Picazoplatin, an azide-containing platinum(II) derivative for target analysis by click chemistry

Article abstract of DOI:10.1021/ja402453k

Despite the broad use of platinum-based chemotherapeutics, identification of their full range of cellular targets remains a significant challenge. In order to identify, visualize, and isolate cellular targets of Pt(II) complexes, we have modified the chemotherapeutic drug picoplatin with an azide moiety for subsequent click reactivity. The new compound picazoplatin readily binds DNA and RNA oligonucleotides and undergoes facile post-labeling click reactions to alkyne-fluorophore conjugates. Pt-fluorophore click reactions in rRNA purified from drug-treated Saccharomyces cerevisiae demonstrate its potential for future in vivo efforts.

Full text of DOI:10.1021/ja402453k

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Communication
Picazoplatin, an Azide-Containing Platinum(II)
Derivative for Target Analysis by Click Chemistry
Jonathan D. White, Maire F. Osborn, Alan D. Moghaddam, Lindsay
E. Guzman, Michael M. Haley, and Victoria Jeanne DeRose
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja402453k • Publication Date (Web): 24 Jul 2013
Downloaded from http://pubs.acs.org on July 27, 2013
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Picazoplatin,ꢀanꢀAzide-ContainingꢀPlatinum(II)ꢀDerivativeꢀforꢀ
TargetꢀAnalysisꢀbyꢀClickꢀChemistryꢀ
ꢀ
ꢀ
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†
†
JonathanꢀD.ꢀWhite, ꢀMaireꢀF.ꢀOsborn, ꢀAlanꢀD.ꢀMoghaddam,ꢀLindsayꢀE.ꢀGuzman,ꢀMichaelꢀM.ꢀHaley,ꢀ
andꢀVictoriaꢀJ.ꢀDeRose*ꢀ
DepartmentꢀofꢀChemistryꢀandꢀInstituteꢀofꢀMolecularꢀBiology,ꢀUniversityꢀofꢀOregon,ꢀEugene,ꢀORꢀ97403-1253ꢀUSA.ꢀ
SupportingꢀInformationꢀPlaceholder
ABSTRACT:ꢀDespiteꢀtheꢀbroadꢀuseꢀofꢀplatinum-basedꢀ
chemotherapeutics,ꢀ identificationꢀ ofꢀ theirꢀ fullꢀ rangeꢀ
ofꢀcellularꢀtargetsꢀremainsꢀaꢀsignificantꢀchallenge.ꢀInꢀ
orderꢀ toꢀ identify,ꢀ visualize,ꢀ andꢀ isolateꢀ cellularꢀ tar-
getsꢀofꢀPt(II)ꢀcomplexes,ꢀweꢀhaveꢀmodifiedꢀtheꢀche-
motherapeuticꢀdrugꢀpicoplatinꢀwithꢀanꢀazideꢀmoietyꢀ
forꢀ subsequentꢀ clickꢀ reactivity.ꢀ Theꢀ newꢀ compoundꢀ
picazoplatinꢀreadilyꢀbindsꢀDNAꢀandꢀRNAꢀoligonucleo-
tidesꢀ andꢀ undergoesꢀ facileꢀ post-labelingꢀ clickꢀ reac-
tionsꢀ toꢀ alkyne-fluorophoreꢀ conjugates.ꢀ Pt-
fluorophoreꢀ clickꢀ reactionsꢀ inꢀ ribosomalꢀ RNAꢀ puri-
fiedꢀ fromꢀ drug-treatedꢀ S.ꢀ cerevisiaeꢀ demonstrateꢀ itsꢀ
potentialꢀforꢀfutureꢀinꢀvivoꢀefforts.ꢀꢀ
candidateꢀ forꢀ post-treatmentꢀ labelingꢀ isꢀ theꢀ azide-
alkyneꢀ dipolarꢀ cycloaddition,ꢀ theꢀ prototypicalꢀ clickꢀ
5
reaction. ꢀClickꢀchemistryꢀhasꢀbeenꢀbroadlyꢀusedꢀforꢀ
localizationꢀ andꢀ functionalꢀ studiesꢀ ofꢀ modifiedꢀ bio-
molecules,ꢀ includingꢀ proteinsꢀ andꢀ nucleicꢀ acids.6,7ꢀ
Despiteꢀ theꢀ importanceꢀ ofꢀ Pt(II)ꢀ compoundsꢀ inꢀ the-
rapeuticsꢀandꢀcatalysis,ꢀapplicationsꢀofꢀclickꢀreactionsꢀ
8
