A homogeneous carbene-based palladium catalyst was conjugated to a cell-penetrating peptide, allowing intracellular delivery of catalytically active Pd complexes that demonstrated bioorthogonal activation of a profluorophore within prostate cancer cells

Bradley, Mark

Chankeshwara, Sunay V.

Clavadetscher, Jessica

Indrigo, Eugenio

Lilienkampf, Annamaria

Megia-fernandez, Alicia

Chemical Communications

English

Megía Fernández, Alicia

Repositorio Institucional Universidad de Granada

6712 | Chem. Commun., 2017, 53, 6712--6715 This journal is©The Royal Society of Chemistry 2017Cite this:Chem. Commun., 2017,53, 6712Intracellular delivery of a catalytic organometalliccomplex†Eugenio Indrigo,‡ Jessica Clavadetscher,‡ Sunay V. Chankeshwara,Alicia Megia-Fernandez, Annamaria Lilienkampf and Mark Bradley *A homogeneous carbene-based palladium catalyst was conjugatedto a cell-penetrating peptide, allowing intracellular delivery ofcatalytically active Pd complexes that demonstrated bioorthogonalactivation of a profluorophore within prostate cancer cells.In recent years, bioorthogonal organometallic reactions havebeen successfully applied in chemical biology.1–8 In particular,Pd-mediated chemical transformations have been used to modifyor synthesise molecules through cleavage of protecting groupsor cross-coupling reactions.5,9 The first use of Pd chemistry inmammalian cells was reported by Bradley,10 who trapped Pdnanoparticles within an inert polymer microsphere for intracel-lular delivery. This ‘‘active’’ transition metal catalyst was usedto mediate C–C bond formation via Suzuki–Miyaura cross-coupling and allyl carbamate cleavage reactions inside livingcells, including the intracellular activation of a prodrug of theantineoplastic agent amsacrine. This approach was also used toconvert propargyl derivatives of 5-fluorouracil and gemcitabine totheir active forms, as well as catalyse Suzuki–Miyaura cross-couplings of drugs in the presence of an extracellular hetero-geneous Pd catalyst.11–13 Pd-mediated transformations have sincebeen used to selectively activate biomolecules in cells. Chendeveloped the chemical activation of proteins based on thedecaging of propargylcarbamate caged lysine with simple Pdsalts, while Pd(NO3)2, Pd(dba)2, and allyl2PdCl2 have also beenused to mediate Sonogashira cross-coupling reactions insidebacteria,14 or to activate a protein of interest inside cells.14–17However, under optimised conditions, 50 equivalents of these Pdcomplexes were required for the completion of the cross-couplingreactions.16 The major drawbacks of all the above methods arethe lack of cell selectivity of the catalyst and the use of stoichio-metric, or even excess (up to 500 eq.), Pd complex and the cellulartoxicity when used in non-complexed/encapsulated form.5,18Targeted localisation of a Pd catalyst, by means of cell selectivedelivery of a homogeneous catalyst, would represent a majoradvance in the field.Herein, we have extended the scope of Pd catalysed chem-istry in living systems by developing a method to deliver andtrack an active organometallic complex inside mammalian cells.These water-soluble and traceable catalysts were based on aPd(II)–carbene complex coupled to a fluorescently labelled homingpeptide for targeted cell delivery. The biocompatible homo-geneous Pd catalyst was based on a previously reported carbenePd ligand coupled to a peptide.19,20 N-Heterocyclic carbenes(NHC) are strong s-electron donating molecules (enhancingthe