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Intracellular delivery of a catalytic organometallic complex

Citation for published version:Indrigo, E, Clavadetscher, J, Chankeshwara, SV, Megia-fernandez, A, Lilienkampf, A & Bradley, M 2017,'Intracellular delivery of a catalytic organometallic complex', Chemical Communications.https://doi.org/10.1039/C7CC02988H

Digital Object Identifier (DOI):10.1039/C7CC02988H

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Published In:Chemical Communications

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https://doi.org/10.1039/C7CC02988Hhttps://doi.org/10.1039/C7CC02988Hhttps://www.research.ed.ac.uk/en/publications/0b47fd4d-8f3a-40c5-8d5a-4755985f7414

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Received00thJanuary20xx,Accepted00thJanuary20xx

DOI:10.1039/x0xx00000x

www.rsc.org/chemcomm

IntracellulardeliveryofacatalyticorganometalliccomplexEugenioIndrigo,†JessicaClavadetscher,†SunayV.Chankeshwara,AliciaMegia-Fernandez,AnnamariaLilienkampfandMarkBradley*a

Ahomogeneouscarbene-basedpalladiumcatalystwasconjugatedto a cell-penetrating peptide, allowing intracellular delivery ofcatalytically active Pd complexes that demonstratedbioorthogonal activation of a profluorophore within prostatecancercells.

In recent years, bioorthogonal organometallic reactions havebeensuccessfullyapplied inchemicalbiology.1–8 Inparticular,Pd-mediated chemical transformations have been used tomodifyorsynthesisemoleculesthroughcleavageofprotectinggroups or cross-coupling reactions.5,9 The first use of PdchemistryinmammaliancellswasreportedbyBradley,10whotrappedPdnanoparticleswithinaninertpolymermicrospherefor intracellular delivery. This “active” transition metal wasused to mediate C–C bond formation via a Suzuki–Miyauracross-coupling and allyl carbamate cleavage reactions insidelivingcells,includingtheintracellularactivationofaprodrugofthe antineoplastic agent amsacrine. This approach was alsoused to convert propargyl derivatives of 5-fluorouracil andgemcitabine to their active forms, aswell as catalyse Suzuki-Miyaura cross-couplings of drugs in the presence of anextracellular heterogeneous Pd catalyst.11–13 Pd-mediatedtransformations have since also been used to selectivelyactivate biomolecules in cells. Chen developed the chemicalactivation of proteins based on the decaging ofpropargylcarbamate caged lysine with simple Pd salts, whilePd(NO3)2, Pd(dba)2, and allyl2PdCl2, have also been used tomediate Sonogashira cross-coupling reactions insidebacteria,14or toactivateaproteinof interest insidecells.14–17However,underoptimisedconditions,50equivalentsofthesePd complexes are required for the completion of the cross-coupling reactions.16 The major drawbacks of all the abovemethodsarethelackofcellselectivityofthecatalystandtheuse of stoichiometric, or even excess (up to 500 eq.), Pd

complex and the cellular toxicity when used in non-complexed/encapsulatedform.5,18TargetedlocalisationofaPdcatalyst,bymeansofcellselectivedeliveryofahomogeneouscatalyst,wouldrepresentamajoradvanceinthefield.Herein,wehaveextendedthescopeofPdcatalysedchemistryin livingsystemsbydevelopingamethodtodeliverandtrackan active organometallic complex inside mammalian cells.Thesewater-soluble and traceable catalystswerebasedon aPd(II)–carbene complex coupled to a fluorescently labelledhoming peptide for targeted cell delivery. The biocompatiblehomogeneousPdcatalystwasbasedonapreviouslyreportedcarbene Pd ligand coupled to a peptide.19,20 N-heterocycliccarbenes (NHC) are strong σ-electron donating molecules(enhancingtherateofoxidativeaddition),coordinatetightlytoPd centres (disfavouring aggregation), and can have bulkysubstituents(increasingtherateofreductiveelimination).21,22PeptideswerechosentodeliverthePdcomplexinsidecellsasthey are biocompatible, easy to prepare by solid-phasepeptide synthesis (SPPS), versatile, readily labelled andfunctionalised,withknowncelldeliveryabilities.23

Scheme1:SyntheticroutetothePd–peptidecatalystsonaRink functionalisedpolystyreneresin.

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Fig.1.CellularuptakeofPd–peptide2.(A)StructureofthecarboxyfluoresceinlabelledPd–peptide2.(B)IntracellularPdcontent(ngPdpermillioncells)ofPC-3cellsincubatedwith10µMPd(OAc)2or2(3µMand30µM)for2h(higherconcentrationsofPd(OAc)2weretoxic).Cellswereharvested,lysedandthecellcontentwassubjectedtoICP-OESmeasurements.(C)Flowcytometryhistogramsshowingtheuptakeof2(30µM)inPC-3cellsafter2h(inblack)anduntreatedcontrolcells(ingrey).(D)PC-3cellswereincubatedwith2(30µM)for2h,fixedwith4%paraformaldehyde,stainedwithDAPI(nucleistain)andimagedbyconfocalmicroscopy.Panels show (i) Pd catalyst2 (green, λex = 488nm) located in the cytoplasmandnucleus, (ii) cell nucleus (blue, λex = 405nm), and (iii)merged image (lightblueindicatesco-localisation).Scalebar20µm.

