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comChemicalmutagenesis:selectivepost-expressioninterconversionofproteinaminoacidresiduesJustinMChalkerandBenjaminGDavisTheabilitytoalterproteinstructurebysite-directedmutagenesishasrevolutionizedbiochemicalresearch.
ControlledmutationsattheDNAlevel,beforeproteintranslation,arenowroutine.
Thesetechniquesallowspecic,highdelityinterconversionlargelybetween20natural,proteinogenicaminoacids.
Nonetheless,thereisaneedtoincorporateotheraminoacids,bothnaturalandunnatural,thatarenotaccessibleusingstandardsite-directedmutagenesisandexpressionsystems.
Post-translationalchemistryoffersaccesstothesesidechains.
Nearlyhalfacenturyago,theideaofa'chemicalmutation'wasproposedandtheinterconversionbetweenaminoacidsidechainswasdemonstratedonselectproteins.
Intheseisolatedexamples,apowerfulproof-of-conceptwasdemonstrated.
Here,werevivetheideaofchemicalmutagenesisanddiscusstheprospectofitsgeneralapplicationinproteinscience.
Inparticular,weconsideraminoacidsthatarechemicalprecursorstoafunctionalsetofothersidechains.
Amongthese,dehydroalaninehasmuchpotential.
Therearemultiplemethodsavailablefordehydroalanineincorporationintoproteinsandthisresidueisanacceptorforavarietyofnucleophiles.
Whenusedinconjunctionwithstandardgenetictechniques,chemicalmutagenesismayallowaccesstonatural,modied,andunnaturalaminoresiduesontranslated,foldedproteins.
AddressDepartmentofChemistry,UniversityofOxford,ChemistryResearchLaboratory,ManseldRoad,OxfordOX13TA,UnitedKingdomCorrespondingauthor:Davis,BenjaminG(ben.
davis@chem.
ox.
ac.
uk)CurrentOpinioninChemicalBiology2010,14:781–789ThisreviewcomesfromathemedissueonMethodsforBiomolecularSynthesisandModicationEditedbyMattFrancisandIsaacCarrico1367-5931/$–seefrontmatter#2010ElsevierLtd.
Allrightsreserved.
DOI10.
1016/j.
cbpa.
2010.
10.
007IntroductionThedevelopmentofsite-directedmutagenesishasrevo-lutionizedproteinscience[1,2].
Specic,highdelitymutationsattheDNAlevelarenowroutineandrecombi-nantexpressionsystemsenablesite-specicincorporationandvariationofaminoacidresiduesinthenalprotein.
Standardsite-directedmutagenesis,however,islargelylimitedto20natural,proteinogenicaminoacidresidues.
Asbiochemicalendeavorsandgoalshaveadvanced,aneedtoincorporatenon-naturalormodiedaminoacidsidechainshasemerged.
Unnaturalaminoacidscanbetargetedforfurtherlabelingaswellasproteintrackingandanalysis[3,4].
Aminoacidsidechainsarealsonaturallymodied—phosphorylation,methylation,acylation,glycosylationareafewsuchexamples—andaccesstothesesidechainsishighlydesirableifwearetoexploretheirrolesmoreprecisely[5].
Ambercodonsuppressionandreprogrammedgeneticcodesaretwostrategiesforincorporatingsomeunnaturalormodiedaminoacidsnotaccessiblethroughnormaltranslation[6–9].
Semi-synthesisbynativechemi-calligationisanotheralternativeandthroughthismethodthetotalsynthesisofproteinshasbecomereality[10].
Anotherstrategyistousechemistryonanexpressedandfoldedproteintoinstalladesiredsidechainatadesiredsite.
Itisthisnalstrategythatisthefocusofthisreview.
Ourintentionisnottoprovideacomprehensivereviewofproteinlabelingmethods.
Rather,wewishtoexplore—andindeedrevive—theconceptof'chemicalmutagen-esis'putforthnearlyhalfacenturyagobyDanielE.
Koshland,Jr.
[11].