rate of oxidative addition), coordinate tightly to Pd centres(disfavouring aggregation), and can have bulky substituents(increasing the rate of reductive elimination).21,22 Peptides werechosen to deliver the Pd complex inside cells as they are bio-compatible, easy to prepare by solid-phase peptide synthesis(SPPS), versatile, readily labelled and functionalised, with knowncell delivery abilities.23The polycationic (tri-lysine) catalyst 1 represented a simplecell penetrating peptide (CPP) which was able to cross the cellmembrane,23 and could be readily labelled to allow tracking ofthe catalyst inside cells.Here, either 5(6)-carboxyfluorescein (FAM, lex/em = 492/517 nm)or sulfonated Cy5 (lex/em = 630/654 nm) were attached to thepeptides to give the fluorescent Pd-catalysts 2 and 3, respectively(Scheme 1). Peptides 1, 2 and 3 were synthesised on a Rinkamide linker functionalised aminomethyl polystyrene resinusing standard Fmoc/tBu SPPS with Oxyma/DIC as the couplingcombination (Scheme 1 and ESI†). Coupling of the fluoro-phores in 2 and 3 was achieved by introducing a bis-N-[1-(4,4-dimethyl-2,6-dioxocyclo hexylidene) ethyl] (Dde) protected Lysas the first amino acid (Fmoc-Lys(Dde)-OH). Prior to Fmocremoval of the terminal Lys residue, the Dde group wasorthogonally removed with hydroxylamine24 and the liberatedamine coupled to the activated dye. The imidazole ligand 4 wascoupled to the amino terminus of the peptides using TBTU asEaStCHEM, School of Chemistry, University of Edinburgh, David Brewster Road,EH9 3FJ Edinburgh, UK. E-mail: Mark.Bradley@ed.ac.uk† Electronic supplementary information (ESI) available: Experimental section,Proc-rhodamine decaging and biological studies. See DOI: 10.1039/c7cc02988h‡ These authors contributed equally.Received 18th April 2017,Accepted 31st May 2017DOI: 10.1039/c7cc02988hrsc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 06 June 2017. Downloaded on 9/18/2018 2:12:51 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is©The Royal Society of Chemistry 2017 Chem. Commun., 2017, 53, 6712--6715 | 6713an activating agent. The imidazole salt was treated with thebase BEMP to form the carbene, which was subsequently trappedby the addition of dichloro(1,5-cyclooctadiene) palladium(II)(PdCl2COD) as the source of Pd.19 The catalysts were cleavedfrom the resin by treatment with TFA (5% water, addition ofconventional scavenging agents destroyed the Pd complex) andpurified by preparative HPLC. To evaluate the catalytic activity ina biological setting, non-fluorescent bis-propargyloxycarbonyl(Proc) rhodamine 110 (5) was treated with Pd catalyst 1 inphosphate buffered saline (PBS) and PC-3 (prostate adeno-carcinoma) cell lysate (see ESI†). Upon Pd catalysed cleavageof the protecting groups with 0.1 eq. of 1 for 24 h, fluorescent 6was generated giving a 200-fold increase in fluorescence. Thereaction in cell lysate resulted in a more modest 9-fold relativeincrease in fluorescence with 0.1 eq. of the catalyst, butdemonstrated that the Pd–peptide hybrid was catalyticallycompetent even in the presence of a complex mixture ofbiomolecules.The cellular uptake of the carboxyfluorescein labelledPd–peptide 2 was investigated with the total quantity of theinternalised Pd determined by inductively coupled plasmaoptical emission spectrometry (ICP-OES) analysis of PC-3 cells(Fig. 1B), giving a total Pd content of 20 ng per million cells(when incubated with 30 mM of 2 for 2 h), showing a highermetal uptake compared to cells