Enhanced Cytotoxicity through Conjugation of a "Clickable" Luminescent Re(I) Complex to a Cell-Penetrating Lipopeptide.

Letter pubs.acs.org/acsmedchemlett “ ” Enhanced Cytotoxicity through Conjugation of a Clickable Luminescent Re(I) Complex to a Cell-Penetrating Lipopeptide Anna Leonidova,† Vanessa Pierroz,†,‡ Luke A. Adams,§ Nicholas Barlow,§ Stefano Ferrari,‡ Bim Graham,*,§ and Gilles Gasser*,† † Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland ‡ Institute of Molecular Cancer Research, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland § Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia * S Supporting Information ABSTRACT: Re(I)tricarbonylpolypyridine-basedcomplexes are particularly attractive metal complexes in the field of inorganicchemicalbiologyduetotheirluminescentproperties, ease of conjugation to targeting biomolecules, and the possibility to prepare their “hot” 99mTc analogues for radioimaging. In this study, we prepared and characterized a novel, “clickable” complex, [Re(2,2′-bipyridine)(3- ethynylpyridine)(CO) ](BF ) ([Re(CO) (bipy)(py- 3 4 3 alkyne)](BF )), exhibiting the characteristic luminescent 4 properties and moderate cytotoxicity of this general class of compound. Using Cu(I)-catalyzed “click” chemistry, the complex was efficiently attached to a lipidated peptide knowntoincreasecellpermeability,namely,themyristoylated HIV-1Tatpeptide(myr-Tat),togiveRe-myr-Tat.Fluorescencemicroscopylocalizationinhumancervicalcancercells(HeLa) confirmedenhancedcellularuptakeofRe-myr-Tatcomparedwith[Re(CO) (bipy)(py-alkyne)](BF ),andcytotoxicitystudies 3 4 showed that this resulted in an increase in potency to a level comparable with cisplatin (13.0 ± 2.0 μM). KEYWORDS: Anticancer, fluorescence microscopy, medicinal organometallic chemistry, peptide, rhenium complexes Re(I) tricarbonyl [2 + 1] complexes based on polypyridine- Inthiswork,weaimedtoimprovethecytotoxicityofaRe(I) derived ligands, such as (Re(bipy)(L)(CO) ) (L = mono- compound, namely, [Re(CO) (bipy)(py-alkyne)]+, whose 3 3 dentate ligand), have attracted considerable attention in the moderate cytotoxicity was discovered by serendipity in the past decade as catalysts,1 photosensitizers in photocatalytic courseofaseparatestudy.Todothis,weenvisagedenhancing water reduction,2,3 and CO-releasing molecules.4 They remain, cellular uptake of the complex via conjugation to a myristoylated HIV-1 Tat peptide (Scheme 1). HIV-1 Tat is a however, best known for their outstanding photochemical membrane translocation sequence from HIV, while myristic properties (large Stokes shifts, long emission lifetimes, and resistance to photobleaching),5 which make them excellent acid is a saturated linear fatty acid that naturally occurs as a candidatesforcellularimagingandionsensingapplications.6−14 post-translational protein modification. Both myristic acid and myristoylated Tat peptide were shown to increase the cellular Their use in these areas has been spurred on by their good uptake of compounds when conjugated to them.35,36 In this biocompatibility15 and the possibility to incorporate targeting study, the Re complex was appended to an azide-modified vectors via the pyridine or bipyridine-based ligands.9,16,17 myristoylated Tat peptide via “click” chemistry. Over the past Significantly,the“hot”99mTcanaloguesoftheseRecompounds few years, click chemistry has been successfully