Luminescent Re(<scp>i</scp>) and Re(<scp>i</scp>)/Au(<scp>i</scp>) complexes as cooperative partners in cell imaging and cancer therapy

{"full_text": " View Article Online\n\n\n\n\nChemical\n View Journal\n\n\n\n\nScience\nAccepted Manuscript\n\n\n\n\n This article can be cited before page numbers have been issued, to do this please use: M. C. Gimeno, V.\n Fern\u00e1ndez-Moreira and I. Marzo, Chem. Sci., 2014, DOI: 10.1039/C4SC01684J.\n\n This is an Accepted Manuscript, which has been through the\n Royal Society of Chemistry peer review process and has been\n accepted for publication.\n\n Accepted Manuscripts are published online shortly after\n acceptance, before technical editing, formatting and proof reading.\n Using this free service, authors can make their results available\n to the community, in citable form, before we publish the edited\n article. 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In no event shall the Royal Society of Chemistry be held\n responsible for any errors or omissions in this Accepted Manuscript\n or any consequences arising from the use of any information it\n contains.\n\n\n\n\n www.rsc.org/chemicalscience\n\f Page 1 of 14 Chemical Science\n View Article Online\n DOI: 10.1039/C4SC01684J\n\n\n Journal Name RSCPublishing\n\n ARTICLE\n\n Luminescent Re(I) and Re(I)/Au(I) complexes as\n cooperative partners in cell imaging and cancer\n\n\n\n\n Chemical Science Accepted Manuscript\n Cite this: DOI: 10.1039/x0xx00000x\n therapy\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Vanesa Fern\u00e1ndez-Moreira,*a Isabel Marzo,b and M. Concepci\u00f3n Gimeno*a\n Received 00th January 2012,\n Accepted 00th January 2012\n\n DOI: 10.1039/x0xx00000x\n A series of luminescent hometallic fac-[Re(bipy)(CO)3(L)]+ and heterometallic fac-[Re(bipy)(CO)3(L-\n AuPPh3)]+ complexes, where L is an imidazole, alkynyl-imidazole or alkynyl-pyridine derivative, have\n www.rsc.org/ been synthesised for the purpose of finding a synergic effect between the excellent photophysical\n properties of rhenium complexes and the good antiproliferative effects of gold compounds. Cytotoxicity\n studies performed in human A549 lung cancer cells revealed the importance of the alkynyl-phosphine-\n gold fragment within the probe to design efficient anticancer agents. Heterometallic Re(I)/Au(I)\n derivatives presented values of IC50 more than 10 times lower than their analogous Re(I) complexes. In\n addition, fluorescent cell microscopy pointed out the different biodistribution behaviour of the\n monometallic and heterometallic families. Whereas the monometallic Re(I) species showed some\n general cytoplasmatic staining with mitochondrial accumulation, the heterometallic Re(I)/Au(I)\n derivatives shifted from localising in the mitochondria to the nucleus and nucleolus upon increasing the\n loading concentration, suggesting a completely differ rent driving force for their localisation pattern.\n These facts revealed that these bimetallic species can be excellent partners in cell imaging and cancer\n therapy.\n\n\n\n\n Introduction activity of metal complexes after introducing akynyl ligands in their\n coordination sphere. For instance, comparative studies on the\n Several studies on gold(I) complexes have shown their anti- cytotoxic effects of [Pt(terpy)Cl]+ and [Pt(terpy)(C\u2261CAr)]+ showed\n inflammatory activity and very recent publications also the latter as the species with the higher activity. Moreover,\n demonstrated their antitumoral, bactericidal and anti-HIV activity. [Pt(terpy)(C\u2261CAr)]+ turned to be about 100 times more cytotoxic\n Gold(I) antiarthritic drugs such as Solganol, Miocrisine, etc. and the than cisplatin.4 In the particular case of alkynyl gold complexes,\n second generation drug Auranofin\u00ae1 have been pioneering there have been also publications suggesting these types of\n compounds for the use of gold(I) in medicine, that has boosted the organometallic compounds as a new kind of chemotherapeutic\n development of new metallodrugs as modern therapeutic and agents.5 However, only a few studies on the biological activity of\n diagnostic agents.2 In particular, Auranofin\u00ae is known to induce alkynyl gold compounds have been reported so far.6\n apoptosis via a selective inhibition of the thioredoxin reductase Rhenium complexes have also attracted growing attention for\n (TrxR), a mitochondrial enzyme. Further gold(I) complexes have their possible use as anticancer drugs.7 To date, the studies reported\n shown similar behaviour, promoting the mitochondria as an in literature regarding the anticancer activity and diagnostic\n extraordinary biological target for anticancer drugs.3 One of the applications of rhenium complexes, contain derivatives of Re(I),\n major features to consider in the design of novel bioactive species is Re(III), Re(V) and very recently Re(IV). In terms of cell\n their stability under physiological conditions. Within this context, visualization, species of the type [Re(bisimine)(CO)3 (L)] where L is\n gold alkynyl complexes can be thought as alternative bioactive a pyridine derivative, have great potential as cell imaging agents.\n agents to conventional gold(I) complexes. They exhibit reasonably Their extraordinary photohysical properties,8 i.e. 3MLCT species\n stable coordinative bonds and the alkynyl ligand itself seems to with large Stokes shifts, long lifetimes and good quantum yields,\n contribute to improve the bioactivity of the species, which is of have led to their successful application in fluorescent microscopy\n extremely importance when it comes to design anticancer agents. cell imaging.9 Moreover, their kinetical inertness due to the low-spin\n Several reports support the idea of an increase of the cytotoxic octahedral d6 character confers these species with a low rate of\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2013 J. Name., 2013, 00, 1-3 | 1\n\f Chemical Science Page 2 of 14\n View Article Online\n ARTICLE Journal Name\n DOI: 10.1039/C4SC01684J\n\n\n ligand exchange, which is crucial in order to modulate the toxicity of literature precedents. This involved initial formation of rhenium\n heavy metal ions. Several reports on the subject have shown that a tricarbonyl bipyridyl chloride derivative, then activation of the\n thoughtful modulation of the coordination sphere allows to tune chloride by exchange to triflate using triflic acid affording fac-\n properties such as uptake and/or localisation10 whereas the [Re(bipy)(CO)3(CF3SO3)].13 Finally, for complexes 1-3 and 5-7,\n photophysical properties remain basically unaltered. In order to take the triflate ligand was displaced for either a pyridine / imidazole\n an step forward in the design of novel bioactive cell imaging agents, derivative or their analogous gold complex under mild\n combination of the two metallic fragments, (a) tricarbonyl bisimine conditions (Scheme 1). Complex 4, which has been already\n rhenium and (b) alkynyl gold species, seems to be an excellent reported in the literature,14 was also synthesized following the\n approach to bring together photophysical properties, cytotoxic same procedure However, a different synthetic route was used\n effects and bioactivity. The synergy of thoroughly selected in the synthesis of complex 8. In this case, coordination of the\n\n\n\n\n Chemical Science Accepted Manuscript\n photophysical and biological properties derived from the merging of gold fragment was performed directly in complex 4, after\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n both metallic fragments in a single species could deliver novel deprotonating the imidazolyl with Cs2CO3 to attain 8.\n heterometallic species with great potential in the area of therapy and\n diagnosis. However, to the best of our knowledge, few examples\n have been published dealing with heterometallic alkynyl\n Re(I)/Au(I)-species, which are focused on the synthesis and\n photophysical properties11 with no mention to their possible\n application in medicine as either therapeutic or diagnostic agents.