onꢀPt(II)ꢀcomplexesꢀhaveꢀbeenꢀlimited. ꢀClickꢀreacti-
Platinumꢀ (II)ꢀ compoundsꢀ compriseꢀ upꢀ toꢀ 50-70%ꢀ
ofꢀanticancerꢀtreatmentꢀregimesꢀinꢀuseꢀtoday. ꢀUnlikeꢀ
Figureꢀ 1.ꢀ (a)ꢀ Theꢀ anticancerꢀ drugsꢀ cisplatinꢀ andꢀ
picoplatin,ꢀ withꢀ theꢀ novelꢀ picoplatinꢀ analogueꢀ 1,ꢀ
picazoplatin.ꢀ(b)ꢀReactionꢀschemeꢀofꢀbiomolecule-boundꢀ
picazoplatinꢀclickingꢀtoꢀaꢀdansylꢀfluorophore.ꢀ
1
otherꢀ classesꢀ ofꢀ commonꢀ therapeuticsꢀ suchꢀ asꢀ anti-
biotics,ꢀhowever,ꢀaꢀmolecular-levelꢀunderstandingꢀofꢀ
2
theꢀ specificꢀ targetsꢀ ofꢀ Pt(II)ꢀ drugsꢀ isꢀ lacking. ꢀ Pt(II)ꢀ
drugsꢀ preferentiallyꢀ targetꢀ purineꢀ nucleobases,ꢀ andꢀ
drugꢀbindingꢀtoꢀgenomicꢀDNAꢀisꢀanꢀacceptedꢀcauseꢀofꢀ
downstreamꢀ apoptoticꢀ signaling. ꢀ Lessꢀ thanꢀ 10%ꢀ ofꢀ
onsꢀhaveꢀbeenꢀusedꢀasꢀaꢀsyntheticꢀrouteꢀtoꢀgenerateꢀ
librariesꢀofꢀPt-basedꢀtherapeutics8d-fꢀ andꢀforꢀfluores-
centꢀpost-labelingꢀofꢀaꢀmonofunctionalꢀPt(II)-acridineꢀ
derivative. ꢀOurꢀgoalꢀisꢀtoꢀmodifyꢀsmallꢀbifunctionalꢀ
Pt(II)ꢀ reagentsꢀ suchꢀ asꢀ cisplatinꢀ andꢀ relatedꢀ com-
poundsꢀ(Figureꢀ1)ꢀforꢀpost-treatmentꢀanalysis.ꢀ
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Ptꢀ (inꢀ theꢀ caseꢀ ofꢀ cisplatin)ꢀ accumulatesꢀ withinꢀ ge-
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nomicꢀ DNA, ꢀ andꢀ questionsꢀ remainꢀ regardingꢀ addi-
tionalꢀtargetsꢀthatꢀcouldꢀimpactꢀcytotoxicityꢀandꢀre-
sistance.ꢀAꢀfacileꢀandꢀsensitiveꢀmethodꢀforꢀdrugꢀtargetꢀ
analysisꢀwouldꢀgreatlyꢀaidꢀsuchꢀinvestigations.ꢀꢀꢀ
Here,ꢀ weꢀ reportꢀ theꢀ synthesisꢀ ofꢀ theꢀ novelꢀ azide-
functionalizedꢀPt(II)ꢀcomplexꢀ1ꢀandꢀpresentꢀDNAꢀoli-
gonucleotideꢀbindingꢀandꢀsubsequentꢀclickꢀreactivityꢀ
withꢀalkyne-modifiedꢀfluorophoresꢀ(Figuresꢀ2ꢀandꢀ3).ꢀ
Weꢀextendꢀtheseꢀstudiesꢀtoꢀshowꢀinꢀvitroꢀlabelingꢀofꢀaꢀ
structuredꢀRNAꢀoligonucleotideꢀ(Figureꢀ4)ꢀandꢀinꢀvivoꢀ
treatmentꢀandꢀpost-labelingꢀofꢀyeastꢀribosomalꢀRNAꢀ
(Figureꢀ 5).ꢀ Thisꢀ tandemꢀ Ptꢀ bindingꢀ andꢀ post-
modificationꢀ techniqueꢀ enablesꢀ visualizationꢀ andꢀ
quantificationꢀ ofꢀ theꢀ cellularꢀ targetsꢀ ofꢀ Pt(II)ꢀ com-
pounds,ꢀandꢀmayꢀbeꢀbroadlyꢀapplicableꢀtowardsꢀiden-
tificationꢀofꢀotherꢀmetal-ionꢀbasedꢀdrugꢀtargets.ꢀ
Oneꢀ synthetic-basedꢀ strategyꢀ forꢀ targetꢀ identifica-
tionꢀ involvesꢀ modifyingꢀ Pt(II)ꢀ complexesꢀ toꢀ containꢀ
stericallyꢀ smallꢀ bioorthogonallyꢀ reactiveꢀ handles.ꢀ
Chemicalꢀ transformationsꢀ atꢀ thisꢀ handleꢀ (suchꢀ asꢀ
reactionꢀ withꢀ aꢀ fluorophore)ꢀ followingꢀ drugꢀ treat-
mentꢀwouldꢀallowꢀforꢀaccurateꢀPtꢀlocalizationꢀinꢀvivoꢀ
2
c
orꢀsubsequentꢀpurificationꢀofꢀcellularꢀtargets. ꢀPost-
treatmentꢀlabelingꢀcircumventsꢀproblemsꢀassociatedꢀ
withꢀusingꢀcompoundsꢀmodifiedꢀwithꢀbulkyꢀorꢀchar-
gedꢀ fluorophores,ꢀ whichꢀ mayꢀ notꢀ accuratelyꢀ repre-
2
d
sentꢀcellularꢀprocessingꢀofꢀtheꢀnativeꢀdrug. ꢀAnꢀidealꢀ
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Weꢀ designedꢀ complexꢀ 1ꢀ withꢀ theꢀ azide-
functionalizedꢀ 2-picolineꢀ ligand,ꢀ similarꢀ toꢀ theꢀ well-