treated with 10 mM of Pd(OAc)2(higher concentration of Pd(OAc)2 showed cytotoxicity). Catalyst 2showed no cytotoxicity even at 200 mM (see ESI†). The cellularuptake and intracellular location of the Pd catalyst 2 was inves-tigated by flow cytometry and confocal microscopy (Fig. 1Cand D). PC-3 cells showed a shift of the whole cell populationScheme 1 Synthetic route to the Pd–peptide catalysts on a Rink func-tionalised polystyrene resin.Fig. 1 Cellular uptake of Pd–peptide 2. (A) Structure of the carboxyfluorescein labelled Pd–peptide 2. (B) Intracellular Pd content (ng Pd per million cells)of PC-3 cells incubated with 10 mM Pd(OAc)2 or 2 (3 mM and 30 mM) for 2 h (higher concentrations of Pd(OAc)2 were toxic). Cells were harvested, lysedand the cell content was subjected to ICP-OES measurements. (C) Flow cytometry histograms showing the uptake of 2 (30 mM) in PC-3 cells after 2 h(in black) and untreated control cells (in grey). (D) PC-3 cells were incubated with 2 (30 mM) for 2 h, fixed with 4% paraformaldehyde, stained with DAPI(nuclei stain) and imaged by confocal microscopy. Panels show (i) Pd catalyst 2 (green, lex = 488 nm) located in the cytoplasm and nucleus, (ii) cellnucleus (blue, lex = 405 nm), and (iii) merged image (light blue indicates co-localisation). Scale bar 20 mm.Communication ChemCommOpen Access Article. Published on 06 June 2017. Downloaded on 9/18/2018 2:12:51 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online6714 | Chem. Commun., 2017, 53, 6712--6715 This journal is©The Royal Society of Chemistry 2017towards higher fluorescence intensity after 2 h incubation withcatalyst 2 (30 mM), with the fluorescence localised within thecytoplasm and, unexpectedly, in the nucleus (cationic peptidesgenerally localise in the cytoplasm and in vesicles25,26).The catalytic activity of the Pd–peptide catalysts in cell-basedassays via Proc-rhodamine 5 activation was investigated withthe Cy5 labelled Pd–peptide 3. PC-3 cells were incubated with 3(30 mM) for 2 h, washed to remove any extracellular catalyst,and subsequently incubated with 5 (50 mM) for 18 h (Fig. 2A).Fluorescence microscopy verified the presence of the Pd-catalyst 3as well as synthesised 6 within the cytoplasm of PC-3 cells(Fig. 2B). Analysis by flow cytometry showed a shift of the wholecell population towards higher fluorescence intensity in the Cy5channel indicating the uptake of the catalyst, while a shift tohigher FITC intensity indicated the formation of 6 (Fig. 2C).In conclusion, we have demonstrated the ability to combinea catalytically active Pd complex with a cell-delivery peptidescaffold, which was able to catalyse a depropargylation reactionin a biologically relevant setting. The non-cytotoxic Pd catalystwas successfully used in the fluorescent labelling of mammaliancells, showing significant catalytic activity in an intracellularenvironment. Future applications will thus include the activationof prodrugs via these Pd catalysts. This new type of catalyticsystem gives the basis for a wide range of biocompatible catalysts,where their facile modification allows the incorporation of avariety of different targeting peptides that could selectively enterdifferent cells or specific tissues.Notes and references1 C. Streu and E. Meggers, Angew. Chem., Int. Ed., 2006, 45, 5645–5648.2 J. Li and P. R. Chen, ChemBioChem, 2012, 13, 1728–1731.3 G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuhara, R. Mout, R. Das, S. T. Kim,Y.