyhavedoneformeandfeelsofortunateto
havebeensurroundedbysomanycompassionatepeople.
I’dalsoliketothankmyfamilyandfriendsfortheirsupportduringthistime.Their
effortsandexistencehavehelpedmetopushforwardthrougheverything.I’m
happythattheyareinmylifeandknowthattheywillbethereformeinthefuture.
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TableofContents
Acknowledgments....................................................................................................................................iii
TableofContents......................................................................................................................................iv
ListofFigures..............................................................................................................................................vListofSchemes...........................................................................................................................................vi
ListofTables..............................................................................................................................................viiAbbreviations..........................................................................................................................................viii
1 Peptidesinchemistry................................................................................................................1
1.1 Usesofpeptides.....................................................................................................................11.2 Synthesisofpeptides...........................................................................................................3
1.3Synthesisofpeptidesderivatives..................................................................................5
2 IsocyanatesandPeptides.........................................................................................................92.1β-alanineandderivatives..................................................................................................9
2.2Isocyanateequivalents.....................................................................................................132.3Reactivityofthioureas......................................................................................................17
2.4ConclusionsandOutlook.................................................................................................21
3 Peptidecouplingconditions.................................................................................................223.1Methodsofactivation........................................................................................................22
3.2Applicationsofactivation................................................................................................253.3ConclusionsandOutlook.................................................................................................31
Experimental.............................................................................................................................................32
Appendix......................................................................................................................................................75References................................................................................................................................................135
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v
ListofFiguresFigure1.Naturallyoccurringpeptides 2
Figure2.Commonprotectinggroups 4
Figure3.Commoncouplingreagents 4
Figure4.Naturallyoccurringpeptidesandpeptidebaseddrugs 6
Figure5.TheN-terminusbeingasecondaryamine 8
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vi
ListofSchemesScheme1.Synthesisofglyclglycine 1
Scheme2.Synthesisofbenzoylglyclglycine 3
Scheme3.GeneralstrategyforSPPS 5
Scheme4.Peptidescanbemodifiedatmultiplelocations 7
Scheme5.Nucleophilicadditionofaminesintoisocyanates 9
Scheme6.β-Alaninederivativesformedfromphenylisocyanate 10
Scheme7.Synthesisofapeptideona2-chlorotritylresin 11
Scheme8.Substitutedβ-alaninesderivativescouplingtopolymerbound 12peptides
Scheme9.Formationofcompound27e,withoutimpurities 13
Scheme10.Reactionandproductsofreactionofpolymerboundpeptide 14withvariousisocyanatesandisothiocyanates.
Scheme11.PolymerboundpeptidewithvariousN-alkylcarbamoylimidazoles 16
Scheme12.Polymerboundpeptidescontainingthioureascanformhydantoins 17
Scheme13.Cyclizationofaβ-alaninethiourea 18
Scheme14.Trappingoutapolymersupportedcarbodiimidewithbenzylamine 19
Scheme15.Trappingoutapolymersupportedcarbodiimidewith 20variousnucleophiles
Scheme16.TheuseofaC-terminusprotectedaminoacidasanucleophile 21allowsfortheorientationofthepeptidetoflip
Scheme17.Reactingthecarboxylicacid18awithvariousamines 30
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vii
ListofTablesTable1.Differentconditionsforcyclizingaβ-alaninethiourea 18
Table2.Reactionofapolymerboundpeptidewithsimpleacylatingagents 23
Table3.Reactionofapolymerboundpeptidewithvariousactivatingagents 24andbenzoicacid
Table4.Polymerboundpeptidereactingwithactivatedaminoacid 25
Table5.Differentactivatingagentswithanelectronpoorcarboxylicacid 26
Table6.Reactionusingdifferentactivatingagentswithanelectronpoor 27carboxylicacid
Table7.Comparisonofabulkycarboxylicacid,53c,couplingsusingHATU 28andEDC
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viii
AbbreviationsAla,A Alanine
Asp,D Asparticacid
br Broad(spectral)
Boc tert-butoxycarbonyl
cm-1 Wavenumber
CDCl3Deuteratedchloroform
CDICarbonyldiimidazole
d Doublet(spectral)
DABCO 1,4-Diazabicyclo[2.2.2]octane
DCM Dichloromethane
DIPEA Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DMSO-d6 Deuterateddimethylsulfoxide
DPPA Diphenylphosphorylazide
dr Dram
δ Chemicalshift
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
ESI+Electrosprayionization
Et2ODiethylether
FmocFluorenylmethylcarbonyl
HATU1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxidehexafluorophosphate
HFIP Hexafluoroisopropanol
HOAt 1-Hydroxy-7-azobenzotriazole
HPLC High-performanceliquidchromatography
HRMS High-resolutionmassspectrometry
IR InfraredSpectroscopy
m Multiplet(spectral)
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ix
MeI Iodomethane
MeOH Methanol
mgMilligram
[M+H]+Molecularionwithhydrogen
MHz Megahertz
mL Milliliter
mmol Millimole
[M+Na]+ Molecularionwithsodium
m.p. Meltingpoint
Mukaiyama’sreagent 2-chloro-1-methylpyridiniumiodide
m/z Mass-to-chargeratio
NaHSodiumhydride
NaOHSodiumhydroxide
NEt3 Triethylamine
NMP N-methyl-2-pyrrolidone
Ph Phenyl
ppm Partspermillion
Pro,P Proline
PS 2-chlorotritylpolymersupport/resin
q Quartet(spectral)
s Singlet(spectral)
Ser,S Serine
SPPS Solid-phasepeptidesynthesis
t Triplet(spectral)tBu tert-butyl
THF Tetrahydrofuran
Trityl Triphenylmethyl
Trp,W Tryptophan
μL Microliter
Val,V Valine
YamaguchiReagent2,4,6-Trichlorobenzoylchloride
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x
1HNMR Protonnuclearmagneticresonance13CNMR Carbonnuclearmagneticresonance
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1
Chapter1Introduction
1PeptidesinChemistry
1.1Thehistoryofpeptides
Peptidesareamidesderivedfromtwoormoreaminoacidsconnectedbyacovalent
amidebondformedbetweentheamineandcarboxylicacidofcontiguousamino
acids,afterthelossofwater.1Theyaredistinguishedfromproteinsduetotheir
relativesmallersize.Justlikeproteins,peptidesexistformanyreasonsandhave
manyuses.
Theinitialclassificationofpeptideswasin1902.Thedipeptideglycylglycinewas
synthesizedbyErnestFourneaufromthehydrolysisof2,5-diketopiperazine
(Scheme1).2Whenhepresentedthisresult,heintroducedthetermpeptide.Before
this,theonlyidentificationofshorterformsofproteinswaspeptones,theproductof
proteinsaftertheyaredigestedinthestomach.Thenewclassificationhelped
establishedthetheorythatproteinsarelongchainsofaminoacidslinkedbyamide
bonds.Sincethen,thisstructuraltheoryhasbeenproventhroughmany
spectroscopicandotheranalyticaltechniques.Forexample,proteinscanbe
characterizedbymassspectrometry,throughtheirfragmentationintopeptide
fragments.3
Scheme1.Synthesisofglyclglycine
HNNH
O
OO
H2N
OHN OH
2,5-diketopiperazine glyclglycine
Hydrolysis
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2
Later,naturallyoccurringpeptideswerediscoveredsuchasinsulin,oxytocin2and
glutathione(Figure1).4Naturallyoccurringpeptidesactaseitherinhibitorsor
activatorsforproteinsinlivingorganisms.5Synthesizingpeptidesallowsusto
understandhowtheyworkandopensuproutesformodificationstoexplorethe
structuralfeaturesrequiredforbiologicalactivity.Modifiedpeptidescanfunction
betterinalivingorganismthantheinitialpeptide,aprocessthatcanbeusefulfor
drugdiscovery.Allofthisisalsotrueforproteins,butduetotheirsizetheyare
naturallymorechallengingtoworkwith.
Figure1.Naturallyoccurringpeptides
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly
S S
SS SS
H
H2NO O
O
NHO
HN NH2
N
S
NH O
HN
OH2N
O
NH
O
NH
O
HO
NH
O
S
NH2O
HO
O
NH2
O
OHHN
OSH
NH O
Insulin
Oxytocin Glutathione
Thr-Lys-Pro-Thr-Tyr-Phe-Phe
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3
1.2Synthesisofpeptides
Thefirstsynthesisofapeptideusedaglycine-silver(I)saltandbenzoylchloride.2
Thiswasbeforetheidentificationofpeptidesasaclassofmolecules.Thedesired
productforthereactionwasbenzoylglycine.Inadditiontomakingthedesired
product,benzoylglycylglycineandlongerchainssuchasbenzoylhexaglycinewere
alsomade(Scheme2).Inthecontextofpeptidechemistrythissynthesisraiseda
fewissues.Duetotheamineandcarboxylicacidfunctionalityoneitherendofany
aminoacid,polymerizationcanoccur.Thiscanbefurtherexacerbatedbyanamino
acidwithreactivesidechains,suchaslysineorcysteine.Inaddition,ahardto
removeprotectinggroup,suchasabenzoylgroup,beingonthepeptidewouldmake
itdifficulttoextendthepeptide.
Scheme2.Synthesisofbenzoylglyclglycine
Themethodologyofpeptidechemistryhasgrownmuchsincethisinitialstudy.
TypicallytheaminoacidsareprotectedoneithertheirC-orN-terminusand
allowedtocouplewithanotheraminoacidthatisprotectedonthecomplementary
end(Figure2).6Theaminoacidsarecoupledtogetherusuallywithanactivating
agentthatreducesthepossibilityforracemization(Figure3).7
OH2N O Ag
Cl
O
OHN OH
O
+ +
OHN
ONH
OH
O
nGlycine-silver(I) Benzoyl Chloride
Benzoylglycine
1
Benzene
Reflux
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4
Figure2.Commonprotectinggroups:Fmoc(2,blue),Boc(3,blue),Trityl(2,red),tBu(3,red)
Figure3.Commoncouplingreagents
In1963,amoreefficientmethodforpeptidesynthesiswasestablished,Solid-phase
peptidesynthesis(SPPS).8Thismethodusesapolymersupportwithanactivated
chainendwhereeithertheC-orN-terminusofanaminoacidcanattach.Inthis
thesis,allpolymer-supportedpeptideswereattachedbytheC-terminustothe
polymer.WithSPPS,couplingsarecarriedoutasnormal,butanywastecanjustbe
washedoffwithfiltrationduetothepolymersupport.Thisreducesthetimefor
synthesiswithouttheneedforlengthypurifications.Solubilityisalsolessofanissue
becausehighlysolubilizingsolvents,suchasDMF,canbeused.Thereisnoconcern
withremovingthesolventsinceitcanalsobefilteredoff.Oncethepeptidehasbeen
completeditcaneasilybecleavedfromthepolymersupport(Scheme3).
OHN
O
O O
OHN
O
O O
2 3
N NN
N
ONCNN
N
NN
NOH
HOAt
PF6
EDCHATU
NN
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5
Scheme3.GeneralstrategyforSPPS
1.3Synthesisofpeptidederivatives
OneproblemwithSPPSisthatnormallythetypesofreactionsperformedarenot
thatdiverse.Thisisduetothemildconditionsneededtokeepalltheprotecting
groupsonthepeptide.Thenarrowscopeofreactionscanbeanissuebecause
naturalproductsarenotalwaysjustsimplepeptides,butcanhavemodifications
(Figure4A).9Thesenaturalproductsarebiosynthesizedaspeptidesandpost
modifiedinthecell.Drugsthatarederivedfrompeptidesarealsooftenpeptides
thathavebeenmodified(Figure4B).10Drugsusuallyhavemorenon-proteinogenic
modificationsthanthenaturalproductitisbaseduponhave.Becauseofthis,new
typesofreactionsforSPPSwouldgreatlyhelpthefieldofmedicinalchemistryand
pharmaceuticaldevelopment.
Whenlookingatapeptidetherearemultiplelocationsthatcanbemodified
(Scheme4).Thehydrogenonanitrogenofanyamidebondcanbereplaced.A
X Linker PolymerSupport+OH
N OH LinkerPolymerSupport
OHN
R R
Linker PolymerSupportOH
N
R
OHN OH
R
ONH
Rn
OHN
RONH
Rn
OH
Cleave
1. Deprotection2.PeptideCoupling
4 5 6
7
89
PG PG
PG
PGPG
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6
Figure4.Naturallyoccurringpeptides(A)andpeptidebaseddrugs(B)withnonproteinogenicaminoacidcomponents(green)
O
O
ON
H
O
HN
O O
HO NH
OCl
HO
Cl
H
HO
O
O
HN
H2NOH
O
NH
O NH
OH O
NH
HN
ON
O
OHHN
O
OH
HN O
HN
O
HN
HN
O
NHO
OO
H
H
H
H
H H
TelomycinNannocystin A
O
N
O NHO
NH
NH
O
HN
NH2
OHN O
HNO
H
O
NHO
NH2
Pasireotide
O
N
O
HN
O
NH
O
HN
O
NH
O
H
H
O
Carfilzomib
A
B
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7
commonreplacementisamethylgroup,whichhasbeenshowntobeaparticularly
importantmodificationforcellpermeability.11Modificationscanalsobeperformed
onthesidechainsofthepeptide.Disulfideformationisanimportantmodification
thatchangestheconformationofthepeptideandincreasesitsstability.12Amidation
ofacarboxylicresiduewithanitrogenonthepeptideoffersinterestingstructures.13
Scheme4.Peptidescanbemodifiedatmultiplelocations
OnematterthatisalwaysaddressedisthenatureoftheN-terminusbecauseitis
usuallythelaststepforthesynthesis.Itcanbeleftasaprimaryaminebefore
cleavagefromresin.ThemostfrequentN-terminalmodificationisacetylation
becauseithassignificantimplicationsonthepeptideinteractionwithproteins.14
TheN-terminusisagoodpointforsinglemodificationonapeptide.Itiseasyto
performanditfollowsthelinearcouplingstrategyofSPPS.AlsoitchangestheN-
terminusfromaprimaryaminetoasecondaryamine.Ifthepeptidewasbasedoffof
aproteinfragment,themodificationoftheN-terminus(R’)ofthepeptidewouldbe
analogoustotheprotein(Figure5).Inthisthesisdifferentreactionsareinvestigated
thatchangetheN-terminusofpeptideswhilestillonthepolymersupport.