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knownꢀcomplexꢀpicoplatinꢀ(Figureꢀ1a). ꢀSinceꢀPt(II)ꢀ
isꢀ capableꢀ ofꢀ catalyzingꢀ hydroaminationꢀ andꢀ otherꢀ
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undesirableꢀsideꢀreactionsꢀwithꢀalkynes, ꢀtheꢀazide-
functionalizedꢀcomplexꢀremainedꢀtheꢀinitialꢀfocusꢀofꢀ
thisꢀwork.ꢀInꢀaddition,ꢀorganoazideꢀcomplexesꢀofꢀPt,ꢀ
whereꢀtheꢀazideꢀmoietiesꢀareꢀconnectedꢀthroughꢀor-
ganicꢀlinkersꢀandꢀnotꢀdirectlyꢀboundꢀtoꢀtheꢀPtꢀcenter,ꢀ
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areꢀ knownꢀ compounds. ꢀ Furthermore,ꢀ anꢀ azide-
Pt(II)ꢀclickꢀreagentꢀallowsꢀforꢀtheꢀfutureꢀuseꢀofꢀnon-
linear,ꢀ ring-strainedꢀ alkynesꢀ andꢀ thusꢀ copper-freeꢀ
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Figureꢀ 3.ꢀ (a)ꢀ DNAꢀ hairpinꢀ withꢀ 1ꢀ boundꢀ andꢀ labeledꢀ
withꢀtheꢀalkyneꢀfluorophore.ꢀ(b)ꢀdPAGEꢀanalysisꢀshow-
ingꢀfluorescentꢀlabelingꢀofꢀDNAꢀhairpinꢀbyꢀCu-catalyzedꢀ
clickꢀ chemistry.ꢀ Laneꢀ 1ꢀ containsꢀ nonplatinatedꢀ DNAꢀ
hairpinꢀandꢀshowsꢀnoꢀnonspecificꢀDNA-fluorophoreꢀin-
teraction.ꢀLanesꢀ2-5ꢀcontainꢀplatinatedꢀDNAꢀunderꢀvary-
ingꢀclickꢀconditionꢀsetsꢀ(SupportingꢀInformation),ꢀallꢀofꢀ
whichꢀshowꢀsuccessfulꢀDNA-fluorophoreꢀlabeling.ꢀ
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clickꢀ chemistry. ꢀ Theꢀ stericꢀ hindranceꢀ providedꢀ byꢀ
theꢀ2-methylꢀgroupꢀofꢀpicoplatinꢀandꢀitsꢀderivativesꢀ
slowsꢀtheꢀkineticsꢀofꢀsubstitutionꢀreactionsꢀonꢀPtꢀandꢀ
isꢀ anꢀ importantꢀ effectorꢀ ofꢀ therapeuticꢀ activity.10,14
Theꢀ presenceꢀ ofꢀ theꢀ 2-methylꢀ substituentꢀ alsoꢀ pre-
ventsꢀtheꢀformationꢀofꢀdipicolineꢀadductsꢀandꢀgener-
allyꢀ yieldsꢀ cleanerꢀ synthesesꢀ comparedꢀ toꢀ otherꢀ
ꢀ
asymmetricallyꢀ substitutedꢀ cisplatinꢀ derivatives.15ꢀ
Complexꢀ 1ꢀ wasꢀ synthesizedꢀ andꢀ fullyꢀ characterizedꢀ
byꢀ Hꢀandꢀ195PtꢀNMR,ꢀIR,ꢀandꢀHRMSꢀ(seeꢀSupportingꢀ
Information).ꢀ
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Cisplatin,ꢀ picoplatin,ꢀ andꢀ similarꢀ Ptꢀ compoundsꢀ
bindꢀ readilyꢀ toꢀ 5’-GG-3’-containingꢀ shortꢀ DNAꢀ oli-
gonucleotidesꢀandꢀhairpinꢀstructures.3a,c,16ꢀBindingꢀofꢀ
1
ꢀ toꢀ double-strandedꢀ DNAꢀ andꢀ clickꢀ labelingꢀ wereꢀ
ꢀ
analyzedꢀ byꢀ dPAGEꢀ (Figureꢀ 2). Resolutionꢀ betweenꢀ
theꢀdenaturedꢀstrandsꢀisꢀclearlyꢀobserved,ꢀandꢀreac-
tionꢀwithꢀtwo-foldꢀexcessꢀ1ꢀ(4ꢀh,ꢀ37ꢀ°C)ꢀresultsꢀinꢀaꢀ
newꢀbandꢀcorrespondingꢀtoꢀPt-boundꢀT
6 5
GGT ꢀ(Figureꢀ
1
b,ꢀ leftꢀ andꢀ centerꢀ lanes)ꢀ inꢀ yieldsꢀ similarꢀ toꢀ thoseꢀ
observedꢀwithꢀtheꢀparentꢀcompoundꢀpicoplatinꢀ(notꢀ
shown).ꢀ Reactionꢀ ofꢀ theꢀ Pt-boundꢀ oligonucleotideꢀ
withꢀ anꢀ alkyneꢀ fluorophoreꢀ (dansylꢀ propargy-
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lamine) ꢀinꢀtheꢀpresenceꢀofꢀCuIꢀ(4ꢀh,ꢀ50ꢀ°C)ꢀresultsꢀinꢀ
fluorescentꢀlabelingꢀofꢀtheꢀPt-boundꢀspeciesꢀ(Figureꢀ
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b,ꢀ rightꢀ lane).ꢀ Observationꢀ ofꢀ theꢀ expectedꢀ clickꢀ
productꢀwithꢀaꢀsingle-strandedꢀT GGT ꢀconstructꢀwasꢀ