VisionarycontributionsfromtheKosh-land[11]andBender[12]laboratoriesdescribed,forthersttime,thechemicalconversionofoneaminoacidsidechaintoanother—achemicalmutation.
Asgenetictech-nology,expressionsystems,andaqueouschemistryhavedeveloped,itisworthwhiletorevisittheideaofachemicalmutation.
Wersttracetheinceptionofthisconceptandthenconsideritsplaceincontemporarychemistryandbiologyasageneralmethodtoalterproteinstructureprecisely.
Chemicalmutagenesis:aseminalconceptinproteinscienceIn1966,thelaboratoriesofDanielE.
Koshland,Jr.
andMyronL.
Benderindependentlyreportedtherstpointmutationofanenzyme[11,12].
Inbothreports,theserineproteasesubtilisinwaschemicallyconvertedtoacysteineprotease.
ThetransformationisdepictedinFigure1.
Theactivesiteserinewasrstselectivelyconvertedintoaleavinggroupbytreatmentwithphe-nylmethanesulfonyluoride(PMSF)andthendisplacedbytheattackofthioacetate.
Theresultingthioacyl-enzymewashydrolyzedthroughtheinnateactivityoftheprotease,providingthethiol-subtilisinproduct.
Themutantenzymewascharacterizedbothchemically(thecysteineproteasecontainsasingle,easilydetectedcysteine)andthroughkineticanalysisofproteaseactivity.
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comCurrentOpinioninChemicalBiology2010,14:781–789TheSertoCysmutationprovidedanear-isostericmutantforstudyoftheproteaseactive-siteandmechanism.
Itshouldbepointedoutthatatthetimeofthesediscov-eries,nomethodsyetexistedforsite-specicmutagenesisattheDNAlevelandthetotalchemicalsynthesisofproteinswasnotyetfeasible.
Remarkably,KoshlandandBenderbothanticipatedtheuseofgeneticmodicationstospecicallycontrolproteinstructurebutuntilthistechnologywasavailable,theyproposedamoreimmedi-atelyaccessiblemethodofmutationperformedonthetranslatedproteinthroughtheuseofchemistry—suchmodicationswerereferredtoas'chemicalmutations'byKoshland[11]and'simulatedmutations'byBender[13].
Thenomenclatureforthesetransformationscanbeamatterofcontentionandconfusion,soafewpointsregardingtheconceptof'chemicalmutation'areperhapsinorder.
Inthisreview,werefertoa'chemicalmutation'asaprocessthatconvertsanaminoacidresidueonatranslatedproteintoanotheraminoacidresidue.
Chemi-calmutationinthiscontextdoesnotrefertochemicalalterationofDNAanditdoesnotrefertocovalentlabelingofproteinswithsyntheticprobes,cofactors,andotherbiochemicaltools.
ToavoidanyconfusionwithDNAmodication,weproposetheterm'post-expressionmutagenesis'forchemicalalterationofthetranslated,foldedprotein[14].
Sinceinthisreviewweconsideronlymutationsonproteins,wewilluse'chemicalmutation'and'post-expressionmutation'interchangeably.
Theproof-of-principlesetforthbyKoshlandandBenderwasalandmarkinproteinscience.
Theconversionofactivesiteserinestocysteineshasbeenappliedtootherproteasesubstratessuchastrypsin[15]andrelatedpro-tocolsforconversiontoselenocysteine[16–19]andeventellurocysteine[20]havebeenreported.
Despitetheseadvances,theserinetocysteinemutationwasonlypossibleinthesecasesbecauseoftheuniquechemicalreactivityofactive-siteserine.
AdifferentapproachwasreportednotlongafterKoshlandandBender'sdisclosures.
Laskowskidescribedan'enzy-maticmutation'onsoybeantrypsininhibitorthatreliedontrypsinandcaboxypeptidaseBtoexiseanaminoacidfromtheproteinandre-ligateanotheraminoacidatthesiteofmutation[21].
