Synthesis of libraries of peptidomimetic compounds containing a 2-oxopiperazine unit in the main chain

Article abstract of DOI:10.1039/c3ob27476d

Peptoid libraries have been shown to be a useful source of protein-binding agents. However, simple linear peptoids lack conformational constraints, which may limit their binding affinity for proteins. Here we report facile chemistry for the assembly of 2-oxopiperazine rings into the main chain of peptoid-like oligomers, thus rigidifying the structure. This modified sub-monomer synthesis is suitable for the creation of high quality combinatorial libraries. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob27476d

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SynthesisꢀofꢀLibrariesꢀofꢀPeptidomimeticꢀCompoundsꢀContainingꢀAꢀ2‐
OxopiperazineꢀUnitꢀInꢀTheꢀMainꢀChainꢀ
ꢀ
SujitꢀSuwalꢀandꢀThomasꢀKodadek*ꢀ
ꢀ
DepartmentsꢀofꢀChemistryꢀ&ꢀCancerꢀBiologyꢀ
TheꢀScrippsꢀResearchꢀInstitute,ꢀScrippsꢀFloridaꢀ
130ꢀScrippsꢀWay,ꢀJupiter,ꢀFLꢀ33458ꢀ
ꢀ
*Toꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed:ꢀKodadek@scripps.eduꢀ
ꢀ
Abstractꢀ
Peptoidꢀlibrariesꢀhaveꢀbeenꢀshownꢀtoꢀbeꢀaꢀusefulꢀsourceꢀofꢀprotein‐bindingꢀagents.ꢀꢀHowever,ꢀsimpleꢀlinearꢀpeptoidsꢀ
lackꢀconformationalꢀconstraints,ꢀwhichꢀmayꢀlimitꢀtheirꢀbindingꢀaffinityꢀforꢀproteins.ꢀꢀHereꢀweꢀreportꢀfacileꢀchemistryꢀ
forꢀtheꢀassemblyꢀofꢀ2‐oxopiperazineꢀringsꢀintoꢀtheꢀmainꢀchainꢀofꢀpeptoid‐likeꢀoligomers,ꢀthusꢀrigidifyingꢀtheꢀstructure.ꢀꢀ
Thisꢀmodifiedꢀsub‐monomerꢀsynthesisꢀisꢀsuitableꢀforꢀtheꢀcreationꢀofꢀhighꢀqualityꢀcombinatorialꢀlibraries.ꢀꢀꢀ
ꢀ
1ꢀ

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ꢀ
Peptoids1ꢀ(oligomersꢀofꢀN‐substitutedꢀglycine)ꢀhaveꢀseveralꢀadvantagesꢀoverꢀpeptidesꢀandꢀmanyꢀotherꢀtypesꢀofꢀpeptide‐
likeꢀoligomersꢀasꢀaꢀpotentialꢀsourceꢀofꢀbioactiveꢀcompounds.ꢀꢀPeptoidsꢀareꢀmoreꢀcellꢀpermeableꢀthanꢀpeptidesꢀ2,ꢀ3ꢀandꢀ
alsoꢀareꢀinsensitiveꢀtoꢀproteasesꢀandꢀpeptidases4.ꢀꢀMostꢀimportantly,ꢀlargeꢀlibrariesꢀofꢀpeptoidsꢀcanꢀbeꢀcreatedꢀeasilyꢀ