confirmedꢀ byꢀ MALDIꢀ massꢀ spectrometryꢀ (Figureꢀ
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c). ꢀ Theꢀ observedꢀ Pt-boundꢀ speciesꢀ (Figureꢀ 2c,ꢀ
black)ꢀ shiftsꢀ toꢀ aꢀ higherꢀ m/zꢀ asꢀ predictedꢀ fromꢀ cy-
cloadditionꢀ ofꢀ theꢀ dansylꢀ fluorophoreꢀ (Figureꢀ 2c,ꢀ
blue).ꢀTheꢀclickꢀreactionꢀefficiencyꢀappearsꢀtoꢀbeꢀhigh,ꢀ
asꢀ theꢀ Pt-boundꢀ peakꢀ disappearsꢀ completelyꢀ inꢀ theꢀ
clickedꢀ(blue)ꢀspectrum.ꢀꢀ
Figureꢀ2.ꢀ(a)ꢀDNAꢀduplexꢀwithꢀpicazoplatinꢀ(1)ꢀboundꢀ
andꢀlabeledꢀwithꢀdansylꢀalkyneꢀfluorophore.ꢀ(b)ꢀdPAGEꢀ
analysisꢀ showingꢀ theꢀ denaturedꢀ complementaryꢀ
strandsꢀ (left)ꢀ andꢀ higherꢀ molecularꢀ weightꢀ Pt-boundꢀ
speciesꢀ (center).ꢀ Clickꢀ reactionꢀ withꢀ theꢀ fluorophoreꢀ
resultsꢀ inꢀ fluorescenceꢀ ofꢀ theꢀ Pt-DNAꢀ bandꢀ underꢀ UVꢀ
lightꢀ(right).ꢀ(c)ꢀMALDIꢀmassꢀspectraꢀofꢀPt-DNAꢀ(black)ꢀ
andꢀPt-DNA-clickꢀproductꢀ(blue)ꢀshowingꢀtheꢀchangeꢀinꢀ
massꢀuponꢀbindingꢀ1ꢀandꢀreactingꢀwithꢀtheꢀfluorophore.ꢀ
Expectedꢀ m/zꢀ ofꢀ 4301.7ꢀ andꢀ 4590.8ꢀ comparedꢀ toꢀ theꢀ
observedꢀ4300.03ꢀandꢀ4587.3,ꢀrespectively.ꢀConditionsꢀ
areꢀoutlinedꢀinꢀtheꢀSupportingꢀInformation.ꢀꢀ
Additionalꢀ workꢀ examiningꢀ nonspecificꢀ interac-
tionsꢀbetweenꢀtheꢀdansylꢀalkyneꢀfluorophoreꢀandꢀtheꢀ
terminalꢀazideꢀofꢀtheꢀPt-boundꢀDNAꢀscaffoldꢀwasꢀper-
formedꢀ withꢀ aꢀ pyrimidineꢀ richꢀ DNAꢀ hairpinꢀ (Figureꢀ
1
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3a). ꢀThisꢀsequenceꢀwasꢀselectedꢀtoꢀminimizeꢀpoten-
tialꢀPtꢀinterstrandꢀcrosslinkꢀformationꢀbetweenꢀhair-
pinsꢀ (asꢀ wasꢀ occasionallyꢀ observedꢀ forꢀ single-
strandedꢀT
6 5
GGT ,ꢀdataꢀnotꢀshown).ꢀAꢀPt-boundꢀspe-
ciesꢀ isꢀ notꢀ resolvedꢀ underꢀ theseꢀ gelꢀ conditions,ꢀ butꢀ
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fluorescenceꢀdetectionꢀofꢀtheꢀsubsequentꢀclickꢀreac- degradationꢀofꢀourꢀSRLꢀRNAꢀconstructꢀunderꢀvariousꢀ
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tionꢀ clearlyꢀ identifiesꢀ aꢀ platinatedꢀ species.ꢀ Noꢀ non-
specificꢀ fluorescentꢀ labelingꢀ isꢀ observedꢀ underꢀ Cu-
freeꢀconditionsꢀ(Figureꢀ3,ꢀlaneꢀ1)ꢀfollowingꢀtheꢀreac-
tionꢀofꢀtheꢀhairpinꢀwithꢀtwo-foldꢀexcessꢀ1ꢀ(18ꢀh,ꢀ37ꢀ
Cu-catalyzedꢀ clickꢀ conditionsꢀ (notꢀ shown).ꢀ Recentꢀ
workꢀhasꢀdemonstratedꢀthatꢀtraceꢀamountsꢀofꢀaceto-
nitrileꢀstabilizeꢀfreeꢀCu(I)ꢀinꢀaqueousꢀsolutionꢀwhileꢀ
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stillꢀ facilitatingꢀ efficientꢀ cycloadditionꢀ catalysis. ꢀ
Here,ꢀ weꢀ demonstrateꢀ substantialꢀ clickꢀ productꢀ for-
mationꢀfromꢀpicazoplatin-boundꢀRNAꢀinꢀtheꢀpresenceꢀ
ofꢀ0.6%ꢀv/vꢀacetonitrileꢀ(Figureꢀ4ꢀandꢀS7).ꢀLanesꢀ1-3ꢀ
ofꢀFigureꢀ4ꢀshowꢀthatꢀtheꢀSRLꢀRNAꢀisꢀstableꢀinꢀupꢀtoꢀ
0.8ꢀmolarꢀequivalentsꢀofꢀCu(I).ꢀInꢀlanesꢀ4-7,ꢀaꢀsuper-
shiftꢀ demonstratesꢀ Pt-adductꢀ formationꢀ followingꢀ
RNAꢀtreatmentꢀwithꢀfour-foldꢀexcessꢀ1ꢀ(18ꢀh,ꢀ37ꢀ°C).ꢀ
Notably,ꢀ lanesꢀ 6ꢀ andꢀ 7ꢀ demonstrateꢀ efficientꢀ Cu(I)-
catalyzedꢀclickꢀlabelingꢀtoꢀdansylꢀalkyneꢀfluorophoreꢀ
(1ꢀ h,ꢀ 50ꢀ °C).ꢀ Thus,ꢀ post-treatmentꢀ labelingꢀ byꢀ clickꢀ