\n For that reason, and with the certainty of being able to select,\n modulate and combine the right photophysical and biological\n properties by a careful design of heterometallic alkynyl Re(I)/Au(I)\n species, this work pretend to be the pioneer to deepen in their\n biological aspects. Consequently, a series of monometallic Re(I) and\n heterometallic alkynyl Re(I)/Au(I) were prepared and their\n luminescent and bioactivity properties against human A549 lung\n cancerous cell line were studied. Furthermore, their application as\n contrast agents was also analysed highlighting the importance of the\n partnership of the two metal centres.\n\n Scheme 1 Depiction of the synthesis of the monometallic and heterometalic\n Results and discussion species 1-8.\n\n Synthetic procedure and characterization\n Spectroscopic characterisation was performed using IR, 1H, 13C-\n The ligands L(1-4) used as linkers between the rhenium and NMR, and UV-vis spectroscopy. Further analytical data for each\n gold metal centres together with their gold phosphine complex was provided through mass spectrometry, which\n derivatives, L(5-6), are illustrated in Fig 1. In particular, the corroborate the accomplishment on the synthesis of the mono- and\n synthesis of 2-PyC\u2261CAuPPh3, L5, and 3-PyC\u2261CAuPPh3, L6, heterometallic species and their purity was determined by elemental\n has been reported elsewhere.12 Consequently, 5- analysis. Additionally, crystals of 2, 5, 7 and 8 suitable for X-ray\n (C\u2261CAuPPh3)ImMe, L7, was synthesised by a similar analysis (SHELX programs) were obtained by slow diffusion of\n procedure, i.e. \u03c0-coordination of [AuClPPh3] to the alkyne either ether or hexane into a solution of the complex in DCM or\n activates the terminal proton to be deprotonated by KOH in a acetone.\n mixture of methanol / acetone. 1\n H-NMR spectra of complexes 1-8 were performed in either\n acetone-d6 or DCM-d2. In all cases, the spectra are well defined and\n exhibit the characteristic downfield shift of the bipyridine protons\n upon coordination to the rhenium metal centre when compared to the\n corresponding proton resonances of the free ligand. This\n phenomenon is generally rationalised in terms of the \u03c3 donation of\n the bipyridine ligand. Moreover, phenyl protons of the fragment -\n AuPPh3 for the heterometallic complexes (5-8) are observed as\n multiplets in the region between 7.46 and 7.77 ppm. As a result of\n such coordination, there is also the disappearance of the terminal\n Fig. 1 Depiction of L1-L7. alkynyl protons in the case of 5, 6 and 7, which is clearly observed in\n their precursors at 4.82, 4.09, 4.27 ppm. Furthermore, there is an up-\n Synthesis of complexes 1-4 and 5-8, i.e. Re(I) and Re(I)/Au(I) field shift of the protons belonging to pyridine and imidazole ligands\n derivatives respectively, was achieved starting from fac- upon coordination to the -AuPPh3 fragment. Such behaviour can be\n [Re(bipy)(CO)3(CF3SO3)] which was synthesised following interpreted in terms of \u03c0-back donation from the Au(I) centre to the\n\n\n\n 2 | J. Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f Page 3 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n ligands. In addition, 1H-NMR-spectroscopic data corroborate the geometry is basically originated from the geometrical\n idea of the facial disposition of CO ligands around the Re metal restrictions of the chelate ligand, with chelate angles of N(1)-\n centre. Only four set of signals are observed in the 1H-NMR Re(1)-N(2) = 75.28(7)\u00ba (complex 2), N(1)-Re(1)-N(2) =\n spectrum for the bipyridine ligand suggesting its symmetry within 75.10(1)\u00ba and N(4)-Re(2)-N(5) = 74.50(1)\u00ba (complex 5), N(1)-\n the complex.15 In the case of 31P-NMR spectroscopy, a single peak Re(1)-N(4) = 74.4(6)\u00ba (complex 7) N(1)-Re(1)-N(2) =\n appears at 41.54, 42.29, 42.19 ppm for species 5, 6 and 7 74.96(13)\u00ba and N(6)-Re(2)-N(5) = 74.81(13)\u00ba (complex 8)\n respectively and at 31.18 ppm the case of 8, where AuPPh3 is instead of the ideal 90\u00ba. Bond distances in the rhenium core are\n coordinated directly to one of the nitrogen of the imidazole.16 Such within the typical values for similar complexes, i.e. Re-C(CO)\n high field chemical shift of complex 8 is due to the paramagnetic distances are between 1.902(3) and 1.94(3), Re-N(py/im)\n shielding tensor contribution \u03c3para, which unlike in 1H-NMR between 2.17(2) \u00c5 and 2.255(4) \u00c5 and Re-N(Bipy) between\n\n\n\n\n Chemical Science Accepted Manuscript\n spectroscopy, is the main contributor to the chemical shift value. 2.166(2) \u00c5 and 2.19(2) \u00c5.20 As commented before, in the\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Therefore, the presence of less basic ligands in trans position to the particular case of complex 5, two independent molecules\n P atom will promote \u03b4p to get shifted towards higher field than the crystallised in the asymmetric unit together with their counter\n more basic ligands.17 Stretching frequencies of CO, C\u2261C and H-C\u2261C ion and two acetone molecules coming from the crystallisation\n are selected in Table 1. The IR spectra of complexes 1-8 showed the solvent. Hydrogen bonds between either the crystallisation\n characteristic strong \u03bd(CO) absorptions in the range between 1892 solvent or the counter ion with the two molecules in the\n and 2029 cm-1, typical of these type of cationic species with C3v asymmetric units are also present, with distances ranging from\n symmetry around the rhenium centre, i.e. facial configuration.18 In 2.487\u00c5 to 2.641\u00c5 (Fig. S1 and Fig. S2). Likewise, complex 8\n addition, complexes 5 and 6 presented a weak \u03bd(C\u2261C) band in the has two independent molecules within the asymmetric unit,\n region of c.a. 2122 cm-1, which is typical for \u03b71 coordination of which crystallises with their corresponding counter ions and an\n alkynyl gold(I) derivatives.11b,19 Moreover, those complexes lack ether molecule from the crystallisation solvent. Molecule 1\n \u03bd(H-C\u2261C) bands, which are seem for their rhenium precursors, presents a short contact between one of the oxygen atoms from\n complexes 1 and 2. the triflate and a hydrogen atom from the phenyl ring\n (O(12)\u00b7\u00b7\u00b7H(25) = 2.668 \u00c5), which can be considered as a\n Table 1 IR stretching frequencies for complexes 1-3 and 5-8\n hydrogen bond (Fig. S3).\n Compound \u03c5(C\u2261O) \u03c5(C\u2261C) \u03c5(H- In addition, heterometallic species, complex 5, 7 and 8,\n (C\u2261C) presented the gold atom in a distorted linear geometry with C-\n [Au(C\u2261CImMe)PPh3] - 2159(w) -\n 1 2028(s),1928(sh), 1904(s) 2113(w) 3205 (m) Au-P angles of c.a. 174\u00b0 for complexes 5 and 7 and an angle of\n 2 2029(s), 1904(bs) 2113(w) 3243(m) N-Au-P of c.a. 177\u00b0 for complex 8. Bond distances within the\n 3 2025(s), 1918(sh), 1894(s) - 3248(m) gold environment are also within the normal values for C\u2261C-\n 5 2026(s) 1915(bs) 2123(m) -\n 6 2028(s), 1903(bs) 2121(w) -\n Au-P derivatives and Im-Au-P.12a,16,21 A summary of bond\n 7 2025(s), 1930(sh), 1893(s) - - distances and angles is presented in Figs. 2-5.\n 8 2020(s), 1921(sh), 1892(s) - -\n\n\n\n X-ray crystallography\n Single crystals suitable for X-ray diffraction analysis of\n complexes 2, 5, 7 and 8 were obtained by slow diffusion.\n Compound 2 crystallised in the monoclinic space group P21/n,\n and presented a single molecule in the asymmetric unit.\n Complexes 5, 7 and 8 crystallised in the triclinic P-1 space\n group with two independent molecules in the asymmetric unit\n of 5 and 8, and only one for complex 7. The X-ray diffraction\n data for complex 7 is not good because of the presence of a Fig. 2 Ortep representation of complex 2. The most relevant bond lengths (\u00c5)\n non-solved twin crystal. Despite several attempts to and angles (deg): Re(1)\u2212C(12) = 1.932(2), Re(1)\u2212C(13) = 1.934(2), Re(1)\u2212C(14) =\n 1.902(3), Re(1)\u2212N(1) = 2.166(2), Re(1)\u2212N(2) = 2.172(2), Re(1)\u2212N(3) = 2.214(2);\n recrystallise complex 7, suitable single crystals could not be N(1)\u2212Re(1)\u2212N(2) = 75.28(3), C(12)\u2212Re(1)\u2212N(3) = 174.40(9).