usingꢀtheꢀsolid‐phaseꢀ“sub‐monomer”ꢀchemistryꢀdevelopedꢀbyꢀZuckermannꢀandꢀco‐workers5,ꢀ6ꢀandꢀtheꢀsplitꢀandꢀpoolꢀ
strategy7,ꢀwhereasꢀmostꢀotherꢀtypesꢀofꢀoligomerꢀlibrariesꢀrequireꢀfarꢀgreaterꢀsyntheticꢀeffort.ꢀꢀTheꢀsub‐monomerꢀ
protocolꢀinvolvesꢀtwoꢀsteps:ꢀacylationꢀofꢀanꢀamineꢀwithꢀ2‐bromoaceticꢀacidꢀfollowedꢀbyꢀdisplacementꢀofꢀtheꢀbromideꢀ
withꢀaꢀprimaryꢀamine.ꢀꢀTheꢀlargeꢀnumberꢀofꢀaminesꢀthatꢀareꢀcommerciallyꢀavailableꢀorꢀsynthesizedꢀreadilyꢀallowꢀlibrariesꢀ
ofꢀtremendousꢀdiversityꢀtoꢀbeꢀcreatedꢀrapidlyꢀwithoutꢀtheꢀneedꢀforꢀsynthesizingꢀandꢀmaintainingꢀextensiveꢀstocksꢀofꢀ
expensiveꢀprecursorsꢀ8‐10.ꢀꢀSeveralꢀstudiesꢀhaveꢀshownꢀthatꢀpeptoidꢀlibrariesꢀcanꢀbeꢀminedꢀtoꢀproduceꢀusefulꢀbioactiveꢀ
compounds11‐17.ꢀꢀHowever,ꢀwithꢀrareꢀexceptions11,ꢀprimaryꢀscreeningꢀhitsꢀthatꢀariseꢀfromꢀpeptoidꢀlibrariesꢀhaveꢀnotꢀ
exhibitedꢀhighꢀaffinityꢀorꢀpotency.ꢀꢀThisꢀmayꢀbeꢀdue,ꢀinꢀpart,ꢀtoꢀtheꢀfactꢀthatꢀcommonꢀpeptoidsꢀdoꢀnotꢀadoptꢀwell‐definedꢀ
conformations.ꢀꢀIndeed,ꢀunlikeꢀpeptides,ꢀbothꢀtheꢀcisꢀandꢀtransꢀisomersꢀofꢀtheꢀamideꢀbondꢀareꢀpopulatedꢀandꢀthereꢀisꢀ
littleꢀorꢀconformationalꢀpreferenceꢀforꢀtheꢀotherꢀtwoꢀtypesꢀofꢀbondsꢀinꢀtheꢀmolecule.ꢀꢀVariousꢀstrategiesꢀhaveꢀbeenꢀ
reportedꢀtoꢀaddressꢀthisꢀlimitationꢀandꢀcreateꢀmoreꢀconformationallyꢀconstrainedꢀpeptoidsꢀorꢀpeptoidꢀanalogues.18‐20ꢀꢀ
However,ꢀuntilꢀrecently21,ꢀnoneꢀofꢀtheseꢀsolutionsꢀwasꢀbasedꢀonꢀchemistryꢀthatꢀwasꢀefficientꢀenoughꢀtoꢀsupportꢀtheꢀ
creationꢀofꢀhighꢀqualityꢀcombinatorialꢀlibraries.ꢀꢀRecently,ꢀweꢀhaveꢀaddressedꢀthisꢀproblemꢀandꢀhaveꢀdemonstratedꢀtheꢀ
synthesisꢀofꢀlibrariesꢀofꢀpeptoid‐likeꢀoligomersꢀwithꢀeitherꢀmainꢀchain22,ꢀ23ꢀorꢀsideꢀchain24,ꢀ25ꢀsub‐monomerꢀunitsꢀthatꢀ
imposeꢀsignificantꢀconformationalꢀrestrictions.ꢀꢀInꢀthisꢀpaper,ꢀweꢀintroduceꢀanotherꢀstrategyꢀforꢀtheꢀcreationꢀofꢀ
conformationally‐restrictedꢀmainꢀchainsꢀviaꢀtheꢀinsertionꢀofꢀ2‐oxopiperazineꢀunitsꢀintoꢀtheꢀoligomerꢀ(Schemeꢀ1).ꢀꢀWeꢀ