chemistryꢀisꢀalsoꢀsuccessfulꢀforꢀtheꢀanalysisꢀofꢀRNA-
Ptꢀadducts.ꢀ
°
C).ꢀProficientꢀfluorescentꢀlabelingꢀofꢀtheꢀ1-boundꢀGGꢀ
DNAꢀ hairpinꢀ isꢀ observedꢀ withꢀ eitherꢀ CuI-catalyzedꢀ
Figureꢀ3,ꢀlanesꢀ2-3)ꢀorꢀCuSO -catalyzedꢀclickꢀproto-
colsꢀ(Figureꢀ3,ꢀlanesꢀ4-5)ꢀfollowingꢀincubationꢀatꢀ50ꢀ
(
4
°
Cꢀforꢀ1ꢀh.ꢀ
0
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TheꢀpresenceꢀofꢀPt(II)ꢀlesionsꢀinꢀcellularꢀRNAsꢀandꢀ
theirꢀ potentialꢀ downstreamꢀ consequencesꢀ haveꢀ be-
comeꢀanꢀincreasinglyꢀimportantꢀconsiderationꢀgivenꢀ
theꢀ manyꢀ regulatoryꢀ rolesꢀ ofꢀ RNAꢀ inꢀ essentialꢀ cellꢀ
19
processes. ꢀ Recentꢀ studiesꢀ inꢀ ourꢀ laboratoryꢀ haveꢀ
demonstratedꢀ thatꢀ inꢀ S.ꢀ cerevisiae,ꢀ cisplatin-derivedꢀ
Ptꢀ speciesꢀ accumulateꢀ toꢀ aꢀ significantlyꢀ greaterꢀ ex-
tentꢀinꢀRNAꢀversusꢀDNA,ꢀwithꢀribosomalꢀRNAꢀactingꢀ
asꢀ aꢀ deꢀ factoꢀ cellularꢀ Ptꢀ sponge.19aꢀ Inꢀ contextꢀ ofꢀ E.ꢀ
coli20ꢀ andꢀ S.ꢀ cerevisiae19aꢀ ribosomalꢀ RNA,ꢀ specificꢀ Ptꢀ
bindingꢀ sitesꢀ haveꢀ beenꢀ identifiedꢀ withinꢀ theꢀ smallꢀ
ribosomalꢀ subunit.ꢀ Ptꢀ adductsꢀ areꢀ alsoꢀ observedꢀ inꢀ
theꢀ sarcin-ricinꢀ loopꢀ (SRL),ꢀ aꢀ universallyꢀ conservedꢀ
exogenousꢀloopꢀessentialꢀforꢀelongationꢀfactorꢀbind-
ingꢀ andꢀ GTP-dependentꢀ translocationꢀ (unpublishedꢀ
Toꢀassessꢀtheꢀfeasibilityꢀofꢀidentifyingꢀcellularꢀpica-
zoplatin-boundꢀ species,ꢀ weꢀ extractedꢀ andꢀ labeledꢀ
ribosomalꢀRNAꢀfromꢀ1-treatedꢀS.ꢀcerevisiaeꢀwithꢀAl-
exaꢀ Fluorꢀ 488ꢀ DIBOꢀ alkyneꢀ (Figureꢀ 5).ꢀ Laneꢀ 1ꢀ con-
tainsꢀ aꢀ 200ꢀ nucleotideꢀ RNAꢀ marker.ꢀ Lanesꢀ 2-4ꢀ con-
tainꢀ 5.8Sꢀ ribosomalꢀ RNAꢀ isolatedꢀ fromꢀ cellsꢀ treatedꢀ
forꢀ6ꢀhꢀwithꢀ0ꢀ(Laneꢀ2),ꢀ250ꢀorꢀ500ꢀµMꢀpicazoplatinꢀ
(Lanesꢀ 3,4)ꢀ andꢀ reactedꢀ withꢀ anꢀ excessꢀ ofꢀ alkyneꢀ
fluorophoreꢀ (18ꢀ h,ꢀ 37°C).ꢀ Laneꢀ 2ꢀ indicatesꢀ theꢀ ab-
senceꢀ ofꢀ nonspecificꢀ labeling.ꢀ Lanesꢀ 3ꢀ andꢀ 4ꢀ clearlyꢀ
demonstrateꢀ increasingꢀ picazoplatinꢀ accumulationꢀ
onꢀ 5.8Sꢀ rRNAꢀ inꢀ vivoꢀ andꢀ successfulꢀ alkyneꢀ fluoro-
phoreꢀlabelingꢀtoꢀanꢀintactꢀazide.ꢀTheseꢀstudiesꢀshowꢀ
theꢀ potentialꢀ forꢀ analysisꢀ ofꢀ Pt(II)ꢀ drugꢀ targetsꢀ
and/orꢀfluorescentꢀimagingꢀofꢀPtꢀtraffickingꢀinꢀvivoꢀinꢀ
2
1
data). ꢀToꢀestablishꢀpotentialꢀforꢀusingꢀPt-clickꢀrea-
gentsꢀ onꢀ cellularꢀ RNA,ꢀ weꢀ investigatedꢀ theꢀ interac-
tionꢀ andꢀ clickꢀ reactivityꢀ ofꢀ 1ꢀ onꢀ aꢀ modelꢀ SRLꢀ RNAꢀ
constructꢀ(Figureꢀ4).ꢀ
Figureꢀ 4.ꢀ Fluorescentꢀ labelingꢀ ofꢀ Pt-boundꢀ RNAꢀ SRLꢀ
hairpinꢀbyꢀclickꢀchemistry.ꢀ(a)ꢀRNAꢀSRLꢀhairpin.ꢀPoten-
tialꢀPtꢀbindingꢀsitesꢀareꢀindicatedꢀbyꢀstarredꢀnucleotides.ꢀ
(
b)ꢀLanesꢀ1-3:ꢀnon-platinatedꢀRNAꢀisꢀstableꢀinꢀclickꢀreac-
tionꢀ conditionsꢀ withꢀ 0.4ꢀ (triangle)ꢀ orꢀ 0.8ꢀ equivalentsꢀ
circle)ꢀCuI.ꢀLanesꢀ4-7:ꢀSRLꢀRNAꢀtreatedꢀwithꢀ4ꢀequiva-
(
lentsꢀ1ꢀshowsꢀincreasingꢀyieldsꢀofꢀfluorophoreꢀlabelingꢀ
followingꢀclickꢀreactionsꢀwithꢀ0.4ꢀ(laneꢀ6)ꢀandꢀ0.8ꢀ(laneꢀ
Figureꢀ5.ꢀFluorescentꢀlabelingꢀbyꢀclickꢀchemistryꢀofꢀ
ribosomalꢀ RNAꢀ fromꢀ 1-treatedꢀ S.ꢀ cerevisiae.ꢀ Laneꢀ 1ꢀ