-C. Yeh, B. Yan, S. Hou and V. M. Rotello, Nat. Chem., 2015, 7, 597–603.4 J. Li and P. R. Chen, Nat. Chem. Biol., 2016, 12, 129–137.5 M. Yang, J. Li and P. R. Chen, Chem. Soc. Rev., 2014, 43, 6511–6526.6 M. Tomás-Gamasa, M. Martı́nez-Calvo, J. R. Couceiro and J. L.Mascareñas, Nat. Commun., 2016, 7, 12538.7 J. Clavadetscher, S. Hoffmann, A. Lilienkampf, L. Mackay, R. M.Yusop, S. A. Rider, J. J. Mullins and M. Bradley, Angew. Chem., Int. Ed.,2016, 55, 15662–15666.8 P. K. Sasmal, C. N. Streu and E. Meggers, Chem. Commun., 2013, 49,1581–1587.9 S. V. Chankeshwara, E. Indrigo and M. Bradley, Curr. Opin. Chem.Biol., 2014, 21C, 128–135.10 R. M. Yusop, A. Unciti-Broceta, E. M. V. Johansson, R. M. Sánchez-Martı́n and M. Bradley, Nat. Chem., 2011, 3, 239–243.11 J. T. Weiss, J. C. Dawson, K. G. Macleod, W. Rybski, C. Fraser,C. Torres-Sánchez, E. E. Patton, M. Bradley, N. O. Carragher andA. Unciti-Broceta, Nat. Commun., 2014, 5, 3277.12 J. T. Weiss, J. C. Dawson, C. Fraser, W. Rybski, C. Torres-Sánchez,M. Bradley, E. E. Patton, N. O. Carragher and A. Unciti-Broceta,J. Med. Chem., 2014, 57, 5395–5404.13 E. Indrigo, J. Clavadetscher, S. V. Chankeshwara, A. Lilienkampf andM. Bradley, Chem. Commun., 2016, 52, 14212–14214.14 J. Li, S. Lin, J. Wang, S. Jia, M. Yang, Z. Hao, X. Zhang and P. R. Chen,J. Am. Chem. Soc., 2013, 135, 7330–7338.Fig. 2 Pd catalysed decaging of Proc-rhodamine 5 in PC-3 cells. (A) Catalysed fluorescence ‘‘switch-on’’ of the caged fluorophore 5 with thePd–peptide complex 3. Cells were incubated with Pd–peptide 3 (30 mM) for 2 h, followed by incubation with 5 (50 mM) for 18 h. (B) Confocal microscopyimages of PC-3 cells fixed with 4% paraformaldehyde and stained with DAPI (nuclei stain). Panels show: (i) cell nucleus (blue, lex = 405 nm) andPd–peptide 3 (magenta, lex = 633 nm), (ii) cell nucleus (blue) and in situ synthesised 6 (green, lex = 488 nm), and (iii) merged image. Scale bar 20 mm.(C) Flow cytometry cytograms showing: (i) untreated control cells, (ii) cells treated with Proc-rhodamine 5, and (iii) cells treated with 5 and 3.ChemComm CommunicationOpen Access Article. Published on 06 June 2017. Downloaded on 9/18/2018 2:12:51 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2017 Chem. Commun., 2017, 53, 6712--6715 | 671515 J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S. Jia,X. Zhang and P. R. Chen, Nat. Chem., 2014, 6, 352–361.16 N. Li, R. K. V. Lim, S. Edwardraja and Q. Lin, J. Am. Chem. Soc., 2011,133, 15316–15319.17 C. D. Spicer and B. G. Davis, Chem. Commun., 2013, 49, 2747–2749.18 C. P. Ramil and Q. Lin, Chem. Commun., 2013, 49, 11007–11022.19 K. Worm-Leonhard and M. Meldal, Eur. J. Org. Chem., 2008, 2008,5244–5253.20 J. F. Jensen, K. Worm-Leonhard and M. Meldal, Eur. J. Org. Chem.,2008, 2008, 3785–3797.21 W. A. Herrmann, Angew. Chem., Int. Ed., 2002, 41, 1290–1309.22 G. Altenhoff, R. Goddard, C. W. Lehmann and F. Glorius, Angew.Chem., Int. Ed., 2003, 42, 3690–3693.23 S. Deshayes, M. C. Morris, G. Divita and F. Heitz, Cell. Mol. Life Sci.,2005, 62, 1839–1849.24 J. J. Dı́az-Mochón, L. Bialy and M. Bradley, Org. Lett., 2004, 6, 1127–1129.25 R. Fischer, K. Köhler, M. Fotin-Mleczek and R. Brock, J. Biol. Chem.,2004, 279, 12625–12635.26 G. Drin, S. Cottin, E. Blanc, A. R. Rees and J. Temsamani, J. Biol.Chem., 2003, 278, 31192–31201.Communication ChemCommOpen Access Article. Published on 06 June 2017. Downloaded on 9/18/2018 2:12:51 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Intracellular delivery of a catalytic organometallic complex