\n grown. Nevertheless, complex 7 will be discussed in this\n section together with complexes 2, 5 and 8 although the values\n for the bond lengths and angles are not as accurate as for the\n rest of complexes. As expected, in all cases the rhenium atom\n presented a distorted octahedral disposition, where the carbonyl\n moieties adopted a facial distribution. The equatorial plane is\n described by the chelate bipyridine ligand and two carbonyls.\n The third carbonyl and the pyridine/imidazole derivative\n occupy the axial plane. Deviation from the ideal octahedron\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 3\n\f Chemical Science Page 4 of 14\n View Article Online\n ARTICLE Journal Name\n DOI: 10.1039/C4SC01684J\n\n\n\n\n Chemical Science Accepted Manuscript\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Fig. 3 Ortep representation of complex 5. Re(2)\u2212C(57) = 1.919(5), Re(2)\u2212C(58) =\n 1.919(5), Re(2)\u2212C(59) = 1.909(5), Re(2)\u2212N(4) = 2.174(3), Re(2)\u2212N(5) = 2.174(4),\n Re(2)\u2212N(6) = 2.242(4); N(5)\u2212Re(2)\u2212N(4) = 74.5(1), N(6)\u2212Re(2)\u2212C(59) = 176.7(2),\n Au(2)\u2212C(78) = 1.995(5), Au(2)\u2212P(2) = 2.278(1).\n\n\n\n\n Fig. 5 Ortep representation of complex 8. The most relevant bond lengths (\u00c5)\n and angles (deg): Re(2)\u2212C(47) = 1.932(4), Re(2)\u2212C(48) = 1.929(5), Re(2)\u2212C(49) =\n 1.915(4), Re(2)\u2212N(6) = 2.169(3), Re(2)\u2212N(7) = 2.175(3), Re(2)\u2212N(5) = 2.181(3),\n N(5)\u2212Re(2)\u2212N(6) = 74.8(1), N(7)\u2212Re(2)\u2212C(48) = 178.3(2), P(2)\u2212Au(2)\u2212N(8) =\n 177.9(1), Au(2)\u2212N(8) = 2.048(4), Au(2)\u2212P(2) = 2.235(1).\n\n\n Photophysical studies\n UV-visible absorption spectra, recorded in a degassed CH3CN\n solution at 298K, show the typical pattern for bisimine Re(I)\n derivatives (Fig. 6 and Fig. S4). There are ligand centred transitions\n (alkynyl, bipyridine, pyridine and imidazole derivatives) at higher\n energies, \u03c0 \u2192 \u03c0* transitions at < 320 nm, and metal-to-ligand-\n charge-transfer transitions (1MLCT), formally attributed to Re(d\u03c0)\n \u2192 L(\u03c0*) transitions, at lower energies 352 - 366 nm, with a tail\n extending up to 420 nm. Moreover, it is noteworthy that\n heterometallic species have a considerable increment in the molar\n extinction coefficient for the \u03c0 \u2192 \u03c0* bands in comparison with the\n monometallic analogue. Such difference could be associated with the\n existence of an intense absorption at c.a. 288 nm, which appears\n depending on the alkynyl ligand present and it is likely assigned as a\n mixture of intraligand \u03c0 \u2192 \u03c0* (C\u2261C) , \u03c0 \u2192 \u03c0* (bipy) and \u03c0 \u2192 \u03c0*\n Fig. 4 Ortep representation of complex 7. The most relevant bond lengths (\u00c5) (PPh3) transitions.11a On the contrary, molar extinction coefficients\n and angles (deg): Re(1)\u2212C(7) = 1.92(2), Re(1)\u2212C(8) = 1.94(3), Re(1)\u2212C(9) = 1.91(2), for the 1MLCT bands are practically the same. Such behaviour could\n Re(1)\u2212N(3) = 2.18(2), Re(1)\u2212N(4) = 2.19(2), Re(1)\u2212N(1) = 2.17(2); N(4)\u2212Re(1)\u2212N(1) be attributed to the fact that the main ligand orbital implicated in the\n = 74.4(6), C(9)\u2212Re(1)\u2212N(1) = 176.5(7), P(1)\u2212Au(1)\u2212C(1) = 173.85, Au(1)\u2212C(1) =\n 2.00(1), Au(1)\u2212P(1) = 2.27(4). MLCT transition is that belonging to the bipyridine ligand.\n Specifically the 1MLCT transition is a Re(d\u03c0) \u2192 bipy(\u03c0*) transition,\n which remains unaltered by the coordination of the gold fragment to\n either a pyridine or a imidazole fragment. The most significant\n absorption data together with the emission maxima values for all\n complexes are collected in Table 2.\n\n\n\n\n 4 | J. Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f Page 5 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n and 7 of 40.15 ns. The similarity within the Re and the\n corresponding Re-Au species lifetime values, together with those\n reported in the literature for rhenium complexes point out that the\n origin of the emission in the complexes is mainly due to the Re\n species and not to the gold alkyne fragment, which agrees with the\n experimental data. 10a,18b\n\n\n\n\n Chemical Science Accepted Manuscript\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Fig. 6 UV-absorption spectra of complexes 2 and 6 recorded in degassed CH3CN\n at 298 K.\n\n\n Table 2 Absorption bands, excitation and emission maxima values.\n\n \u03bbexc \u03bbem\n species UV-Vis (\u00d7104 \u03b5/dm3mol-1cm-1)\n (nm) (nm)\n 240 277 321 357\n 1 398 577\n (2.28) (1.82) (1.20) (0.51) Fig. 7 Emission spectra of complexes 2 and 6 recorded in degassed CH3CN at 298\n 236 268 318 360 K, excitation at 404 and 400 nm, respectively.\n 2 404 574\n (3.04) (1.40) (0.94) (0.32)\n 242 276 318 352\n 3 412 604 Cellular studies and fluorescence microscopy\n (2.49) (1.36) (1.08) (0.38)\n 248 318 356\n 4\n (1.20) (1.07) (0.34)\n 417 600 In view of the favourable emissions properties of complexes 1-\n 243 265 305 366 8, and of the good stability in solution of both the rhenium and\n 5 400 592\n (3.98) (2.91) (2.16) (0.34) the rhenium-gold complexes, as the 1H and 31P NMR signals\n 240 278 3.19 357\n 6 400 576 remain the same with time, a series of experiments were\n (4.26) (4.16) (1.68) (0.37)\n 237 271 318 352 undertaken to test their cytotoxicity and viability as contrast\n 7 402 607\n (4.45) (3.42) (1.10) (0.33) agents in human A549 lung carcinoma cells. As commented\n 237 318 356 before the gold-alkynyl bond is amongst the strongest Au-\n 8 410 628\n (3.04) (0.94) (0.31)\n ligand bonds, the same happen with the Au-P bond. Study of\n Degassed CH3CN, 298 K the cytotoxic effect was performed by an annexin-V analysis\n and revealed a great cytotoxic difference between the\n Luminescence spectra of complexes (1-4) and (5-8) were recorded in\n heterometallic (4-8) and monometallic complexes (1-4). Table\n degassed CH3CN solution at 298 K (Fig 7 and Fig S6). All of them\n 3 summarises the IC50 found for all complexes pointing out the\n showed a broad emission between 577 - 628 nm which is attributed\n higher cytotoxicity of heterometallic species in comparison\n to the phosphorescence from the Re(d\u03c0) \u2192 bipy(\u03c0*) 3MLCT excited\n with their rhenium analogues.\n state and it has been previously reported in many rhenium(I) diimine\n tricarbonyl complexes.8, 22 It is interesting to note, that emission of 2- Table 3 Values of IC50 for species 1-8\n PyC\u2261CAuPPh3, L5, which has been already studied in our group12b Monometallic: 1 2 3 4\n and with a maximum at 450 nm, does not appear when this fragment IC50 (\u00b5M) 120\u00b129 200\u00b156 - -\n is coordinated to [Re(bipy)(CO)3CF3SO3] to give 5. Such behaviour Heterometallic: 5 6 7 8\n IC50 (\u00b5M) 9.5\u00b11.0 9.7\u00b11.1 4.4\u00b10.5 19\u00b17.9\n could be interpreted as an efficient of intramolecular energy transfer\n process from the Au(I) unit to the Re(I) unit, specifically from the\n 3\n [\u03c3(Au-P)\u2192\u03c0*(C\u2261CR)] to the lower-lying 3MLCT 3[d\u03c0(Re)\u2192\n \u03c0*(bipy)] excited state and it is in agreement with optical studies of Whereas complexes 1-4 showed IC50 values over 100 \u00b5M and\n analogous mixed gold(I)-rhenium(I) complexes performed by Yam even some of them did not seem to be cytotoxic at the studied\n and coworkers.11a Luminescent lifetime measurements have been concentrations, the heterometallic complexes 5-8 presented IC50\n performed for the Re complexes 2 and 3, and for the corresponding values lower than 20 \u00b5M. Such cytotoxic difference between\n mixed Re-Au species 6 and 7 in degassed CH3CN solution. They the monometallic and the heterometallic species might be\n present values from 40.15 to 148 ns, with very similar values within associated with the role of Au-phosphine fragment rather than a\n the Re and the corresponding Re-Au species. For