demonstrateꢀthatꢀthisꢀchemistryꢀisꢀefficientꢀenoughꢀforꢀtheꢀcreationꢀofꢀhighꢀqualityꢀcombinatorialꢀlibrariesꢀbyꢀsolid‐phaseꢀ
splitꢀandꢀpoolꢀsynthesis.ꢀ
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ꢀTheꢀsynthesisꢀofꢀ2‐oxopiperazine‐containingꢀpeptoidsꢀwasꢀreportedꢀpreviouslyꢀbyꢀworkersꢀatꢀChiron26,ꢀ27.ꢀꢀHowever,ꢀtheꢀ
routeꢀemployedꢀresultedꢀinꢀaꢀmixtureꢀofꢀstereoisomersꢀandꢀdidꢀnotꢀallowꢀfacileꢀextensionꢀofꢀtheꢀoligomerꢀfollowingꢀ
formationꢀofꢀtheꢀ2‐oxopiperazineꢀring.ꢀꢀBalasubramanianꢀandꢀcolleaguesꢀpublishedꢀaꢀdiastereoselectiveꢀsynthesisꢀthatꢀ
employedꢀaꢀchiralꢀaldehydeꢀinꢀtheꢀkeyꢀstepꢀ28ꢀandꢀGolebiowski,ꢀetꢀal.ꢀdevelopedꢀaꢀsolid‐pahseꢀsynthesisꢀofꢀ2‐
oxopiperazine‐containingꢀβ‐turnꢀmimetics29.ꢀꢀButꢀneitherꢀschemeꢀwasꢀadaptedꢀforꢀembeddingꢀtheꢀmoleculesꢀintoꢀ
oligomers.ꢀꢀOurꢀproposedꢀapproachꢀ(Schemeꢀ1)ꢀinvolvesꢀadditionꢀofꢀmono‐protectedꢀ1,2‐diaminoethaneꢀtoꢀtheꢀendꢀofꢀaꢀ
growingꢀpeptoidꢀchain.ꢀꢀAnotherꢀ2‐haloꢀacidꢀisꢀthenꢀaddedꢀtoꢀtheꢀunprotectedꢀnitrogen,ꢀfollowedꢀbyꢀdeprotectionꢀandꢀ
ringꢀclosureꢀtoꢀcreateꢀtheꢀ2‐oxopiperazineꢀunit.ꢀꢀTheꢀoligomerꢀchainꢀcanꢀthenꢀbeꢀextendedꢀbyꢀacylationꢀofꢀtheꢀsecondaryꢀ
amineꢀinꢀtheꢀringꢀ(Schemeꢀ1).ꢀ
ꢀ
ꢀꢀꢀꢀꢀToꢀ testꢀ thisꢀ strategy,ꢀ diisopropylꢀ carbodiimideꢀ (DIC)‐activatedꢀ bromoaceticꢀ acidꢀ (BAA)ꢀ wasꢀ coupledꢀ toꢀ Rinkꢀ amideꢀ
MBHAꢀresinꢀ(Schemeꢀ1).ꢀTheꢀhalideꢀ2ꢀwasꢀtreatedꢀwithꢀmono‐N‐alloc‐protectedꢀ1,2‐diaminoethaneꢀandꢀtheꢀresultantꢀ
secondaryꢀamineꢀ3ꢀwasꢀcoupledꢀwithꢀDIC‐activatedꢀ2‐chloropropionicꢀacidꢀtoꢀobtainꢀcompoundꢀ4.ꢀTheꢀallocꢀgroupꢀwasꢀ
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thenꢀremovedꢀusingꢀpalladiumꢀtetrakisꢀtriphenylphosphineꢀinꢀtheꢀpresenceꢀofꢀphenylsilaneꢀasꢀaꢀscavengerꢀtoꢀaffordꢀtheꢀ
primaryꢀamine.ꢀCyclizationꢀwasꢀeffectedꢀunderꢀbasicꢀconditionsꢀ(10%ꢀN,ꢀN’ꢀdiisopropylethylamine,ꢀDIEA)ꢀtoꢀaffordꢀtheꢀ2‐
oxopiperazineꢀringꢀ5.ꢀChainꢀextensionꢀfromꢀtheꢀsecondaryꢀamineꢀinꢀ5ꢀwasꢀcarriedꢀoutꢀbyꢀcouplingꢀwithꢀ2‐bromo‐aceticꢀ
