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Organometallics 2011, 30, 20–27 DOI: 10.1021/om100964h
Bioorganometallics: Future Trends in Drug Discovery, Analytical Chemistry, and Catalysis†,‡ Elizabeth A. Hillard* and Gerard Jaouen* Chimie ParisTech (Ecole Nationale Sup erieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France Received October 5, 2010
Bioorganometallic chemistry’s beginnings 25 years ago were timid, modest and sporadic, overshadowed by the supremacy of organometallic catalysis at that time. Its development since the beginning of the present century has, however, been exponential. We provide examples of particularly innovative results that may serve as a springboard for future developments. Medicinal organometallic chemistry is illustrated by antimetastatic drugs, kinase inhibitors, and antiproliferative agents with redox activity. We also describe organometallic bioprobes for nuclear medicine, with Re-PNA conjugates that are non-toxic and photo-stable, and the related development of photothermal induced resonance. Particular stress is placed on organometallic enzymes, both the natural enzymes that are a source of inspiration for hydrogenproducing experimental systems, and artificial enzymes that mimic a different evolutionary path from the one created by the oxidizing atmosphere of the earth, itself the result of photosynthesis. These significant contemporary results may serve to shed light on future developments in this multidisciplinary field.
Introduction Creativity is often closely related to technological advances. For instance, Impressionist art benefited from new paints that could be used outdoors, and the French cinematic New Wave took advantage of new, more sensitive 400 ASA film stock. Likewise, the birth of modern bioorganometallic chemistry can be linked to the advent of FT-IR spectroscopy, which permitted the study of metal carbonyls bound to proteins.1 However, important breakthroughs only occur when the time, place, and conditions are right for taboos to be overcome. Indeed, bioorganometallics, a multidisciplinary field dedicated to the synthesis and study of biologically interesting organometallic complexes has had, under other names, some scattered, sporadic, and isolated precedents, some of them, such as the structural resolution of vitamin B12, very well-known. However, at the start of the 1980s organometallic catalysis was at its height, leaving little room for other angles of attack. In addition, it was assumed that organometallic complexes were essentially incompatible with oxygen and water, a belief that dismissed as illusory any hope of using these complexes in biology. Yet, as has subsequently been demonstrated, nature itself had long ago selected the organometallic option in the form of, for example, hydrogenases and CO dehydrogenases. †
Part of the special issue Future of Organometallic Chemistry. See the end of the text for a glossary of biological terms. *To whom correspondence should be addressed. E.A.H.: tel, þ33 (0)1 44 27 66 98; e-mail,
[email protected]. G.J.: tel, þ33 (0)1 43 26 95 55; e-mail,
[email protected]. (1) Jaouen, G.; Vessieres, A.; Top, S.; Ismail, A. A.; Butler, I. S. J. Am. Chem. Soc. 1985, 107 (16), 4778–4780. (2) Top, S.; Jaouen, G.; Vessieres, A.; Abjean, J. P.; Davoust, D.; Rodger, C. A.; Sayer, B. G.; McGlinchey, M. J. Organometallics 1985, 4, 2143–2150.
Bioorganometallic chemistry, defined as a topic in 1985,2,3 has enjoyed a steady growth in the last 20 years, as evinced by histograms (Figure 1) summarizing the number of publications and their citations found in the ISI Web of Science since 1990. Bioorganometallics encompasses aspects of environmental science, toxicology, metallomics, energy, biosensors, radiopharmaceuticals, natural and artificial enzymes, and bioanalysis; our discussion will be limited to a few highlights of recent innovations in drug discovery, analytical chemistry, and catalysis.
Organometallic Medicinal Chemistry Although the organometallic pharmaceutical Salvarsan was used as a treatment for syphilis in the early 20th century,4 very few organometallic compounds have reached the clinic in the meantime. One of the rare examples is the use of ferrocene as an alternative to iron salts for administration of Fe(II) for the treatment of iron-deficiency anemia.5 Although no organometallic therapeutics are currently used in the clinical setting, the ferrocenyl derivative of chloroquine is now in advanced phase II trials for the treatment of chloroquine-resistant malaria.6 Research in organometallic pharmaceuticals began to gather momentum in the 1990s and now enjoys visibility, particularly in Europe, via such programs as the Biological Function of Organometallic Compounds research group
‡
pubs.acs.org/Organometallics
Published on Web 01/04/2011
(3) Jaouen, G.; Vessieres, A. Pure Appl. Chem. 1985, 57, 1865–1874. (4) Jaouen, G.; Top, S.; Vessieres, A. In Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim, Germany, 2006; pp 65-95. (5) Nesmeyanov, A. N.; Bogomolova, L. G.; Viltchevskaya, V.; Palitsyne, N.; Andrianova, I.; Belozerova, O. Patent Ferrocerone 119,356, 1971. (6) Dive, D.; Biot, C. ChemMedChem 2008, 3 (3), 383–391. r 2011 American Chemical Society
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Figure 1. Number of articles published and their citations for the last 20 years using the query “bioorganometallic” for a search of the Web of Science database (September 2010).