O
NH2HS
ONH
OHO
OHN
SH
OH
10 11
NNS S
O
O NH2
O OH
O
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8
Figure5.TheN-terminusbeingasecondaryamine(12)givesitsimilarpropertiestoaprotein(13)
OHN PeptideR
Peptide
OHN PeptideR
R'
12 13
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9
Chapter2Results&Discussion
2IsocyanatesandPeptides
2.1β-alanineandderivatives
Thenucleophilicadditionofanamineintoanisocyanateisawell-established
reactionthatproducesaurea(Scheme5).15Thisreactionisinterestinginthe
perspectiveofpeptidechemistrybecausethisreactioncanworkwiththeN-
terminusofapeptideandureasareasimilarfunctionalgrouptoamides.Inthis
chapterthereactionandformationofisocyanatesandsimilarfunctionalgroups
withβ-aminoacidsisinvestigated.
Scheme5.Nucleophilicadditionofaminesintoisocyanates
β-aminoacidsarepresentinmanypharmaceuticalandagrochemicaltarget
molecules.16Themoleculestheyarepresentinhaveshownawiderangeof
biologicalpropertiessuchasantibacterial,antiketogenicandinsecticidalactivity.
Since,β-aminoacidsarehomologstoα-aminoacidstheirreactivityisexpectedtobe
quitesimilar.Thismakesthemagoodstartingpointwhendivergingfromnormalα-
aminoacidpeptidesynthesis.
AnumberofN-substitutedβ-alaninesderivativesweresynthesizedfromβ-alanine
andrelatedprecursors,usinggeneralexperimentalprocedureA.Mostofthe
reactionsproceededsmoothly,providingacceptableyieldsoftheproductsafter
+NHR’N
H
ORNH2R’C ONR
14 15 16
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10
purification(Scheme6).Inthecaseofthe3,3-dimethylsubstitutedβ-alaninethe
desiredproductwasnotobtained.Theincreasedsterichindrancebytheadjacent
methylgroupsreducedthenucleophilicityoftheamineoftheβ-alanine.This
resultedinithavingsimilarreactivitytotheweakercarboxylicacidnucleophile,
leadingtotheformationof18einlowyield.
Scheme6.β-alaninederivativesformedfromphenylisocyanate
Next,apeptideona2-chlorotritylpolymersupportwasprepared.Thegeneral
experimentalproceduresBandCwereusedtomakeaL-valyl-L-prolyl-L-
trypotophanpeptideona2-chlorotritylresin(Scheme7).Oncetheresinbound
peptidewasmade,thecompounds18werecoupledtothepeptideusinggeneral
experimentalprocedureD(Scheme8).Inmostinstancesthereactionproceededas
expected.Mostofthecompoundswereobtainedinhighpurity,butlowyield.The
NCO
H2N OH
O
R+
NH
OH
O
RNH
OTHF
3 days
Phenylisocyanate(1.0 equiv)
17(1.0 equiv)
18
NH
OH
O
NH
O
NH
OH
O
NH
O
NH
OH
O
NH
O
18a95%
18b22%
18c24%
NH
OH
O
NH
O
NH
O
O
NH
O
18d52%
18e2%
NH
O
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11
lowyieldismostlikelyduetoresidualamountsofthepeptidenotbeingableto
cleavefromtheresin.Thisisanissuethatcameupinmostinstancesofcleavageof
thepeptidefromthepolymersupportinthisthesis.
Scheme7.Synthesisofapeptideona2-chlorotritylresin
Oneofthestartingcompounds,compound18e,whichinthepreviousreactionwas
notthedesiredproduct,didnotreactinthesamewayascompounds18a-d.Wehad
thoughtthatthecarbamatefunctionalityon18ewouldhaveasimilarreactivityas
theactivatedcarboxylicacidofcompounds18a-d.However,thecarbamategroup
interferedwiththereactionafteritwaseliminatedfromthecarbonyl.Thisyielded
sideproductsinadditiontothedesiredproduct,resultinginlowpurity.
O
OHNH
PSCl+ PSO
OHNDIPEA (10.0 equiv)
DCM, 5 h
RFmoc
RFmoc
DMF/Piperidine (4:1 v/v)15 min x2
PSO
OH2N
R
O
OHNH
RFmoc
HATU (3.0 equiv)DIPEA (6.0 equiv)
NMP, 2 h x2
PSO
OHN
RONH
RFmoc
PSO
OHN
ROH2N
Rn
19(3.0 equiv)
20 21
22
19(3.0 equiv)
23
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12
Scheme8.Substitutedβ-alaninesderivativescouplingtopolymerboundpeptides
Duetotheseissues,adifferentapproachwastakentomakecompound27e
(Scheme9).Thecyclicdimethylatedmaleicanhydrideunderwentnucleophilic
attackbytheN-terminusofapeptideboundresin.Followingaliteratureprocedure,
theresultantcarboxylicacidreactedwithDPPA,formingtheacylazide.17ACurtius
rearrangementcouldthenbeperformedandtheresultantisocyanatereactedwith
anilinetoformcompound27e.
VPW PS VPW
O
+ PSNHOH
O
RNH
O
NH R
NH
O
NH
VPW
O
NH
O
NH
VPW
O
NH
O
NH
VPW
O
NH
O
27a22% (91% purity)
27b23% (82% purity)
27c30% (80% purity)
NH
VPW
O
NH
O
NH
VPW
O
NH
O
27d23% (91% purity)
27e16% (31% purity)
OH OH OH
OHOH
VPW
O
NH R
NH
O
OH
18(3.0 equiv)
25 26
27
HATU (3.0 equiv)DIPEA (6.0 equiv)
NMP, 16 h
DCM/HFIP (4:1 v/v)1 h x2
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13
Scheme9.Formationofcompound27e,withoutimpurities
2.2Isocyanateequivalents
Thenextapproachwastosynthesizetheureafunctionalitybyreactionof30with
variousisocyanates,usingthegeneralexperimentalprocedureE(Scheme10).The
productcompounds32weremadesuccessfullyinhighpurity,butonceagainlow
yields.Notably,theisocyanatesdidnothaveanyadverseeffectonthepolymer
support.Thisreactionperformedwellevenwithelectrondonatinggroupsonthe
aromaticring,suchasmethoxy,whichreducetheelectrophilicityoftheisocyanate.
Inaddition,replacingtheisocyanatewithisothiocyanateswasalsosuccessful,
leadingtothiourea-terminatedpeptides.
VPW PSVPW
O+
PS
O OOHO
O
VPW
O
PSNCO
VPW
O
PSNHNH
O
26eI
27e
VPW
O
NH
NH
O
OH
28(3.0 equiv)
25
26e
THF
2 h
DPPA (10. 0 equiv)DIPEA (10.0 equiv)
Toluene, 2 hNH2
Aniline(10. equiv)
50oC, 40 h
DCM/HFIP (4:1 v/v)1 h x2
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14
Scheme10.Reactionandproductsofreactionofpolymerboundpeptidewithvariousisocyanatesandisothiocyanates.
PS VPW
O
+ PSNHNH
XR
VPW
O
H2NNCX
R
NH
VPW
O
NH
O
NH
VPW
O
NH
O
NH
VPW
O
NH
S
32a35% (100% purity)
32b27% (100% purity)
32f48% (100% purity)
F3C Cl
NH
VPW
O
NH
O
NH
VPW
O
NH
S
NH
VPW
O
NH
O
32c41% (94% purity)
32d50% (96% purity)
32g18% (100% purity)
N
O2N
NH
VPW
O
NH
O
32e43% (97% purity)
O
VPW
O
NH
NH
XR
OH
OH OH
OH
OH
OH
OH
OH
29(3.0 equiv)
THF
19.5 h
30 31
32
DCM/HFIP (4:1 v/v)1 h x2
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15
AnotherwaytoformureaswithanamineiswithN-alkylcarbamoylimidazoles,
derivativesofCDI.18Thesecompoundsarenotassimpletoworkwith,butoffera
largersubstratescopethanisocyanates.Thisscopeisonlylimitedtoanyamine
nucleophilethatcanreactwithCDIandnottheresultantN-alkylcarbamoyl
imidazoles.Thereactioneffectivelychangesanucleophilicaminegroupintoan
electrophilicisocyanateequivalent.TheN-alkylcarbomylimidazoleswereprepared
byanothermemberofourgroupfromvariousaminesandCDI,usinggeneral
experimentalprocedureF(Scheme11A).Thepolymerboundpeptidewasreacted
withtheN-alkylcarbamoylimidazoles,usinggeneralexperimentalprocedureG.
Thisyieldedthedesiredureasinhighpurity,butagaininlowyield(Scheme11B).
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16
Scheme11.PolymerboundpeptidewithvariousN-alkylcarbamoylimidazoles
PS
VPW
O
+
PSNHNH
O
VPW
O
H2NR N
HN
O
N
R
NH
VPW
O
NH
O
37d10% (100% purity)
NH
VPW
O
NH
O
NH
VPW
O
NH
O
NH
VPW
O
NH
O
37a2% (100% purity)
37b2% (100% purity)
37e13% (100% purity)
NH
VPW
O
NH
O
37c10% (100% purity)
Cl
Cl
O
Cl
N N
O
NN + R NHN
O
NR NH2
R NH
N
O
N
VPW
O
NH
NH
O
R OH
OH OH
OH
OH
OH
A
B
CDI(1.1 equiv)
DMAP (0.1 equiv)
DCM, 0OC -> rt3 h33 34
34(3.0 equiv)
MeI (15.0 equiv)
MeCN, 24 h35 30
3637
NEt3 (6.0 equiv)DCM, 20 h
DCM/HFIP (4:1 v/v)1 h x2
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17
2.3Reactivityofthioureas
PreviouslyintheBateygroup,itwasshownthatthioureasattachedtopolymer
boundpeptidesundergocyclizationtoformhydantoins,inthepresenceof
Mukaiyama’sreagent(Scheme12).19Usingtheβ-alaninethioureapreviouslymade
Scheme12.Polymerboundpeptidescontainingthioureascanformhydantoins
32f,thisapproachwasattemptedtoforma6-memberedversionofthehydantoin,
usinggeneralexperimentalprocedureH(Scheme13).Whenfirstattemptingthis
reactiontheformationofthecarbodiimideintermediate40andtheureaside
product42wasobserved,butthedesired6-memberedringproduct41wasnot
(Table1,entry1).Thepresenceoftheureaindicatesthatthecarbodiimide
underwentnucleophilicattackbywater.Thereactionwasattemptedagainunder
dryerconditions,toattempttoreducetheamountofureaformation(Table1,entry
2).ByLCMSthepresenceoftheproductplus2m/z,indicatedasmallquantityofthe
startingmaterialhadlikelybeenreduced.Thisismostlikelyduetotheincreasein
peptideHN PS
Mukaiyama’sReagent
(10.0 equiv)
NEt3 (10.0 equiv)DMF, 20 hO
NH
R
NH
SR' peptide
HN PS
ON
RCN’R
peptideN PSHN
O
N
R
R'
38
39
N ClI
-
18
Scheme13.Cyclizationofaβ-alaninethiourea
Entry Base Product(LCMS,%)***
1* NEt3 0
2 NEt3 0
3 Piperidine 0
4 DABCO 0
5** NaOH 0
6 NaH 0
*Entry1usedDMF,Mukaiyama’sreagent,NEt3andDCMwithoutspecialpreparation.Entries2-6useddistilledDMFandDCM,Mukayaima’sreagentfromadesiccatorandNEt3withmolecularsieves.**IRindicatedcleavedfromresin,nobandscorrespondingtothepeptidewerepresent(Seeappendix)***LCMSisfromasmallamountofthepeptidethatwascleavedfromthepolymersupport.IRswereobtainedwiththepeptidestillattachedtothepolymersupport.
Table1.Differentconditionsforcyclizingaβ-alaninethiourea
VPW PS
O
NH
NH
S
VPW PS
O
NCN
PW PS
O
HN
N N O
32f
Mukaiyama’sReagent
(10.0 equiv)
Base (10.0 equiv)DMF, 20 h
N ClI
41
40
VPW PS
O
NH
NH
O
42
-
19
purityoftheMukaiyama’sreagentfromthedrying,resultinginahigheramountof
equivalentsofthereagentrelativetothepreviousentry.Toincreasethe
nucleophilicityofthenitrogenonthevalineofthepeptide,theaminoacidadjacent
tothecarbodiimide,differentbaseswereexperimentedwith(Table1,entry3-6).
Thesetestsdidnotresultinformationoftheproduct,andinthecaseofNaOHit
appearedtohavecleavedthepeptidefromthepolymer.
Sinceitappearedthatthecarbodiimidewasbeingformed,trappingitoutwitha
nucleophilewasattempted.Anewpeptideonresinwaspreparedusinggeneral
experimentalprocedureB,CandE,yielding43.Usinggeneralexperimental
procedureI,thisreactionworkedwithbenzylaminegivinganalmostpureproduct
andsurprisinglyhighyield(Scheme14).