example for the synergic effect with the [Re(bipy)(CO)3L] core. The probed\n rhenium complex 2 the lifetime is 148 ns and for the corresponding cytotoxic activity of different gold(I) phosphine fragments\n rhenium-gold 6, 134 ns. Similarly, 3 presents a value of 52.56 ns reported on several cell lines supports this idea.6,23 The\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 5\n\f Chemical Science Page 6 of 14\n View Article Online\n ARTICLE Journal Name\n DOI: 10.1039/C4SC01684J\n\n\n cytotoxic activity in A549 cells have been reported only for a cell region with more intensive luminescence in specific spots\n few gold(I) complexes, and none of them in the same within the cytoplasm, (Fig. 9). Such, distinct granular\n conditions that those reported here. Auranofin gives a value of localization pattern might be indicative of mitochondrial\n 0.72 \u00b5M after 72 h of incubation for the MTT method, but this staining. The only area in the cell that seems not to uptake the\n value could be considerably higher at 24 h.3f We have reported complexes resembles that of the nucleus. Superimposition of\n values as low as 0.4 \u00b5M after 24 h of incubation for the MTT the images obtained upon excitation at 405 nm and 647 nm\n method for a thiazolylalanine carbene gold species.24 These gave crucial information to elucidate the possible localisation\n facts probes that cytotoxic activity comes mainly from the gold pattern. Hence, (Fig. 9A) clearly showed as complexes 1-4\n fragment rather than the rhenium moiety. were taken up by the cell (red colour), with an emission coming\n A closer look to this result revealed that among the from the cytoplasmatic area except for the nucleus, which is\n\n\n\n\n Chemical Science Accepted Manuscript\n monometallic complexes those bearing an imidazole derivative lighted up in blue due to the specific staining of DRAQ5, (Fig\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n are the least cytotoxic species, i.e. complexes 3 and 4. In 9B). Such localisation pattern has been previously seen for\n contrast, among the heterometallic complexes, those having an several tricarbonyl bisimine rhenium derivatives and it is the\n alkynyl in their chemical structure turned to be the most typical localisation of monocationic Re complexes; some\n cytotoxic species reaching IC50 values of even as low as 4 \u00b5M, general cytoplasmatic staining with more intense mitochondrial\n (Fig. 8 and Fig. S5). localisation and no emission from the nucleus.10(a-c),25\n Negligible emission from the nuclei indicates the exclusion of\n the complexes from the nucleous, which seems to be also an\n common feature for monocationic Re complexes.10f,26\n\n\n\n\n Fig. 8 Representation of toxicity values of complexes 1-8 obtained by annexin-V\n analysis.\n\n\n This result is in concordance with the idea that the presence of\n alkynyl groups increases the cytotoxic activity of the probe.4 In\n addition, examination of the cell morphology suggested that\n cellular death might be due to apoptosis for species 1 and 2,\n with cell shrinking and blebbing. For species 3-4 and 5-8 some\n apoptotic cells can be observed but cellular death could be due\n to both apoptosis and necrosis (Fig. S6).\n In an attempt to assess whether these complexes could be used\n as contrast agents, they were incubated with A549 lung\n carcinoma cells and their emission was examined by\n fluorescent confocal microscopy. Complexes were loaded at\n concentrations below their IC50 values, i.e. monometallic\n species 1-4 at 150 \u00b5M and complexes 5-8 at 10, 10, 5 and 15\n \u00b5M respectively. In addition, a known DNA co-staining\n fluorescent dye, DRAQ5, was used as an internal standard in\n order to ascertain the site of cellular localisation. Confocal\n fluorescent images were taken after excitation at 405 nm and\n 647 nm. Using a 405 nm laser excitation, only emission from Fig. 9 Images of complexes 1-4 incubated with A549 cells for 4 h at 150 \u00b5M. (A)\n the complexes 1-8 was observed, whereas exciting at 647 nm Images upon excitation at 405 nm. (B) Superimposed image upon excitation at\n the emission displayed was that of the known fluorescent dye 405 nm and 647 nm. Please note that images from complex 2 and 3 were taken\n with an objective of \u00d710 which might lower the resolution of the picture.\n localised in the nucleus, where the DNA is concentrated.\n Therefore, upon excitation at 405 nm cells incubated with\n complexes 1-4 showed an emission coming from almost all the\n\n\n 6 | J. Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f Page 7 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n Heterometallic species 5-8 showed a similar pattern to that of\n monometallic species, with the strongest luminescence\n emission coming from specific spots in the cytoplasm and none\n of complexes seemed to enter the nucleus either, (Fig 10).\n Several factors within these species, such as their cationic\n nature, their lipophilicity and being thiol and selenol reactive\n because of the presence of gold(I), point out the possibility of\n considering the mitochondria as the possible target.\n Comparison with published reports of Au(I) complexes\n supports the mitochondrial localisation hypothesis, as\n\n\n\n\n Chemical Science Accepted Manuscript\n cancerous cells bear an elevated expression of thioredoxin\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n reductase which is known to be inhibited by Au(I) species.27 It\n is worth pointing out that even though the loading\n concentration was much lower for the heterometallic species,\n the emission intensity was similar, which is an indication of the\n ease internalization of the dye. Consequently, it could be\n rationalised that the fragment \u2013AuPPh3 confers higher\n lipophilicity to the probe, and facilitates the cell membrane\n permeability.\n\n\n\n\n Fig. 11 Images of complexes 5-7 incubated with A549 cells for 4 h at 10, 10 and 4\n \u00b5M respectivelly. (A) Images upon excitation at 405 nm. (B) Superimposed image\n upon excitation at 405 nm and 647 nm.\n\n\n Moreover, these heterometallic species bearing an alkynyl\n group, complexes 5-7, seemed to present a different localisation\n pattern than this lacking of an alkynyl group, complex 8 (Fig\n 12). Therefore, complex 8 showed emission spread out along\n the cell including the nucleus whereas complexes 5-7 apart\n from displaying an emission dispersed all along the cell,\n seemed to accumulate in a specific organelle in the nucleus.\n\n Fig. 10 Images of complexes 6-7 incubated with A549 cells for 4 h at 150 \u00b5M. (A)\n Images upon excitation at 405 nm. (B) Superimposed image upon excitation at\n 405 nm and 647 nm.\n\n\n Finally, the cell imaging assay of the heterometallic species 5-8\n was repeated using the same loading concentration as in the\n case of monometallic species 1-4, i.e. 150 \u00b5M. The aim of this\n new experiment was to make a fair comparison between both\n Fig. 12 Images of complexes 8 incubated with A549 cells for 4 h at 15 \u00b5M\n set of complexes regarding their uptake level as well as to\n respectivelly. (A) Images upon excitation at 405 nm. (B) Superimposed image\n analyse the possibility of another localisation pattern. Indeed, upon excitation at 405 nm and 647 nm.\n all of them revealed a completely different behaviour than the\n previously observed (Fig. 11). Complexes 5-7, i.e. the The superimposed image of Fig. 11B revealed a clear overlap\n heterometallic species bearing an alkynyl group, presented of the red emission belonging to species 5-7 with the blue\n higher internalisation than the monometallic analogues 1-4, and emission coming from DRAQ5, which is a clear indication of\n also than the mixed Re(I)/Au(I) complex lacking of alkynyl, the nuclear staining. Moreover, a closer look to an amplified\n complex 8. The laser power for visualising the cells incubated image revealed that complexes 5-7 not only get accumulated in\n with those alkynyl heterometallic derivatives (5-7) had to be the nucleus but also, they seem to localise preferentially in an\n lowered from 40% to c.a. 2% in order not to damage the area inside the nucleus that it is assumed to be the nucleolus\n detector, which revealed their higher concentration in the cell. (Fig. 13).