Thepolycationic(tri-lysine)catalyst1representedasimplecellpenetrating peptide (CPP) which was able to cross the cellmembrane,23andcouldbereadilylabelledtoallowtrackingofthecatalystinsidecells.Here, either 5(6)-carboxyfluorescein (FAM, λEx/Em = 492/517nm)orsulfonatedCy5(λEx/Em=630/654nm)wereattachedtothe peptides to give the fluorescent Pd-catalysts 2 and 3,respectively(Scheme1).Peptides1,2and3weresynthesisedonaRinkamidelinkerfunctionalisedaminomethylpolystyreneresin using standard Fmoc/tBu SPPS with Oxyma/DIC as thecoupling combination (Scheme 1 and ESI). Coupling of thefluorophores in2 and3wasachievedby introducingabis-N-[1-(4,4-dimethyl-2,6-dioxocyclo hexylidene) ethyl] (Dde)protectedLysasthefirstaminoacid(Fmoc-Lys(Dde)-OH).Priorto Fmoc removal of the terminal Lys residue, the Dde groupwas orthogonally removed with hydroxylamine24 and theliberated amine coupled to the activated dye. The imidazoleligand4 was coupled to the amino terminus of the peptides

using TBTU as an activating agent. The imidazole salt wastreatedwith thebaseBEMP to form the carbene,whichwassubsequently trapped by the addition of dichloro(1,5-cyclooctadiene)palladium(II)(PdCl2COD)asthesourceofPd.

19The catalystswere cleaved from the resin by treatmentwithTFA (5 % water, addition of conventional scavenging agentsdestroyed thePdcomplex)andpurifiedbypreparativeHPLC.To evaluate the catalytic activity in a biological setting, non-fluorescentbis-propargyloxycarbonyl(Proc)rhodamine110(5)was treated with Pd catalyst 1 in phosphate buffered saline(PBS)andPC-3(prostateadenocarcinoma)celllysate(seeESI).UponPdcatalysedcleavageoftheprotectinggroupswith0.1eqof1for24h,fluorescent6wasgeneratedgivinga200-foldincreaseinfluorescence.Thereactionincelllysateresultedinamoremodest9-foldrelativeincreaseinfluorescencewith0.1eq of the catalyst, but demonstrated that the Pd–peptidehybridwas catalytically competent even in thepresenceof acomplexmixtureofbiomolecules.

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Fig.2.Pdcatalyseddecagingofproc-rhodamine5 inPC-3cells.(A)Catalysedfluorescence“switch-on”ofthecagedfluorophore5withthePd–peptidecomplex3.CellswereincubatedwithPd–peptide3(30µM)for2h,followedbyincubationwith5(50µM)for18h.(B)ConfocalmicroscopyimagesofPC-3cellsfixedwith4%paraformaldehydeandstainedwithDAPI(nucleistain).Panelsshow:(i)cellnucleus(blue,λex=405nm)andPd-peptide3(magenta,λex=633nm),(ii)cellnucleus(blue)andinsitusynthesised6(green,λex=488nm),and(iii)mergedimage.Scalebar20µm.(C)Flowcytometrycytogramsshowing:(i)untreatedcontrolcells,(ii)cellstreatedwithproc-rhodamine5,and(iii)cellstreatedwith5and3.

The cellular uptake of the carboxyfluorescein labelled Pd–peptide 2 was investigated with the total quantity of theinternalised Pd determined by inductively coupled plasmaoptical emission spectrometry (ICP-OES) analysisofPC-3 cells(Fig. 1b), giving a total Pd content of 20 ng permillion cells(when incubatedwith 30µMof2 for 2 h), showing a highermetal uptake compared to cells treated with 10 µM ofPd(OAc)2 (higher concentration of Pd(OAc)2 showedcytotoxicity). Catalyst 2 showed no cytotoxicity even at 200µM (seeESI). Thecellularuptakeand intracellular locationofthe Pd catalyst 2 was investigated by flow cytometry andconfocalmicroscopy (Figure 1c and 1d). PC-3 cells showed ashiftofthewholecellpopulationtowardshigherfluorescenceintensityafter2hincubationwithcatalyst2(30µM),withthefluorescencelocalisedwithinthecytoplasmand,unexpectedly,in the nucleus (cationic peptides generally localise in thecytoplasmandinvesicles25,26).Thecatalyticactivityof thePd–peptidecatalysts incell-basedassaysvia proc-rhodamine5 activationwas investigatedwiththeCy5labelledPd–peptide3.PC-3cellswereincubatedwith3(30µM)for2h,washedtoremoveanyextracellularcatalyst,

and subsequently incubatedwith 5 (50 µM) for 18 h (Figure2a).FluorescencemicroscopyverifiedthepresenceofthePd-catalyst3aswellassynthesised6withinthecytoplasmofPC-3cells(Fig.2b).AnalysisbyflowcytometryshowedashiftofthewholecellpopulationtowardshigherfluorescenceintensityintheCy5channel indicating theuptakeof thecatalyst,whileashifttohigherFITCintensityindicatedtheformationof6(Fig.2c).Inconclusion,wehavedemonstratedtheabilitytocombineacatalytically active Pd complex with a cell-delivery peptidescaffold, which was able to catalyse a depropargylationreactioninabiologicallyrelevantsetting.Thenon-cytotoxicPdcatalyst was successfully used in the fluorescent labelling ofmammalian cells, showing significant catalytic activity in anintracellularenvironment.FutureapplicationswillthusincludetheactivationofprodrugsviathesePdcatalysts.Thisnewtypeof catalytic system gives the basis for a wide range ofbiocompatiblecatalysts,wheretheirfacilemodificationallowsthe incorporation of a variety of different targeting peptidesthatcouldselectivelyenterdifferentcellsorspecifictissues.

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