Themutationiscarriedoutonthenativeprotein,post-expression,inthesamespiritasthechemicalmutationdescribedbyKoshlandandBender,butLas-kowski'smethoddiffersconceptuallysincetheproteinbackboneisaltered,ratherthansidechain.
Moreover,Laskowskihimselfconcededthatthismethodrelieson'abitofluck'thatallthenecessaryenzymescanco-existunderthereactionconditionswithoutdeleteriousproteol-ysis[21].
Nevertheless,thisconceptwasfurtherdevelopedbyTschesche,whodescribedsequentialaminoacidexci-sion,chemicalcoupling,andelastaseligationasamethodtomutatesoybeantrypsininhibitor[22,23].
Tschescheconsideredtheseaminoacidexchangesaformof'chemicalmutation'[23].
Importantly,theseexamplesillustratethatsomealterationscanbeaccomplishedenzymaticallyandthatsuchamutationdoesnotnecessarilyrelysolelyonselectivesmall-moleculechemistry.
Collectively,themutationsdescribedbyKoshland,Bender,andLaskowskiwerethemostprecisemanipulationsofproteinstructurethatprecededthegeneticandbiotechnologicalrevolution.
Chemicalmutagenesis:expandingscopefornaturalresiduesAdecadeaftertherstexamplesofchemicalmutagenesiswereputforthbyKoshlandandBender,PeterClarkandGordonLowereportedtheuseofcysteineasaprecursortomultipleaminoacidsidechains.
Their'chemicalmutationsofpapain'canbeconsideredmoregeneralthanpreviouseffortsinchemicalmutagenesissincethecysteineresidueneednotbeinaprotease,cysteineresidueneednotbeactivatedbyitspresenceinanactivesite,thoughinthiscasetheresiduewasindeedthenucleophileofthecysteineproteasepapain[24,25].
Inthesereports,cysteinewasalkylatedwithaphenacylbromide(Figure2).
Photolysisofthisintermediateledto782MethodsforBiomolecularSynthesisandModificationFigure1ThefirstpointmutationofanenzymewasreportedindependentlybyKoshlandandBenderin1966[11,12].
Theserine-to-cysteinemutationwasaccomplishedchemically.
Themorereactiveserineoftheactivesiteofsubtilisinisselectivelyconvertedintoaleavinggroupusingasulfonylfluoride(BnSO2F).
Displacementbythioacetateprovidesathioacyl-enzymeintermediatethatishydrolyzedtothefreethiolofcysteine.
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comirreversibleNorrishtypeIIcleavage,providingthethioal-dehydedehydrocysteine.
HydrationofthethiocarbonylandlossofH2Sresultsintheformationofformylglycine.
FormylglycinewasinturnreducedwithNaBH4topro-videserine.
Inthisway,theoveralltransformationisamutationfromcysteinetoserine.
Moreover,formylgly-cinecanalsobeconvertedtoglycineafterprolongedincubationatpH9.
0(Figure2).
Lowe'sextensionoftheconceptofchemicalmutagenesiswasthereforeuniqueinthatitcouldprovidemultiplemutationsfromasingle,commonprecursorand,inprinciple,couldbeappliedtoanyfreecysteine.
SinceLowe'sreport,therehavebeennotablyfewpub-licationsconcernedwithchemicalmutations.
Geneticandrecombinanttechnologywasmovingforwardatastrikingrateandaminoacidinterconversionusingpost-translationalchemistrymayhaveseemedobsolete.
Someillustrativereportsofchemicalmutationshavenonethe-lessappearedthatindicatestrategicadvantagesofchemi-calmutations.
Forinstance,Venkateshshowedthatglutamineandasparagineresiduescanbehydrolyzedwithacidto'chemicallymutate'thesidechaintoglu-tamicandasparticacid,respectively.
ItwasshownthatthesemutationsinuencedthekineticsoffoldinginRNaseA[26].
Inthereversemutation,Imotoshowedthatamidationofglutamateandaspartateresiduescouldbeusedinthestudyoftheseresiduesascatalyticsidechains[27].
Themutationinthiscaseisfromglutamateandaspartatetoglutamineandasparagine,respectively.