Scheme14.Trappingoutapolymersupportedcarbodiimidewithbenzylamine
AW PS
O
NH
NH
S
AW PS
O
NCN
NH2
AW PS
O
NH
N
NHAW
O
NH
N
NH
45a90% (90% pure)
OH
Mukaiyama’sReagent
(10.0 equiv)
NEt3 (10.0 equiv)DMF, 20 h
N ClI
43
Benzylamine(10.0 equiv)
44a
DCM/HFIP (4:1 v/v)1 h x2
-
20
Thiswasapromisingresult,asaguanidinefunctionalityisoftenfoundinbiological
moleculesandnotjustintheformoftheaminoacid,arginine.20Thisreactionwas
attemptedagainwithanumberofdifferentnucleophiles46(Scheme15).
Unfortunately,mostofthesereagentswerenotstrongenoughnucleophilestolead
toadditionproducts44.Thetert-butylesteralanineyieldedlowamountsofthe
desiredproduct.Thisisofinterest,becauseifaprotectinggroupsimilartoFmoc
wereplacedonthecarboxylicacidthepeptidewouldhavethepotentialtobegrown
outfurtherinareverseorder.Thus,changingthechaingrowthfromC-terminusto
N-terminusintoN-terminustoC-terminus,whilestillbeingcompatiblewiththe2-
chlorotritylresin(Scheme16).
Scheme15.Trappingoutapolymersupportedcarbodiimidewithvariousnucleophileswithamountconvertedtoproduct(LCMS)shown
AW PS
O
NH
NH
S
AW PS
O
NCN
AW PS
O
NH
N
Nuc
Nuc46
(10.0 equiv)
44
Mukaiyama’sReagent
(10.0 equiv)
NEt3 (10.0 equiv)DMF, 20 h
N ClI
43
NH2 NH2 HNFmoc OH
O
O
ONH2OH
46b0%
46c0%
46d0%
46e0%
46f28%
-
21
Scheme16.TheuseofaC-terminusprotectedaminoacidasanucleophileallowsfortheorientationofthepeptidetoflip
2.4Conclusionandoutlook
ThismethodologyshowedanumberofwaystoformureasontheN-terminusof
polymer-supportedpeptides.Isocyanatescanreactwithunprotectedaminoacids
ontheN-terminusandthentheC-terminuscanbecoupledtoapolymer-supported
peptide.AlternativelytheN-terminusofapeptidealreadyonresincanreactwithan
isocyanate.AnotherwayofmakingtheseureasisreactingtheN-terminuswith
carbamoylimidazoles.Theisocyanatesofferasimpleandcommerciallyavailable
syntheticroute,whilethecarbamoylimidazolesofferapotentialwiderscope.
ItwasalsoshownthataN-terminalgroupbearingacarboxylicacidonapolymer-
supportedpeptidecouldundergotheCurtiusrearrangementtoformureas.Thisis
knowntogothroughanisocyanateintermediateontheN-terminus.Thethiourea
derivativesofpolymer-supportedpeptidesofferaninterestingnewwaytoform
guanidines,throughacarbodiimideintermediateontheN-terminus.Boththese
reactionswarrantfurtheroptimizationandnucleophilicscopeforpotential
practicaluses.
peptide PS
OHN
R
HN
NR''R’O
O
peptide PS
OHN
R
HN
NR’’R’peptide
O
N CC N CC
47 48
-
22
Chapter3Results&Discussion
3PeptideCouplingConditions
3.1Methodsofactivation
Herecouplingconditionsforpeptidesandtheirrelativeeffectivenesswas
investigated.Fortheseexperimentsaprolineterminalpolymerboundpeptidewas
used.Proline,beingtheonlynaturalsecondaryaminoacid,isknowntobemore
difficulttocouplethroughacylationoftheN-terminus.Thepolymer-supported
peptide,50,waspreparedusinggeneralexperimentalprocedureBandC.We
startedthisstudybyreactingthepeptidewithtwoactivatedacylatingcompounds
usinggeneralexperimentalprocedureJ(Table2).Theacylchloride,benzoyl
chloride,andthesymmetricalanhydride,benzoicanhydride,bothgavetheproduct
inhighpuritybutlowyield.
Wenextactivatedbenzoicacidwithdifferenttypesofreagentstoundergopeptide
couplingusinggeneralexperimentalproceduresKandL(Table3).Thebulkofthe
peptidewasnotcleavedfromtheresin,exceptforasmallportionthatwasusedfor
LCMSanalysis.Inthiscasetheacylatingintermediates(Table3,entry1-2)were
showntoresultinhigherproportionofproductthantheanhydrides(Table3,entry
3-6.Oneissueinparticularwastheunsymmetricalnatureoftheanhydrides,
isobutylchloroformate,Yamaguchireagent,anddiethylcarbamylchloride,whichhad
issueswiththeactivatingagentreactingdirectlywiththepeptideforming52.Itwas
alsoshownthatoxalylchlorideisabetteractivatorofcarboxylicacidsthanthionyl
chloride,duetotheamountofproductpresentineachreaction.
-
23
Entry Electrophile Purity(%) Yield(%)
1
99 4
2
99 13
Table2.Reactionofapolymerboundpeptidewithsimpleacylatingagents
Withtheseresultswemovedontoactivationofaminoacidsusinggeneral
experimentalproceduresMandN.Fmocprotectedglycinewasactivatedand
allowedtoreactwiththepolymerboundpeptide(Table4).Onceagainoxalyl
chloridewasshowntobethebestactivatingagent.Thecompoundsshowedhigh
purity,butwereisolatedinpooryields.
PW PS PW+ PS
OO
X
51
PW OH
O
52
49(3.0 equiv)
50
DIPEA (6.0 equiv)
DCM, 1 h
DCM/HFIP (4:1 v/v)1 h x2
Cl
O
O
O
O
-
24
Entry ActivatingAgent X 51(LCMS,%) 52(LCMS,%)
1
98 N/A
2
100 N/A
3
95 0
4
53 43
5
38 61
6
0 10
Table3.Reactionofapolymerboundpeptidewithvariousactivatingagentsandbenzoicacid
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.6 equiv)
DCM, 1 h
O
HO51
Benzoic Acid(3.0 equiv)
49
DIPEA (6.0 equiv)
NMP, 1 h50
+
PW PS
O
X
52
SO
ClCl+ DMF Cl
Cl
O
OH
Cl+ DMF Cl
O
Cl
O Cl
OO
O
Cl
Cl
Cl Cl
Cl
Cl Cl
O
ClNN
-
25
Entry ActivatingAgent X 54(purity,%) Yield(%) 52
1
60 N/A N/A
2
95 9 N/A
3
94 4 4
Table4.Polymerboundpeptidereactingwithactivatedaminoacid
3.2Applicationsofactivation
Withthebestactivatingconditionsfound,comparisonwithstandardcoupling
conditionscouldbeperformed.Twomorepolymerboundpeptides,55and57,
weremadefollowinggeneralexperimentalproceduresBandC.Usingoxalyl
chloridewithacarboxylicacid,thatwaspreviouslyshowntonotcoupletothe
peptideusingHATU,wewereabletogetsuccessfulconversiontotheDVVSV-based
product56a,usinggeneralexperimentalprocedureP(Table5).
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.0-3.6 equiv)
DCM, 0.5 h
O
HO
HNFmoc
NHFmoc
NHFmoc
54
Fmoc-glycine(3.0 equiv)
DIPEA (6.0 equiv)
NMP, 1 h53 50
+
PW PS
O
X
52
SO
ClCl+ DMF Cl
Cl
O
OH
Cl+ DMF Cl
O
Cl
-
26
Entry ActivatingAgent 56a(%)
1
0
2
96
Table5.Differentactivatingagentswithanelectronpoorcarboxylicacid
Tofurthershowtheeffectivenessofthisreagent,wetriedwithanothercompound
polymerboundVVSV57,whichalsodidnotcouplethecorrespondingacidusing
HATU.Inthiscase,theoxalylchlorideconditionsdidnotwork,butitwasfoundthat
EDCconditionsdid,usinggeneralexperimentalprocedureQ(Table6).Thissuccess
ofEDCrelativetoHATUmaybeduetothelargestericsandelectronpoor
characteristicsofthecarboxylicacid.Thiswouldmakeitharderforthecarboxylic
acidtoaddintoHATU,whileEDCwouldberelativelyeasier,duetotherelative
stericsofthesereagents.
SO ONActivating
Agent(3.0 equiv)
DCM, 0-1 hDVVSV PS
DVVSVO
+
PS
SO O
N
O
ClO
X Cl
O
SO ON
O
OH Cl
O
56a53a(3.0 equiv)
54a
55
DIPEA (6.0 equiv)
NMP, 1 h
N NN
N
O
PF6N
N
Cl
O
OH
Cl+ DMF
-
27
Entry ActivatingAgent 56b(%)
1
0
2
0
3
92
Table6.Reactionusingdifferentactivatingagentswithanelectronpoorcarboxylicacid53b
Thispromisingresultencouragedustousethismethodwithonemorecarboxylic
acidthathadpreviouslygivenpoorerresultswithHATU(Table7).Onceagainit
wasshownthatEDCwasmoreeffectiveasacouplingagent.
SO OHN
HO
O SO OHN
X
OVVSV PS VVSV
O
SO
ONH
+ PS
56b53b(3.0 equiv)
54b57
ActivatingAgent
(3.0 equiv)
DCM, 0-1 h
DIPEA (6.0 equiv)
NMP, 1 h
N NN
N
O
PF6N
N
Cl
O
OH
Cl+ DMF
NCNN
N
NN
NOH+
-
28
Entry ActivatingAgent 56c(%)
1
60
32
98
Table7.Comparisonofabulkycarboxylicacid,53c,couplingsusingHATUandEDC
UsingthisnewinformationabouttheeffectivenessofEDC,anumberofdifferent
amineswereinvestigated,usinggeneralexperimentalprocedureR(Scheme17).
Since,wewereinvestigatingawidescopeofamineswithoutaC-terminus,forthis
experimenttheconditionswereinsolutionphasewithoutapolymersupport.Inthis
caseEDCwasshowntonotbeaseffectiveasHATU.Amines57d-f,whichwere
weakernucleophilesthantheotheramines,didnotcoupletothecarboxylicacidat
allwithEDC,butworkedwithHATU.Thisreactionislessdependentonthesterics
oftheEDCandHATUbecausethesamecarboxylicacid,whichisnotbulky,isused
witheachamine.Thepartofthecouplingthatisaffectedbytheamineinvolvesthe
HOAtcomponentofthereaction,whichHATUreleasesafterthefirststepgivinga
greaterentropicdrivingforcerelativetotheEDCconditions.Inadditionthistime
DMAPwasusedinsteadofHOAtfortheEDCconditionsandwasusedinlessthan
SO ONHActivating
Agent(3.0 equiv)
DCM, 0-1 hDVVSV PS
DVVSVO
+
PS
SO O
NH
BrO
X Br
SO ONH
O
OH Br
56c53c(3.0 equiv)
54c
55
DIPEA (6.0 equiv)
NMP, 1 h
Cl
O
OH
Cl+ DMF
NCNN
N
NN
NOH+
-
29
oneequivalent,furthershowingtheimportanceoftheDMAP/HOAtcomponentof
thereaction.Allthecompoundsweremadeinlowyieldsduetotheweak
nucleophilicpropertiesoftheaminesandtheratioofaminetocarboxylicacid
comparedtopreviousreactions.
.
O
+ NH
NH
OO
NH
R NH
ROHNH
O
H2NR
18a(1.0 equiv)
57 58
EDC (1.4 equiv)DMAP (0.5 equiv)
DIPEA (1.5 equiv)DCM, 16 h
N
S
H2N
Ph
PhN
SH2NPh
OH2N
HN
O
HN
OH2N N
N
H2N
N
O
H2N
Ph
Ph
N
NHH2N
N
N
O
HN
OH2N
N
N
O
HN
OH2N
N
NHH2N
Ph
O
N
N
OHN
H2NHN
O
H2N N
N
Ph
57c3%
57b9%
57i6%
57g30%
57d7%
57j30%
57l35%
57k51%
57f2%
57e31%
57a40%
57h16%
* **
-
30
*UsedHATUinsteadofEDCandDMAP,whichresultedinnoproductformationScheme17.Reactingthecarboxylicacid18awithvariousamines
NHN
OH2N
NH2NO F F
F
NH
HN
OH2N
ON
O
H2N
F
O
NNH2
H2NN
O Br
H2NHN
O
N
H3NO
O
O
H3N
N
O
NH2 HN
O
N
H2N N
O
57u8%
57m51%
57v8%
57o37%
57r44%
57t48%
57w19%
Cl
57q47%
Cl
57s45%
57p4%
57n42%
-
31
3.3ConclusionandoutlookOptimalcouplingconditionswerediscoveredforthereactionofpolymer-supported
peptides.Withthelesstypicalactivatingconditionsitwasfoundthatacylchlorides
workedbetterthananhydridesforcoupling.Theanhydrideshadworseleaving
groupsandhadtheissueofthedesiredhalfoftheanhydridebecomingthe
carboxylateleavinggroup,whichisnotlikelytooccurforacylchlorides.
Thedifferencesbetweenstandardcouplingconditionswerealsoinvestigated.HATU
andEDCwereshowntobothworkwelldependingonthesubstratetheywere
activating.Duetotheirdifferencesthereisnotonebestcouplingagentforpolymer
supportedpeptides,butitdependsonthesituation.Thesedifferenceswarrant
furtherinvestigation,asitwouldbeneficialtonowhatarethedifferentparameters
forthesedifferentinstances.