\n Such result, suggested some sort of relationship between\n cellular uptake and heterometallic akynyl derivatives.\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 7\n\f Chemical Science Page 8 of 14\n View Article Online\n ARTICLE Journal Name\n DOI: 10.1039/C4SC01684J\n\n\n The presence or absence of an alkynyl moiety in the ligand\n structure, did not affect these results, proposing the\n heterometallic character as the key point for the membrane\n permeabilisation. Such analysis agrees with the cytotoxicity\n levels found for each complex, being the heterometallic\n complexes 5-8 those with the lowest IC50 values. Hence, in\n view of these results, it can be proposed that the higher toxicity\n might promote the disruption of the cellular membrane and in\n consequence those probes get internalized easily, i.e. species 5-\n 7. Moreover, it is also worth mentioning that all of them bear\n\n\n\n\n Chemical Science Accepted Manuscript\n the fragment \u2013AuPPh3, which should increase the lipophilicity\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n of the probe. In addition, species 8, the only synthesized\n heterometallic species lacking of an alkynyl group and the less\n cytotoxic (IC50 of 19 \u00b5M), does not have the same level of\n internalisation as their analogous complexes 5-7, but its\n localisation pattern is alike. Such findings go along with the\n Fig. 13 Amplified image of complex 7 (red colour: emission from the synthesised idea that the localisation pattern could be related to the presence\n complexes, blue colour: emission from DRAQ5).\n of the fragment \u2013AuPPh3. Some examples published by Ott and\n coworkers on naphthalimide phophine gold derivatives as\n On the other hand, complex 8 seemed to behave in an anticancer metallodrugs, emphasises that uptake increases upon\n intermediate manner between the monometallic (1-4) and exposure to toxic concentrations as it is supposed to be\n heterometallic alkynyl derivatives (5-7) as the internalisation associated with a breakdown of the cell membrane integrity.28\n level is similar to that seen for complexes 1-4 but instead its They observed that localisation of those gold phosphine\n localisation pattern resembles to that of species 5-7. Hence, derivatives is preferably in the cell nuclei, which is the same\n complex 8 exhibited a luminescence spread over the cell, behaviour displayed by complexes 5-8. Further investigations\n including the nucleus, although no clear accumulation of the on the bio-activity of alkynyl phosphine gold derivatives also\n probe seems to take place in the nucleolus (see Fig 12). In an suggest the fragment \u2013AuPPh3 as the responsible to bring the\n attempt to shed a bit of light to the behaviour of complexes 1-8 molecules to the site of action within the cell.6f This\n incubated with A549 cells, the integrity of the cell membrane rationalization together with the high toxicity associated with\n was analysed by a cell viability test. Trypan blue, a dye widely these species might explain the reason why the heterometallic\n used for selective staining of dead tissues or cells, was added complexes 5-8 have a different localisation pattern than\n after incubation in A549 cells for 4 h. Fig. 14 shows that cells complexes 1-4. In addition to these examples on gold\n incubated with complexes 5-8 at concentrations of 150 \u00b5M complexes in cell imaging, some others metallic bioprobes such\n allowed Trypan blue to cross the cell membrane, which is an as luminescent lanthanide complexes published by Parker and\n indication of membrane permeabilisation. In contrast, cell co-workers pointed out that disrupting the cell permeability\n incubated with complexes 1-4 at the same concentration with surfactants or increasing the probe concentration or\n remained healthy. incubation times induce localisation in the nucleoli and\n ribosomes.29 They also demonstrated that localisation patterns\n can be influenced by the loading concentration. Consequently,\n some reported lanthanide species incubated at concentration\n beyond their IC50 revealed nucleolar staining, whereas\n incubation of those at lower concentration presented a different\n pattern.30 These findings completely agrees with the behaviour\n seem for the heterometallic complexes described in here,\n complexes 5-8, corroborating their nucleolar localisation. It is\n worth mentioning that although the highest level emission of\n complexes 5-7 is coming from the nucleus and nucleolus, there\n is also a weak luminescence throughout the entire cell. Such\n luminescence spread over the entire cell was observed as well\n in some alkynyl phosphine gold complexes reported by Dyson\n and coworkers.6d In this case, the low fluorescence intensity\n observed prevented the subcellular compartment from being\n Fig. 14 Cell viability test (Blue colour: emission from Trypan blue indicating that identified. This drawback has been overcome in the present\n the cell membrane has been disrupted upon incubation with the different\n complexes. publications with the synthesis of heterometallic Re(I)/Au(I)-\n species, where the Re fragment is assisting to increase the\n\n\n\n 8 | J. Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f Page 9 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n luminescence intensity whereas the Au fragment is controlling using an Olympus FV10-i Oil type compact confocal laser\n the main biological aspects, i.e. localisation and cytotoxicity. microscope using an \u00d710 or \u00d760 objective, with excitation\n wavelength at 405 nm and 647 nm.\n Experimental The integrity of the plasmatic membrane was analysed by the\n Trypan-blue exclusion test. Cells were treated for 4 h with\n General Measurements and Analysis Instrumentation compounds 1-8 at a concentration of 150 \u00b5M in 24-well plates.\n C, H, and N analysis were carried out with a PERKIN-ELMER 2400 Then, 50 \u00b5L of Trypan blue solution (0.4 % w/v in NaCl 0.15 M)\n microanalyzer. Mass spectra were recorded on a BRUKER were added to wells and cells were observed and photographed in an\n ESQUIRE 3000 PLUS, with the electrospray (ESI) technique and on optical microscope at 400x magnification.\n a BRUKER (MALDI-TOF). 1H, 13C{H} and 31P NMR, including 2D Cell death after treatment with compounds 1-8 was analysed by\n\n\n\n\n Chemical Science Accepted Manuscript\n experiments, were recorded at room temperature on a BRUKER measuring exposure of phosphatidylserine. Cells were treated for 24\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n AVANCE 400 spectrometer (1H, 400 MHz, 13C, 100.6 MHz, 31P, h with different concentrations (0.1-150 \u00b5M) of compounds. Cell\n 162 MHz) with chemical shifts (\u03b4, ppm) reported relative to the morphology after treatment with compounds was evaluated by\n solvent peaks of the deuterated solvent. Infrared spectra were optical microscopy (400 x magnification) and representative fields\n recorded in the range 4000\u2013250 cm\u22121 on a Perkin-Elmer Spectrum were photographed. Then, cells were trypsinized and incubated at\n 100 FTIR spectrometer. Room temperature steady-state emission 37\u00baC for 15 minutes in ABB (140 mM NaCl, 2.5 mM CaCl2, 10 mM\n and excitation spectra were recorded with a Jobin-Yvon-Horiba Hepes / NaOH, pH 7.4) containing 0.5 \u00b5g/ml AnnexinV-PE. Finally,\n fluorolog FL3-11 spectrometer fitted with a JY TBX picosecond cells were diluted to 0.5 ml with ABB and analysed by flow\n detection module. Lifetime measurements were recorded with a cytometry (FACScan, BD Bioscience, Spain).\n Datastation HUB-B with a nanoLED controller and DAS6 software.