acid,ꢀfollowedꢀbyꢀdisplacementꢀofꢀbromideꢀwithꢀR‐(+)‐methylꢀbenzylꢀamineꢀ(Nmba)ꢀtoꢀaffordꢀ6,ꢀwhichꢀwasꢀauthenticatedꢀ
byꢀMALDI‐TOFꢀmassꢀspectrometryꢀ(MS).ꢀꢀNMRꢀandꢀHPLCꢀanalysisꢀofꢀtheꢀproductꢀindicatedꢀhighꢀpurityꢀ(seeꢀsupplementalꢀ
material).ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Figureꢀ1:ꢀLeft:ꢀStructureꢀofꢀtheꢀcompoundꢀ6b,ꢀshowingꢀcisꢀandꢀtrans‐amideꢀbondꢀgeometries.ꢀRight:ꢀPortionꢀofꢀtheꢀ1H‐NMRꢀspectrumꢀ
ofꢀ6bꢀtakenꢀatꢀ25oCꢀandꢀ45oC,ꢀhighlightingꢀtheꢀresonanceꢀcorrespondingꢀtoꢀtheꢀC5ꢀmethylꢀgroup.ꢀꢀꢀꢀ
ꢀ
Obviously,ꢀtheꢀ2‐oxopiperazineꢀringꢀinꢀ6ꢀenforcesꢀaꢀrelativelyꢀrigidꢀmainꢀchainꢀconformationꢀforꢀthisꢀpartꢀofꢀtheꢀmolecule.ꢀꢀ
However,ꢀitꢀwasꢀnotꢀclearꢀifꢀtheꢀamideꢀbondꢀonꢀtheꢀN‐terminalꢀsideꢀofꢀtheꢀringꢀwouldꢀadoptꢀeitherꢀpredominantlyꢀtheꢀcisꢀ
orꢀtransꢀconformationꢀdueꢀtoꢀtheꢀdifferentꢀsubstitutionꢀonꢀC3ꢀandꢀC4ꢀ(Figureꢀ1).ꢀꢀToꢀprobeꢀthisꢀpoint,ꢀNMRꢀanalysisꢀwasꢀ
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carriedꢀout.ꢀFigureꢀ1ꢀdisplaysꢀpartꢀofꢀtheꢀNMRꢀspectrumꢀofꢀcompoundꢀ6b,ꢀhighlightingꢀtheꢀresonancesꢀofꢀtheꢀC5ꢀmethylꢀ
group.ꢀꢀTwoꢀdoubletsꢀatꢀδꢀ1.2ꢀandꢀ1.3ꢀlikelyꢀrepresentꢀtheꢀsignalsꢀfromꢀtheꢀcisꢀandꢀtransꢀamideꢀisomersꢀofꢀ6b,ꢀwhichꢀ
convertꢀslowlyꢀonꢀtheꢀNMRꢀtimeꢀscaleꢀatꢀroomꢀtemperature.ꢀꢀThisꢀwasꢀconfirmedꢀbyꢀheatingꢀtheꢀsampleꢀtoꢀ45oC,ꢀwhichꢀ
resultedꢀinꢀtheꢀmergingꢀofꢀtheꢀsignals.ꢀꢀItꢀisꢀdifficultꢀtoꢀdetermineꢀwhichꢀisomerꢀisꢀpredominant,ꢀbutꢀtheꢀapproximatelyꢀ
2:1ꢀratioꢀofꢀtheꢀdoubletsꢀinꢀtheꢀspectrumꢀshowꢀthatꢀbothꢀareꢀwellꢀpopulated.ꢀꢀTheꢀfactꢀthatꢀonlyꢀoneꢀdoubletꢀforꢀtheꢀC5ꢀ
methylꢀisꢀobservedꢀatꢀ45oCꢀdemonstratesꢀthatꢀonlyꢀaꢀsingleꢀenantiomerꢀofꢀcompoundꢀ6bꢀisꢀpresentꢀandꢀthatꢀtheꢀstepsꢀ
employedꢀtoꢀcreateꢀtheꢀ2‐oxopiperazineꢀringꢀproceededꢀwithoutꢀracemizationꢀ(SupplementalꢀinformationꢀS2,ꢀS2.1).ꢀ
ꢀ
Chiralꢀ2‐halocarboxylicꢀacidsꢀcanꢀbeꢀmadeꢀfromꢀα‐aminoꢀacidsꢀinꢀaꢀsingleꢀstepꢀwithꢀhighꢀstereochemicalꢀpurity30,ꢀ
soꢀitꢀshouldꢀbeꢀpossibleꢀtoꢀincorporateꢀaꢀnumberꢀofꢀdifferentꢀsubstituentsꢀatꢀtheꢀchiralꢀcenterꢀofꢀtheꢀ2‐oxopiperazine.ꢀꢀToꢀ