We show the intracellular delivery of a homogeneous palladium–peptide catalyst able to bioorthogonally activate a profluorophore inside living prostate cancer cells.</p

Eugenio Indrigo

Jessica Clavadetscher

Sunay V. Chankeshwara

Alicia Megia-Fernandez

Annamaria Lilienkampf

Mark Bradley

Crossref

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Intracellular delivery of a catalytic organometallic complexCitation 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/C7CC02988HDigital Object Identifier (DOI):10.1039/C7CC02988HLink:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished In:Chemical CommunicationsGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 11. May. 2020Journal	Name	 	COMMUNICATION	This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	 J.	Name.,	2013,	00,	1-3	|	1 		Please	do	not	adjust	margins	Please	do	not	adjust	margins	Received	00th	January	20xx,	Accepted	00th	January	20xx	DOI:	10.1039/x0xx00000x	www.rsc.org/chemcomm	Intracellular	delivery	of	a	catalytic	organometallic	complex	Eugenio	Indrigo,†	Jessica	Clavadetscher,†	Sunay	V.	Chankeshwara,	Alicia	Megia-Fernandez,	Annamaria	Lilienkampf	and	Mark	Bradley*a	A	homogeneous	carbene-based	palladium	catalyst	was	conjugated	to	 a	 cell-penetrating	 peptide,	 allowing	 intracellular	 delivery	 of	catalytically	 active	 Pd	 complexes	 that	 demonstrated	bioorthogonal	 activation	 of	 a	 profluorophore	 within	 prostate	cancer	cells.	In	 recent	 years,	 bioorthogonal	 organometallic	 reactions	 have	been	successfully	applied	 in	chemical	biology.1–8	 In	particular,	Pd-mediated	 chemical	 transformations	 have	 been	 used	 to	modify	or	synthesise	molecules	through	cleavage	of	protecting	groups	 or	 cross-coupling	 reactions.5,9	 The	 first	 use	 of	 Pd	chemistry	in	mammalian	cells	was	reported	by	Bradley,10	who	trapped	Pd	nanoparticles	within	an	inert	polymer	microsphere	for	 intracellular	 delivery.	 This	 “active”	 transition	 metal	 was	used	 to	 mediate	 C–C	 bond	 formation	 via	 a	 Suzuki–Miyaura	cross-coupling	 and	 allyl	 carbamate	 cleavage	 reactions	 inside	living	cells,	including	the	intracellular	activation	of	a	prodrug	of	the	 antineoplastic	 agent	 amsacrine.	 This	 approach	 was	 also	used	 to	 convert	 propargyl	 derivatives	 of	 5-fluorouracil	 and	gemcitabine	 to	 their	 active	 forms,	 as	well	 as	 catalyse	 Suzuki-Miyaura	 cross-couplings	 of	 drugs	 in	 the	 presence	 of	 an	extracellular	 heterogeneous	 Pd	 catalyst.11–13	 Pd-mediated	transformations	 have	 since	 also	 been	 used	 to	 selectively	activate	 biomolecules	 in	 cells.	 