employed to could also be prepared, making them promising multimodal couple other organometallic compounds to peptides, either by agents.16,18−20 solid-phase or solution-phase methods.37−40 While Re(I) tricarbonyl polypyridine-based complexes are [Re(CO) (bipy)(py-alkyne)](BF ) was synthesized follow- 3 4 normally onlymoderately toxic, or essentially nontoxic, several ing anestablished literature procedure employed for Re(I)fac- other Re(I) compounds have been reported to be as active or tricarbonyl bipyridyl complexes with a substituted pyridine evenmorepotentthancisplatin.8,21−28Givenhowactivemany ligand.41 In brief, the initially formed Re tricarbonyl bipyridyl organometalliccompoundsareknowntobeagainstcancercells lines,29−34 it is surprising that the cytotoxic potential of these Received: April23, 2014 Re(I) complexes has not yet been fully explored, especially in Accepted: May15, 2014 light of their aforementioned advantages. Published: May15, 2014 ©2014AmericanChemicalSociety 809 dx.doi.org/10.1021/ml500158w|ACSMed.Chem.Lett.2014,5,809−814 ACS Medicinal Chemistry Letters Letter Scheme 1. Bioconjugation of [Re(CO) (bipy)(py-alkyne)]+ to a Myristoylated Tat Peptide 3 chloride complex was activated via halide abstraction as the itself can be solubilized in pure water up to 0.79 ± 0.13 mM correspondingacetonitrilecomplex.Theacetonitrileligandwas (25 °C). then displaced by 3-ethynylpyridine. The formation and purity Since some studies have previously noted the loss of the of the desired product was confirmed by 1H and 13C NMR, monodentate pyridine ligand in solution for this type of HR-MS (Figures S1−S3, Supporting Information) and complex,16,18−20 the stability of [Re(CO) (bipy)(py-alkyne)]- 3 elemental analysis. Of note, loss of the monodentate pyridine (BF ) in water and human blood plasma was assessed. 4 ligand (m/z 427.0 [M-py]+) during ionization was observed in Followinganexperimental proceduresimilartothatpreviously the MS spectrum (Figure S3, Supporting Information). reportedbyourgroup,28,55−57thecomplexanddiazepam(used p [R hy e s ( i C ca O l ) p 3 r ( o b p i e p r y t ) ie ( s py fo -a r lk th yn is e t ) y ] p ( e BF o 4 f ) R e e x ( h I i ) bi c ts om th p e le t x y . p 10 ic ,1 a 7 l ,2 p 5, h 42 o − to 50 - a p s las a m n a in an te d rn w a a l te st r a ) n w da e r r d e i d n u cu e ba to ted its in k h n u o m w a n n s p ta la b s i m lit a y o i r n d b o l u o b o l d e TheUV−visabsorptionspectrashowedintensebandsat250− distilled water for up to 72 h. The aqueous phase was then 330nm(absorptioncoefficientsaround15000M −1cm −1)and extractedwithdichloromethaneandtheorganicphaseanalyzed a weaker shoulder at 350−360 nm (about 4500 M −1cm −1) by UPLC-MS. Two peaks corresponding to [Re(CO) (bipy)- 3 (Figure S5, Supporting Information). The former are normally (py-alkyne)]+ (1.7 min, m/z 530.1 [M]+ and 427.1 [M − attributed to spin-allowed intraligand transitions (1IL = (π → ligand]+) and diazepam (2.1 min, m/z 285.2 [M + H]+) could π*) (bipyridine and pyridine), while the latter is assigned to beclearlyidentified(FiguresS9−S12,SupportingInformation). spin-allowedmetal-to-ligandchargetransfer(1MLCT)[dπ(Re) Thepercentageofdecomposed[Re(CO) (bipy)(py-alkyne)]+ → π*(bipy)]. Upon irradiation in the MLCT band (355 nm), 3 wasthencalculatedusingdiazepamastheinternalstandard.As [Re(CO) 3 (bipy)(py-alkyne)](BF 4 ) displayed