containsꢀ aꢀ 200ꢀ nucleotideꢀ RNAꢀ marker.ꢀ Lanesꢀ 2-4ꢀ
containꢀ 5.8Sꢀ ribosomalꢀ RNAꢀ (approx.ꢀ 160ꢀ nucleo-
tides)ꢀ extractedꢀ fromꢀ S.ꢀ cerevisiaeꢀ treatedꢀ withꢀ 0,ꢀ
250,ꢀorꢀ500ꢀμMꢀpicazoplatinꢀandꢀclickedꢀtoꢀanꢀexcessꢀ
ofꢀAlexaꢀFluorꢀ488ꢀDIBOꢀalkyneꢀ(18ꢀh,ꢀ37ꢀ°C).ꢀProfi-
cientꢀ fluorescentꢀ labelingꢀ ofꢀ theꢀ 5.8Sꢀ rRNAꢀ isꢀ ob-
servedꢀfromꢀinꢀvivoꢀ1-treatedꢀsamples.ꢀConditionsꢀareꢀ
givenꢀinꢀtheꢀSupportingꢀInformation.ꢀ
7
)ꢀequivalentsꢀCuIꢀandꢀtheꢀdansylꢀalkyne.ꢀConditionsꢀareꢀ
givenꢀinꢀtheꢀSupportingꢀInformation.ꢀ
Theꢀsensitivityꢀofꢀunmodifiedꢀ(i.e.,ꢀ2’-OH)ꢀRNAꢀspe-
ciesꢀtowardsꢀdegradationꢀbyꢀCu-mediatedꢀchemistryꢀ
hasꢀpreviouslyꢀlimitedꢀstudiesꢀofꢀclickꢀreactionsꢀwithꢀ
RNAꢀsequencesꢀlikeꢀtheꢀSRLꢀinꢀtheꢀabsenceꢀofꢀprotec-
tiveꢀ ligandsꢀ suchꢀ asꢀ TBTAꢀ orꢀ underꢀ O
tions. ꢀWeꢀhaveꢀindependentlyꢀobservedꢀsignificantꢀ
2
-freeꢀ condi-
2
2
3
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fixedꢀandꢀpermeabilizedꢀcells.ꢀꢀ
Journal of the American Chemical Society
(1)ꢀ(a)ꢀHarper,ꢀB.ꢀW.;ꢀKrause-Heuer,ꢀA.ꢀM.;ꢀGrant,ꢀM.ꢀP.;ꢀ
1
2
3
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5
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9
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1
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6
Manohar,ꢀM.;ꢀGarbutcheon-Singh,ꢀK.ꢀB.;ꢀAldrich-Wright,ꢀ
J.ꢀR.ꢀChem.ꢀEur.ꢀJ.ꢀ2010,ꢀ16,ꢀ7064-7077.ꢀ(b)ꢀDyson,ꢀP.ꢀJ.;ꢀ
Sava,ꢀG.ꢀDaltonꢀTrans.ꢀ2006,ꢀ16,ꢀ1929-1933.ꢀ
Inꢀsummary,ꢀweꢀreportꢀtheꢀsuccessfulꢀsynthesisꢀofꢀ
picazoplatin,ꢀ anꢀ azide-containingꢀ picoplatinꢀ deriva-
tiveꢀforꢀPt-boundꢀdrugꢀtargetꢀanalysisꢀbyꢀclickꢀchem-
istry.ꢀ Picazoplatinꢀ readilyꢀ bindsꢀ DNAꢀ oligonucleo-
tidesꢀ andꢀ undergoesꢀ high-yieldingꢀ clickꢀ reactionsꢀ
withꢀ alkyneꢀ fluorophores.ꢀ Moreover,ꢀ successfulꢀ
fluorophoreꢀ labelingꢀ viaꢀ acetonitrile-assistedꢀ clickꢀ
reactionsꢀ wereꢀ demonstratedꢀ onꢀ anꢀ RNAꢀ mimicꢀ ofꢀ
theꢀ ribosomalꢀ sarcin-ricinꢀ loopꢀ construct,ꢀ demon-
stratingꢀviabilityꢀofꢀthisꢀapproachꢀinꢀtheꢀpresenceꢀofꢀ
unmodifiedꢀ (i.e.ꢀ 2’-OH-intact)ꢀ RNA.ꢀ Preliminaryꢀ inꢀ
vivoꢀapplicationsꢀofꢀthisꢀtechniqueꢀidentifiedꢀtheꢀ5.8Sꢀ
ribosomalꢀRNAꢀasꢀaꢀcellularꢀPt(II)ꢀdrugꢀtarget.ꢀ
(
2)(a)ꢀ Sava,ꢀ G.;ꢀ Jaouen,ꢀ G.;ꢀ Hillard,ꢀ E.ꢀ A.;ꢀ Bergamo,ꢀ A.ꢀ
Daltonꢀ Trans.ꢀ 2012,ꢀ 41,ꢀ 8226-8234.ꢀ (b)ꢀ Casini,ꢀ A.;ꢀ Re-
edijk,ꢀJ.ꢀChem.ꢀSci.ꢀ2012,ꢀ3,ꢀ3135-3144.ꢀ(c)ꢀGuggenheim,ꢀ
E.R.;ꢀXu,ꢀD.;ꢀZhang,ꢀC.X.;ꢀChang,ꢀP.V.;ꢀLippard,ꢀS.J.ꢀChem.ꢀ
Bio.ꢀChem.ꢀ2009,ꢀ10,ꢀ141-57.ꢀ(d)ꢀWexselblatt,ꢀE.;ꢀYavin,ꢀ
E.;ꢀGibson,ꢀD.ꢀInorg.ꢀChim.ꢀActaꢀ2012,ꢀ393,ꢀ75-83.ꢀꢀ
0
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3
4
5
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7
8
9
0
(
2
3)ꢀ (a)ꢀ Wang,ꢀ D.;ꢀ Lippard,ꢀ S.ꢀ J.ꢀ Nat.ꢀ Rev.ꢀ Drugꢀ Discov.ꢀ
005,ꢀ4,ꢀ307-320.ꢀ(b)ꢀBoulikas,ꢀT.;ꢀVougiouka,ꢀM.ꢀOncol.ꢀ
Rep.ꢀ2003,ꢀ10,ꢀ1663-1682.ꢀ(c)ꢀJamieson,ꢀE.ꢀR.;ꢀLippard,ꢀ
S.ꢀJ.ꢀChem.ꢀRev.ꢀ1999,ꢀ99,ꢀ2467-2498.ꢀ