-
32
Chapter4Experimental
Copiesof1HNMR,13CNMRandIRspectraforallsynthesizedcompoundscanbe
foundintheappendix.THF,diethyletherandpentanewerefreshlydistilledfrom
sodium/benzophenoneketylundernitrogen.DCMandtoluenewerefreshlydistilled
fromsodiumundernitrogen.AnhydrousDMFwasstoredundernitrogenwith4Å
molecularsieves.AllothersolventswereobtainedasACSgradeorbetterfrom
commercialsuppliers.Allstartingmaterialsandreagentswerepurchasedfrom
Aapptec,AlfaAesarorSigma-Aldrich.Flashchromatographyonsilicagel(60Å,230-
400mesh,obtainedfromSilicycle)wasperformedwithreagentgradesolvents.
AnalyticalTLCwasperformedonMerckSilicagel60F254pre-coatedplatesand
visualizedwithaUVlamp.Reversephasecolumnchromatographywasperformed
withaBiotageSNAPUltraC18column.IRspectrawereobtainedonaPerkinElmer
100SeriesFTIRequippedwithadiamond/ZnSeATRaccessory.UVspectrawere
obtainedusingaLambda1050UV/VisSpectrometer.LCMSdatawasobtainedfrom
AgilentHPLCsystems(1100,1200,1260).Massspectrawereobtainedbythe
UniversityofTorontoAIMSmassspectrometryfacility;HRMSwererecordedonan
Agilent6538UHDAccurate-MassQ-TOFLC/MS.Meltingpointswereobtainedona
MEL-TEMPcapillarymeltingpointapparatusandareuncorrected.AllNMRspectra
wereobtainedon400and500MHzspectrometersassolutionsindeuterated
solvents(obtainedfromCambridgeIsotopeLabs).Chemicalshiftsarereportedasδ
values.1HNMRchemicalsshiftswereinternallyreferencedtotetramethylsilane(δ
0.00)forCDCl3ortotheresidualprotonresonanceinMeOH-d4(δ3.31)andDMSO-
d6(δ2.49).Carbonchemicalshiftswereinternallyreferencedtothesolvent
resonancesinCDCl3(δ77.16),MeOH-d4(δ49.15)andDMSO-d6(δ39.51).Peak
multiplicitiesaredesignatedbythefollowingabbreviations:s,singlet;d,doublet;t,
triplet;q,quartet;m,multiplet;br,broad;J,couplingconstantinHzandroundedto
thenearest0.5Hz.
-
33
Generalexperimentalprocedures
GeneralexperimentalprocedureA
Toaflamedried25mLroundbottomflaskundernitrogengasaβ-aminoacid(1.0
equiv),phenylisocyanate(1.0equiv)anddistilledTHF(5-28mL)wereadded.The
reactionmixturewasstirredfor3daysthenpurifiedusingautomatedflashsilicagel
columnchromatography(10gcolumn,0%-50%ethylacetateinhexane,0%-20%
MeOHinDCM)yieldingtheproduct18.
GeneralexperimentalprocedureB
Toafrittedroundbottomreactionvessel2-chlorotritylchlorideresin(1.0equiv)
wasadded.ThereactionvesselwasattachedtoanAapptecEndeavor90tabletop
peptidesynthesizer.Undernitrogengastheresinwasswelledwithpeptidegrade
DMF(10mL/gofresin)for15min.Afterthesolventwasdrained,asolutionofan
Fmoc-protectedaminoacid(5.0equiv)andDIPEA(10.0equiv)indistilledDCM(10
mL/gofresin).Theresinsolutionwasshakenfor5h.Thesolutionwasdrainedand
theresinwascappedbytheadditionofHPLCgradeMeOH.Theresinsolutionwas
shakenfor15min,drainedandwashedwithpeptidegradeDMF(10mL/gofresin
x2),HPLCgradeMeOH(10mL/gofresinx2)anddistilledDCM(10mL/gofresin
NCO
H2N OH
O
R+
NH
OH
O
RNH
OTHF
3 days
Phenyl Isocyanate(1.0 equiv)
17(1.0 equiv)
18
O
OHNH
PSCl+ PSO
OHNDIPEA (10.0 equiv)
DCM, 5 h
RFmoc
RFmoc
19(3.0 equiv)
20 21
-
34
x2)yieldingtheFmoc-protectedaminoacidattachedtoa2-chlorotritylresin.The
resinwasdriedfor24hundervacuum.Asmallamountoftheresin(10.0mg)was
weighedoutina0.5drvial.TheresinwasswelledinDMF(0.800mL)for15min.To
theresinsolutionpiperidine(0.200mL)wasaddedandtheresinsolutionwas
shakenfor15minutes.Asmallamount(0.050mL)ofthesolutionwastaken
withouttheresinandwasaddedtoaDMF(3mL)ina0.5drvial.TheUV
absorbanceofthissolutionwasmeasuredandusedtocalculatetheloading
efficiencyoftheFmoc-protectedaminoacidontothe2-chlorotritylresin.
GeneralexperimentalprocedureC
ToafrittedroundbottomreactionvesselanFmoc-protectedaminoacidattachedto
a2-chlorotritylresin(1.0equiv)wasadded.Thereactionvesselwasattachedtoan
AapptecEndeavor90tabletoppeptidesynthesizer.Undernitrogengastheresin
wasswelledwithpeptidegradeDMF(10mL/gofresin)for15min.Afterthe
solventwasdrained,amixtureofpeptidegradeDMFandpiperidine(4:1v/v)was
addedandshakenwiththeresinfor15min.Afterdrainingthisprocesswas
repeatedwiththemixtureforanother15min.Themixturewasdrainedandthe
PSO
OHN
RFmoc
DMF/Piperidine (4:1 v/v)15 min x2
PSO
OH2N
R
O
OHNH
RFmocHATU (3.0 equiv)
DIPEA (6.0 equiv)NMP, 2 h x2
PSO
OHN
RONH
RFmoc
PSO
OHN
ROH2N
Rn
21 22
19(3.0 equiv)
2324
-
35
resinwashedwithpeptidegradeDMF(10.0mL/gofresinx2).Nextasolutionofan
Fmoc-protectedaminoacid(3.0equiv),HATU(3.0equiv)andDIPEA(6.0equiv)in
NMP(10mL/gofresin)wasadded.Theresinsolutionwasshakenfor2h.The
solutionwasdrainedandthisprocesswasrepeatedwiththemixtureforanother2
h.TheresinsolutionwasdrainedandwashedwithpeptidegradeDMF(10mL/gof
resinx2),HPLCgradeMeOH(10mL/gofresinx2)anddistilledDCM(10mL/gof
resinx2)yieldingaFmoc-protecteddipeptideattachedtoa2-chlorotritylresin.This
entireprocessisrepeatedtoaddadditionalaminoacidstothepeptides.
GeneralexperimentalprocedureD
ToaRediSepdisposablecolumn(22.4mL)L-valyl-L-prolyl-L-trypotophanattached
toa2-chlorotritylresin(1.0equiv)wasswelledinpeptidegradeDMF(5mL)for15
minutes.Thesolventwasdrainedandasolutionofcarboxylicacid(3.0equiv),
HATU(3.0equiv)andDIPEA(6.0equiv)inNMP(3mL)wasadded.Theresin
solutionwasshakenovernight.Thesolutionwasdrainedandtheresinwashedwith
peptidegradeDMF(5mLx2),HPLCgradeMeOH(5mLx2)anddistilledDCM(5mL
x2).DistilledDCM(2.4mL)andHFIP(0.6mL)wereaddedtotheresinandthe
reactionmixtureshakenfor1h.Thesolutionwascollectedandthisprocesswas
repeatedwiththeresin,moreHFIPandmoreDCM.AsolutionofdistilledEt2Oand
distilledpentane(3mL,1:1v/v)wasaddedtotheresultingoilformingasolid.The
solidwastrituratedthroughsonicationandthenfilteredundervacuumyieldingthe
productasasolid27.
VPW PS VPW
O
+ PSNHOH
O
RNH
O
NH R
NH
O
VPW
O
NH R
NH
O
OH
18(3.0 equiv)
25 26
27
HATU (3.0 equiv)DIPEA (6.0 equiv)
NMP, 16 h
DCM/HFIP (4:1 v/v)1 h x2
-
36
GeneralexperimentalprocedureE
ToaRediSepdisposablecolumn(22.4mL)L-valyl-L-prolyl-L-trypotophanattached
toa2-chlorotritylresinsolutionwasswelledinpeptidegradeDMF(5mL)for15
minutes.Totheresinaphenylisocyanate(3.0equiv)anddistilledTHF(3mL)was
added.Theresinwasshakeninthesolutionovernight(19.5h).Thesolutionwas
drainedandtheresinwashedwithpeptidegradeDMF(5mLx2),HPLCgradeMeOH
(5mLx2)anddistilledDCM(5mLx2).DistilledDCM(2.4mL)andHFIP(0.6mL)
wasaddedtotheresinandthereactionmixturewasshakenfor1h.Thesolution
wascollectedandthisprocesswasrepeatedwiththeresin,moreHFIPandmore
DCM.AsolutionofdistilledEt2Oanddistilledpentane(3mL,1:1v/v)wasaddedto
theresultingoilformingasolid.Thesolidwastrituratedthroughsonicationand
thenfilteredundervacuumyielding32.
GeneralexperimentalprocedureF
Toaflameddry25mLroundbottomflask,undernitrogengasat0°C,anamine(1.0
equiv),CDI(1.1equiv),DMAP(0.1equiv)anddistilledDCM(10mL)wasadded.The
solutionwasallowedtocometoroomtemperature,stirredfor3handthenpurified
usingflashsilicagelcolumnchromatography(0%-5%MeOHinDCM)yielding34.
PS VPW
O
+ PSNHNH
XR
VPW
O
H2NNCX
R
VPW
O
NH
NH
XR
OH
29(3.0 equiv)
THF
19.5 h
30 31
32
DCM/HFIP (4:1 v/v)1 h x2
N N
O
NN + R NHN
O
NR NH2
CDI(1.1 equiv)
DMAP (0.1 equiv)
DCM, 0OC -> rt3 h33 34
-
37
GeneralexperimentalprocedureG
Toa20mLvial,undernitrogengasanN-alkylcarbamoylimidazole(3.0equiv),MeI
(15.0equiv)anddistilledacetonitrile(1mL)wasaddedandstirredfor24h.The
reactionmixturewasthendried.NEt3(6.0equiv)anddistilledDCM(3mL)were
addedtothemixture.ThesolutionwasthenaddedtoaRediSepdisposablecolumn
(22.4mL)containing3-aminopropanamido-L-valyl-L-prolyl-L-trypotophan
attachedtoa2-chlorotritylresin(1.0equiv)wasswelledinpeptidegradeDMF(5
mL)for15minutes.Theresinsolutionwasshakenfor20h.Thesolutionwas
drainedandwashedwithpeptidegradeDMF(5mLx2),HPLCgradeMeOH(5mL
x2)anddistilledDCM(5mLx2).DistilledDCM(2.4mL)andHFIP(0.6mL)were
addedtotheresinandthereactionmixturewasshakenfor1h.Thesolutionwas
collectedandthisprocesswasrepeatedwiththeresin,moreHFIPandmoreDCM.A
solutionofdistilledEt2Oanddistilledpentane(3mL,1:1v/v)wasaddedtothe
resultingoilformingasolid.Thesolidwastrituratedthroughsonicationandthen
filteredundervacuumyieldingthe37.
PS
VPW
O
+
PSNHNH
O
VPW
O
H2NR N
HN
O
N
R
R NH
N
O
N
VPW
O
NH
NH
O
R OH
34(3.0 equiv)
MeI (15.0 equiv)
MeCN, 24 h35 30
3637
NEt3 (6.0 equiv)DCM, 20 h
DCM/HFIP (4:1 v/v)1 h x2
-
38
GeneralexperimentalprocedureH
ToaRediSepdisposablecolumn(22.4mL)containingL-valyl-L-prolyl-L-
trypotophanattachedtoa2-chlorotritylresin(1.0equiv,0.017mmol,0.34mmol/g)
wasaddedandallowedtoswellinpeptidegradeDMF(5mL)for15minutes.Tothe
resinsolutionMukaiyama’sreagent(10equiv,0.17mmol),base(10equiv,0.17
mmol)anddriedDMF(3mL)wasadded.Theresinsolutionwasshakenovernight
(19h).DistilledDCM(0.8mL)andHFIP(0.2mL)wereaddedtoasmallamountof
theresin(1mg)andthereactionmixturewasshakenfor1h.Theresultingsolid
wasusedforLCMSanalysis.
VPW PS
O
NH
NH
S
VPW PS
O
NCN
PW PS
O
HN
N N O
32f
Mukaiyama’sReagent
(10.0 equiv)
Base (10.0 equiv)DMF, 20 h
N ClI
41
40
-
39
GeneralexperimentalprocedureI
ToaRediSepdisposablecolumn(22.4mL)L-valyl-L-prolyl-L-trypotophanattached
toa2-chlorotritylresin(1.0equiv)wasswelledinpeptidegradeDMF(5mL)for15
minutes.TotheresinsolutionMukaiyama’sreagent(10.0equiv),NEt3(10.0equiv)
anddriedDMF(3mL)wereadded.After1hanamine(10.0equiv)wasadded.The
resinsolutionwasshakenovernight(19h).Thesolutionwasdrainedandwashed
withpeptidegradeDMF(5mLx2),HPLCgradeMeOH(5mLx2)anddistilledDCM
(5mLx2).DistilledDCM(2.4mL)andHFIP(0.6mL)wereaddedtotheresinand
thereactionmixturewasshakenfor1h.Thesolutionwascollectedandthisprocess
wasrepeatedwiththeresin,moreHFIPandmoreDCM.AsolutionofdistilledEt2O
anddistilledpentane(3mL,1:1v/v)wasaddedtotheresultingoilformingasolid.
Thesolidwastrituratedthroughsonicationandthenfilteredundervacuumyielding
the45.