(Deutsche Forschungsgemeinshaft) and the current European Union COST Action D39 (Metallo-Drug Design and Action). A sign of the growing maturity of this subject is the current emphasis on mechanistic studies. Indeed, the following examples demonstrate the search for novel mechanisms in cancer therapy, where the classical cell culture antiproliferation tests often play only a small role. RAPTA Antimetastatics. Although metastatic tumors are generally the most resistant to treatment, there exist very few drugs that show selective antimetastatic activity. The coordination complex imidazolium trans-[tetrachloro(S-dimethyl sulfoxide)(1H-imidazole)ruthenate(III)] (NAMI-A)7 was recently found to exhibit such activity and is currently undergoing clinical trials. Paul Dyson’s group discovered an organometallic ruthenium(II) compound, [(η6-toluene)Ru(pta)Cl2] (RAPTA-T, where pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane; Figure 2), which demonstrates a selectivity toward metastasis similar to that of NAMI-A.8 The stability of the organometallic scaffold provides an ideal foundation for rational drug design, and a series of RAPTA derivatives have been prepared and studied. Like NAMI-A, RAPTA compounds are not very cytotoxic and their antimetastatic effects were established from in vitro assays that mimic steps in metastatic progression and on in vivo models.9,10 Notably, RAPTA compounds alter the expression and the activity of numerous key proteins involved in metastatic progression and in the regulation of the cell cycle. Unlike classical metal-based cytotoxins, DNA does not seem to be a target; RAPTA compounds instead induce multiple effects on the cell and appear to act on both extraand intracellular targets. Indeed, a high affinity for proteins, particularly clinically relevant sulfur-rich enzyme targets, has been observed.11 Specific binding seems to be determined by steric and hydrophobic effects endowed by the arene ligand, and modification of this ligand appears to be a promising direction for the development of new RAPTA drugs. (7) Pacor, S.; Zorzet, S.; Cocchietto, M.; Bacac, M.; Vadori, M.; Turrin, C.; Gava, B.; Castellarin, A.; Sava, G. J. Pharmacol. Exp. Ther. 2004, 310, 737–744. (8) Dyson, P. J.; Sava, G. Dalton Trans. 2006, 16, 1929–1933. (9) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Gelbach, T. J.; Sava, G.; Dyson, P. J. J. Med. Chem. 2005, 48, 4161–4171. (10) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Int. J. Oncol. 2008, 33, 1281–1289. (11) Casini, A.; Gabbiani, C.; Sorrentino, F.; Rigobello, M. P.; Bindoli, A.; Geldbach, T. J.; Marrone, A.; Re, N.; Hartinger, C. G.; Dyson, P. J.; Messori, L. J. Med. Chem. 2008, 51, 6773–6781.
Figure 2. (a) Structure of RAPTA-C (C corresponds to p-cymene). (b) X-ray crystallographic analysis of an ethacrynate-RAPTA compound bound to human GST P1-1.
In one such example, the arene group in the RAPTA structure was modified by ethacrynic acid, a potent inhibitor of glutathione-S-transferase (GST), a drug-resistant enzyme often overexpressed in metastatic tumors as well as primary tumors.12 Indeed, it appears that this compound inhibits GST, thereby sensitizing the cell toward apoptosis, and subsequent release of the ruthenium ion from the enzyme instigates apoptosis. The ethacrynate-RAPTA compound bound to two (12) Ang, Wee H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Angew. Chem., Int. Ed. 2009, 48 (21), 3854–3857.
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Hillard and Jaouen Scheme 1. Oxidative Activation of Ferrocenyl Phenol to Toxic Quinone Methide Form
Figure 3. (a) Illustration of an octahedral pyridocarbazole metal complex bound to the active site of a protein kinase. The coordinating ligands A-D are capable of controlling kinase affinities and selectivities, if arranged properly. (b) Two examples of very selective organometallic protein kinase inhibitors.
cysteine residues in human GSTP1-1 with the ethacrynate ligand directed into the enzyme active site has been obtained (Figure 2). Kinase Inhibitors. Eric Meggers’ group has designed metal complexes that interact with the ATP-binding site of protein kinases by mimicking the binding of ATP (Figure 3). The pyridocarbazole moiety forms two key hydrogen bonds with the hinge region of the ATP binding site, while additional ligands in the coordination sphere of the metal center interact with other areas of the ATP binding site.13 Through traditional medicinal chemistry approaches, such as combinatorial chemistry and structure-based design, a series of highly potent kinase inhibitors have been developed. While initial studies dealt mainly with half-sandwich scaffolds, recent work has focused on pure octahedral coordination spheres to economically access structures with defined and rigid shapes to occupy enzyme active sites. For example, the octahedral organoruthenium complex Λ-FL172 was designed as a selective inhibitor for the p21-activated kinase 1 (PAK-1).14 PAK-1 possesses a particularly open ATP-binding site, making it difficult to target with typical organic scaffolds but particularly suitable for bulky and rigid octahedral complexes. However, the large number of possible stereoisomers not only provides new structural opportunities but also poses a formidable challenge due to the limited ability to control the stereochemistry in the course of ligand exchange reactions. This issue was addressed in a recent publication in which the bioactive octahedral iridium(III) complex AW63 was synthesized through a stereoselective oxidative addition as the key synthetic step.15 This compound functions as a selective nanomolar inhibitor of the vascular endothelial growth factor (13) Meggers, E.; Atilla-Gokcumen, G. E.; Bregman, H.; Maksimoska, J.; Mulcahy, S. P.; Pagano, N.; Williams, D. S. Synlett 2007, 8, 1177–1189. (14) Maksimoska, J.; Feng, L.; Harms, K.; Yi, C.; Kissil, J.; Marmorstein, R.; Meggers, E. J. Am. Chem. Soc. 2008, 130 (47), 15764–15765. (15) Wilbuer, A.; Vlecken, D. H.; Schmitz, D. J.; Kr€aling, K.; Harms, K.; Bagowski, C. P.; Meggers, E. Angew. Chem., Int. Ed. 2010, 49, 3839– 3842.