Inthesereports,multi-sitemutationsareaccomplished.
Inthecaseofamidation,thesemutationsarecarriedoutonthefoldedprotein.
Itisconceivablethatifthesemutationswereintroducedatthegeneticleveltheproteinwouldnotfoldproperlyandsothisexamplehighlightsastrategicadvantageofpost-expressionmuta-genesiswheredifferencesinactivitybetweentheproteinmutantscanbeattributedtothemutationanddecoupledfrommisfoldingduringexpression.
Otherexamplesofchemicalmutationshavebeentheproductsofeffortstogeneralizenativechemicalligation(NCL)andovercomeitsinherentrelianceoncysteine[10].
AnumberofcysteinedesulfurizatonprocesseshavebeenputforththatallowtheuseofNCLinthesynthesisofpeptidesandproteinsthatdonotcontaincysteine.
DawsonhasdisclosedamethodtoreducecysteinetoalanineusingpalladiumorRaneynickel[28].
Relatedtransformationshavesincebeenreportedtoprovidephenylalanine[29]andvaline[30,31]atthesiteofligation.
OfnoteisDanishefsky'smild,radicalbasedmethodforthedesulfurizationofcysteine[32].
Intheseexamplestheapplicationofchemicalmutagenesisinnativechemicalligationisapparent:achemicalmutationofcysteinetoanotherresidueconstitutesaligation,intheformalsense,atthenalresidue.
ChemicalmutationtoanaminoacidanalogChemicalmutagenesisinitspurestformconstitutestheconversionofsomeprecursorsidechaintoanaturalChemicalmutagenesisChalkerandDavis783Figure2ClarkandLowe's'ChemicalMutationsofPapain.
'Cysteinewasusedasacommonprecursortomultipleaminoacids:formylglycine,glycine,andserine[24,25].
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Insomecases,however,thenaturalresiduemaynotbeeasilyobtainedbychemicalmethods.
Intheseinstances,amimicoranalogmaysufce[33].
Indeed,someoftheearliestexamplesofchemicalproteinmodi-cationweretheconversionoflysinetohomoarginineandtheconversionofcysteinetothialysine.
Asearlyas1949,itwasshownthattreatmentofhumanserumalbuminwithO-methylisoureaprovidedhomoarginineanalogsthatfacilitatedproteincrystallization[34].
Suchatransformationhasalsobeenusedtoinvestigateactive-sitearginineresidues[35,36].
Inthe1950s,aminoethyla-tionofcysteinewasusedtoinstallasyntheticsiteofproteolysisfortrypsin,whichrecognizeslysineresidues[37–39].
Althoughthistechniquewasusedmainlyfortrypticanalysisandsequencingproteins,theunderlyingenablingtechnologyistheconversionofcysteinetothialysine.
Althoughthispseudo-mutationdoesnotpro-videanaturalresidue,thesimilaritytolysinehassincebeenusedmanytimesintheinvestigationofcatalyticlysineresidues,highlightingtheclearfunctionalutilityoftheseanalogs[40–44].
Similaralkylationsofcysteinehavealsoprovidedarginineanalogs[45].
Recentinterestincysteineaminoethylationhasintensiedsinceaccesstomethylatedlysineresiduesisrequired,especiallyonhistoneproteins[46].
Thispseudo-mutationhasallowedanincreasedunderstandingoftheeffectsofhistonemodicationandtheoverarchingepigeneticcode[47–49].
Otherpseudo-mutationshaveallowedaccesstodifferentpost-translationalmimics,suchassulfatedandphosphorylatedtyrosine[3,50].
Althoughtheseaminoacidanalogshaveprovenusefulinanumberofcontexts,theyarestillonlyanalogs,indicatingperhapsasmuchabouttheexibilityofthesystemthatrecognizesthem.
Flexiblemethodstoaccesstheresidue,modiedorotherwise,initsnaturalstateremainsanongoingchal-lengeinchemicalmutagenesisandinproteinscienceingeneral.