testꢀtheꢀscopeꢀofꢀthisꢀprocess,ꢀfiveꢀadditionalꢀcompoundsꢀ(Schemeꢀ1,ꢀ6a‐f)ꢀwereꢀsynthesizedꢀinꢀwhichꢀtheꢀsubstituentꢀatꢀ
theꢀchiralꢀcenterꢀinꢀtheꢀ2‐oxopiperazineꢀringꢀwasꢀvaried.ꢀꢀTheꢀproductsꢀwereꢀsubsequentlyꢀauthenticatedꢀbyꢀMALDI‐TOFꢀ
andꢀtheirꢀpurityꢀtestedꢀbyꢀHPLC.ꢀꢀTheꢀpurityꢀofꢀtheꢀcompoundsꢀrangedꢀfromꢀ85‐98%ꢀ(supportingꢀinformationꢀTableꢀT1ꢀ
andꢀSupplementaryꢀFiguresꢀS11‐S15).ꢀꢀꢀ
ꢀ
Therefore,ꢀ weꢀ exploredꢀ theꢀ useꢀ ofꢀ thisꢀ chemistryꢀ inꢀ theꢀ constructionꢀ ofꢀ aꢀ combinatorialꢀ library.ꢀ ꢀ Aꢀ generalꢀ
structureꢀofꢀtheꢀone‐bead‐one‐compoundꢀ(OBOC)ꢀlibraryꢀisꢀshownꢀinꢀFigureꢀ2a.ꢀꢀFollowingꢀanꢀinvariantꢀlinkerꢀshownꢀinꢀ
red,ꢀ aꢀ tetramericꢀ 2‐oxopiperazine‐containingꢀ peptoidꢀ libraryꢀ containingꢀ 20,000ꢀ compoundsꢀ wasꢀ createdꢀ usingꢀ 15ꢀ
differentꢀprimaryꢀaminesꢀ(linearꢀorꢀα‐branched)ꢀandꢀfiveꢀ2‐chloroꢀacids.ꢀSinceꢀtheꢀlibraryꢀwasꢀcreatedꢀonꢀtentagel‐MB‐
NH2,ꢀN‐tbocꢀprotectedꢀethylenediamineꢀ(EDA)ꢀwasꢀusedꢀratherꢀthanꢀtheꢀN‐alloc‐protectedꢀEDAꢀthatꢀwasꢀusedꢀwithꢀRinkꢀ
amideꢀ resinꢀ (Schemeꢀ 1).ꢀ Theꢀ qualityꢀ ofꢀ theꢀ libraryꢀ wasꢀ testedꢀ byꢀ pickingꢀ 20ꢀ beadsꢀ atꢀ randomꢀ fromꢀ theꢀ library.ꢀ Theꢀ
compoundsꢀ wereꢀ releasedꢀ fromꢀ theꢀ beadsꢀ byꢀ cleavageꢀ atꢀ methionineꢀ inꢀ theꢀ invariantꢀ linkerꢀ withꢀ CNBr.ꢀ 80%ꢀ ofꢀ theꢀ
compoundsꢀwereꢀsequencedꢀsuccessfullyꢀbyꢀtandemꢀMALDIꢀmassꢀspectrometryꢀ(supplementalꢀinformation,ꢀS30‐S46),ꢀaꢀ
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resultꢀcomparableꢀtoꢀthatꢀseenꢀwithꢀstandardꢀpeptoidꢀlibraries31.ꢀThisꢀdemonstratesꢀhighꢀqualityꢀcombinatorialꢀlibrariesꢀ
withꢀaꢀ2‐oxopiperazineꢀunitꢀinsertedꢀintoꢀaꢀpeptoidꢀchainꢀcanꢀbeꢀcreated.ꢀ
ꢀ
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Figureꢀ2:ꢀ(a)ꢀGeneralꢀstructureꢀofꢀaꢀ2‐oxopiperazineꢀring‐containingꢀpeptoidꢀlibrary.ꢀ(b)ꢀTop‐left:ꢀMSꢀofꢀaꢀpeptoidꢀ([MH+]=1179.4)ꢀ