\n Materials and Procedures\n The nanoLED employed for lifetime measurements was one of 390\n nm with pulse lengths of 0.8\u20131.4 ns. The lifetime data were fitted The intermediate fac-[Re(bipy)(CO)3(CF3SO3)] was prepared\n with the Jobin-Yvon software package. UV/vis spectra were according to literature procedures.12b,c Complex 4, specifically\n recorded with a 1cm quartz cells on an Evolution 600 fac-[Re(bipy)(CO)3(ImH)](CF3SO3), has been prepared using a\n spectrophotometer. modified method to that one reported in the literature for the\n synthesis of the analogous hexafluorophospate salts,13 see\n Crystal Structure Determinations. below. All other starting materials and solvents were purchased\n Crystals were mounted in inert oil on glass fibers and transferred to from commercial suppliers and used as received unless\n the cold gas stream of an Xcalibur Oxford Diffraction diffractometer otherwise stated.\n equipped with a low-temperature attachment. Data were collected [Au(C\u2261CImMe)PPh3]: 5-ethynyl-1-methyl-1H-imidazole (36\n using monochromated MoK\u03b1 radiation (\u03bb= 0.71073 \u00c5). Scan type \u03c9. \u00b5l, 0.35 mmol) and KOH (59 mg, 1.05 mmol) were stirred in\n Absorption correction based on multiple scans were applied using methanol (5 ml) for 5 minutes under an argon atmosphere.\n spherical harmonics implemented in SCALE3 ABSPACK scaling Then, [AuClPPh3] (183 mg, 0.37 mmol) dissolved in a mixture\n algorithm. The structures were solved by direct methods and refined of methanol acetone (1:1) was added and the reaction was kept\n on F2 using the program SHELXL-97.31 All non-hydrogen atoms stirring for 12 hours. The white solid precipitated was filtered\n were refined anisotropically, with the exception of complex 7. and washed with further methanol to furnish 140 mg, 75 %\n Further details on the crystal refinements are collected in Table 4. yield. 1H NMR (400 MHz, Acetone) \u03b4 7.69-7.55 (m, 15H, 3Ph),\n 7.47 (s, 1H CH(2) Im), 6.93 (d, J = 1.0 Hz, 1H,CH(4) Im), 3.65\n Human Cell Studies and Cell Microscopy. (s, 3H, CH(Me)). ). 31P NMR (162 MHz, Acetone) \u03b4 42.46. 13C\n European Collection of Cell Cultures, were maintained in Hepes NMR (101 MHz, Acetone) \u03b4 137.9 (C(2)), 134.9 (d, 2J = 13.1\n modified minimum essential medium (HMEM) supplemented with Hz, Cortho, Ph), 132.7 (d, 4J = 2.4 Hz, Cpara, Ph), 132.3\n 10% fetal bovine serum, penicillin, and streptomycin. Cells were (C(4)), 130.8 (C(CCAu-)), 133.3(d, 3J = 11.3 Hz, Cmeta, Ph),\n detached from the plastic flask using trypsin-EDTA solution and 119.1 (C(5)), 90.1 (C(CCAu-)), 31.9 (C(Me)). Cipso(Ph) not\n suspended in an excess volume of growth medium. The observed. IR (solid, cm-1): 2159w (C\u2261C), MS ES m/z:\n homogeneous cell suspension was then distributed into 1 mL calculated for C24H20AuN2P (M+) 564.1, found 565.2 (MH)\n aliquots over a cover slip in a 24-well plate, with each aliquot being Complex 1: A solution of [Re(bipy)(CO)3(CF3SO3)] (100 mg,\n subject to incubation with the different complexes, final 0.17 mmol) and 2-PyC\u2261CH (72 \u00b5l, 0.68 mmol) in DCM (3 ml)\n concentrations varies from 125 \u00b5M to 4 \u00b5M, at 37 \u00b0C for 4 h. Cells were stirred for 5 days at room temperature under an argon\n were finally washed three times in phosphate buffer saline (PBS, pH atmosphere. Then, addition of ether afforded the precipitation\n 7.2). Then, 0.5 ml of paraformaldehyde 4% was added to fix the of a dark brown gel which was separated by decantation.\n cells at 37 \u00b0C for 15 min. Eventually, cells were washed three times Finally, a trituration process with ether afforded complex 1 as\n with phosphate buffer saline (PBS, pH 7) and they were mounted on light brown solid (32 mg, 27 % yield). 1H NMR (400 MHz,\n a slide for imaging where previously was added 5\u00b5l of Flouromont Acetone) \u03b4 9.56 (ddd, J = 5.5, 1.5, 0.7 Hz, 2H, CH(6) bipy),\n and DRAQ5 (1, 5-bis-[2-(di-methylamino)ethyl]amino]-4, 8- 8.73 (d, J = 8.2 Hz, 2H CH(3) bipy), 8.49 \u2013 8.41 (m, 3H CH(4)\n dihydroxyanthracene-9,10-dione) (2 \u00b5M). Preparations were viewed bipy, CH(6) Py), 8.03 \u2013 7.94 (m, 3H, CH(5) bipy, CH(4) Py),\n 7.77 (ddd, J = 7.9, 1.5, 0.7 Hz, 1H, CH(3) Py), 7.42 (ddd, J =\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 9\n\f Chemical Science Page 10 of 14\n View Article Online\n ARTICLE Journal Name\n DOI: 10.1039/C4SC01684J\n\n\n 7.5, 5.9, 1.6 Hz, 1H, CH(5) Py), 4.82 (s, 1H, CCH). 13C NMR obtained as a yellow solid (167 mg, 91 %).1H NMR (400 MHz,\n (101 MHz, Acetone) \u03b4, 196.3 (CO), 191.8(CO), 157.1 (C(2) CD2Cl2) \u03b4 11.89 (s, br, 1H, NH), 9.10 (ddd, J = 5.5, 1.5, 0.8 Hz,\n bipy), 156.4 (C(6) bipy), 153.6 (C(6) py), 146.3 (C(2) py), 2H, CH(6) bipy), 8.32 (d, J = 8.2 Hz, 2H, CH(3) bipy), 8.28 \u2013\n 142.34(C(4) bipy), 140.9(C(4) py), 133.4(C(3) py), 129.3 (C(5) 8.19 (m, 2H, CH(4) bipy), 7.70 (ddd, J = 7.6, 5.5, 1.3 Hz, 2H,\n bipy), 127.1(C(5) py), 125.6(C(3) bipy), 91.8 (CCH), 82.6 CH(5) bipy), 7.11 (s, 1H, CH(2)ImH), 6.93 (t, J = 1.4 Hz, 1H,\n (CCH). IR (solid, cm-1): 2028s (CO), 1928sh (CO), 1904s CH(4)ImH), 6.72 (t, J = 1.4 Hz, 1H, CH(5)ImH).14\n (CO), 2113w (C\u2261C), 3205m (H-CC), MS ES m/z: calculated for Complex 5: [Re(bipy)(CO)3(CF3SO3)] (98 mg, 0.17 mmol) and\n C20H13N3O3Re+ (M+) 530.1, found 529.8. [Au(C\u2261CPy-2)PPh3] (120 mg, 0.21 mmol) were stirred in DCM\n Complex 2: [Re(bipy)(CO)3(CF3SO3)] (200 mg, 0.34 mmol) (10 ml) for 72 h at room temperature under an argon\n and 3-PyC\u2261CH (118 mg, 1.14 mmol) were stirred in DCM (15 atmosphere. Then, the solvent was removed under reduced\n\n\n\n\n Chemical Science Accepted Manuscript\n ml) for 12 h at room temperature under an argon atmosphere. pressure and methanol was added in order to precipitated the\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Then, the volume of the reaction was reduced up to 1/3 under non-reacted pyridine derivative. The mixture was passed\n vacuum and ether was added affording the precipitation of 2 as through celite and the solvent was removed again. The\n a yellow solid (135 mg, 57 % yield). 1H NMR (400 MHz, brownish slurry left was redisolved in THF and further addition\n Acetone) \u03b4 9.52 (ddd, J = 5.5, 1.4, 0.7 Hz, 2H, CH(6) bipy), of ether afforded the precipitation of 5 as a brownish solid (100\n 8.78 (d, J = 8.2 Hz, 2H, CH(3) bipy), 8.63 \u2013 8.61 (m, 1H, mg, 52 % yield) 1H NMR (400 MHz, Acetone) \u03b4 9.73 (dd, J =\n CH(2) py), 8.57 (dd, J = 5.7, 1.0 Hz, 1H, CH(6) py), 8.48 (td, J 5.5, 0.7 Hz, 2H, CH(6) bipy), 8.62 (d, J = 8.2 Hz, 2H, CH(3)\n = 8.0, 1.5 Hz, 2H, CH(4) bipy), 8.09 \u2013 8.05 (m, 1H, CH(4) py), bipy), 8.39 (td, J = 8.0, 1.5 Hz, 2H, , CH(4) bipy), 8.35 \u2013 8.31\n 8.02 (ddd, J = 7.7, 5.5, 1.2 Hz, 2H, CH(5) bipy), 7.52 (ddd, J = (m, 1H, , CH(6) py), 7.89 \u2013 7.83 (m, 2H, CH(5) bipy), 7.80 (td,\n 8.0, 5.7, 0.7 Hz, 1H, CH(5) Py ), 4.09 (s, 1H, CCH). 13C NMR J = 7.8, 1.6 Hz, 1H, , CH(4) py), 7.77 \u2013 7.61 (m, 15H, Ph), 7.51\n (101 MHz, Acetone) \u03b4 196.1 (CO), 192.3 (CO), 156.9 (C(2) (dd, J = 8.0, 0.7 Hz, 1H, CH(3) py), 7.19 (ddd, J = 7.5, 6.0, 1.5\n bipy), 155.2 (C(2) py), 155.0 (C(6) bipy), 152.7 (C(6) py), Hz, 1H, , CH(5) py). 13C NMR (101 MHz, Acetone) \u03b4 196.5\n 143.7 (C(4) py), 142.4 (C(4) bipy), 130.0 (C(5) bipy), 127.7 (CO), 192.3 (CO), 157.1 (C(6) bipy), 156.8 (C(2) bipy), 152.5\n (C(5) py), 126.0 (C(3) bipy), 123.2 (C(3) py), 85.5 (CCH), 78.4 (C(6) py), 148.6 (C(2) py), 142.1 (C(4) bipy), 139.9 (C(4) py),\n (CCH). IR (solid, cm-1, \u03bd(CO)): 2029, 1904, MS ES m/z: 135.2 (d, 2J = 13.9 Hz, Cortho, ph), 133.1 (d, 4J = 1.9 Hz\n calculated for C20H13N3O3Re+ (M+) 530.0, found 530.2. Cpara, ph), 132.7 (C(3) py), 130.5 (d, 3J = 11.4 Hz, Cmeta, ph),\n Elemental analysis for C21H13F3N3O6ReS required C, 37.17; H, 130.0 (s, br, CCAu), 129.0 (C(5) bipy), 125.1, (C(3) bipy),\n 1.93; N, 6.19 %, found C, 37.25; H, 2.27; N, 6.24 %. 124.3 (C(5) py), 101.9 ((d, 3J = 26 Hz, CCAu). Cipso(Ph) not\n Complex 3: To a solution of 5-ethynyl-1-methyl-1H-imidazole observed. 