Chen	 developed	 the	 chemical	activation	 of	 proteins	 based	 on	 the	 decaging	 of	propargylcarbamate	 caged	 lysine	 with	 simple	 Pd	 salts,	 while	Pd(NO3)2,	 Pd(dba)2,	 and	 allyl2PdCl2,	 have	 also	 been	 used	 to	mediate	 Sonogashira	 cross-coupling	 reactions	 inside	bacteria,14	or	 to	activate	a	protein	of	 interest	 inside	cells.14–17	However,	under	optimised	conditions,	50	equivalents	of	these	Pd	 complexes	 are	 required	 for	 the	 completion	 of	 the	 cross-coupling	 reactions.16	 The	 major	 drawbacks	 of	 all	 the	 above	methods	are	the	lack	of	cell	selectivity	of	the	catalyst	and	the	use	 of	 stoichiometric,	 or	 even	 excess	 (up	 to	 500	 eq.),	 Pd	complex	 and	 the	 cellular	 toxicity	 when	 used	 in	 non-complexed/encapsulated	form.5,18	Targeted	localisation	of	a	Pd	catalyst,	by	means	of	cell	selective	delivery	of	a	homogeneous	catalyst,	would	represent	a	major	advance	in	the	field.	Herein,	we	have	extended	the	scope	of	Pd	catalysed	chemistry	in	 living	systems	by	developing	a	method	to	deliver	and	track	an	 active	 organometallic	 complex	 inside	 mammalian	 cells.	These	water-soluble	 and	 traceable	 catalysts	were	based	on	 a	Pd(II)–carbene	 complex	 coupled	 to	 a	 fluorescently	 labelled	homing	 peptide	 for	 targeted	 cell	 delivery.	 The	 biocompatible	homogeneous	Pd	catalyst	was	based	on	a	previously	reported	carbene	 Pd	 ligand	 coupled	 to	 a	 peptide.19,20	 N-heterocyclic	carbenes	 (NHC)	 are	 strong	 σ-electron	 donating	 molecules	(enhancing	the	rate	of	oxidative	addition),	coordinate	tightly	to	Pd	 centres	 (disfavouring	 aggregation),	 and	 can	 have	 bulky	substituents	(increasing	the	rate	of	reductive	elimination).21,22	Peptides	were	chosen	to	deliver	the	Pd	complex	inside	cells	as	they	 are	 biocompatible,	 easy	 to	 prepare	 by	 solid-phase	peptide	 synthesis	 (SPPS),	 versatile,	 readily	 labelled	 and	functionalised,	with	known	cell	delivery	abilities.23		Scheme	1:	Synthetic	route	to	the	Pd–peptide	catalysts	on	a	Rink	 functionalised	polystyrene	resin.		Journal	Name	 	COMMUNICATION	This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	 J.	Name.,	2013,	00,	1-3	|	2 		Please	do	not	adjust	margins	Please	do	not	adjust	margins	Fig.	1.	Cellular	uptake	of	Pd–peptide	2.	(A)	Structure	of	the	carboxyfluorescein	labelled	Pd–peptide	2.	(B)	Intracellular	Pd	content	(ng	Pd	per	million	cells)	of	PC-3	cells	incubated	with	10	µM	Pd(OAc)2	or	2	(3	µM	and	30	µM)	for	2	h	(higher	concentrations	of	Pd(OAc)2	were	toxic).	Cells	were	harvested,	lysed	and	the	cell	content	was	subjected	to	ICP-OES	measurements.	(C)	Flow	cytometry	histograms	showing	the	uptake	of	2	(30	µM)	in	PC-3	cells	after	2	h	(in	black)	and	untreated	control	cells	(in	grey).	(D)	PC-3	cells	were	incubated	with	2	(30	µM)	for	2	h,	fixed	with	4	%	paraformaldehyde,	stained	with	DAPI	(nuclei	stain)	and	imaged	by	confocal	microscopy.	Panels	 show	 (i)	 Pd	 catalyst	2	 (green,	 λex	 =	 488	nm)	 located	 in	 the	 cytoplasm	and	nucleus,	 (ii)	 cell	 nucleus	 (blue,	 λex	 =	 405	nm),	 and	 (iii)	merged	 image	 (light	blue	indicates	co-localisation).	