AW PS
O
NH
NH
S
AW PS
O
NCN
AW PS
O
NH
N
Nuc
Nuc46
(10.0 equiv)
44
Mukaiyama’sReagent
(10.0 equiv)
NEt3 (10.0 equiv)DMF, 20 h
N ClI
43
AW
O
NH
N
Nuc
45
OH
DCM/HFIP (4:1 v/v)1 h x2
-
40
GeneralexperimentalprocedureJ
ToaRediSepdisposablecolumn(22.4mL)prolyl-L-trypotophanattachedtoa2-
chlorotritylresin(1.0equiv)wasaddedandallowedtoswellinpeptidegradeDMF
(5mL)for15minutes.Totheresinsolutionactivatedbenzoicacid(3.0equiv),
DIPEA(6.0equiv)andNMP(3mL)wereadded.Theresinsolutionwasshakenfor1
h.ThesolutionwasdrainedandwashedwithdistilledDCM(5mLx2).DistilledDCM
(2.4mL)andHFIP(0.6mL)wasaddedtotheresinandshakenfor1h.Thesolution
wascollectedandthisprocesswasrepeatedwiththeresin,moreHFIPandmore
DCM.AsolutionofdistilledEt2Oanddistilledpentane(3mL,1:1v/v)wasaddedto
theresultingoilformingasolid.Thesolidwastrituratedthroughsonicationand
thenfilteredundervacuumyielding52.
GeneralexperimentalprocedureK(acylchlorides)
PW PS PW+ PS
OO
X
51
PW OH
O
52
49(3.0 equiv)
50
DIPEA (6.0 equiv)
DCM, 1 h
DCM/HFIP (4:1 v/v)1 h x2
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.6 equiv)
DCM, 0.5-1 h
O
HO51
Benzoic Acid(3.0 equiv)
49
DIPEA (6.0 equiv)
NMP, 1 h50
+
PW PS
O
X
52
-
41
Toaflamedried25mLroundbottomflaskundernitrogengasbenzoicacid(3.0
equiv),activatingagent(3.6equiv),DMF(3.0equivlants)anddistilledDCM(7mL)
wasadded.After30minutesthereactionmixturewasdriedundervacuum.The
resultantoilwasdissolvedinDIPEA(6.0equiv)andNMP(3mL).Thesolutionwas
addedtoaRediSepdisposablecolumn(22.4mL)withprolyl-L-trypotophan
attachedtoa2-chlorotritylresin(1.0equiv)thatwasswelledinpeptidegradeDMF
(5mL)for15minutes.Theresinsolutionwasshakenfor1h.Thesolutionwas
drainedandwashedwithdistilledDCM(5mLx2)yielding51.DistilledDCM(0.8
mL)andHFIP(0.2mL)wereaddedtoasmallamountoftheresin(1mg)andthe
reactionmixturewasshakenfor1h.TheresultingsolidwasusedforLCMSanalysis.
GeneralexperimentalprocedureL(anhydrides)
Toaflamedried25mLroundbottomflaskundernitrogengasbenzoicacid(3.0
equiv),activatingagent(3.6equiv),anddistilledDCM(7mL)wasadded.After30
minutesthereactionmixturewasdriedundervacuum.Theresultantoilwas
dissolvedinDIPEA(6.0equiv)andNMP(3mL).Thesolutionwasaddedtoa
RediSepdisposablecolumn(22.4mL)withprolyl-L-trypotophanattachedtoa2-
chlorotritylresin(1.0equiv)thatwasswelledinpeptidegradeDMF(5mL)for15
minutes.Theresinsolutionwasshakenfor1h.Thesolutionwasdrainedand
washedwithdistilledDCM(5mLx2)yielding51.DistilledDCM(0.8mL)andHFIP
(0.2mL)wereaddedtoasmallamountoftheresin(1mg)andthereactionmixture
wasshakenfor1h.TheresultingsolidwasusedforLCMSanalysis.
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.6 equiv)
DCM, 0.5-1 h
O
HO51
Benzoic Acid(3.0 equiv)
49
DIPEA (6.0 equiv)
NMP, 1 h50
+
PW PS
O
X
52
-
42
GeneralexperimentalprocedureM(acylchlorides)
Toaflamedried25mLroundbottomflaskundernitrogengasFmoc-glycine(3.0
equiv),activatingagent(3.6equiv),DMF(3.0equiv)anddistilledDCM(7mL)was
added.After30minthereactionmixturewasdriedundervacuum.Theresultantoil
wasdissolvedinDIPEA(6.0equiv)andNMP(3mL).Thesolutionwasaddedtoa
RediSepdisposablecolumn(22.4mL)withprolyl-L-trypotophanattachedtoa2-
chlorotritylresin(1.0equiv)thatwasswelledinpeptidegradeDMF(5mL)for15
min.Theresinsolutionwasshakenfor1h.Thesolutionwasdrainedandwashed
withdistilledDCM(5mLx2)yieldingthe54.DistilledDCM(0.8mL)andHFIP(0.2
mL)wereaddedtoasmallamountoftheresin(1mg)andthereactionmixturewas
shakenfor1h.TheresultingsolidwasusedforLCMSanalysis.
GeneralexperimentalprocedureN(anhydrides)
Toaflamedried25mLroundbottomflaskundernitrogengasFmoc-glycine(3.0
equiv),activatingagent(3.0equiv),anddistilledDCM(7mL)wasadded.After30
minthereactionmixturewasdriedundervacuum.Theresultantoilwasdissolved
inDIPEA(6.0equiv)andNMP(3mL).ThesolutionwasaddedtoaRediSep
disposablecolumn(22.4mL)withprolyl-L-Trypotophanattachedtoa2-chlorotrityl
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.0-3.6 equiv)
DCM, 0.5 h
O
HO
HNFmoc
NHFmoc
NHFmoc
54
Fmoc-glycine(3.0 equiv)
DIPEA (6.0 equiv)
NMP, 1 h53 50
+
PW PS
O
X
52
PW PS
PW
+
PS
O
O
X
ActivatingAgent
(3.0-3.6 equiv)
DCM, 0.5 h
O
HO
HNFmoc
NHFmoc
NHFmoc
54
Fmoc-glycine(3.0 equiv)
DIPEA (6.0 equiv)
NMP, 1 h53 50
+
PW PS
O
X
52
-
43
resin(1.0equiv)thatwasswelledinpeptidegradeDMF(5mL)for15min.The
resinsolutionwasshakenfor1h.Thesolutionwasdrainedandwashedwith
distilledDCM(5mLx2)yieldingtheproduct.DistilledDCM(0.8mL)andHFIP(0.2
mL)wereaddedtoasmallamountoftheresin(1mg)andthereactionmixturewas
shakenfor1h.TheresultingsolidwasusedforLCMSanalysis.
GeneralexperimentalprocedureO(HATU)
ToaRediSepdisposablecolumn(22.4mL)L-aspartyl-L-valyl-L-valyl-L-seryl-L-
valineattachedtoa2-chlorotritylresin(1.0equiv)wasswelledinpeptidegrade
DMF(5mL)for15minutes.Totheresinacarboxylicacid(3.0equiv),HATU(3.0
equiv),DIPEA(3.0equiv)anddistilledDCM(3mL)wereadded.Theresinsolution
wasshakenfor1h.ThesolutionwasdrainedandwashedwithdistilledDCM(5mL
x2).DistilledDCM(2.4mL)andHFIP(0.6mL)wereaddedtotheresinandthe
reactionmixturewasshakenfor1h.Thesolutionwascollectedandthisprocess
wasrepeatedwiththeresin,moreHFIPandmoreDCM.AsolutionofdistilledEt2O
anddistilledpentane(3mL,1:1v/v)wasaddedtotheresultingoilformingasolid.
Thesolidwastrituratedthroughsonicationandthenfilteredundervacuumyielding
56.
GeneralexperimentalprocedureP(oxalylchloride)
Toaflamedried25mLroundbottomflaskundernitrogengasacarboxylicacid(3.0
equiv),oxalylchloride(3.0equiv),DMF(3.0equiv)anddistilledDCM(7mL)was
DVVSV PS DVVSV+ PSHO R
O
X R
OO
R
ActivatingAgent
(3.0 equiv)
DCM, 0-1 h
DIPEA (6.0 equiv)
NMP, 1 h53
(3.0 equiv)54 55 56
DVVSV PS DVVSV+ PSHO R
O
X R
OO
R
ActivatingAgent
(3.0 equiv)
DCM, 0-1 h
DIPEA (6.0 equiv)
NMP, 1 h53
(3.0 equiv)54 55 56
-
44
added.After30minutesthereactionmixturewasdriedundervacuum.The
resultantoilwasdissolvedinDIPEA(3.0equiv)andNMP(3mL).Thesolutionwas
addedtoaRediSepdisposablecolumn(22.4mL)L-aspartyl-L-valyl-L-valyl-L-seryl-
L-valineattachedtoa2-chlorotritylresin(1.0equiv)thatswelledinpeptidegrade
DMF(5mL)for15minutes.Theresinsolutionwasshakenfor2h.Thesolutionwas
drainedandwashedwithdistilledDCM(5mLx2).DistilledDCM(2.4mL)andHFIP
(0.6mL)wereaddedtotheresinandthereactionmixturewasshakenfor1h.The
solutionwascollectedandthisprocesswasrepeatedwiththeresin,moreHFIPand
moreDCM.AsolutionofdistilledEt2Oanddistilledpentane(3mL,1:1v/v)was
addedtotheresultingoilformingasolid.Thesolidwastrituratedthrough
sonicationandthenfilteredundervacuumyielding56.
GeneralexperimentalprocedureQ(EDC)
ToaRediSepdisposablecolumn(22.4mL)L-aspartyl-L-valyl-L-valyl-L-seryl-L-
valineattachedtoa2-chlorotritylresin(1.0equiv)wasswelledinpeptidegrade
DMF(5mL)for15minutes.Totheresinacarboxylicacid(3.0equiv),EDC(3.0
equiv),HOAt(3.0eqviv),NEt3(6.0equiv)anddistilledDCM(3mL)wasadded.The
resinsolutionwasshakenfor1h.Thesolutionwasdrainedandwashedwith
distilledDCM(5mLx2).DistilledDCM(2.4mL)andHFIP(0.6mL)wereaddedto
theresinandthereactionmixturewasshakenfor1h.Thesolutionwascollected
andthisprocesswasrepeatedwiththeresin,moreHFIPandmoreDCM.Asolution
ofdistilledEt2Oanddistilledpentane(3mL,1:1v/v)wasaddedtotheresultingoil
formingasolid.Thesolidwastrituratedthroughsonicationandthenfilteredunder
vacuumyielding56.
DVVSV PS DVVSV+ PSHO R
O
X R
OO
R
ActivatingAgent
(3.0 equiv)
DCM, 0-1 h
DIPEA (6.0 equiv)
NMP, 1 h53
(3.0 equiv)54 55 56
-
45
GeneralexperimentalprocedureR
Toa20mLvial3-(3-phenylureido)propanoicacid(1.0equiv),EDC(1.4equiv),
DMAP(0.5equiv),NEt3(1.5equiv)anddistilledDCM(0.8mL)wasadded.After
stirringfor1min,anamine(1.0equiv)wasaddedandthereactionmixturewas
allowedtostirovernight.Thereactionmixturewasthenpurifiedusingautomated
flashsilicagelcolumnchromatography(10gcolumn,0%-10%MeOHinDCM)
yieldingtheproduct58.
3-(3-Phenylureido)propanoicacid(18a)
UsingthegeneralexperimentalprocedureAwith3-aminopropanoicacid(500mg,
5.6mmol.0.2M),phenylisocyanate(0.67mg,5.6mmol,0.61mL)anddistilledTHF
(28mL)yielded18a,whichwasisolatedasawhitesolid(1.10g,95%).Rf:0.13
(DCM:MeOH9:1).1HNMR(500MHz,DMSO-d6)(ppm)δ12.32(1H,brs),8.58(1H,
brs),7.36(2H,dd,J=7.5,4.6Hz),7.20(2H,t,J=8.0Hz),6.87(1H,tt,J=7.4,1.2Hz),
6.23(1H,brt,J=5.7Hz),3.28(2H,q,J=6.3Hz),2.38(2H,t,J=6.4Hz).13CNMR
(126MHz,CDCl3)(ppm)δ173.4,155.1,140.5,128.6,120.9,117.5,35.2,34.8.HRMS
(ESI+)m/zcalc’dforC10H13N2O3[M+H]+:209.09262,found:209.09258.
(S)-3-(3-Phenylureido)butanoicacid(18b)
O
+ NH
NH
OO
NH
R NH
ROHNH
O
H2NR
18a(1.0 equiv)
57 58
EDC (1.4 equiv)DMAP (0.5 equiv)
DIPEA (1.5 equiv)DCM, 16 h
NH
O
OHNH
O
NH
O
OHNH
O
-
46
UsingthegeneralexperimentalprocedureAwith(S)-3-aminobutanoicacid(100mg,
0.968mmol,0.2M),phenylisocyanate(115.4mg,0.968mmol,0.106mL)and
distilledTHF(4.85mL)yielded18b,whichwasisolatedasawhitesolid(47.9mg,
22%).Rf:0.26(DCM:MeOH9:1).1HNMR(500MHz,MeOH-d4)(ppm)δ7.33(2H,d,
J=8.5Hz),7.23(2H,t,J=8.0Hz),6.96(1H,td,J=7.5,1.1Hz),4.19(1H,hextet,J=
6.7Hz,1H),2.49(2H,dq,J=41.1,6.2Hz),1.25(3H,d,J=6.7Hz).13CNMR(126MHz,
CDCl3)(ppm)δ172.9,154.4,140.5,128.6,120.9,117.5,42.2,41.0,20.6.HRMS
(ESI+)m/zcalc’dforC11H15N2O3[M+H]+:223.10827,found:223.10871.