Wenowturntothechallengesandrecenteffortsthathaveadvancedthenotionofachemicalmutation.
TowardsageneralchemicalmutagenesisForchemicalmutagenesistobeageneralstrategyforproteinaminoacidinterconversion,itisdesirabletouseaprecursor,naturalorunnatural,thatcanprovideaccesstoafunctionalsetofsidechains.
Ideally,thisuniversalprecursorwouldprovidedivergentaccesstothe20mostcommonnaturalsidechains,alongwithmanyunnaturalresidues.
Forexample,intheworkofKoshland[11]andBender[12]suchaprecursorwastheactiveserineofsubtilisin.
Thisresiduecanbeselectivelyconvertedintoaleavinggroupanddisplacedwithanucleophile.
Intheircasetheconversiontocysteinewastheirtarget,yieldinganearisostericmutanttoinvestigatethecatalyticactivesiteoftheseproteases.
Yetonecanimagineothernucleo-philesthatcoulddisplacethesulfonylatedserineofsubtilisin.
Suchatransformationcouldprovideotherchemicalmutants,andindeedhassincebeenextendedtoSe-andTellurocysteinevariants[16,20].
Nonetheless,whensuchtransformationsarepossible,theyrelyontheinnatereactivityoftheproteasetorenderthehydroxylmoietyoftheserinealeavinggroup.
Themethodcouldnotbeeasilyextendedtootherproteinsubstrates.
Forgeneralapplication,adifferenthandleisneeded.
ArecentexampleofaprecursortootheraminoacidswasreportedbytheSchultzgroup.
Althoughtheirmethodwasnotpresentedinthecontextofchemicalmutagenesis,theoveralltransformationscouldbeconsideredchemicalmutations.
Theincorporationofp-boronophenylalanineinresponsetotheamberstopcodoncreatedaproteinwithanunnaturalaminoacidthatcanbeconvertedtophenyl-alanineandtyrosinewhenreducedoroxidized,respect-ively(Figure3)[51].
Toaidthiswork,theauthorselegantlytookadvantageoftheboronicacidasanafnitytagsinceitbindstopolyhydroxylatedresin.
Elutionwithoxidantorreductantprovidedthenativeprotein,freeoftag.
Fullconversionswereobservedintheoxidationafter2hourswhileovernightincubationwasrequiredforthereduction.
Whilethemainapplicationofthistechnologyistracelessafnitypurication,thechemicalconversionofacommonprecursortootheraminoacidscanbeconsideredachemicalmutation.
Moreover,thechemicalhandleformutation(theboronicacid)canbeincorporatedatapre-determinedsiteoftheproteininresponsetoauniquecodonanddoesnotrelyontheinnatefunctionalityorsequenceoftheproteinofinterest.
Theprecedingexampleprovidesaccesstophenylalanineandtyrosinefromacommonprecursor.
Adifferentpre-cursorisneededforaliphaticsidechains.
Koshlandagainprovidesinspirationforsuchanaminoacidprecursor.
Inaproteasesystemsimilartotheonewherehereportedtherstpointmutationofanenzyme,Koshlandshowedthatthesulfonylatedserineofchymotrypsincanbeelimi-natedunderbasicconditionstogivedehydroalanine[52,53].
Thisenamideindehydroalaninecanbecon-sideredtobeanelectrophile,andvariousthiolswereaddedtothisresidue.
Koshlandrecognizedthatthisresiduecould,inprinciple,beaprecursortootheraminoacidsidechains[53].
AdditionofH2S,forinstance,wouldprovidecysteine.
Indeedtheadditionofthiolstodehydroalaninehassinceprovidedaccesstomultiplecysteinederivativesandaminoacidanalogs.
Theseexampleshavereliedlargelyonthemultiplemethodsthathavebecomeavailablefortheincorporationofdehy-droalanineintoproteins.
Forinstance,theSchultzgrouphasdescribedtheuseofphenylselenocysteineasaprecursortodehydroalanineonproteins[54].
Oxidativeeliminationusinghydrogenper-oxideprovideddehydroalanine.