receptor 3 (VEGFR-3), and it was demonstrated that this nontoxic organometallic iridium compound can inhibit the development of blood vessels in vivo in two different zebrafish angiogenesis models. Redox-Activated Antiproliferative Agents. One particular characteristic of many organometallic compounds is a reversible redox behavior. This property lends itself to the conception of redox-activated compounds, some of which show selectivity for cancer cells, presumably due to the altered redox environment of the continually dividing cancer cell versus a normal cell. As we will see below, some compounds can be considered as redox-activated prodrugs, while other compounds act as redox catalysts, thus driving the cell further from redox homestasis. Gerard Jaouen’s group has been studying a class of oxidatively activated ferrocenyl prodrugs. It appears that the reversible and mild oxidation chemistry of ferrocene allows it to act as an intramolecular oxidant of a distant phenol group, when linked by a conjugated system,16 thereby lowering the oxidation potential necessary for toxic quinone methide formation (Scheme 1). These compounds are selectively active on cancer cells, with approximately 100 less activity on normal cells,17 probably due to their prodrug features. This behavior was also observed in an intracerebral glioma model, where the ferrocenyl compounds acted as radiosensitizers but showed no activity in the absence of ionizing radiation in the reducing tissues of the brain.18 Peter Sadler’s group has recently published an article concerning catalytic “piano-stool” ruthenium compounds for cancer treatment.19 Although the complexes, [(η6-arene)Ru(azpy)I]þ (where arene = p-cymene, biphenyl and azpy= N,N-dimethylphenyl- or hydroxyphenyl-azopyridine), are inert to ligand hydrolysis in aqueous solution, they are highly cytotoxic to human ovarian A2780 and human lung A549 cancer cells, with IC50 values in the low micromolar range. The cytotoxicity seems to be a result of an increase in reactive oxygen species. Specifically, it appears that the ruthenium complex acts as a catalyst in the oxidation reaction of glutathione (GSH) to yield millimolar concentrations of glutathione disulfide in the presence of micromolar ruthenium concentrations. The proposed catalytic mechanism (Scheme 2) involves the addition of GSH over the azo double bond, followed by reduction of this bond concomitant with GSH oxidation. The double bond is then regenerated via hydrogenation of O2 (16) Hillard, E. A.; Vessieres, A.; Thouin, L.; Jaouen, G.; Amatore, C. Angew. Chem., Int. Ed. 2006, 45, 285–290. (17) Allard, E.; Passirani, C.; Garcion, E.; Pigeon, P.; Vessieres, A.; Jaouen, G.; Benoit, J. P. J. Contr. Release 2008, 130 (2), 146–153. (18) Allard, E.; Jarnet, D.; Vessieres, A.; Vinchon-Petit, S.; Jaouen, G.; Benoit, J.-P.; Passirani, C. Pharm. Res. 2010, 27 (1), 56–64. (19) Dougan, S. J.; Habtemariam, A.; McHale, S. E.; Parsons, S.; Sadler, P. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (33), 11628–11633.