(R)-3-(3-Phenylureido)butanoicacid(18c)
UsingthegeneralexperimentalprocedureAwith(R)-3-
aminobutanoicacid(100mg,0.968mmol,0.2M),phenylisocyanate(115.4mg,
0.968mmol,0.106mL)anddistilledTHF(4.85mL)yielded18c,whichwasisolated
asawhitesolid(52.2mg,24%).Rf:0.22(DCM:MeOH9:1).1HNMR(500MHz,
MeOH-d4)(ppm)δ7.33(2H,dd,J=8.7,1.1Hz),7.23(2H,t,J=8.0Hz),6.96(1H,t,J=
7.2,1.2Hz),4.19(1H,hextet,J=6.5Hz),2.48(2H,dq,J=40.7,6.1Hz),1.25(3H,d,J
=6.7Hz).13CNMR(126MHz,CDCl3)(ppm)δ172.2,154.4,140.5,128.6,120.9,
117.5,42.2,40.9,20.6.HRMS(ESI+)m/zcalc’dforC11H15N2O3[M+H]+:223.10827,
found:223.10897.
2-Methyl-3-(3-phenylureido)propanoicacid(18d)
UsingthegeneralexperimentalprocedureAwith(RS)-3-aminoisobutanoicacid
(100mg,0.968mmol,0.2M),phenylisocyanate(115.4mg,0.968mmol,0.106mL)
anddistilledTHF(4.85mL)yielded18d,whichwasisolatedasawhitesolid(111.5
mg,52%).Rf:0.26(DCM:MeOH9:1).1HNMR(500MHz,DMSO-d6)(ppm)δ12.35
(1H,brs),8.64(1H,brs),7.37(2H,dd,J=8.7,1.1Hz),7.20(2H,t,J=8.0Hz),6.87
NH
O
OHNH
O
NH
O
OHNH
O
-
47
(1H,tt,J=7.5,1.2Hz),6.26(1H,t,J=5.6Hz),3.20(2H,t,J=6.2Hz),2.52(1H,m),
1.08(3H,d,J=7.1Hz).13CNMR(126MHz,CDCl3)(ppm)δ176.8,155.6,140.9,
129.1,121.4,117.9,42.3,40.1,15.1.HRMS(ESI+)m/zcalc’dforC11H15N2O3[M+H]+:
223.10827,found:223.10860.
3-Methyl-3-(3-phenylureido)butanoicacid(18e)
UsingthegeneralexperimentalprocedureAwith
3-amino-3-amino-butanoicacid(200mg,1.707mmol,0.2M),phenylisocyanate
(203.2mg,1.707mmol,0.185mL)anddistilledTHF(8.53mL)yielded18e,which
wasisolatedasayellowoil(8.9mg,2%).
N-(3-(3-phenylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan(27a)
FollowedthegeneralexperimentalprocedureD
using3-(3-phenylureido)propanoicacid(61.4mg,0.295mmol),L-valyl-L-prolyl-L-
trypotophanattachedtoa2-chlorotritylresin(60mg,0.098mmol)HATU(112.2
mg,0.295mmol),DIPEA(0.103mL,0.590mmol)andNMP(3mL)yielded27a,
whichwasisolatedasalightbrownsolid(12.7mg,22%).1HNMR(500MHz,DMSO-
d6)(ppm)δ12.55(1H,brs),10.84(1H,brs),8.56(1H,s),8.09(1H,d,J=8.5Hz),
7.98(1H,d,J=7.5Hz),7.52(1H,d,J=8.0Hz),7.36(2H,dd,J=8.7,1.1Hz),7.32(1H,
dt,J=8.1,0.9Hz),7.20(3H,m),7.05(1H,ddd,J=8.1,7.0,1.1Hz),6.97(1H,ddd,J=
8.0,7.0,1.0Hz),6.87(1H,tt,J=7.5,1.2Hz),6.15(1H,t,J=5.8Hz),4.44(2H,m),
4.32(1H,t,J=8.4Hz),3.73(1H,m),3.54(1H,m),3.26(2H,qd,J=6.6,2.0Hz),3.10
(2H,qd,J=14.7,6.1Hz),2.35(2H,qt,J=15.2,6.6Hz),2.00(1H,m),1.86(1H,m)
0.86(6H,dd,J=23.2,6.7Hz).13CNMR(126MHz,CDCl3)(ppm)δ173.2,171.4,
170.8,170.1,155.0,140.5,136.0,128.6,127.3,123.8,120.9,120.8,118.3,118.2,
117.5,111.3,109.4,59.1,55.6,53.0,47.1,35.6,35.3,30.0,29.0,27.0,24.4,19.1,18.5.
HRMS(ESI+)m/zcalc’dforC31H39N6O6[M+H]+:591.2926,found:223.2923.
NH
O
O
NH
O
NH
O
NH
O
Val-Pro-Trp-OHNH
O
-
48
(S)-N-(3-(3-phenylureido)butanamido)-L-valyl-L-prolyl-L-trypotophan(27b)
UsingthegeneralexperimentalprocedureD
with(S)-3-(3-phenylureido)butanoicacid(47.9mg,0.222mmol),L-valyl-L-prolyl-L-
trypotophanattachedtoa2-chlorotritylresin(60mg,0.098mmol)HATU(112.2
mg,0.295mmol),DIPEA(0.103mL,0.590mmol)andNMP(3mL)yielded27b,
whichwasisolatedasabrownsolid(13.9mg,23%).1HNMR(500MHz,DMSO-d6)
(ppm)δ12.48(1H,brs),10.83(1H,s),8.47(1H,s),8.10(1H,d,J=8.6Hz),7.96(1H,
brd,J=4.6Hz),7.53(1H,d,J=8.0Hz),7.36(2H,d,J=8.7,1.1Hz),7.32(1H,dt,J=
8.1,0.8Hz),7.20(3H,m),7.05(1H,ddd,J=8.1,7.1,1.1Hz),6.97(1H,ddd,J=7.9,
7.0,1.0Hz),6.87(1H,tt,J=7.5,1.1Hz),6.18(1H,brd,J=7.3Hz),4.43(2H,m),4.33
(1H,t,J=8.5Hz),4.00(1H,heptet,J=6.6Hz)3.73(1H,dt,J=9.7,6.7Hz),3.53(1H,
m),3.10(1H,dd,m),2.33(2H,m),1.99(1H,m),1.86(4H,m),1.06(3H,d,J=6.6Hz),
0.85(6H,dd,J=20.4,6.7Hz).13CNMR(126MHz,CDCl3)(ppm)δ173.2,171.4,
170.2,170.0,154.4,140.6,136.0,128.6,127.3,123.8,120.9,120.8,118.3,118.2,
117.5,111.3,109.5,59.1,55.5,53.0,47.1,42.7,41.7,30.0,29.0,27.0,24.4,20.6,19.1,
18.5.HRMS(ESI+)m/zcalc’dforC32H41N6O6[M+H]+:605.3082,found:605.3082.
(R)-N-(3-(3-phenylureido)butanamido)-L-valyl-L-prolyl-L-trypotophan(27c)
UsingthegeneralexperimentalprocedureD
with(R)-3-(3-phenylureido)butanoicacid(52.2mg,0.242mmol),L-valyl-L-prolyl-
L-trypotophanattachedtoa2-chlorotritylresin(60mg,0.098mmol)HATU(112.2
mg,0.295mmol),DIPEA(0.103mL,0.590mmol)andNMP(3mL)yielded27c,
whichwasisolatedasalightorangesolid(18.0mg,30%).1HNMR(500MHz,
DMSO-d6)(ppm)δ12.56(1H,brs),10.84(1H,s),8.53(1H,s),8.12(1H,d,J=8.5
Hz),7.98(1H,d,J=7.5Hz),7.53(1H,d,J=8.0Hz),7.36(2H,m),7.32(1H,d,J=8.1,
0.9Hz),7.19(3H,m),7.05(1H,ddd,J=8.1,7.1,1.1Hz),6.97(1H,ddd,J=8.0,7.0,
1.0Hz),6.86(1H,tt,J=7.5,1.2Hz),6.23(1H,d,J=8.3Hz),4.44(2H,m),4.33(1H,t,
NH
O
Val-Pro-Trp-OHNH
O
NH
O
Val-Pro-Trp-OHNH
O
-
49
J=8.4Hz),3.99(1H,m)3.73(1H,dt,J=9.7,6.9Hz),3.54(1H,m),3.10(2H,m),2.42
(1H,dd,J=14.1,5.7Hz)2.25(1H,dd,J=14.1,6.3Hz),1.99(1H,m),1.86(4H,m),
1.06(3H,d,J=6.6Hz),0.86(6H,dd,J=18.4,6.7Hz).13CNMR(126MHz,CDCl3)
(ppm)δ173.1,171.4,170.4,170.0,154.3,140.6,136.0,128.6,127.3,123.8,120.8,
118.3,118.2,117.5,111.3,109.4,59.1,55.6,53.0,47.1,42.7,41.3,30.1,29.0,27.0,
24.4,20.6,19.1,18.4,17.2.HRMS(ESI+)m/zcalc’dforC32H41N6O6[M+H]+:
605.3082,found:605.3082.
N-(2-Methyl-3-(3-phenylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan
(27d)
UsingthegeneralexperimentalprocedureD
with2-Methyl-3-(3-phenylureido)propanoicacid(63.6mg,0.295mmol),L-valyl-L-
prolyl-L-trypotophanattachedtoa2-chlorotritylresin(60mg,0.098mmol)HATU
(112.2mg,0.295mmol),DIPEA(0.103mL,0.590mmol)andNMP(3mL)yielded
27d,whichwasisolatedasalightbrownsolid(13.6mg,23%).1HNMR(500MHz,
DMSO-d6)(ppm)δ12.51(1H,brs),10.83(1H,s),8.51(1H,s),8.04(1H,dd,30.3,8.5
Hz),7.97(1H,dd,J=11.8,6.5Hz),7.52(1H,dd,J=8.0,3.4Hz),7.33(3H,m),7.19
(3H,m,J=7.9Hz),7.05(1H,dddd,J=8.0,7.1,3.6,1.1Hz),6.96(1H,dddd,J=8.0,
7.0,3.0,1.0Hz),6.87(1H,tq,J=7.6,1.1Hz),6.11(1H,dt,J=38.0,5.6),4.44(2H,m),
4.32(1H,dt,J=31.7,8.3Hz),3.70(1H,m),3.53(1H,brm),3.11(4H,m),1.92(5H,
m),0.99(3H,dd,J=29.7,7.0Hz),0.85(3H,ddd,J=22.9,6.7,3.7Hz).13CNMR(126
MHz,CDCl3)(ppm)δ174.4,173.2,171.4,170.1,155.1,140.5,136.0,128.6,127.3,
123.8,120.9,120.8,118.3,118.2,117.4,111.3,109.4,59.1,55.6,53.0,48.5,47.0,
42.2,41.9,35.8,30.1,29.0,27.0,24.4,19.1*,18.5*,18.2*,17.2*,16.1*,15.4*.HRMS
(ESI+)m/zcalc’dforC32H41N6O6[M+H]+:605.3082,found:605.3082.
*diasteriomers
NH
O
Val-Pro-Trp-OHNH
O
-
50
(3-Carboxy-3-methyl-3-butanamido)-L-valyl-L-prolyl-L-trypotophan(26eI)
ToafrittedcolumnL-valyl-L-prolyl-L-trypotophan
attachedtoa2-chlorotritylresin(100mg,0.061mmol)wasaddedandallowedto
swellinpeptidegradeDMFfor15minutes.Totheresin2,2-dimethylsuccinic
anhydride(0.207mL,0.183mmol)anddistilledTHF(3mL)wasadded.Theresin
wasthereactionmixturewasshakeninthesolutionfor2h.
N-(3-Methyl-3-(3-phenylureido)butanamido)-L-valyl-L-prolyl-L-trypotophan(27e)
Toafrittedcolumn(3-carboxy-3-methyl-3-
butanamido)-L-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(100
mg,0.061mmol)wasaddedandallowedtoswellinpeptidegradeDMFfor15
minutes.Theresinwasthentransferredtoa2drvial.DIPEA(0.106mL,0.610
mmol)anddistilledtoluene(2mL)wasaddedfollowedbyDPPA(0.131mL,0.610
mmol)wasaddedandthesolutionwasstirred.After2haniline(0.056mL,0.610
mmol)wasaddedandthesolutionwasallowedtostirfor16hat50°C.Theresin
wastransferredbacktoafrittedcolumnandthesolutionwasdrained.Another
equivalentofaniline(0.610mmol)andDIPEA(0.610mmol)wasaddedindistilled
Toluene(2mL)totheresinandthereactionmixturewasshakenfor24hatroom
temperature.ThesolutionwasdrainedandwashedwithpeptidegradeDMF(5mL
x2),HPLCgradeMeOH(5mLx2)anddistilledDCM(5mLx2).DistilledDCM(2.4
mL)andHFIP(0.6mL)wasaddedtotheresinandthereactionmixturewasshaken
for1h.Thesolutionwascollectedandthisprocesswasrepeatedwiththeresin,
moreHFIPandmoreDCM.Thecrudewasthenpurifiedwithreversephasecolumn
chromatography(water,acetonitrile)yielded27e,,whichwasisolatedasawhite
solid(1.3mg,3%).HRMS(ESI+)m/zcalc’dforC33H43N6O6[M+H]+:619.3239,found:
619.3233.