AdditionofglycosylthiolsandaliphaticthiolsprovidedS-hexadecylcysteineandS-mannosylcysteine.
OurownlabhasreportedanovelchemicalmethodtooxidativelyeliminatecysteinedirectlytodehydroalanineusingO-mesitylenesulfonylhy-784MethodsforBiomolecularSynthesisandModificationCurrentOpinioninChemicalBiology2010,14:781–789www.
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comdroxylamine(MSH)[55].
Inthisreportitwasshownthatdehydroalanineisaprecursortoglycosylcysteines,phos-phocysteine,andfarnesylcysteine—allresiduesthatarefoundinnature.
Wealsoreportedaccesstomethylatedlysineanalogs;thisuseofDhaasaprecursortolysineanalogsisacomplementarymethodtothemorecommonaminoethylationprotocol[46].
Similarly,theSchultzgrouplateruseddehydroalanineasaprecursortomethylandacyllysineanalogsonhistoneH3[56].
Intheirreport,theydemonstratedtherstuseofN-acetyl-thialysineasasub-strateforhistonedeacetylaseenzymes.
Inthisway,dehydroalanineisausefulprecursortocysteinederivatives,includingnaturalresiduessuchasglycosyl-,phosphoryl-,andfarnesylcysteine.
Thialysineanalogs,however,areonlymimicsofthenaturalsidechain.
Whilemimickingthisfunctionalitymaysufceinmanycases,itisdesirabletoaccessnativestructures.
Withthedevelopmentofahostofaqueousandasym-metrictransformations,thepotentialofdehydroalanineasageneralhandleinchemicalmutagenesisisnotoutofthequestionandwemayspeculateonfuturedirections.
Forinstance,arylsidechainscanbeaccessedfromdehydroa-laninebytheRh(I)catalyzedconjugateadditionofarylboronicacids.
Thisreactioniscompatiblewithwater,hasseveralasymmetricvariants[57],anditsuseonpeptidesubstrateshasalsobeeninvestigated[58,59].
Foraliphaticsidechains,radicaladditionstounsaturatedsystemsarepossibleinwater[60].
Thedeploymentofsuchtrans-formationsatdehydroalaninecan,inprinciple,provideaccesstomanydiversesidechains.
Applyingasimilarretrosyntheticanalysistoothersidechainsrevealsthatasignicantnumberofresiduescanbeconsideredacces-siblefromadehydroalanineprecursor.
Alanine,forinstancecanbeaccessedbycatalytic,andperhapsasym-metric,hydrogenation.
Asimilarexerciseinretrosynth-esismayrevealothertransformationsthatwillconstitutea'chemicalmutation.
'AproposalforseveralsidechainsisdepictedinFigure4.
Itisworthnotingthediversefunctionalityaccessiblefromdehydroalanine.
Aromatic,aliphatic,acidic,basic,andpost-translationallymodiedsidechainsareallaccessible,inprinciple,fromthisprecursor.
Figure4onlydepictsnaturalresidues.
Itisclearthatmanyothercomplementaryunnaturalaminoacidsshouldbeaccessiblethroughasimilarroute.
Dehydroalanineasahandleforchemicalmutagenesisisnotwithoutpotentialpitfallsandcautionarynotes.
Therstistherequirementfordehydroalanine.
Althoughsev-eralmethodsforitsincorporationintoproteinsareknown,noneisentirelygeneral.
Asimple,selective(orbetteryet,specic)installationofdehydroalanineisrequired.
Second,thetransformationsproposedinFigure4havenotbeenreportedonanyproteinsubstrates,muchlessfragile,pHsensitiveproteinsamples.
Thechallengeofwatercompa-tible,chemoselectivemutationsofDhatootherresiduesisthesecondoutstandingproblem.
Inaddition,manyoftheseproposedtransformationsmayrequireanasymmetricvariantorsomeunderstandingofsubstratecontrolofthediastereoselectivity.
Theveryanalysisofthechemicalandstereochemicaloutcomeoftheproposedtransformationisalsonosmallfeat.