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Scheme 2. Proposed Mechanism for Catalytic Oxidation of GSH to GSSG by Ruthenium(II) Arene Phenylazopyridine Complexes
Scheme 3. Metal-Mediated Retro Diels-Alder Reaction to Cyclopentadienyl Complexes of 99mTc with Pendant, Biological Functionsa
to produce H2O2. The depletion of the intracellular antioxidant GSH and H2O2 production stimulate a state of oxidative stress, presumably involved in cell death. Such redox catalysts have the potential to be selective against cancer cells, due to their particular sensitivity to reactive oxygen species.20 Other promising antitumor ruthenium complexes are now under mechanistic investigation.21
[Tc] refers to [99mTc(OH2)3(CO)3]þ, but the reactions can also be performed directly from [99mTcO4]- in a one-pot reaction. The right side shows a rhenium model complex with a melanoma-targeting R group.24
Organometallic Bioprobes Cardiolite is probably the most widely used organometallic compound in medicine. A radioactive 99mTc octahedral complex, it is used in single photon emission computed tomography imaging of the myocardium. Although nuclear imaging by organometallics remains an important topic, other properties, such as spectroscopic features, make organometallics valuable biological probes. Nuclear Medicine. The example of the ferrocifen-tamoxifen hybrid is probably the best demonstration that cyclopentadienyl complexes can structurally mimic a phenyl group.22 In extension of this principle, one could imagine access to biomolecules modified by radioactive organometallics to be used for therapeutic or diagnostic purposes. However, cyclopentadienyl transition-metal complexes are normally prepared in organic solvents under inert conditions and are unstable and insoluble in water, while radioactive compounds used in the clinic must be prepared directly in aqueous solution. For many years this opinion hampered attempts to consider (Cp-R)99mTc(CO)3 as an option in radiopharmaceutical chemistry, despite the advantages of using R as a targeting vector and its small size compared to “Werner-type” complexes. It was recently shown by Roger Alberto’s group that Diels-Alder dimerized cyclopentadiene scaffolds, bearing biologically active substituents, can be used as precursors for (20) Pelicano, H.; Carney, D.; Huang, P. Drug Resist. Update 2004, 7, 97–110. (21) Meng, X. J.; Leyva, M. L.; Jenny, M.; Gross, I.; Benosman, S.; Fricker, B.; Harlepp, S.; Hebraud, P.; Boos, A.; Wlosik, P.; Bischoff, P.; Sirlin, C.; Pfeffer, M.; Loeffler, J. P.; Gaiddon, C. Cancer Res. 2009, 69 (13), 5458–5466. (22) Top, S.; Vessieres, A.; Leclercq, G.; Quivy, J.; Tang, J.; Vaissermann, J.; Huche, M.; Jaouen, G. Chem. Eur. J. 2003, 9 (23), 5223–5236.
a
a quantitative, one-step synthesis of [(Cp-R)99mTc(CO)3] starting from [99mTcO4]- in water (Scheme 3).23 This synthetic strategy provides access not only to aqueous-phase cyclopentadienyl complexes with 99mTc for diagnosis but also to their corresponding radioactive rhenium analogues for therapy. The potential of rhenium bioorganometallics for therapy needs to be further explored, and direct visualization of the biological in vitro and in vivo pathways of the corresponding 99mTc compounds can complement this work. Organometallic PNA. Peptide nucleic acids (PNA) are DNA mimics,25 in which N-(2-aminoethyl)glycine units form a pseudopeptide chain bearing the four nucleobases. Because the backbone is neutral, PNAs exhibit stronger and more selective binding affinity for complementary nucleic acid (DNA and RNA) strands than natural nucleic acids. PNAs also show higher mismatch selectivity and noticeable chemical and enzymatic stability compared to natural oligonucleotides.26 To use PNA for DNA detection, it is necessary to provide PNAs of appropriate analytical probes (electrochemical,27,28 fluorescent,29 radioactive,30,31 etc.). A number of metal (23) Liu, Y.; Spingler, B.; Schmutz, P.; Alberto, R. J. Am. Chem. Soc. 2008, 130, 1554–1555. (24) N’Dongo, H. W. P.; Raposinho, P. D.; Fernandes, C.; Santos, I.; Can, D.; Schmutz, P.; Spingler, B.; Alberto, R. Nucl. Med. Biol. 2010, 37, 255–264. (25) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998, 37, 2796–2823. (26) Peptide Nucleic Acids: Protocols and Applications. 2nd ed.; Horizon Bioscience: Wymondham, U.K., 2004. (27) Baldoli, C.; Rigamonti, C.; Maiorana, S.; Licandro, E.; Falciola, L.; Mussini, P. Chem. Eur. J. 2006, 12, 4091–4100. (28) Metzler-Nolte, N. In Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim, Germany, 2006; p 125. (29) Oquare, B. Y.; Taylor, J.-S. Bioconjugate Chem. 2008, 19, 2196– 2204and references therein. (30) Wickstrom, E.; Tian, X.; Amirkhanov, N. V.; Chakrabarti, A.; Aruva, M. R.; Rao, P. S.; Qin, W.; Zhu, W.; Sauter, E. R.; Thakur, M. L. Methods Mol. Med. 2005, 106, 135–191and references therein.