(4)ꢀ (a)ꢀ Akaboshi,ꢀ M.;ꢀ Kawai,ꢀ K.;ꢀ Ujeno,ꢀ Y.;ꢀ Takada,ꢀ S.;ꢀ
Miyahara,ꢀ T.ꢀ Jpn.ꢀ J.ꢀ Can.ꢀ Res.ꢀ 1994,ꢀ 85,ꢀ 106-111.ꢀ (b)ꢀ
Akaboshi,ꢀ M.;ꢀ Kawai,ꢀ K.;ꢀ Maki,ꢀ H.;ꢀ Akuta,ꢀ K.;ꢀ Ujeno,ꢀ Y.;ꢀ
Miyahara,ꢀT.ꢀJpn.ꢀJ.ꢀCan.ꢀRes.ꢀ1992,ꢀ83,ꢀ522-526.ꢀ(c)ꢀDe-
Conti,ꢀ R.ꢀ C.;ꢀ Toftness,ꢀ B.ꢀ R.;ꢀ Lange,ꢀ R.ꢀ C.;ꢀ Creasy,ꢀ W.ꢀ A.ꢀ
CancerꢀRes.ꢀ1973,ꢀ33,ꢀ1310-1315.ꢀ
Futureꢀ applicationsꢀ ofꢀ Pt-clickꢀ reactionsꢀ couldꢀ in-
cludeꢀtheꢀisolation,ꢀpurification,ꢀandꢀidentificationꢀofꢀ
inꢀ vivoꢀ targetsꢀ ofꢀ Pt(II)ꢀ therapeuticsꢀ usingꢀ variousꢀ
alkyne-functionalizedꢀ scaffolds.ꢀ Reversalꢀ ofꢀ Ptꢀ cova-
lentꢀ linkagesꢀ couldꢀ beꢀ achievedꢀ byꢀ saturationꢀ withꢀ
thiourea,18aꢀ releasingꢀ Pt-boundꢀ constituentsꢀ forꢀ
analysisꢀviaꢀhigh-throughputꢀsequencing,ꢀproteomics,ꢀ
(
5)ꢀKolb,ꢀH.ꢀC.;ꢀFinn,ꢀM.ꢀG.;ꢀSharpless,ꢀK.ꢀB.ꢀAngew.ꢀChem.ꢀ
Int.ꢀEd.ꢀ2004,ꢀ40,ꢀ2004-2021.ꢀ
6)ꢀ(a)ꢀJewett,ꢀJ.ꢀC.;ꢀBertozzi,ꢀC.ꢀR.ꢀChem.ꢀSoc.ꢀRev.ꢀ2010,ꢀ
9,ꢀ1272-1279.ꢀ(b)ꢀSletten,ꢀE.ꢀM.;ꢀBertozzi,ꢀC.ꢀR.ꢀAngew.ꢀ
Chem.ꢀ2009,ꢀ48,ꢀ6974-6998.ꢀ
7)ꢀ(a)ꢀEl-Sagheer,ꢀA.ꢀH.;ꢀBrown,ꢀT.ꢀAcc.ꢀChem.ꢀRes.ꢀ2012,ꢀ
(
3
2
c
orꢀ otherꢀ analyticalꢀ methods. ꢀ Theꢀ abilityꢀ ofꢀ Pt(II)ꢀ
compoundsꢀtoꢀcrosslinkꢀRNAꢀandꢀRNA-proteinꢀcom-
2
3
plexes ꢀsuggestsꢀadditionalꢀapplicationsꢀinꢀRNAꢀandꢀ
ribonucleoproteinsꢀstructure-functionꢀanalyses.ꢀSuchꢀ
effortsꢀtowardsꢀusingꢀPt-mediatedꢀclickꢀchemistryꢀtoꢀ
isolateꢀ andꢀ quantifyꢀ Pt-boundꢀ speciesꢀ areꢀ currentlyꢀ
underwayꢀinꢀourꢀlaboratory.ꢀ
(
4
5,ꢀ1258-1267.ꢀ(b)ꢀJao,ꢀC.ꢀY.;ꢀSalic,ꢀA.ꢀPNASꢀ2008,ꢀ105,ꢀ
1
5779-15784.ꢀ
(
8)ꢀ (a)ꢀ Struthers,ꢀ H.;ꢀ Mindt,ꢀ T.ꢀ L.;ꢀ Schibli,ꢀ R.ꢀ Daltonꢀ
Trans.ꢀ2010,ꢀ39,ꢀ675-696.ꢀ(b)ꢀClough,ꢀM.ꢀC.;ꢀZeits,ꢀP.ꢀD.;ꢀ
Bhuvanesh,ꢀN.;ꢀGladysz,ꢀJ.ꢀA.ꢀOrganometallicsꢀ2012,ꢀ31,ꢀ
ASSOCIATEDꢀCONTENTꢀꢀ
5
231-5234.ꢀ(c)ꢀStengel,ꢀI.;ꢀStrassert,ꢀC.ꢀA.;ꢀPlummer,ꢀE.ꢀ
A.;ꢀ Chien,ꢀ C.-H.;ꢀ Deꢀ Cola,ꢀ L.;ꢀ Bäuerle,ꢀ P.ꢀ Eur.ꢀ J.ꢀ Inorg.ꢀ
Chem.ꢀ2012,ꢀ1795-1809.ꢀ(d)ꢀDing,ꢀS.;ꢀQiao,ꢀX.;ꢀKucera,ꢀG.ꢀ
L.;ꢀ Bierbach,ꢀ U.ꢀ J.ꢀ Med.ꢀ Chem.ꢀ 2012,ꢀ 55,ꢀ 10198-10203.ꢀ
SupportingꢀInformationꢀAvailable:ꢀExperimentalꢀpro-
cedures,ꢀsynthesesꢀofꢀcomplexes,ꢀ1Hꢀ&ꢀ195PtꢀNMR,ꢀcom-
pleteꢀ MALDI,ꢀ andꢀ high-resolutionꢀ massꢀ spectrometryꢀ
data.ꢀ Thisꢀ materialꢀ isꢀ availableꢀ freeꢀ ofꢀ chargeꢀ viaꢀ theꢀ
Internetꢀatꢀhttp://pubs.acs.org.ꢀ
(
3
e)ꢀUrankar,ꢀD.;ꢀKošmrlj,ꢀJ.ꢀInorg.ꢀChim.ꢀActaꢀ2010,ꢀ363,ꢀ
817-3822.ꢀ (f)ꢀ Struthers,ꢀ H.;ꢀ Mindt,ꢀ T.ꢀ L.;ꢀ Schibli,ꢀ R.ꢀ
DaltonꢀTrans.ꢀ2010,ꢀ39,ꢀ675-696.ꢀ
9)ꢀDing,ꢀS.;ꢀQiao,ꢀX.;ꢀSuryadi,ꢀJ.;ꢀMarrs,ꢀG.ꢀS.;ꢀKucera,ꢀG.ꢀ
L.;ꢀ Bierbach,ꢀ U.ꢀ Angew.ꢀ Chem.ꢀ Int.ꢀ Ed.ꢀ 2013,ꢀ 52,ꢀ 3350-
354.ꢀ
10)ꢀ(a)ꢀKelland,ꢀL.ꢀNat.ꢀRev.ꢀCancerꢀ2007,ꢀ7,ꢀ573-584.ꢀ
AUTHORꢀINFORMATIONꢀ
(
CorrespondingꢀAuthorꢀ
3
(
derose@uoregon.eduꢀ
AuthorꢀContributionsꢀ
(b)ꢀRaynaud,ꢀF.ꢀI.;ꢀBoxall,ꢀF.ꢀE.;ꢀGoddard,ꢀP.ꢀM.;ꢀValenti,ꢀ
M.;ꢀ Jones,ꢀ M.;ꢀ Murrer,ꢀ B.ꢀ A.;ꢀ Abrams,ꢀ M.;ꢀ Kelland,ꢀ L.ꢀ R.ꢀ
Clin.ꢀCanc.ꢀRes.ꢀ1997,ꢀ3,ꢀ2063-2074.ꢀ
†ꢀTheseꢀauthorsꢀcontributedꢀequallyꢀtoꢀthisꢀwork.