ChemicalmutagenesisChalkerandDavis785Figure3Schultz'sboronicacid:achemicalprecursortoPheandTyrmutantproteins[51].
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comCurrentOpinioninChemicalBiology2010,14:781–789Whenthesechallengesaremetusingdiverseanddiver-gentintermediatessuchasdehydroalanine,weenvisionmanyadvantagesandapplicationsofchemicalmutagen-esis.
Forinstance,asinglemutantderivedfromroutinesite-directedmutagenesiscanbeachemicalprecursorformultiplemutantproteins.
Thus,asingleroundofexpres-sionandpuricationcouldyieldadiverselibraryofmutantproteinsafterchemicalmutation.
Furthermore,thenalchemicalmutationcouldbecarriedoutonthefoldedprotein,soproteinactivitycanbeattributedtothe786MethodsforBiomolecularSynthesisandModificationFigure4XX=SH,SePhEliminationDehydroalanine"RH"RRH=nucleophileChemicalMutantNHONHONHOOHNHOHNNHONHNNHONHOSMeNHOOHONHOOH2NNHONH2NHONHNHH2NNHONHAcNHONHnMe(3-n)n=0-2NHONHNNMeMe+NHONHNHnMe(2-n)H2N+HHn=0,1AromaticHydrophobiccisaBcidicAAcylatedandMethylatedNHORR,reductant(R=aliphatic)R-Rh(I)Ln(R=Ar)ChemicalMutantDehydroalanineorPolarCurrentOpinioninChemicalBiologyAproposal:dehydroalanineasageneralchemicalmutagenesishandle.
Arylandaliphaticnucleophilesmayallowaccesstoadiversefunctionalsetofaminoacidsidechains.
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commutationitselfandnotmisfoldingduringexpression.
Anothersignicantapplicationwouldbeaccesstounna-turalaminoacidsandmodiednaturalaminoacidsthatarenotyetaccessibleinstandardexpressionsystems.
Ultimately,chemicalmutagenesismayresolvesupplyissuesforproteinsbearingthesepost-translationalmodi-cations,severalexamplesofwhichareshowninFigure4.
Finally,effortsinchemicalmutagenesisareattractivechemicalchallengesthatmayinspirenewchemistrythatismildandcompatiblewithbiologicalsystems.
ThesechallengesandpotentialapplicationsareguidingourcurrenteffortswhilewerevisitKoshland'sideaofchemicalmutations.
ConclusionsTheconceptofchemicalmutagenesisdatesbacktotherstpointmutationofanenzymebyKoshlandandBenderin1966.
However,asDNAandrecombinantexpressiontechnologydeveloped,theuseofchemistryinaminoacidconversionwaslargelyreplaced.
Wehaverevisitedtheideaofchemicalmutagenesisagaininanefforttoreviveitsuseinproteinscience.
Wearenotinanywaypittingchemistryagainstbiologybutratherencourageadualandreinforcinguseofcurrentmethodsinbiosyntheticincorporationofnaturalandunnaturalaminoacidsandtheirpost-expression'mutation'usingchemistry.
Itisourhypothesisthatmutationoftranslated,foldedproteinsusingchemistrycanallowrapidaccesstonatural,unnatural,andmodiednaturalsidechains.
Thechemistryandbiologyrequiredforageneral'chemicalmutagenesis'isrifewithopportunity.
Meetingthechal-lengesdescribedabovewillhelpbuildtheconceptualandpracticalfoundationforageneralmethodofproteinconstructionfreeofmanyofthecurrentlimitsofpurelybiologicalstrategies.
AcknowledgementsTheauthorsthanktheirgeneroussourcesofsupport.
JustinM.
ChalkerisaRhodesScholarandNationalScienceFoundationGraduateResearchFellow.
BenjaminG.
DavisisarecipientofaRoyalSocietyWolfsonResearchMeritAwardandissupportedbyanEngineeringandPhysicalSciencesResearchCouncilLifeSciencesInterfacePlatformGrant.
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