a strong emis- showninFigure1,decompositionproceededsignificantlyfaster sion centered at 550 nm (Figure S6, Supporting Information). in human blood plasma (half-life of approximately 22 h) than Long, submicrosecond luminescence lifetimes (Table 1) and pure water (half-life of approximately 5 days), probably due to thesubstitutionofthepyridineligandbystongerdonorgroups Table 1. Emission Lifetimes and Quantum Yields of present in blood plasma proteins (e.g., histidine or cysteine). [Re(CO) (bipy)(py-alkyne)](BF ) 3 4 These results are consistent with the recent study by the solvent aerated degassed quantumyields(aerated) Valliant group, which showed a marked dependence of the HO 117±2ns 128±3ns 0.0048±0.0005 2 acetonitrile 168±6ns 329±1ns 0.011±0.001 large Stokes shift indicate the phosphorescent nature of the emission,51which canbeascribed toatriplet 3MLCT [dπ(Re) → π*(bipy)] transition, as observed for similar complexes.4,12 Typical emission lifetimes and quantum yields of Re- (CO) (bipy)(L) complexes encompass a relatively broad 3 range(0.05−9.6μsand0.002−0.59,respectively).52Thevalues measured for [Re(CO) (bipy)(py-alkyne)](BF ) are, in fact, 3 4 very close to those of structurally similar Re(CO) (bipy)(L) 3 complexes, such as isothiocyanate-substituted pyridine com- plexes.17 Of note, shorter lifetimes in water compared to acetonitrile can be explained by the greater polarity of water, which stabilizes the 3MLCT excited state, thus lowering its Figure 1. [Re(CO)(bipy)(py-alkyne)]+ stability in human blood 3 energyandfacilitatingnonradiativeprocesses.53,54Thecomplex plasma andwater (double distilled) at 37 °C. 810 dx.doi.org/10.1021/ml500158w|ACSMed.Chem.Lett.2014,5,809−814 ACS Medicinal Chemistry Letters Letter Figure2.FluorescencemicroscopyofHeLacellsfixedafter2h:(A)untreatedcells;(B)treatedwith[Re(CO) (bipy)(py-alkyne)](BF )at100 3 4 μM; and(C) treated with Re-myr-Tatat 20μM. pyridine ligand lability on its basicity/leaving group ability.16 compounds in human cervical cancer cells (HeLa) by The plasma stability of [Re(CO) (bipy)(py-alkyne)](BF ) is fluorescence microscopy. The cells were first incubated for 2 3 4 on par with the most stable compounds reported by Valliant h with an appropriate concentration of each compound, then and co-workers.16 fixed with formaldehyde and finally imaged. Figure 2 shows a The azide-modified myristoylated Tat peptide (myr-Tat) pronounced difference in emission intensity between the cells was prepared via standard solid-phase peptide synthesis treated with [Re(CO) (bipy)(py-alkyne)](BF ) (B) or Re- 3 4 techniques and then purified by preparative HPLC to give a myr-Tat (C). Indeed, while the concentration of Re-myr-Tat sticky yellow solid, which was unambiguously characterized by in the cell medium was five times lower than that of the HR-MS and HPLC (Figures S13 and S14, Supporting complex, the cells incubated with Re-myr-Tat appeared much Information). It was then successfully conjugated to [Re- brighter, indicating significantly higher uptake (it is important (CO) (bipy)(py-alkyne)](BF ) via Cu(I)-catalyzed “click” to keep in mind that the intensity of the emission signal of a 3 4 chemistry using similar experimental conditions to those compoundinthecellssometimesfailstocorrelatewithuptake, reported by Fokin and co-workers.58 The crude compound as luminescence can be quenched in the cellular