31P NMR (162 MHz, Acetone) \u03b4 41.54. IR (solid, cm-\n 1\n (0.176 ml, 1.73 mmol) in dry DCM (15 ml) was added , \u03bd(CO)): 2026, 1915, MS ES m/z: calculated for\n [Re(bipy)(CO)3(CF3SO3)] (100 mg, 0.17 mmol) affording a C38H38AuN3O3PRe+ (M+) 989.1, found 988.3. Elem. Anal. for\n brown-orange suspension. The suspension was stirring at room C39H27AuF3N3O6PReS, required C, 41.20; H, 2.39; N, 3.70 %,\n temperature under an argon atmosphere for 12 hours. Then, the found C, 41.38; H, 2.62; N, 3.61%.\n volume of the solution was reduced up to 2/3 and ether was Complex 6: [Re(bipy)(CO)3(CF3SO3)] (52 mg, 0.09 mmol) and\n added dropwise to give an orange gel which was separated from [Au(C\u2261CPy-3)PPh3] (51 mg, 0.09 mmol) were stirred in DCM\n the mother liquid by decantation. Trituration of the gel with (10 ml) for 12 h at room temperature under an argon\n ether afforded complex 3 as a dark yellow solid (92 mg, 87% atmosphere. Then, the volume of the reaction was reduced up to\n yield). 1H NMR (400 MHz, Acetone) \u03b4 9.37 (dd, J = 18.7, 5.3 1/3 under vacuum and ether was added to precipitate 6 as a\n Hz, 2H, CH(6) bipy), 8.79 (d, J = 8.1 Hz, 2H, CH(3) bipy), yellow solid (45 mg, 44 % yield). 1H NMR (400 MHz,\n 8.46 (t, J = 7.9 Hz, 2H, CH(4) bipy), 8.05 (s, 1H, CH(2)Im), Acetone) \u03b4 9.54 (d, J = 4.8 Hz, 2H, CH(6) bipy), 8.76 (d, J =\n 7.94 (dd, J = 7.1, 6.1 Hz, 2H, CH(5) bipy), 7.05 (s, 1H, 8.2 Hz, 2H, CH(3) bipy), 8.48 (td, J = 8.0, 1.5 Hz, 2H, CH(4)\n CH(5)Im), 4.27 (s, 1H, CCH), 3.64 (s, 3H, CH3). 13C NMR bipy), 8.39 (d, J = 1.8 Hz, 1H, CH(2) py), 8.33 (d, J = 4.9 Hz,\n (101 MHz, Acetone) \u03b4 157.7 (C(2) bipy), 155.7 (C(6) bipy), 1H, CH(6) bipy), 8.03 (ddd, J = 7.6, 5.5, 1.2 Hz, 2H, CH(5)\n 143.3(C(2) M), 143.1 (C(4) bipy), 135.2 (C(4) Im), 130.7 (C(5) bipy), 7.90 \u2013 7.84 (m, 1H, CH(4) py), 7.72 \u2013 7.56 (m, 15H,\n bipy), 126.8 (C(3) bipy), 89.8 (CCH), 70.7 (CCH), 34.5 (CH3). 3Ph), 7.36 (dd, J = 7.7, 6.0 Hz, 1H, CH(5) py). 31P NMR (162\n CO and (C(5) Im) not observed. IR (solid, cm-1, \u03bd(CO)): 2025, MHz, Acetone) \u03b4 42.29. 13C NMR (101 MHz, Acetone) \u03b4\n 1918, 1894, MS ES m/z: calculated for C18H14N4O3Re+ (M+) 196.3 (CO), 192.4 (CO), 156.8 (C(2) bipy), 155.0 (C(6) bipy),\n 533.0, found 532.8. Elem. Anal. for 154.9 (C(2) py), 149.9 (C(6) py), 143.2 (C(4) py), 142.4 (C(4)\n C20H14F3N4O6PReS.CH3CN required C, 36.56; H, 2.37; N, 9.69 bipy), 134.5 (d, 2J = 13.8 Hz, Cortho, Ph), 132.9 (d, 4J = 2.5 Hz,\n %, found C,36.99 ; H, 2.22; N, 9.35%. Cpara, Ph), 130.4 (d, 3J = 11.4 Hz, Cmeta, Ph), 130.3 (d, J =\n Complex 4: [Re(bipy)(CO)3(CF3SO3)] (164 mg, 0.28 mmol) 56.8 Hz, Cipso, Ph 130.0(C(5) bipy), 127.3 (C(5) py), 126.7\n was added to a solution of imidazole (193 mg, 2.8 mmol) in (C(3) py), 125.9 (C(3) bipy), 97.0 (s, br, CCAu). CCAu not\n DCM (10ml). The mixture was stirred for 48 h at room observed. IR (solid, cm-1, \u03bd(CO)): 2028, 1903, \u03bd(CC): 2121.\n temperature under an argon atmosphere. Then, the volume of MS ES m/z: calculated for C38H38AuN3O3PRe+ (M+) 989.1,\n the reaction was reduced up to 2/3 under vacuum, and ether was found 988.4. Elemental analysis for\n added to force the precipitation of the desired product. 4 was\n\n\n 10 | J. Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f Page 11 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n C39H27AuF3N3O6PReS.2H2O required C, 39.44; H, 2.66; N, Complex 8: KOH (6.5 mg, 0.11 mmol) dissolved in MeOH (2\n 3.58 %, found C, 39.8; H, 2.44; N, 3.82 %. ml) was added dropwise to a solution of 4 (50 mg, 0.07 mmol)\n Complex 7: [Re(bipy)(CO)3(CF3SO3)] (100 mg, 0.17 mmol) and [AuClPPh3] (42 mg, 0.08 mmol) in THF (5ml). The\n was added to a solution of [Au(C\u2261CImMe)PPh3] (100 mg, 0.18 mixture was stirred at room temperature under an argon\n mmol) in DCM (5ml). The mixture was stirred for 12 h at room atmosphere for 1 hour. Then, the bulk reaction was filtered\n temperature under an argon atmosphere. Then, 2/3 of the through celite to eliminate the KCl formed during the reaction\n solvent was removed under vacuum to afford an orange gel, and the volume of the reaction was reduced until 1/3 under\n which was triturated with ether to give complex 7 as an orange vacuum. Further addition of ether forced the precipitation of\n solid (92 mg, 54 %). 1H NMR (400 MHz, Acetone) \u03b4 9.38 \u2013 complex 8 as a yellow solid (75 mg, 88 % yield). 1H NMR (400\n 9.29 (m, 2H, CH(6) bipy), 8.80 (d, J = 8.1 Hz, 2H, CH(3) bipy), MHz, CD2Cl2) \u03b4 9.09 (ddd, J = 5.5, 1.6, 0.8 Hz, 2H, CH(6)\n\n\n\n\n Chemical Science Accepted Manuscript\n 8.46 (td, J = 8.0, 0.8 Hz, 2H, CH(4) bipy), 8.01 \u2013 7.91 (m, 2H, bipy), 8.61 (dd, J = 7.3, 0.9 Hz, 2H, CH(3) bipy), 8.31 \u2013 8.25\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n CH(5) bipy), 7.89 (s, 1H, CH(2)Im), 7.69 \u2013 7.51 (m, 15H), 6.57 (m, 2H, CH(4) bipy), 7.67 (ddd, J = 7.7, 5.5, 1.2 Hz, 2H, CH(5)\n (s, 1H, CH(4)Im), 3.57 (s, 3H, CH3). 13C NMR (101 MHz, bipy), 7.62 \u2013 7.55 (m, 3H, CH(para) Ph), 7.54 \u2013 7.46 (m, 12H,\n Acetone) \u03b4 196.7 (CO), 192.9 (CO), 156.7 (C(2) bipy), 154.7 CH(ortho, meta) Ph), 7.16 (t, J = 1.0 Hz, 1H, CH(2)Im ), 6.78\n (C(6) bipy), 142.1 (C(4) bipy), 139.9 (C(2)Im), 135.0 (d, 2J = (t, J = 1.2 Hz, 1H, CH(Im)), 6.41 (t, J = 1.2 Hz, 1H, CH(Im)).\n 31\n 13.9 Hz, Cortho, Ph), 132.9 (s, br, Cpara, Ph), 131.0 (C(4)Im), P NMR (162 MHz, CD2Cl2) \u03b4 31.18. 13C NMR (101 MHz,\n 130.0 (s, br, CCAu), 130.4 (d, 3J = 11.3 Hz, Cmeta, Ph), 129.7 CD2Cl2) \u03b4 197.3 (CO), 192.8(CO), 156.3 (C(2) bipy), 153.4\n (C(5) bipy) , 125.8 (C(3) bipy), 121.5 (C(5)Im), 86.9 (d, 3J = (C(6) bipy), 144.2 (C(2) Im), 141.4 (C(4) bipy), 134.7 (d, 2J =\n 21.6 Hz, CCAu), 33.1 (CH3). Cipso(Ph) not observed. 31P NMR 13.5 Hz, Cortho, ph) 132.9 (d, 4J = 2.2 Hz, Cpara, ph), 130.0\n (162 MHz, Acetone) \u03b4 42.19. IR (solid, cm-1, \u03c5(CO)): 2025, (d, 3J = 11.9 Hz, Cortho, ph), 128.7 (C(5) bipy), 127.0 (C(Im)),\n 1930, 1893 MS ES m/z: calculated for C37H28AuN4O3PRe+ 126.8, (C(Im)), 125.41 (C(3) bipy). Cipso(Ph) not observed. IR\n (M+) 991.1, found 990.9. Elem. Anal. for (solid, cm-1, \u03bd(CO)): 2020, 1921, 1892-. MS ES m/z: calculated\n C38H28AuF3N4O6PReS required C, 40.04; H, 2.48; N, 4.92 %, for C34H24AuN4O3PRe+ (M+) 953.1, found 952.8. Elem. Anal.\n found C, 39.46; H, 2.44; N 4.99 % for C35H26AuF3N4O6PReS required C, 38.15; H, 2.38; N, 5.08\n %, found C, 38.52; H, 2.56; N, 4.94 %.\n\n\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 11\n\f Chemical Science Page 12 of 14\n View Article Online\n DOI: 10.1039/C4SC01684J\n\n\n Journal Name RSCPublishing\n\n\n Table 4 X-ray crystallographic data of complexes 2, 5, 7 and 8.\n\n Compound 2 5 7 8\n Formula C21H13N3F3O3RePS C41H33AuF6N3O4P2Re C21H13F3N3O6ReS C37H26AuN4F3O6.5RePS\n\n Mr 678.62 1190.81 1139.86 1133.81\n\n\n\n\n Chemical Science Accepted Manuscript\n Crystal size (mm) 0.42 \u00d7 0.20 \u00d7 0.18 0.42 \u00d7 0.18 \u00d7 0.04 0.44 x 0.14 x 0.04 0.36 \u00d7 0.34 \u00d7 0.22\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n Crystal system Monoclinic Triclinic Triclinic Triclinic\n\n Space group P2(1)/n P-1 P-1 P-1\n Cell constants:\n a (\u00c5) 13.1254(2) 9.46830(10) 8.9054(4) 13.0494(2)\n b (\u00c5) 13.0009(2) 16.4296(2) 9.0770(5) 13.4519(2)\n c (\u00c5) 13.7872(2) 29.0868(3) 22.9630(8) 22.5256(4)\n \u03b1 (\u00b0) 90 92.4970(10) 84.156(4) 95.9470(10)\n \u03b2 (\u00b0) 109.269(2) 92.6600(10) 88.572(3) 99.8470(10)\n \u03b3 (\u00b0) 90 91.2060(10) 88.527(4) 102.1570(10)\n V (\u00c53) 2220.88(6) 4748.77(8) 1845.48(15) 3768.28(10)\n Z 4 4 2 4\n Dx (Mg m-3) 2.030 1.954 2.051 1.999\n \u00b5(mm-1) 5.636 6.759 7.415 7.264\n F(000) 1304 2280 1088 2160\n T (K) 100(2) 100(2) 100(2) 173(2)\n 2\u03b8max 51 51 51 51\n No. of refl.:\n Measured 21904 60411 27131 72134\n Independent 4116 15009 6799 13940\n Transmissions 0.4303 \u2013 0.2006 0.7738 - 0.1635 0.7558-0.1388 0.2979 \u2013 0.1796\n Rint 0.0215 0.0429 0.0664 0.0299\n Parameters 316 1049 227 982\n Restraints 0 0 0 0\n 2\n Goodness of fit on F 1.085 1.037 1.142 1.103\n wR(F2, all Refl.) 0.0352 0.0641 0.2280 0.0551\n R(I >2\u03c3(I)) 0.0144 0.0272 0.0873 0.0244\n max. \u2206\u03c1 (e \u00c5-3) 0.871 1.212 7.266 1.155\n\n\n\n\n heterometallic Re(I)/Au(I) derivatives presented values of IC50\n Conclusions more than 10 times