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complexes have been conjugated to PNA oligomers with the aim of imparting new biochemical and spectroscopic properties.27,28,32,33 A novel family of dinuclear tricarbonyl rhenium(I) derivatives, of general formula [Re2(μ-X)(μ-Y)(μ-1,2-diazine)(CO)6], which exhibit intense emission in the range 550-620 nm, originating from 3MLCT excited states,34-36 has been used as a promising tool for PNA labeling for biomedical applications. Emanuela Licandro’s group has conjugated such dinuclear rhenium(I) complexes to a homothymine PNA decamer, through the formation of an amide bond between the carboxyl group of the diazine ligand and the terminal NH2 group of PNA (Chart 1). A reliable solid-phase synthetic methodology has been established, which provided a luminescent rhenium-PNA decamer conjugate that exhibits intense emission (quantum yield 12%), centered at 586 nm in toluene solution, with a long excited-state lifetime.37 The ability of the new Re-PNA conjugate to recognize and bind the cDNA strand was evaluated by measuring the melting point, indicating the formation of a stable triple helix of type PNA2/DNA. Cell samples were imaged using twophoton excitation, showing that the Re-PNA conjugate penetrates the cell membrane, differentially staining the cytoplasm and the nucleus, suggesting a possible use for highlighting environments with different lipophilicity or rigidity. These preliminary results indicate that the nontoxic and photostable Re-PNA conjugate is viable as a fluorophore for cell imaging, although more detailed experiments are needed in order to establish the kinetics and mechanism of the process, the influence of the kind of cells used, and the lower limits of complex and DMSO concentration. So far, no PNA with such photophysical properties has been reported, and this study could open new perspectives for biological PNA applications. Infrared Microscopy. Metal carbonyls show many advantages in imaging, such as small size, stability in biological environments, and intense absorption in the 1800-2200 cm-1 region, where biological material is transparent. Vibrational spectroscopy38,39 is currently used for two-dimensional analysis of tissues. However, in classical optical microscopy, submicrometric resolutions are not attainable in the IR range, because the best resolutions cannot surpass λ/2 (i.e., 2.5 μm at 2000 cm-1), as imposed by the diffraction law.40 (31) Gallazzi, F.; Wang, Y.; Jia, F.; Shenoy, N.; Landon, L. A.; Hannink, M.; Lever, S. Z.; Lewis, M. R. Bioconjugate Chem. 2003, 14, 1083–1095. (32) Sosniak, A. M.; Gasser, G.; Metzler-Nolte, N. Org. Biomol. Chem. 2009, 7, 4992–5000. (33) Baldoli, C.; Cerea, P.; Giannini, C.; Licandro, E.; Rigamonti, C.; Maiorana, S. Synlett 2005, 1984–1994. (34) Panigati, M.; Donghi, D.; D’Alfonso, G.; Mercandelli, P.; Sironi, A.; Mussini, P.; D’Alfonso, L. Inorg. Chem. 2006, 45, 10909– 10921. (35) Donghi, D.; D’Alfonso, G.; Mauro, M; Panigati, M.; Mercandelli, P.; Sironi, A.; Mussini, P.; D’Alfonso, L. Inorg. Chem. 2008, 47, 4243– 4255. (36) Mauro, M.; Procopio, E. Q.; Sun, Y.; Chien, C. H.; Donghi, D.; Panigati, M.; Mercandelli, P.; Mussini, P.; D’Alfonso, G.; Cola, L. D. Adv. Funct. Mater. 2009, 19, 2607–2614. (37) Ferri, E.; Donghi, D.; Panigati, M.; Prencipe, G.; D’Alfonso, L.; Zanoni, I.; Baldoli, C.; Maiorana, S.; D’Alfonso, G.; Licandro, E. Chem. Commun. 2010, 46, 6255–6257. (38) Dumas, P.; Sockalingum, G. D.; Sule-Suso, J. Trends Biotechnol. 2006, 25, 40–44. (39) Meister, K.; Niesel, J.; Schatzschneider, U.; Metzler-Nolte, N.; Schmidt, D. A.; Havenith, M., Angew. Chem., Int. Ed. 49(19), 33103312. (40) Lasch, P.; Naumann, D. Biochim. Biophys. Acta, Biomembr. 2006, 1758 (7), 814–829.
Hillard and Jaouen Chart 1
Photothermal induced resonance (PTIR) is an emerging technique using a system patented by Alexandre Dazzi and co-workers,41 coupling an ATM and a tunable infrared pulsed laser to record spatially resolved absorption measurements. The laser is tuned to an IR absorption band of the probe, and the sample is heated upon administration of the laser pulse. The AFM tip detects local deformations due to the change in temperature and oscillates with an amplitude corresponding to the local absorbance. A recent study by Clotilde Policar’s and Anne Vessieres’ groups42 demonstrated the utility of this method in detecting metal carbonyls inside a eukaryotic cell. The molecule selected was the cyclopentadienyl rhenium tricarbonyl derivative Re-2OH (Chart 2), and its uptake and distribution in MDA-MB-231 human breast cancer cells was studied. In one experiment, cells were incubated with various concentrations of Re-2OH and deposited on a nitrocellulose disk stage. Using a calibration curve, the cellular content of Re-2OH was quantified by PTIR, showing that the cellular content increases with the incubation concentration. In order to map intracellular distribution, cells were incubated with 10 μM Re-2OH and then washed and deposited on a ZnSe prism. Cells were mapped at different wavelengths: 1925 and 2017 cm-1 (corresponding to the organometallic probe) and 1240 and 1650 cm-1 (corresponding to PO2- and amide vibrations characteristic of DNA). These experiments suggested that Re-2OH accumulates in the nucleus. This interpretation was validated by recording the IR spectrum with the AFM tip on the nucleus, which reproduced the FTIR spectrum of the CO stretches at 1925 and 2017 cm-1.