environ- was purified by preparative HPLC to yield Re-myr-Tat as a ment).28,64Intermsofsubcellularlocalization,thecomplexand pale yellow solid, which was characterized by UPLC-MS and the bioconjugate were visualized in the cytoplasm (although MALDI-TOF (Figures S15 and S16, Supporting Information). weak signals were sometimes detected in the nucleoli of cells A single peak was observed in the UPLC-MS chromatogram, incubated with the complex), a localization typically observed andconsistentwiththeMSspectrumofthecomplex,boththe for this type of Re complex.10,12,15,16 Of note, while the HIV-1 desired product and fragments resulting from pyridine ligand Tat peptide sequence has been reported to promote nuclear dissociation appeared in the MS spectrum (m/z 338.9 [M − localization,65 with a myristoylated Tat derivative dual-labeled Re(CO) (bipy)+6H]6+,406.4[M−Re(CO) (bipy)+5H]5+, withGd-DOTAandfluoresceinpreviouslyshowntostainboth 3 3 491.6[M+4H]5+,507.8[M−Re(CO) (bipy)+4H]4+,614.2 nucleoliandnuclearmembranes,36thiswasnotthecaseforRe- 3 [M + 3H]4+, and 818.7 [M + 2H]3+). In the MALDI-TOF myr-Tat. Our microscopy images were recordered at a spectrum, only the fragmented product (m/z 2026.4 [M − relatively low Re-myr-Tat concentration that left most of the Re(CO) (bipy)+H]+) was detected due to the harsher cells alive after 2 h. The Gd-DOTA myr-Tat derivative, 3 ionization conditions compared to ESI-MS. The presence of however, was imaged at a much higher concentration (260 the fac-Re(CO) core in the Re-myr-Tat bioconjugate was μM), at which about 60% of the cells were already dying.36 3 unambiguouslyconfirmedbythepresenceoftheCOstretching When HeLa cells were incubated with a higher concentration bandsintheIRspectrum,whichappearedinthesamerangeas (2.5-fold increase) of Re-myr-Tat, localization in nuclear that of [Re(CO) (bipy)(py-alkyne)](BF ) (Figure S17, membrane and nucleoli was also observed (Figure S18, 3 4 SupportingInformation).Theinfluenceofthemyr-Tatmoiety Supporting Information). However, the cells appeared to be on the lipophilicity of the resulting Re-myr-Tat bioconjugate already slightly affected by the conjugate at this concentration, wasevaluatedbymeasuringthedistributioncoefficientbetween so this change in localization could be due to toxin stress. octanol and phosphate buffer, pH 7.01 (logD ), using a Next,thecytotoxicityof[Re(CO) (bipy)(py-alkyne)](BF ) 7.01 3 4 similarproceduretooneusedbyourgroupforRucomplexes.59 andRe-myr-TattowardHeLacellswasevaluatedbyincubating Interestingly, although myr-Tat contains both a long lipophilic the cells with increasing concentrations of the compounds for fatty acid chain and a highly positively charged peptide 48 h and quantifying cell viability using the resazurin assay.66 sequence, the net effect is an increase in lipophilicity, namely, The toxicity of our compounds was compared with that of an from a logD of −0.36 ± 0.05 for [Re(CO) (bipy)(py- established metal-containing anticancer drug, namely, cisplatin. 