lower than their analogous Re(I)\n complexes. In addition, among the heterometallic species, the\n In the search of new anti-cancer and diagnosis agents, two new\n presence of alkynyl groups increases the toxicity of the\n families of luminescent fac-[Re(bipy)(CO)3(L)]+ and fac-\n bioprobes. Although this statement is made by comparison of\n [Re(bipy)(CO)3(L-AuPPh3)]+, where L is an imidazole,\n three alkynyl species, complexes 5, 6 and 7, with the single\n alkynyl-imidazole or alkynyl-pyridine derivative, have been\n species lacking of an alkynyl group, complex 8, it agrees with\n synthesised and characterised. Cytotoxicity studies performed\n published reports that support the same idea.4,6 These results\n in human A549 lung cancer cells revealed that the\n revealed the feasibility of easily modulate the cytotoxicity of\n\n\n This journal is \u00a9 The Royal Society of Chemistry 2013 J. Name., 2013, 00, 1-3 | 12\n\f Page 13 of 14 Chemical Science\n View Article Online\n Journal Name ARTICLE\n DOI: 10.1039/C4SC01684J\n\n\n the probes upon their chemical structure, reaching their cooperative partners in the fields of drug biodistribution,\n maximum cytotoxic effect when an alkynyl group and a visualization and cancer therapy.\n phosphine gold fragment are brought together. Moreover,\n fluorescence cell imaging pointed out the different bio- Acknowledgements\n distribution as well as uptake level depending on the\n Authors thank the Ministerio de Econom\u00eda y Competitividad\n concentration loadings and nature of the probes. Therefore,\n (MINECO/FEDER CTQ2010-20500-C02-01 and SAF2010-\n whereas rhenium species 1-4 followed the typical\n 14920) and DGA-FSE (E77 and B16) for financial support and\n biodistribution of monocationic rhenium species, i.e. general\n J. M. L\u00f3pez-de-Luzuriaga for lifetime measurements.\n cytoplasmatic staining and likely mitochondrial localisation, the\n heterometallic Re(I)/Au(I) species 5-8 displayed a more intense\n Notes and references\n\n\n\n\n Chemical Science Accepted Manuscript\n luminescence suggesting a higher uptake level and a\nPublished on 08 July 2014. Downloaded by University of Pittsburgh on 09/07/2014 17:45:27.\n\n\n\n\n complicated localisation pattern. Loadings at concentrations a\n Departamento de Qu\u00edmica Inorg\u00e1nica, Instituto de S\u00edntesis Qu\u00edmica y\n beyond their IC50 value promoted a high uptake level possibly Cat\u00e1lisis Homog\u00e9nea (ISQCH), Universidad de Zaragoza-CSIC, E-50009\n due to the disruption of the cell membrane. Then, accumulation Zaragoza, Spain. E-mail: gimeno@unizar.es; Fax: +34 976761187; Tel: +34\n in the nucleus and nucleolus, where proteins are concentrated, 976762291 and vanesa@unizar.es; Tel: +34 976763523.\n could be proposed because of the great affinity of Au(I) species b\n Departamento de Bioqu\u00edmica y Biolog\u00eda Celular, Universidad de Zaragoza,\n for S donor ligands. The exact mechanism according to which\n E-50009 Zaragoza, Spain.\n these heterometallic species present such behaviour is not clear. \u2020Electronic supplementary information (ESI) available: Structural features\n The reaction with thiol residues (RSH) could afford either (fac- secondary interactions and hydrogen bonds of 5 and 8 (Fig. S1\u2013S3); UV-Vis\n [Re(bipy)(CO)3(L-Au-SR)]+) species, if the phosphine fragment and emission spectra of complexes 1, 3-5, 7 and 8 (Fig. S4); Annexin-V\n is displaced by the thiol residue or (fac-[Re(bipy)(CO)3(L)]+) analysis graph of complexes 1-8 (Fig. S5); Cell morphology pictures of\n and (RS-Au-PR3), if the alkynyl group is displaced instead. complexes 1-8. (Fig. S6); CCDC 996045\u2013996048.\n Taking in mind that the bond length of Au-P is longer than Au-\n 1 E. R. T. Tiekink, Inflammopharmacology, 2008, 16, 138.\n C, which can be used as an indication of bond strength, and also\n 2 (a) I. Ott, Coord. Chem. Rev., 2009, 253, 1670; (b) B. Bertrand and\n knowing that photoelectron spectroscopy measurements\n A. Casini, Dalton trans., 2014, 43, 4209.\n together with theoretical studies have shown that the Au-C 3 (a) L. Ortego, F. Cardoso, S. Martins, M. F. Fillat, A. Laguna, M.\n bond in Au-alkynyl complexes represent one of the strongest Meireles, M. D. Villacampa and M. C. Gimeno, J. Inorg. Biochem.,\n gold-ligand bonds,32 the formation of -[Re(bipy)(CO)3(L-Au- 2014, 130, 32; (b) M. P. Rigobello, L. Messori, G. Marcon, M. A.\n SR)]+) seems to be more likely. Moreover, the nucleous and Cinellu, M. Bragadin, A. Folda, G. Scutari, A. Bindoli, J. Inorg.\n nucleolus remained lighted up during the experiment, which Biochem., 2004, 10, 1634; (c) P. J. Barnard, S. J. Berners-Price, Coord.\n suggest that the fragment of the bioprobe providing the Chem. Rev., 2007, 251 1889; (d) K. Yan, C.-N. Lok, K. Bierlab and C.-\n luminescence remains trapped. Again, the mechanism where - M. Che, Chem. Commun., 2010, 46, 7691; (e) E. Vergara, A. Casini, F.\n [Re(bipy)(CO)3(L-Au-SR)]+) is formed seemed to be the Sorrentino, O. Zava, E. Cerrada, M. P. Rigobello, A. Bindoli, M. Laguna\n expected as nothing would prevent (fac-[Re(bipy)(CO)3(L)]+) and P. J. Dyson, ChemMedChem, 2010, 5, 96; (f) V. Gandin, A. P.\n to leave the nucleus and nucleolus. In contrast, loadings at Fernandes, M. P. Rigobello, B. Dani, F. Sorrentino, F. Tisato, M.\n concentrations below their IC50 revealed a possible Bj\u00f6rnstedt, A. Bindoli, A. Sturaro, R. Rella and C. Marzano, Biochem.\n mitochondrial localisation. This result is in agreement with the Pharmacol, 2010, 79, 90; (g) J. L. Hickey, R. A. Ruhayel, P. J. Barnard,\n affinity of Au(I) inhibiting the mitochondrial thioredoxin M. V. Baker, S. J. Berners-Price and A. Filipovska, J. Am. Chem. Soc.,\n reductase. The same mechanism than the one described 2008, 130, 12570; (h) S. Urig, K. Fritz-Wolf, R. R\u00e9au, C. Herold-Mende,\n previously can be proposed for the interaction with the K. T\u00f3th, E. Davioud-Charvet and K. Becker, Angew. Chem. Int. Ed.,\n thioredoxin reductase. In view of this results, it could be 2006, 45, 1881.\n postulated that (fac-[Re(bipy)(CO)3(L-)]+) fragment emerges\n 4 D.-L. Ma, T. Y.-T. Shum, F. Zhang, C.-M. Che and M. Yang, Chem.\n not only as an excellent luminescent associate, able to light up\n Commun., 2005, 4675.\n the cell, but also can easily host bioactive species without\n 5 J. C. Lima and L. Rodriguez, Anticancer Agents Med. Chem., 2011,\n interfering in biological role and allowing to visualise their\n 11, 921.\n biodistribution. Moreover, the synthetic feasibility of alkynyl\n 6 (a) A. Meyer, A. Guti\u00e9rrez, I. Ott and L. Rodriguez, Inorg. Chim.\n gold complexes together with their demonstrated bioactivity\n Acta, 2013, 398, 72; (b) A. Meyer, C. P. Bagowski, M. Kokoschka,\n opens a wide variety of possibilities for the design of more\n M. Stefanopoulou, H. Alborzinia, S. Can, D. H. Vlecken, W. S.\n sophisticated heterometallic bioprobes. Although these are\n Sheldrick S. W\u00f6lfl and I. Ott, Angew. Chem. Int. Ed., 2012, 51, 8895;\n preliminary results and no other reports dealing with the\n (c) R. G. Balasingham, C. F. Williams, H. J. Mottram, M. P. Coogan\n biological aspects of heterometallic alkynyl Re(I)/Au(I) species\n and S. J. A. Pope, Organometallics, 2012, 31, 5835; (d) E. Vergara,\n have been published yet, there is a remarkable future for these\n E. Cerrada, A. Casini, O. Zava, M. Laguna and P. J. Dyson,\n type of heterometallic complexes. The synergy effect attained\n Organometallics, 2010, 29, 2596; (e) C.-H. Chui, R. S.-M. Wong, R.\n by the luminescent rhenium fragment (fac-[Re(bipy)(CO)3(L-\n Gambari, G. Y.-M. Cheng, M. C.-W. Yuen, K.-W. Chan, S.-W.\n )]+) and the bioactive gold fragment (-C\u2261CAuPPh3) is unique\n Tong, F.-Y. Lau, P. B.-S. Lai, K.-H. Lam, C.-L. Ho, C.-W. 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Name., 2012, 00, 1-3 This journal is \u00a9 The Royal Society of Chemistry 2012\n\f", "pages_extracted": 15, "text_length": 178109}

Luminescent Re(i) and Re(i)/Au(i) complexes as cooperative partners in cell imaging and cancer therapy