obtainedꢀ afterꢀ CNBrꢀ mediatedꢀ singleꢀ beadꢀ cleavage;ꢀ Top‐right:ꢀ Structureꢀ ofꢀ theꢀ peptoidꢀ identifiedꢀ afterꢀ MALDI‐TOFꢀ basedꢀ
sequencing;ꢀBottom:ꢀMS/MSꢀofꢀaꢀpeptoidꢀobtainedꢀfromꢀMALDI‐TOFꢀwithꢀtheꢀprecursorꢀmassꢀ1179.4.ꢀꢀꢀ
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ToꢀstudyꢀtheꢀscopeꢀofꢀchainꢀextensionꢀwithꢀrespectꢀtoꢀtheꢀunitꢀN‐terminalꢀtoꢀtheꢀoxopiperazineꢀring,ꢀtheꢀadditionꢀ
ofꢀ L‐aminoꢀ acidsꢀ wasꢀ attempted.ꢀ Weꢀ presumedꢀ thatꢀ thisꢀ wouldꢀ beꢀ aꢀ relativelyꢀ difficultꢀ couplingꢀ givenꢀ theꢀ crowdingꢀ
aroundꢀtheꢀnitrogen,ꢀbutꢀworthꢀassessingꢀsinceꢀitꢀmightꢀbiasꢀtheꢀamideꢀbondꢀmoreꢀtowardsꢀtheꢀtransꢀgeometry.ꢀꢀThus,ꢀaꢀ
seriesꢀ ofꢀ compoundsꢀ wereꢀ madeꢀ inꢀ whichꢀ variouslyꢀ substitutedꢀ 2‐oxopiperazinesꢀ wereꢀ coupledꢀ withꢀ fmoc‐protectedꢀ
aminoꢀacids,ꢀwhichꢀwereꢀthenꢀacylatedꢀwithꢀactivatedꢀchiralꢀ2‐haloꢀacidsꢀfollowedꢀbyꢀhalideꢀdisplacementꢀwithꢀanꢀamineꢀꢀ
ꢀ
ꢀ
Figureꢀ3:ꢀ(a)ꢀGeneralꢀstructureꢀofꢀaꢀ2‐oxopiperazine‐containingꢀhybridꢀlibraryꢀinꢀwhichꢀtheꢀringꢀisꢀfollowedꢀbyꢀanꢀaminoꢀacidꢀunitꢀandꢀ
thenꢀcappedꢀwithꢀaꢀpeptideꢀtertiaryꢀamide.ꢀ(b)ꢀTop‐left:ꢀMSꢀofꢀaꢀpeptoidꢀ([MH+]=1137.5)ꢀobtainedꢀafterꢀCNBrꢀmediatedꢀsingleꢀbeadꢀ
cleavage;ꢀTop‐right:ꢀStructureꢀofꢀtheꢀpeptoidꢀidentifiedꢀafterꢀMALDI‐TOFꢀbasedꢀsequencing;ꢀBottom:ꢀMS/MSꢀofꢀaꢀpeptoidꢀobtainedꢀ
fromꢀMALDI‐TOFꢀwithꢀtheꢀprecursorꢀmassꢀ1137.5.ꢀꢀꢀ
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ꢀ
(seeꢀ Supplementaryꢀ Schemeꢀ Sch1).ꢀ ꢀ Sixꢀ differentꢀ DIC/HOAt‐activatedꢀ Fmoc‐aminoꢀ acidsꢀ wereꢀ coupledꢀ toꢀ aꢀ 2‐
oxopiperazineꢀmoietyꢀcontainingꢀmethylꢀandꢀisobutylꢀsubstituents.ꢀꢀTheꢀFmocꢀgroupꢀwasꢀremovedꢀusingꢀ20%ꢀpiperidineꢀ
inꢀDMFꢀandꢀtheꢀresultingꢀprimaryꢀamineꢀwasꢀcoupledꢀtoꢀ2‐bromopropionicꢀacidꢀ(2‐chloro‐4‐methyl‐pentanoicꢀacidꢀwasꢀ
alsoꢀ usedꢀ successfully;ꢀ seeꢀ supplementalꢀ information,ꢀ Schemeꢀ Sch1).ꢀ Theꢀ halidesꢀ wereꢀ thenꢀ displacedꢀ byꢀ aꢀ primaryꢀ
amine.ꢀ Theꢀ compoundsꢀ wereꢀ cleavedꢀ fromꢀ theꢀ Rinkꢀ amideꢀ MBHAꢀ resinꢀ andꢀ analyzedꢀ byꢀ tandemꢀ MSꢀ andꢀ HPLCꢀ