7.01 3 alkyne)]+ to 0.86 ± 0.15 for Re-myr-Tat. This change could Although several studies report on the cytotoxicity of Re(I) potentially improve the cellular uptake of Re-myr-Tat, as tricarbonyl bipyridine−pyridine complexes, their antiprolifer- higherlipophilicitycanfavortheentryofcompoundsintocells ativeeffecthasgenerallybeenfoundtorangefromnonexistent and hence enhance the cytotoxicity.59−63 to moderate; [Re(CO) (bipy)(py-alkyne)](BF ) does not 3 4 With the desired organometallic complex and bioconjugate deviatefromthistrend(Table2).Aseriesofsimilarcomplexes, in hand, we investigated the intracellular fate of both namely, Re(phen)(diaminopy)(CO) ,display IC values 2−3- 3 50 811 dx.doi.org/10.1021/ml500158w|ACSMed.Chem.Lett.2014,5,809−814 ACS Medicinal Chemistry Letters Letter Table 2. Cytotoxicity Data (IC Values) for NMR, nuclear magnetic resonance; HR-MS, high-resolution 50 [Re(CO) (bipy)(py-alkyne)](BF ), Re-myr-Tat and mass spectroscopy; MALDI-TOF, matrix-assisted laser desorp- Cisplatin 3 Towards HeLa Cells a 4 tion ionization time-of-flight; MLCT, metal-to-ligand charge transfer;Py,pyridine;Tat,trans-activatoroftranscription(here, compd HeLaIC (μM) 50 a cell-permeating peptide sequence derived from HIV-1 Tat [Re(CO) 3 (bipy)(py-alkyne)]+ 29.9±6.1 protein); UPLC, ultraperformance liquid chromatography; Re-myr-Tat 13.0±2.0 UV−vis, ultraviolet−visible light ■ cisplatin 9.1±2.8 aExperiments were performed intriplicate. REFERENCES (1)Kutal,C.;Weber,M.A.;Ferraudi,G.;Geiger,D.Amechanistic foldhigherthancisplatin.25Significantly,however,thecoupling investigation of the photoinduced reduction of carbon dioxide of [Re(CO) (bipy)(py-alkyne)]+ to the myristoylated Tat mediated by tricarbonylbromo(2,2′-bipyridine)rhenium(I). 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Recent exploitation of material is available free of charge via the Internet at http:// luminescent rhenium(I) tricarbonyl polypyridine complexes as p■ubs.acs.org. biomolecular and cellular probes. Eur. J. Inorg. Chem. 2011, 3551− 3568. AUTHOR INFORMATION (8)Louie,M.-W.;Choi,A.W.-T.;Liu,H.-W.;Chan,B.T.-N.;Lo,K. Corresponding Authors K.-W. Synthesis, emission characteristics, cellular studies, and *(G.G.)E-mail:gilles.gasser@chem.uzh.ch.Fax:+414463568 bioconjugation properties of luminescent rhenium(I) polypyridine complexes with a fluorous pendant. Organometallics 2012, 31, 5844− 03. Tel: +41 44 635 46 30. Website: www.gassergroup.com. 5855. *(B.G.) E-mail: bim.graham@monash.edu. Fax: +61 3 (9)Louie,M.-W.;Liu,H.-W.;Lam,M.H.-C.;Lam,Y.-W.;Lo,K.K.- 99039582. Tel +61 3 9903 9706. Website: www. W. Luminescent rhenium(I) polypyridine complexes appended with bimgrahamgroup.com. an α-D-glucose moiety as novel biomolecular and cellular probes. Funding Chem.Eur. J. 2011, 17,8304−8308. This work was supported by the Swiss National Science (10)Fernaǹ dez-Moreira,V.;Ortego,M.L.;Williams,C.F.;Coogan, M. P.; Villacampa, M. D.; Gimeno, M. C. Bioconjugated rhenium(I) Foundation (Professorship No. PP00P2_133568 and Research complexes with amino acid derivatives: synthesis, photophysical GrantsNo.200021_129910andNo.200020_146776toG.G.), the University of Zurich (G.G and S.F.), the Stiftung für 5 p 9 ro 5 p 0 e − r 5 ti 9 e 5 s, 7. and cell imaging studies. Organometallics 2012, 31, wissenschaftliche Forschung of the University of Zurich (G.G (11) Thorp-Greenwood, F. L.; Balasingham, R. G.; Coogan, M. P. and S.F.), the Stiftung zur Krebsbekam̈ pfung (S.F.), the Organometallic complexes of transition metals in luminescent cell Huggenberger-BischoffStiftung(S.F.),theUniversityofZurich imaging applications. J. Organomet. Chem. 2012,714, 12−21. Priority Program (S.F.), and the Australian Research Council (12) Choi, A. 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