Scale	bar	20	µm.	The	polycationic	(tri-lysine)	catalyst	1	represented	a	simple	cell	penetrating	 peptide	 (CPP)	 which	 was	 able	 to	 cross	 the	 cell	membrane,23	and	could	be	readily	labelled	to	allow	tracking	of	the	catalyst	inside	cells.		Here,	 either	 5(6)-carboxyfluorescein	 (FAM,	 λEx/Em	 =	 492/517	nm)	or	sulfonated	Cy5	(λEx/Em	=	630/654	nm)	were	attached	to	the	 peptides	 to	 give	 the	 fluorescent	 Pd-catalysts	 2	 and	 3,	respectively	(Scheme	1).	Peptides	1,	2	and	3	were	synthesised	on	a	Rink	amide	linker	functionalised	aminomethyl	polystyrene	resin	 using	 standard	 Fmoc/tBu	 SPPS	 with	 Oxyma/DIC	 as	 the	coupling	 combination	 (Scheme	 1	 and	 ESI).	 Coupling	 of	 the	fluorophores	 in	2	 and	3	was	achieved	by	 introducing	a	bis-N-[1-(4,4-dimethyl-2,6-dioxocyclo	 hexylidene)	 ethyl]	 (Dde)	protected	Lys	as	the	first	amino	acid	(Fmoc-Lys(Dde)-OH).	Prior	to	 Fmoc	 removal	 of	 the	 terminal	 Lys	 residue,	 the	 Dde	 group	was	 orthogonally	 removed	 with	 hydroxylamine24	 and	 the	liberated	 amine	 coupled	 to	 the	 activated	 dye.	 The	 imidazole	ligand	4	 was	 coupled	 to	 the	 amino	 terminus	 of	 the	 peptides	using	 TBTU	 as	 an	 activating	 agent.	 The	 imidazole	 salt	 was	treated	with	 the	base	BEMP	 to	 form	 the	 carbene,	which	was	subsequently	 trapped	 by	 the	 addition	 of	 dichloro(1,5-cyclooctadiene)	palladium(II)	(PdCl2COD)	as	the	source	of	Pd.19	The	 catalysts	were	 cleaved	 from	 the	 resin	 by	 treatment	with	TFA	 (5	 %	 water,	 addition	 of	 conventional	 scavenging	 agents	destroyed	 the	Pd	complex)	and	purified	by	preparative	HPLC.	To	 evaluate	 the	 catalytic	 activity	 in	 a	 biological	 setting,	 non-fluorescent	bis-propargyloxycarbonyl	(Proc)	rhodamine	110	(5)	was	 treated	 with	 Pd	 catalyst	 1	 in	 phosphate	 buffered	 saline	(PBS)	and	PC-3	(prostate	adenocarcinoma)	cell	lysate	(see	ESI).	Upon	Pd	catalysed	cleavage	of	the	protecting	groups	with	0.1	eq	of	1	for	24	h,	fluorescent	6	was	generated	giving	a	200-fold	increase	in	fluorescence.	The	reaction	in	cell	lysate	resulted	in	a	more	modest	9-fold	relative	increase	in	fluorescence	with	0.1	eq	 of	 the	 catalyst,	 but	 demonstrated	 that	 the	 Pd–peptide	hybrid	was	 catalytically	 competent	 even	 in	 the	presence	of	 a	complex	mixture	of	biomolecules.		Journal	Name	 	COMMUNICATION	This	journal	is	©	The	Royal	Society	of	Chemistry	20xx	 J.	Name.,	2013,	00,	1-3	|	3 		Please	do	not	adjust	margins	Please	do	not	adjust	margins	Fig.	2.	Pd	catalysed	decaging	of	proc-rhodamine	5	 in	PC-3	cells.	(A)	Catalysed	fluorescence	“switch-on”	of	the	caged	fluorophore	5	with	the	Pd–peptide	complex	3.		Cells	were	incubated	with	Pd–peptide	3	(30	µM)	for	2	h,	followed	by	incubation	with	5	(50	µM)	for	18	h.	(B)	Confocal	microscopy	images	of	PC-3	cells	fixed	with	4	%	paraformaldehyde	and	stained	with	DAPI	(nuclei	stain).	