Organometallic Enzymes It is now accepted that the first living organisms appeared at a time when Earth’s atmosphere was devoid of molecular oxygen. This reducing environment would have been very (41) Dazzi, A.; Reading, M.; Rui, P.; Kjoller, K. WO/2008/143817, 2008. (42) Policar, C.; Waern, J. B.; Plamont, M.-A.; Clede, S.; Mayet, C.; Prazeres, R.; Ortega, J.-M.; Vessieres, A.; Dazzi, A., Angew. Chem., Int. Ed. 2010, DOI: 10.1002/anie.201003161.
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suitable for organometallic chemistry and may explain the evolution of the organometallic hydrogenase enzyme. With the development of photosynthesis, the atmosphere became more oxidizing, and biologically important metals are now often found in high oxidation states. Thanks to their original structure based on soft ligands and low-oxidation-state metals, organometallic artificial enzymes represent an innovative deviation from the reactivity exhibited by post-photosynthesis natural enzymes. Hydrogenase. The reversible interconversion of protons and hydrogen is becoming important, due to the interest in H2 as a unique energy vector. Transformation of water to H2, through electrolysis, is an efficient way to store energy in a stable chemical form, and oxidation of H2 to water using atmospheric oxygen a simple way to recover the energetic input in fuel cells, without production of greenhouse gases or atmospheric pollutants.43,44 However, these reactions are complex multielectronic processes which require catalysts to be practically useful. Available technological devices, such as proton exchange membrane (PEM) electrolyzers and fuel cells, integrate unsustainable and expensive metals such as platinum.45 On the other hand, a number of microorganisms are able to produce or metabolize hydrogen thanks to hydrogenases.46 These enzymes catalyze both hydrogen uptake and hydrogen evolution at very high rates (one molecule of hydrogenase produces 1500-9000 molecules of H2 per second at pH 7 and 37 °C in water).47,48 When careful examination of these enzymes49-51 revealed that the metals involved were nonprecious metals such as nickel and iron, it became obvious that a solution to the catalytic problem could be found in nature.44 Since the demonstration that the catalytic active site of [FeFe]-hydrogenase possesses a [2Fe2S] cluster coordinated by CO and CN- ligands as well as an azadithiolato cofactor,49,51 the chemistry tremendously influenced by (43) Crabtree, G. W.; Dresselhaus, M. S. MRS Bull. 2008, 33, 421–428. (44) Artero, V.; Fontecave, M. Coord. Chem. Rev. 2005, 249, 1518– 1535. (45) Gordon, R. B.; Bertram, M.; Graedel, T. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1209–1214. (46) Vignais, P. M.; Billoud, B.; Meyer, J. FEMS Microbiol. Rev. 2001, 25, 455–501. (47) Pershad, H. R.; Duff, J. L. C.; Heering, H. A.; Duin, E. C.; Albracht, S. P. J.; Armstrong, F. A. Biochemistry 1999, 38, 8992–8999. (48) Jones, A. K.; Sillery, E.; Albracht, S. P. J.; Armstrong, F. A. Chem. Commun. 2002, 866–867. (49) Nicolet, Y.; Piras, C.; Legrand, P.; Hatchikian, C. E.; FontecillaCamps, J. C. Structure 1999, 7, 13–23. (50) Volbeda, A.; Fontecilla-Camps, J. C. Dalton Trans. 2003, 4030– 4038. (51) Peters, J. W.; Lanzilotta, W. N.; Lemon, B. J.; Seefeldt, L. C. Science 1998, 282, 1853–1858. (52) Hieber, W.; Spacu, P. Z. Anorg. Allg. Chem. 1937, 233, 353–364. (53) Hieber, W.; Beck, W. Z. Anorg. Allg. Chem. 1960, 305, 265–273.