ꢀ
(
11)ꢀBelluco,ꢀU.;ꢀBertani,ꢀR.;ꢀMichelin,ꢀR.ꢀA.;ꢀMozzon,ꢀM.ꢀJ.ꢀ
Organomet.ꢀChem.ꢀ2000,ꢀ600,ꢀ37-55.ꢀ
12)ꢀKane,ꢀS.ꢀA.;ꢀLippard,ꢀS.ꢀJ.ꢀBiochem.ꢀ1996,ꢀ35,ꢀ2180-
188.ꢀ
13)ꢀSletten,ꢀE.ꢀM.;ꢀBertozzi,ꢀC.ꢀR.ꢀAcc.ꢀChem.ꢀRes.ꢀ2011,ꢀ
4,ꢀ666-676.ꢀ
14)ꢀ(a)ꢀChen,ꢀY.;ꢀGuo,ꢀZ.;ꢀParsons,ꢀS.;ꢀSadler,ꢀP.ꢀJ.ꢀChem.ꢀ
Notesꢀ
Theꢀauthorsꢀdeclareꢀnoꢀcompetingꢀfinancialꢀinterests.ꢀ
(
2
(
4
ACKNOWLEDGMENTꢀꢀ
WeꢀthankꢀtheꢀNationalꢀScienceꢀFoundationꢀforꢀsupportꢀ
ofꢀ thisꢀ researchꢀ (CHE-1153147)ꢀ asꢀ wellꢀ asꢀ supportꢀ inꢀ
theꢀ formꢀ ofꢀ anꢀ instrumentationꢀ grantꢀ (CHE-0923589).ꢀ
Weꢀ gratefullyꢀ acknowledgeꢀ Dr.ꢀ Robertꢀ Spitaleꢀ atꢀ Stan-
fordꢀUniversityꢀforꢀadviceꢀonꢀinꢀvivoꢀRNAꢀclickꢀchemistryꢀ
protocols.ꢀ
(
Eur.ꢀJ.ꢀ1998,ꢀ4,ꢀ672-676.ꢀ(b)ꢀChen,ꢀY.;ꢀGuo,ꢀZ.;ꢀParkinson,ꢀ
J.ꢀ A.;ꢀ Sadler,ꢀ P.ꢀ J.ꢀ Daltonꢀ Trans.ꢀ 1998,ꢀ 3577-3585.ꢀ (c)ꢀ
Kelland,ꢀL.ꢀR.;ꢀSharp,ꢀS.ꢀY.;ꢀO’Neill,ꢀC.ꢀF.;ꢀRaynaud,ꢀF.ꢀI.;ꢀ
Beale,ꢀP.ꢀJ.;ꢀJudson,ꢀI.ꢀR.ꢀJ.ꢀInorg.ꢀBiochem.ꢀ1999,ꢀ77,ꢀ111-
1
ꢀ
15.ꢀ
(15)ꢀBattle,ꢀA.ꢀR.;ꢀChoi,ꢀR.;ꢀHibbs,ꢀD.ꢀE.;ꢀHambley,ꢀT.ꢀW.ꢀ
Inorg.ꢀChem.ꢀ2006,ꢀ45,ꢀ6317-6322.ꢀ
REFERENCESꢀ
4
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Page 6 of 6
(
16)ꢀChen,ꢀY.;ꢀParkinson,ꢀJ.ꢀA.;ꢀGuo,ꢀZ.;ꢀBrown,ꢀT.;ꢀSadler,ꢀ
(20)ꢀKeshab,ꢀR.;ꢀChow,ꢀC.ꢀS.ꢀChem.ꢀCommun.ꢀ2009,ꢀ107-
109.ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
P.ꢀJ.ꢀAngew.ꢀChem.ꢀInt.ꢀEd.ꢀ1999,ꢀ38,ꢀ2060-2063.ꢀ
(
17)ꢀ Bolletta,ꢀ F.;ꢀ Fabbri,ꢀ D.;ꢀ Lombardo,ꢀ M.;ꢀ Prodi,ꢀ L.;ꢀ
Trombini,ꢀC.;ꢀZaccheroni,ꢀN.ꢀOrganometallics,ꢀ1996,ꢀ15,ꢀ
415-2417.ꢀ
18)ꢀ(a)ꢀChapman,ꢀE.ꢀG.;ꢀDeRose,ꢀV.ꢀJ.ꢀJ.ꢀAm.ꢀChem.ꢀSoc.ꢀ
010,ꢀ132,ꢀ1946-1952.ꢀ(b)ꢀHostetter,ꢀA.ꢀA.;ꢀChapman,ꢀE.ꢀ
G.;ꢀDeRose,ꢀV.ꢀJ.ꢀJ.ꢀAm.ꢀChem.ꢀSoc.ꢀ2009,ꢀ131,ꢀ9250-9257.ꢀ
19)ꢀ(a)ꢀHostetter,ꢀA.ꢀA.;ꢀOsborn,ꢀM.ꢀF.;ꢀDeRose,ꢀV.ꢀJ.ꢀACSꢀ
(21)ꢀ(a)ꢀShi,ꢀX.;ꢀKhade,ꢀP.ꢀK.;ꢀSanbonmatsu,ꢀK.ꢀY.;ꢀJoseph,ꢀ
S.ꢀJ.ꢀMol.ꢀBiol.ꢀ2012,ꢀ419,ꢀ125-138.ꢀ(b)ꢀMoazed,ꢀD.;ꢀRob-
ertson,ꢀJ.ꢀM.;ꢀMoller,ꢀH.ꢀF.ꢀNatureꢀ1988,ꢀ334,ꢀ362-364.ꢀ
(22)ꢀ(a)ꢀParedes,ꢀE.;ꢀDas,ꢀS.ꢀR.ꢀBioorg.ꢀMed.ꢀChem.ꢀLett.ꢀ
2012,ꢀ 12,ꢀ 5313-5316.ꢀ (b)ꢀ Paredes,ꢀ E.;ꢀ Das,ꢀ S.ꢀ R.ꢀ Chem.ꢀ
Bio.ꢀChem.ꢀ2011,ꢀ12,ꢀ125-131.ꢀ
2
(
2
(
23)ꢀDufour,ꢀE.;ꢀReinbolt,ꢀJ.;ꢀCastroviejo,ꢀM.;ꢀEhresmann,ꢀ
(
B.;ꢀ Litvak,ꢀ S.;ꢀ Tarrago-Litvak,ꢀ L.;ꢀ Andreola,ꢀ M.-L.ꢀ J.ꢀ Mol.ꢀ
Biol.ꢀ1999,ꢀ285,ꢀ1339-1346.ꢀ
Chem.ꢀBio.ꢀ2012,ꢀ7,ꢀ218-225.ꢀ(b)ꢀGuan,ꢀL.;ꢀDisney,ꢀM.ꢀACSꢀ
Chem.ꢀBio.ꢀ2012,ꢀ7,ꢀ73-86.ꢀ(c)ꢀHedman,ꢀH.K.;ꢀKirpeker,ꢀ
F.;ꢀ Elmroth,ꢀ S.ꢀ K.ꢀ J.ꢀ Am.ꢀ Chem.ꢀ Soc.ꢀ 2011,ꢀ 133,ꢀ 11977-
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