Panels	show:	(i)	cell	nucleus	(blue,	λex	=	405	nm)	and	Pd-peptide	3	(magenta,	λex	=	633	nm),	(ii)	cell	nucleus	(blue)	and	in	situ	synthesised	6	(green,	λex	=	488	nm),	and	(iii)	merged	image.	Scale	bar	20	µm.	(C)	Flow	cytometry	cytograms	showing:	(i)	untreated	control	cells,	(ii)	cells	treated	with	proc-rhodamine	5,	and	(iii)	cells	treated	with	5	and	3.	The	 cellular	 uptake	 of	 the	 carboxyfluorescein	 labelled	 Pd–peptide	 2	 was	 investigated	 with	 the	 total	 quantity	 of	 the	internalised	 Pd	 determined	 by	 inductively	 coupled	 plasma	optical	 emission	 spectrometry	 (ICP-OES)	 analysis	of	PC-3	 cells	(Fig.	 1b),	 giving	 a	 total	 Pd	 content	 of	 20	 ng	 per	million	 cells	(when	 incubated	with	 30	µM	of	2	 for	 2	 h),	 showing	 a	 higher	metal	 uptake	 compared	 to	 cells	 treated	 with	 10	 µM	 of	Pd(OAc)2	 (higher	 concentration	 of	 Pd(OAc)2	 showed	cytotoxicity).	 Catalyst	 2	 showed	 no	 cytotoxicity	 even	 at	 200	µM	 (see	ESI).	 The	cellular	uptake	and	 intracellular	 location	of	the	 Pd	 catalyst	 2	 was	 investigated	 by	 flow	 cytometry	 and	confocal	microscopy	 (Figure	 1c	 and	 1d).	 PC-3	 cells	 showed	 a	shift	of	the	whole	cell	population	towards	higher	fluorescence	intensity	after	2	h	incubation	with	catalyst	2	(30	µM),	with	the	fluorescence	localised	within	the	cytoplasm	and,	unexpectedly,	in	 the	 nucleus	 (cationic	 peptides	 generally	 localise	 in	 the	cytoplasm	and	in	vesicles25,26).		The	catalytic	activity	of	 the	Pd–peptide	catalysts	 in	cell-based	assays	via	 proc-rhodamine	5	 activation	was	 investigated	with	the	Cy5	labelled	Pd–peptide	3.	PC-3	cells	were	incubated	with	3	(30	µM)	for	2	h,	washed	to	remove	any	extracellular	catalyst,	and	 subsequently	 incubated	with	 5	 (50	 µM)	 for	 18	 h	 (Figure	2a).	Fluorescence	microscopy	verified	the	presence	of	the	Pd-catalyst	3	as	well	as	synthesised	6	within	the	cytoplasm	of	PC-3	cells	(Fig.	2b).	Analysis	by	flow	cytometry	showed	a	shift	of	the	whole	cell	population	towards	higher	fluorescence	intensity	in	the	Cy5	channel	 indicating	 the	uptake	of	 the	catalyst,	while	a	shift	to	higher	FITC	intensity	indicated	the	formation	of	6	(Fig.	2c).		In	conclusion,	we	have	demonstrated	the	ability	to	combine	a	catalytically	 active	 Pd	 complex	 with	 a	 cell-delivery	 peptide	scaffold,	 which	 was	 able	 to	 catalyse	 a	 depropargylation	reaction	in	a	biologically	relevant	setting.	The	non-cytotoxic	Pd	catalyst	 was	 successfully	 used	 in	 the	 fluorescent	 labelling	 of	mammalian	 cells,	 showing	 significant	 catalytic	 activity	 in	 an	intracellular	environment.	Future	applications	will	thus	include	the	activation	of	prodrugs	via	these	Pd	catalysts.	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Intracellular delivery of a catalytic organometallic complex

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