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Chart 3. [Fe2(CO)4(PMe3)(μ-dmpdt)] Showing the Rotated State
Hieber52,53 and Seyferth54,55 attracted new attention. Novel carbonyl diiron dithiolato complexes have been synthesized and investigated according to their electrocatalytic ability to reduce protons to dihydrogen.56-59 Further efforts were directed to the protonation behavior of these complexes,60-63 mimicking the enzymatic surroundings,64-67 the importance of sulfur as bridgehead atoms,68,69 and the formation of a rotated state.70 The rotated state (formation of a semibridging or bridging CO ligand and a vacant coordination site at on iron atom) was found to be a particularly essential geometry of the natural enzyme, providing high reactivity for the dihydrogen formation.63,71 In model complexes, this species was not present, due to the missing stabilization enforced by the secondary coordination sphere present in the enzyme. Recently, Marcetta Darensbourg’s group was able to synthesize and characterize a mixed-valent FeIFeII complex as a model for the Hox state, showing a “rotated” geometry (Chart 3)70 by oxidation of [Fe2(CO)4(PMe3)(μ-dmpdt)] (μ-dmpdt = 2, 2-dimethyl-1,3-propanedithiolate) with ferricenium hexafluorophosphate. The molecular structure revealed the (54) Cowie, M.; DeKock, R. L.; Wagenmaker, T. R.; Seyferth, D.; Henderson, R. A.; Gallagher, M. K. Organometallics 1989, 8, 119–132. (55) Seyferth, D.; Henderson, R. S. J. Am. Chem. Soc. 1979, 101, 508– 509. (56) Felton, G. A. N.; Mebi, C. A.; Petro, B. J.; Vannucci, A. K.; Evans, D. H.; Glass, R. S.; Lichtenberger, D. L. J. Organomet. Chem. 2009, 694, 2681–2699and references therein. (57) Canaguier, S.; Artero, V.; Fontecave, M. Dalton Trans. 2008, 315–325. (58) Canaguier, S.; Field, M.; Oudart, Y.; Pecaut, J.; Fontecave, M.; Artero, V. Chem. Commun. 2010, 46, 5876–78. (59) Tard, C.; Pickett, C. J. Chem. Rev. 2009, 109, 2245–2274. (60) Li, P.; Wang, M.; Chen, L.; Liu, J.; Zhao, Z.; Sun, L. Dalton Trans. 2009, 1919–1926. (61) Ezzaher, S.; Capon, J.-F.; Gloaguen, F.; Petillon, F. Y.; Schollhammer, P.; Talarmin, J. Inorg. Chem. 2009, 48, 2–4. (62) Gao, W.; Sun, J.; Akermark, T.; Li, M.; Eriksson, L.; Sun, L.; Akermark, B. Chem. Eur. J. 2010, 16, 2537–2546. (63) Barton, B. E.; Rauchfuss, T. B. Inorg. Chem. 2008, 47, 2261– 2263. (64) He, C.; Wang, M.; Zhang, X.; Wang, Z.; Chen, C.; Liu, J.; Akermark, B.; Sun, L. Angew. Chem., Int. Ed. 2004, 43, 3571–3574. (65) de Hatten, X.; Bothe, E.; Merz, K.; Huc, I.; Metzler-Nolte, N. Eur. J. Inorg. Chem. 2008, 4530–4537. (66) Jones, A. K.; Lichtenstein, B. R.; Dutta, A.; Gordon, G.; Dutton, P. L. J. Am. Chem. Soc. 2007, 129, 14844–14845. (67) Apfel, U.-P.; Rudolph, M.; Apfel, C.; Robl, C.; Langenegger, D.; Hoyer, D.; Jaun, B.; Ebert, O.; Alpermann, T.; Seebach, D.; Weigand, W. Dalton Trans. 2010, 39, 3065–3071. (68) Harb, M. K.; Niksch, T.; Windhager, J.; G€ orls, H.; Holze, R.; Lockett, L. T.; Okumura, N.; Evans, D. H.; Glass, R. S.; Lichtenberger, D. L.; El-katheeb, M.; Weigand, W. Organometallics 2009, 28, 1039– 1048. (69) Song, L. C.; Gao, W.; Feng, C. P.; Wang, D. F.; Hu, Q. M. Organometallics 2009, 28, 6121–6130. (70) Singleton, M. L.; Bhuvanesh, N.; Reibenspies, J. H.; Darensbourg, M. Y. Angew. Chem., Int. Ed. 2008, 47, 9492–9495. (71) Ezzaher, S.; Capon, J.-F.; Gloaguen, F.; Petillon, F. Y.; Schollhammer, P.; Talarmin, J.; Pichon, R.; Kervarec, N. Inorg. Chem. 2007, 46, 3426–3428.
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Figure 4. Electrografting of amine groups on MWCNTs and postfunctionalization with the Ni complex through amide links. This is a simplified representation of the structure of the material for sake of clarity: the number of phenylene residues is indeed arbitrary, and attachment of the nickel complex to two or more surface amine groups is not excluded.