O
Val-Pro-Trp-OHO
HO
NH
O
Val-Pro-Trp-OHNH
O
-
51
N-(3-(3-(4-Trifluoromethyl)phenylureido)propanamido)-L-valyl-L-prolyl-L-
trypotophan(32a)
Usingthegeneralexperimentalprocedure
EwithL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(50mg,
0.031mmol),p-(trifluoromethyl)phenylisocyanate(17.1mg,0.092mmol,0.0131
mL)anddistilledTHF(3mL)yielded32a,whichwasisolatedasalightpinksolid
(7.0mg,35%).1HNMR(500MHz,DMSO-d6)(ppm)δ12.55(1H,brs),10.82(1H,s),
9.01(1H,s),8.09(1H,d,J=8.4Hz),7.95(1H,brm),7.54(5H,m),7.30(1H,d,J=8.1
Hz),7.19(1H,d,J=2.3Hz),7.03(1H,ddd,J=8.1,7.2,1.0Hz),6.95(1H,ddd,J=7.9,
7.0,0.8Hz),6.33(1H,t,J=5.9Hz),4.42(2H,m),4.30(1H,t,J=8.4Hz),4.13(m,2H),
3.70(1H,dt,J=9.7,6.7Hz),3.53(1H,m),3.27(2H,q,J=6.7Hz),3.08(2H,m),2.35
(2H,m),1.98(1H,m),1.83(4H,m),0.84(6H,dd,J=24.1,6.7Hz).13CNMR(126
MHz,DMSO-d6)(ppm)δ173.2,171.4,170.8,170.0,154.7,144.2,136.0,127.3,126.0,
125.8,123.8,121.0,120.8,118.3,118.2,117.0,111.3,109.4,59.1,55.7,53.0,47.1,
35.6,35.1,30.0,28.9,27.0,24.4,19.1,18.4.(ESI+)m/zcalc’dforC32H38N6O6F3
[M+H]+:659.2799,found:659.2787.
N-(3-(3-(4-Chloro)phenylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan
(32b)
UsingthegeneralexperimentalprocedureE
withL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(50mg,0.031
mmol),p-chlorophenylisocyanate(14.1mg,0.092mmol)anddistilledTHF(3mL)
yielded32b,whichwasisolatedasalightpinksolid(5.2mg,27%).1HNMR(500
MHz,DMSO-d6)(ppm)δ12.34(1H,brs),10.83(1H,s),8.74(1H,s),8.09(1H,d,J=
8.5Hz),7.95(1H,brs),7.53(1H,d,J=7.7Hz),7.39(1H,d,J=9.0Hz),7.32(1H,d,J=
Hz),7.24(2H,d,J=9.0Hz)7.20(1H,d,J=2.2Hz),7.05(1H,ddd,J=8.1,7.2,1.0Hz),
6.96(1H,ddd,J=7.8,7.0,0.8Hz),6.22(1H,brs),4.43(2H,m),4.31(1H,t,J=8.4
NH
O
Val-Pro-Trp-OHNH
OF3C
NH
O
Val-Pro-Trp-OHNH
OCl
-
52
Hz),3.71(1H,m),3.53(1H,m),3.26(2H,q,J=6.8Hz),3.10(2H,m),2.34(2H,qt,J=
15.4,6.6),1.99(1H,m),1.84(4H,m),0.85(6H,dd,J=23.5,6.7Hz).13CNMR(126
MHz,DMSO-d6)(ppm)δ173.2,171.4,170.8,170.1,154.9,139.5,136.0,129.3,128.4,
127.3,124.3,123.8,120.8,118.9,118.3,118.2,111.2,59.1,55.7,53.0,47.1,35.6,
35.2,30.0,28.9,27.1,24.4,19.1,18.4.(ESI+)m/zcalc’dforC31H38N6O6Cl[M+H]+:
625.2536,found:625.2537.
N-(3-(3-Tolylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan(32c)
UsingthegeneralexperimentalprocedureE
withL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(50mg,0.031
mmol),p-tolylisocyanate(12.2mg,0.092mmol,0.0115mL)anddistilledTHF(3
mL)yielded32c,whichwasisolatedasalightpinksolid(7.5mg,41%).1HNMR
(500MHz,DMSO-d6)(ppm)δ12.52(1H,brs),10.83(1H,s),8.44(1H,s),8.08(1H,
d,J=8.5Hz),7.96(1H,m),7.52(1H,d,J=7.9Hz),7.32(1H,d,J=8.1Hz),7.24(2H,
m)7.21(1H,d,J=2.3Hz),7.05(1H,ddd,J=8.1,7.0,1.1Hz),7.00(2H,d,J=7.9Hz),
6.96(1H,dddJ=7.9,7.0,1.0Hz),6.10(1H,t,J=5.8Hz),4.43(2H,m),4.31(1H,t,J=
8.4Hz),3.72(1H,dt,J=9.5,6.9Hz),3.53(1H,m),3.25(2H,qd,J=6.8,1.8Hz),3.09
(2H,m),2.33(2H,ddt,J=22.0,15.3,7.5Hz),2.20(3H,s),2.00(1H,m),1.84(4H,m),
0.85(6H,dd,J=23.0,6.7Hz)..13CNMR(126MHz,DMSO-d6)(ppm)δ173.2,171.4,
170.8,170.1,155.1,138.0,136.0,129.6,129.0,127.3,123.8,120.8,118.3,118.2,
117.6,111.2,109.4,59.1,55.6,53.0,47.1,35.6,35.3,30.8,30.0,28.9,27.0,24.4,19.1,
18.5.(ESI+)m/zcalc’dforC32H41N6O6[M+H]+:605.3082,found:605.3079.
N-(3-(3-(4-Dimethylamino)phenylthioureido)propanamido)-L-valyl-L-prolyl-L-
trypotophan(32d)
Usingthegeneralexperimentalprocedure
EwithL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(50mg,
NH
O
Val-Pro-Trp-OHNH
O
NH
O
Val-Pro-Trp-OHNH
SN
-
53
0.031mmol),p-dimethylaminophenylisothiocyanate(14.8mg,0.092mmol)and
distilledTHF(3mL)yielded32d,whichwasisolatedasalightorangesolid(9.9mg,
50%).1HNMR(500MHz,DMSO-d6)(ppm)δ12.48(1H,brs),10.83(1H,s),9.30
(1H,s),8.08(1H,d,J=5.6Hz),7.95(1H,s),7.52(1H,d,J=7.9Hz),7.32(1H,d,J=
8.1Hz),7.26(1H,m),7.20(1H,d,J=1.4Hz),7.05(3H,t,J=8.2Hz),6.96(1H,t,J=
7.0Hz),6.67(1H,d,J=9.1Hz),4.43(2H,m),4.28(1H,t,J=8.3Hz),3.70(1H,dt,J=
9.7,7.0Hz),3.62(2H,m),3.52(1H,m),3.10(2H,m),2.87(6H,s),2.43(2H,s),1.99
(1H,m),1.85(4H,m),0.84(6H,dd,J=22.6,6.6Hz).13CNMR(126MHz,DMSO-d6)
(ppm)δ180.2,173.2,171.4,170.7,170.0,148.2,136.0,127.3,125.7,125.6,123.8,
120.8,118.3,118.2,112.5,111.2,109.5,59.1,55.7,53.0,47.1,40.8,35.8,34.1,30.0,
28.9,27.0,24.4,19.0,18.5.(ESI+)m/zcalc’dforC33H44N7O5S[M+H]+:650.3119,
found:650.3111.
N-(3-(3-(4-Methoxy)phenylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan
(32e)
Usingthegeneralexperimentalprocedure
EwithL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(50mg,
0.031mmol),p-methoxyphenylisocyanate(13.6mg,0.092mmol,0.0119mL)and
distilledTHF(3mL)yielded32e,,whichwasisolatedasalightorangesolid(8.1mg,
43%).1HNMR(500MHz,DMSO-d6)(ppm)12.54(1H,brs),10.84(1H,s),8.34(1H,
s),8.08(1H,d,J=8.5Hz),7.97(1H,m),7.52(1H,d,J=8.0Hz),7.32(1H,d,J=8.1
Hz),7.26(2H,d,J=9.1Hz),7.21(1H,dJ=2.3Hz),7.05(1H,ddd,J=8.1,7.1,1.1Hz),
6.97(1H,ddd,J=7.9,7.1,1.0Hz),6.79(2H,d,J=9.1Hz),6.03(1H,t,J=5.8Hz),4.44
(2H,m),4.31(1H,t,J=8.4Hz),3.71(1H,m),3.53(1H,m),3.25(2H,qd,J=6.7,1.7
Hz),3.09(2H,m),2.33(2H,m),1.99(1H,m),1.86(4H,m),0.85(6H,dd,J=22.9,6.7
Hz).13CNMR(126MHz,DMSO-d6)(ppm)δ173.2,171.4,170.8,170.1,155.3,153.8,
136.0,133.7,127.3,123.8,120.8,119.2,118.3,118.2,113.8,111.3,109.4,59.1,55.6,
55.1,53.0,47.1,35.6,35.1,30.0,28.9,27.0,24.4,19.1,18.5.(ESI+)m/zcalc’dfor
C32H41N6O7[M+H]+:621.3031,found:621.3028.
NH
O
Val-Pro-Trp-OHNH
OO
-
54
N-(3-(3-Phenylthioureido)propanamido)-L-valyl-L-prolyl-L-trypotophan(32f)
UsingthegeneralexperimentalprocedureE
withL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin(100mg,
0.034mmol),phenylisothiocyanate(13.8mg,0.102mmol,0.0122mL)anddistilled
THF(3mL)yielded32f,whichwasisolatedasalightyellowsolid(9.8mg,48%).1H
NMR(500MHz,DMSO-d6)(ppm)δ12.56(1H,brs),10.83(1H,s),9.65(1H,s),8.12
(1H,d,J=6.2Hz),7.97(1H,m),7.73(1H,brs),7.53(1H,d,J=8.0Hz),7.40(1H,d,J
=7.6Hz),7.31(3H,m),7.20(1H,d,J=2.3Hz),7.10(1H,tt,J=7.7,1.2Hz),7.05(1H,
ddd,J=8.1,7.1,1.1Hz),6.97(1H,t,J=7.9,7.0,1.0Hz),4.43(2H,m),4.31(1H,t,J=
8.4Hz),3.68(3H,m),3.54(1H,m),3.11(2H,m),2.47(2H,m),2.00(1H,m),1.85
(4H,m),0.85(6H,dd,J=22.8,6.7Hz).13CNMR(126MHz,DMSO-d6)(ppm)δ180.1,
173.2,171.4,170.8,170.0,136.0,128.6,127.3,124.1,123.8,122.9,120.8,118.3,
118.2,111.3,109.4,59.1,55.7,54.9,53.0,47.1,35.8,34.4,30.0,28.9,27.0,24.4,19.0,
18.5.(ESI+)m/zcalc’dforC31H39N6O5S[M+H]+:607.2697,found:607.2702.
N-(3-(3-(4-Nitro)phenylureido)propanamido)-L-valyl-L-prolyl-L-trypotophan
(32g)
Followedthegeneralexperimental
procedureCusingL-valyl-L-prolyl-L-trypotophanattachedtoa2-chlorotritylresin
(50mg,0.031mmol),p-nitrophenylisocyanate(15.0mg,0.092mmol)anddistilled
THF(3mL)yielded32g,whichwasisolatedasayellowsolid(3.4mg,18%).1H
NMR(500MHz,DMSO-d6)(ppm)δ12.57(1H,brs),10.83(1H,s),9.41(1H,s),8.12
(3H,m),7.97(1H,brs),7.60(2H,d,J=9.3Hz),7.52(1H,d,J=7.8Hz),7.32(1H,d,J
=8.1Hz),7.20(1H,d,J=2.1Hz),7.05(1H,ddd,J=8.1,6.9,1.2Hz),6.96(1H,ddd,J=
7.9,7.0,1.0Hz),6.50(1H,brs),4.43(2H,m),4.32(1H,t,J=8.4Hz),3.72(1H,dt,J=
10.1,6.7Hz),3.54(1H,m),3.30(2H,q,J=6.4Hz),3.09(2H,m),2.37(2H,m),2.00
(1H,m),1.85(4H,m),0.85(6H,dd,J=24.7,6.7Hz).13CNMR(126MHz,DMSO-d6)
NH
O
Val-Pro-Trp-OHNH
S
NH
O
Val-Pro-Trp-OHNH
OO2N
-
55
(ppm)δ173.2,171.4,170.7,170.0,154.2,147.1,140.3,136.0,127.3,125.1,123.8,
120.8,118.3,118.2,116.7,111.3,109.4,59.1,55.7,53.0,47.1,35.7,34.9,30.0,29.0,
27.0,24.4,19.1,18.4.(ESI+)m/zcalc’dforC31H38N7O6[M+H]+:636.2776,found:
636.2775.
3-(3-Benzylureido)propanamido-L-valyl-L-prolyl-L-trypotophan(37a)
UsingthegeneralexperimentalprocedureG
with3-aminopropanamido-L-valyl-L-prolyl-L-trypotophanattachedtoa2-
chlorotritylresin(50mg,0.031mmol),N-benzyl-1H-imidazole-1-carboxamide(18.4
mg,0.091mmol),MeI(64.9mg,0.458mmol,28.4μL),NEt3(18.5mg,0.183mmol,
25.5μL),distilledAcetonitrile(1.0mL)anddistilledDCM(3mL)yielded37a,which
wasisolatedasalightyellowsolid(0.4mg,2%).1HNMR(500MHz,DMSO-d6)
(ppm)δ10.84(1H,s),8.04(1H,d,J=8.5Hz),7.96(1H,m),7.53(1H,d,J=8.0Hz),
7.31(3H,m),7.22(3H,m),7.06(1H,ddd,J=8.1,7.1,1.1Hz),6.97(1H,ddd,J=7.9,
7.0,1.0Hz),6.45(1H,t,J=6.0Hz),5.92(1H,t,J=5.9Hz),4.43(2H,m),4.33(t,J=15
Hz,1H),4.30(1H,t,J=8.4Hz),4.19(2H,dd,J=5.9,2.8Hz),3.72(1H,dt,J=9.4,6.8
Hz),3.52(1H,m),3.20(2H,ddt,J=10.3,6.7,3.8Hz),3.11(2H,m),2.30(2H,m),1.99
(1H,m),1.86(4H,m),0.85(6H,dd,J=22.1,6.7Hz).13CNMR(126MHz,DMSO-d6)
(ppm)δ173.6,171.8,171.2,170.6,158.4,141.4,136.4,128.6,127.4,126.9,124.2,
121.6,118.7,118.6,111.7,109.9,59.6,56.1,53.5,47.5,43.3,36.4,36.1,30.4,29.4,
27.5,24.8,19.5,18.9.(ESI+)m/zcalc’dforC32H41N6O6[M+H]+:605.3082,found:
605.3085.