(SupplementaryꢀFiguresꢀS15‐S29).ꢀTheꢀpurityꢀofꢀtheꢀfinalꢀproductsꢀrangedꢀfromꢀ80‐91%ꢀ(supplementalꢀinformation,ꢀTableꢀ
T2).ꢀ
Givenꢀthisꢀencouragingꢀresult,ꢀweꢀproceededꢀtoꢀconstructꢀaꢀcombinatorialꢀlibraryꢀofꢀcompoundsꢀinꢀwhichꢀtheꢀ2‐
oxopiperazineꢀunitꢀisꢀfollowedꢀbyꢀanꢀaminoꢀacid.ꢀꢀAfterꢀremovalꢀofꢀFmoc,ꢀaꢀchiralꢀα‐haloꢀacidꢀwasꢀthenꢀcoupledꢀtoꢀtheꢀ
aminoꢀacidꢀandꢀtheꢀbromideꢀwasꢀthenꢀdisplacedꢀwithꢀanꢀamine.ꢀThisꢀlibraryꢀ(Figureꢀ3ꢀandꢀsupplementalꢀinformationꢀ
FigureꢀL2)ꢀwasꢀsynthesizedꢀonꢀtentagelꢀresinꢀutilizingꢀ12ꢀprimaryꢀamines,ꢀ5ꢀaminoꢀacidsꢀandꢀ5ꢀdifferentꢀtypesꢀofꢀα‐haloꢀ
acidsꢀtoꢀaffordꢀaꢀlibraryꢀofꢀapproximatelyꢀ18,000ꢀcompounds.ꢀTheꢀFmoc‐aminoꢀacidsꢀwereꢀcoupledꢀtoꢀtheꢀsecondaryꢀ
amineꢀofꢀtheꢀ2‐oxopiperazineꢀringꢀusingꢀHOAt/DICꢀatꢀelevatedꢀtemperatureꢀ(50°C).ꢀꢀTheꢀqualityꢀofꢀtheꢀtetramerꢀlibraryꢀ
wasꢀverifiedꢀbyꢀCNBr‐mediatedꢀcleavageꢀofꢀ20ꢀbeadsꢀrandomlyꢀselectedꢀfromꢀtheꢀpool.ꢀ80%ꢀofꢀtheꢀcompoundsꢀwereꢀ
identifiedꢀbyꢀMALDI‐MS/MSꢀ(supplementalꢀinformation,ꢀS47‐64),ꢀconfirmingꢀtheꢀutilityꢀofꢀthisꢀapproachꢀinꢀgeneratingꢀtheꢀ
pureꢀ2‐oxopiperazineꢀcontainingꢀOBOCꢀpeptideꢀtertiaryꢀamideꢀlibrary.ꢀꢀꢀ
Conclusionsꢀ
Inꢀsummary,ꢀweꢀhaveꢀidentifiedꢀanꢀeffectiveꢀsolid‐phaseꢀsyntheticꢀrouteꢀtoꢀincorporateꢀ2‐oxopiperazineꢀmoietiesꢀ
intoꢀpeptoidsꢀusingꢀα‐haloacidsꢀandꢀprotectedꢀ1,2‐diaminoethane.ꢀꢀTheꢀringꢀcanꢀbeꢀsubstitutedꢀwithꢀaꢀvarietyꢀofꢀsideꢀ
chainsꢀderivedꢀfromꢀchiralꢀ2‐haloacidsꢀthatꢀareꢀthemselvesꢀderivedꢀfromꢀα‐aminoꢀacids.ꢀꢀWhenꢀtheꢀfollowingꢀresidueꢀisꢀaꢀ
peptoidꢀunit,ꢀbothꢀtheꢀcis‐ꢀandꢀtrans‐amideꢀbondꢀgeometriesꢀonꢀtheꢀN‐terminalꢀsideꢀofꢀtheꢀringꢀareꢀobserved.ꢀꢀWorkꢀ
focusedꢀonꢀminingꢀlibrariesꢀofꢀtheseꢀcompoundsꢀforꢀproteinꢀligandsꢀisꢀunderway.ꢀ
ꢀ
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Acknowledgementsꢀ
ThisꢀworkꢀwasꢀsupportedꢀbyꢀaꢀcontractꢀfromꢀtheꢀNationalꢀHeartꢀLungꢀandꢀBloodꢀInstituteꢀ(NHLBIꢀProteomicsꢀCenter,ꢀ
NO1‐HV‐00242).ꢀ
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