NH
O
Val-Pro-Trp-OHNH
O
-
56
3-(3-(3,4-Dichlorobenzyl)ureido)propanamido-L-valyl-L-prolyl-L-trypotophan
(37b)
Usingthegeneralexperimentalprocedure
Gwith3-aminopropanamido-L-valyl-L-prolyl-L-trypotophanattachedtoa2-
chlorotritylresin(50mg,0.031mmol),N-(3,4-dichlorobenzyl)-1H-imidazole-1-
carboxamide(24.7mg,0.091mmol),MeI(64.9mg,0.458mmol,28.4μL),NEt3(18.5
mg,0.183mmol,25.5μL),distilledacetonitrile(1.0mL)anddistilledDCM(3mL)
yielded37b,whichwasisolatedasalightyellowsolid(0.5mg,2%).1HNMR(500
MHz,DMSO-d6)(ppm)δ10.83(1H,s),8.03(1H,d,J=8.5Hz),7.92(1H,d,J=8.6
Hz),7.56(1H,d,J=8.2Hz),7.53(1H,m),7.46(1H,d,J=2.0Hz),7.32(1H,d,J=8.9
Hz),7.22(1H,m),7.05(1H,ddd,J=8.1,7.1,1.1Hz),6.96(1H,ddd,J=7.9,7.1,1.0
Hz),6.58(1H,t,J=6.0Hz),6.03(1H,t,J=5.8Hz),4.41(2H,m),4.30(1H,t,J=8.3
Hz),4.18(2H,dd,J=6.1,1.8Hz),3.71(1H,dt,J=9.4,7.0Hz),3.51(1H,m),3.19(2H,
qd,J=6.7,2.2Hz),3.10(2H,ddd,J=35.7,14.8,6.3Hz),2.29(2H,m),1.99(1H,m),
1.85(4H,m),0.85(6H,dd,J=23.7,6.7Hz).13CNMR(126MHz,DMSO-d6)(ppm)δ
173.2,171.3,170.8,170.1,157.8,142.5,136.0,130.7,130.3,128.9,127.3,123.8,
120.8,118.2,118.2,111.2,109.5,59.2,55.6,53.1,47.1,41.8,36.0,35.6,30.0,28.9,
27.0,24.3,19.1,18.4.(ESI+)m/zcalc’dforC32H39N6O6Cl2[M+H]+:673.2303,found:
673.2308.
3-(3-(4-Methoxybenzyl)ureido)propanamido-L-valyl-L-prolyl-L-trypotophan(37c)
Usingthegeneralexperimental
procedureGwith3-aminopropanamido-L-valyl-L-prolyl-L-trypotophanattachedto
a2-chlorotritylresin(50mg,0.031mmol),N-(4-methoxybenzyl)-1H-imidazole-1-
carboxamide(21.2mg,0.091mmol),MeI(64.9mg,0.458mmol,28.4μL),NEt3(18.5
mg,0.183mmol,25.5μL),distilledacetonitrile(1.0mL)anddistilledDCM(3mL)
NH
O
Val-Pro-Trp-OHNH
OCl
Cl
NH
O
Val-Pro-Trp-OHNH
O
O
-
57
yielded37c,whichwasisolatedasalightyellowsolid(2.0mg,10%).1HNMR(500
MHz,DMSO-d6)(ppm)δ10.84(1H,s),8.03(1H,d,J=8.4Hz),7.95(1H,d,J=7.6
Hz),7.53(1H,d,J=7.9Hz),7.34(1H,d,J=8.8Hz),7.21(1H,d,J=2.3Hz),7.13(2H,
d,J=7.6Hz),7.05(1H,ddd,J=8.1,7.2,1.1Hz),6.97(1H,ddd,J=7.9,7.2,0.9Hz),
6.85(2H,d,J=8.7Hz),6.35(1H,t,J=6.0Hz),5.87(1H,t,J=5.9Hz),4.42(2H,m),
4.29(1H,t,J=8.4Hz),4.10(2H,dd,J=5.9,2.4Hz),3.51(1H,dt,J=12.0,6.4Hz),
3.18(2H,m),3.10(2H,m),2.28(2H,m),1.98(1H,m),1.84(1H,m),0.85(6H,dd,J=
22.4,6.7Hz).13CNMR(126MHz,DMSO-d6)(ppm)δ173.7,171.8,170.6,158.5,
158.3,136.4,133.2,128.7,127.8,124.2,121.2,118.7,118.6,114.0,111.7,110.0,
59.6,56.1,55.5,53.5,47.5,42.8,36.4,36.2,30.4,29.4,27.5,24.8,19.5,18.9.(ESI+)
m/zcalc’dforC32H43N6O7[M+H]+:635.3188,found:635.3189.
3-(3-(4-Methylbenzyl)ureido)propanamido-L-valyl-L-prolyl-L-trypotophan(37d)
Usingthegeneralexperimentalprocedure
Gwith3-aminopropanamido-L-valyl-L-prolyl-L-trypotophanattachedtoa2-
chlorotritylresin(50mg,0.031mmol),N-tolyl-1H-imidazole-1-carboxamide(19.7
mg,0.091mmol),MeI(64.9mg,0.458mmol,28.4μL),NEt3(18.5mg,0.183mmol,
25.5μL),distilledacetonitrile(1.0mL)anddistilledDCM(3mL)yielded37d,which
wasisolatedasalightorangesolid(1.9mg,10%).1HNMR(500MHz,DMSO-d6)
(ppm)δ10.85(1H,s),8.03(1H,d,J=8.5Hz),7.97(1H,d,J=7.1Hz),7.53(1H,d,J=
7.9Hz),7.33(1H,d,J=8.1Hz),7.21(1H,d,J=2.3Hz),7.11(3H,s),7.06(1H,ddd,J=
8.1,7.1,1.1Hz),6.97(1H,ddd,J=7.9,7.0,0.9Hz),6.38(1H,t,J=5.9Hz),5.89(1H,t
J=5.9Hz),4.44(2H,m),4.30(1H,t,J=8.4Hz),4.13(2H,dd,J=5.9,3.2Hz),3.72
(1H,dt,J=9.8,6.8Hz),3.53(1H,m),3.19(2H,m),3.09(2H,m),2.31(2H,m),2.27
(3H,s),1.99(1H,m),1.85(4H,m),0.85(6H,dd,J=22.1,6.7Hz).13CNMR(126MHz,
DMSO-d6)(ppm)δ173.7,171.8,171.2,170.6,158.4,138.3,136.5,135.9,129.2,
127.8,127.4,124.3,121.3,118.7,118.6,111.7,109.9,59.6,56.1,53.4,47.5,43.0,
36.4,36.2,30.4,29.4,27.5,24.8,21.1,19.5,18.9.(ESI+)m/zcalc’dforC33H43N6O6
[M+H]+:619.3239,found:619.3233.
NH
O
Val-Pro-Trp-OHNH
O
-
58
3-(3-(3-Chlorobenzyl)ureido)propanamido-L-valyl-L-prolyl-L-trypotophan(37e)
Usingthegeneralexperimentalprocedure
Gwith3-aminopropanamido-L-valyl-L-prolyl-L-trypotophanattachedtoa2-
chlorotritylresin(50mg,0.031mmol),N-(3-chlorobenzyl)-1H-imidazole-1-
carboxamide(21.6mg,0.091mmol),MeI(64.9mg,0.458mmol,28.4μL),NEt3(18.5
mg,0.183mmol,25.5μL),distilledacetonitrile(1.0mL)anddistilledDCM(3mL)
yielded37e,whichwasisolatedasalightyellowsolid(2.5mg,13%).1HNMR(500
MHz,DMSO-d6)(ppm)δ10.83(1H,s),8.04(1H,d,J=8.5Hz),7.95(1H,d,J=7.2
Hz),7.53(1H,d,J=8.0Hz),7.33(2H,m),7.27(2H,m),7.20(2H,m),7.05(1H,ddd,J
=8.1,7.1,1.1Hz),6.97(1H,ddd,J=7.9,7.0,1.0Hz),6.54(1H,t,J=6.1Hz),5.98(1H,
t,J=5.8Hz),4.42(2H,m),4.30(1H,t,J=8.4Hz),4.19(2H,dd,J=6.0,2.5Hz),3.71
(1H,dt,J=9.3,6.7Hz),3.52(1H,dt,J=12.1,6.2Hz),3.20(2H,ddt,J=9.7,6.8,3.0
Hz),3.31(2H,m),2.29(2H,m),1.99(1H,m),1.85(4H,m),0.85(6H,dd,J=22.4,6.7
Hz).13CNMR(126MHz,DMSO-d6)(ppm)δ173.7,171.8,171.2,170.6,158.3,144.2,
136.4,133.3,130.5,127.8,127.1,126.8,126.1,124.2,121.3,118.7,111.7,109.9,
59.6,56.1,53.5,47.5,42.7,36.5,36.1,30.4,29.4,24.8,19.5,18.9.(ESI+)m/zcalc’d
forC32H40N6O6Cl[M+H]+:639.2692,found:639.2697.
3-(3-Benzyl-2phenylguanidino)propanamido-L-allyl-L-trypotophan(44a)
UsingthegeneralexperimentalprocedureI
with3-aminopropanamido-L-allyl-L-trypotophanattachedtoa2-chlorotritylresin
(50mg,0.031mmol),Mukaiyama’sreagent(79.2mg,0.310mmol),benzylamine
(33.2mg,0.310mmol,33.9μL),NEt3(31.4mg,0.310mmol,43.2μL)anddistilled
DMF(3mL)yielded44a,whichwasisolatedasanorangesolid(15.4mg,90%).1H
NMR(400MHz,DMSO-d6)(ppm)δ12.61(1H,brs),10.89(1H,s),8.46(1H,brd,J=
NH
O
Val-Pro-Trp-OHNH
OCl
Ala-Trp
O
NH
N
NH OH
-
59
7.4Hz),8.27(1H,d,J=7.5Hz),8.15(1H,d,J=7.5Hz),7.96(1H,brs),7.52(1H,d,J=
7.9Hz),7.36(3H,m),7.21(1H,t,J=7.4Hz),7.16(3H,dd,J=4.8,2.4Hz),7.05(1H,
ddd,J=8.1,7.0,1.1Hz),6.96(1H,t,J=7.9,7.0,1.0Hz),4.50(2H,brs),4.44(1H,td,
J=7.8,5.4Hz),4.37(1H,t,J=7.3Hz),3.47(2H,brq,J=6.7Hz),3.30(2H,q,J=7.1
Hz),3.11(2H,m),2.46(2H,m),1.19(3H,d,J=7.1Hz).13CNMR(101MHz,DMSO-
d6)(ppm)δ173.2,172.1,169.9,153.9,137.0,136.0,129.5,128.5,127.4,127.3,
127.2,123.9,123.7,120.8,118.3,118.2,111.3,109.7,48.9,48.0,30.1,29.0,27.0,
18.4,17.2.(ESI+)m/zcalc’dforC31H35N6O4[M+H]+:555.2719,found:555.2714.
Benzoyl-L-prolyl-L-trypotophan(52)
UsingthegeneralexperimentalprocedureJwithL-prolyl-L-
trypotophanattachedtoa2-chlorotritylresin(100mg,0.058mmol),benzoyl
chloride(24.5mg,0.174mmol,20.2μL),DIPEA(45.0mg,0.348mmol,60.6μL)and
NMP(3mL)yielded52,whichwasisolatedasalightpinksolid(1mg,4%).1HNMR
(400MHz,DMSO-d6)(ppm)δ12.23(1H,brs),10.84(1H,d,J=22.0Hz),8.07(1H,d,
J=25.6Hz),7.49(4H,m),7.33(2H,m),7.21(2H,m),7.05(1H,m),6.97(1H,m),
4.49(1H,m),4.28(1H,m),3.56(1H,m),3.17(2H,m),2.95(1H,m),2.06(1H,m),
1.86(3H,m).(ESI+)m/zcalc’dforC23H24N3O4[M+H]+:406.1764,found:406.1761.
Fmoc-glycyl-L-prolyl-L-trypotophan(54)
UsingthegeneralexperimentalprocedureMwithL-prolyl-
L-trypotophanattachedtoa2-chlorotritylresin(100mg,0.061mmol),Fmoc-
glycine(54.4mg,0.183mmol),oxalylchloride(27.8mg,0.210mmol,18.8μL),DMF
(13.4mg,0.183mmol,13.4μL),DIPEA(47.3mg,0.366mmol,63.7μL),distilled
DCM(3mL)andNMP(3mL)yielded54,whichwasisolatedasalightorangesolid
(3.3mg,9%).1HNMR(400MHz,DMSO-d6)(ppm)δ10.82(1H,s),8.00(1H,d,J=7.6
Hz),7.88(2H,d,J=8.2Hz),7.72(2H,d,J=6.8Hz),7.54(1H,q,J=6.1,5.5Hz),7.42
Pro-Trp
O
OH
Pro-Trp
OHN
Fmoc
OH
-
60
(2H,m),7.32(3H,m),7.18(1H,m),7.04(1H,q,J=8.1,7.6Hz)6.96(1H,m),4.46