expected [2Fe2S] cluster with a semibridging carbonyl ligand and a vacant coordination site. This vacant site was blocked by the sterically demanding disulfur bridge. Further proof for the establishment of a rotated state in solution was provided by IR spectroscopy, whereby a new CO band could be observed at 1859 cm-1. EPR investigations as well as DFT calculations suggested that the FeI center is the rotated iron atom. In comparison with [Fe2(CO)4(PMe3)(μ-pdt)] (μ-pdt= 1,3propanedithiolate) steric bulk was proclaimed to be a necessity for the formation of a rotated state in model complexes.68,69,72 This can be achieved either by bulky S-to-S linkers or by appropriate substituent ligands.68,69,73 Due to the absence of a stabilizing enzymatic environment, the use of sterically bulky substituents is favorable. Further investigations into the electrochemical properties of sterically bulky complexes, as well as their protonation behavior, will be a new challenge. Elegant work by D. L. DuBois74-76 has shown that active complexes could be produced by combining features of both [NiFe]- and [FeFe]-hydrogenases. When these compounds were assayed in nonaqueous solvents, they displayed remarkable catalytic properties for both the electroreduction of protons to hydrogen and the electrooxidation of hydrogen in the presence of triethylamine. Inspired by DuBois’ complexes, Vincent Artero’s and Marc Fontecave’s groups, in collaboration with Serge Palacin’s team, decided to evaluate the potential of hydrogenase-bioinspired catalysts for the construction of heterogeneized electrode materials.77 They synthesized an analogue of DuBois complexes in which an activated ester group was introduced to allow a covalent (72) Zhao, X.; Georgakaki, I. P.; Miller, M. L.; Yarbrough, J. C.; Darensbourg, M. Y. J. Am. Chem. Soc. 2001, 123, 9710–9711. (73) Thomas, C. M.; Liu, T.; Hall, M. B.; Darensbourg, M. Y. Inorg. Chem. 2008, 47, 7009–7024. (74) Curtis, C. J.; Miedaner, A.; Ciancanelli, R.; Ellis, W. W.; Noll, B. C.; DuBois, M. R.; DuBois, D. L. Inorg. Chem. 2003, 42, 216–227. (75) Wilson, A. D.; Newell, R. H.; McNevin, M. J.; Muckerman, J. T.; DuBois, M. R.; DuBois, D. L. J. Am. Chem. Soc. 2006, 128, 358–366. (76) Wilson, A. D.; Shoemaker, R. K.; Miedaner, A.; Muckerman, J. T.; DuBois, D. L.; DuBois, M. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6951–6956. (77) Tran, P. D.; Artero, V.; Fontecave, M. Energy Environ. Sci. 2010, 3, 727–47. (78) Le Goff, A.; Artero, V.; Jousselme, B.; Tran, P. D.; Guillet, N.; Metaye, R.; Fihri, A.; Palacin, S.; Fontecave, M. Science 2009, 326, 1384–1387.
attachment, through the formation of an amide bond, to a carbon nanotube (CNT) decorated with amine groups at their surface (Figure 4).78 The amine-functionalized CNTs were produced during electrochemical reduction of the 4-(2-aminoethyl)phenyldiazonium salt, generating aryl radicals which react with the carbon surface to form covalent C-C bonds between the grafted group and the carbon nanotube. It should be mentioned that CNTs were chosen not only for their outstanding electron conductivity but also due to their large surface area, from which one can expect high catalyst loading. Deposition of a thin film of these electroactive Ni-functionalized CNTs onto a carbon substrate generated a cheap, stable, air-resistant cathode material with remarkably unique performance, especially under the strongly acidic conditions required in the expanding PEM technology. To the best of our knowledge, this was the first report of a molecular-engineered and noblemetal-free electrode material that is capable of achieving hydrogen evolution/oxidation with no or little overpotential. Artificial Metalloenzymes. Ever since enzymes have been employed out of their “normal” environment to perform, for instance, organic synthesis, there have been studies dedicated to the manipulation of their functional properties. Progress in molecular biology and especially in genetic engineering has contributed to a large extent to the manipulation of enzymes’ catalytic properties, from site-directed mutations to highly evolved directed evolution techniques. Nevertheless, these approaches remain inefficient for imparting a totally new functionality to a protein. Conversely, protein engineering by chemical approaches can bring an almost infinite number of new functionalities, including new catalytic properties. Among the now extensive studies carried out in this area, those connected to the design of metalloproteins are currently very active, since metal complexes display a wealth of unique functions, including highly valuable catalytic properties.79,80 These studies may eventually help to fill the gap between enzymatic and organometallic catalysis which are in many points complementary. (79) Heinisch, T.; Ward, T. R. Curr. Opin. Chem. Biol. 2010, 14, 184– 199. (80) Lu, Y.; Yeung, N.; Sieracki, N.; Marshall, N. M. Nature 2009, 460, 855–862.
Review
Three approaches are currently pursued to prepare artificial metalloenzymes, namely the covalent, the dative, and the supramolecular strategies.81 In the first approach, an artificial metal cofactor is covalently anchored to one or two amino acid side chains of the protein host by one of the ligands coordinating the metal ion. For example, a Mn(salen) complex was introduced within the protein scaffold of myoglobin in lieu of the native prosthetic group by covalent anchoring to two judiciously positioned cysteine residues. The resulting adduct was shown to catalyze the sulfoxidation of thioanisole with high rate and moderate enantioselectivity.82 In the dative approach, native or (chemically/genetically) added coordination sites are used to introduce metal ions into protein scaffolds. In this series, the facile replacement of the ZnII ion in carbonic anhydrase (a metalloenzyme that catalyzes the hydration of carbon dioxide) by MnII conferred peroxidase activity to this enzyme, which catalyzed the epoxidation of olefins with low yield but promising enantiomeric excess.83 Finally, in the supramolecular approach, high affinity association between a ligand/inhibitor and a protein is exploited to introduce metallic entities within the protein environment. With this strategy, very efficient hybrid catalysts were constructed from the biotin/(strept)avidin system. The lowmolecular-weight biotin molecule binds to the tetrameric protein (strept)avidin with very high affinity (Ka ≈ 1015 M), and derivatization of the carboxylic acid function of biotin only moderately alters its affinity. Several metal-containing biotin derivatives were synthesized and assembled with avidin or streptavidin or mutants of these proteins. The hybrid ensembles were able to catalyze allylic alkylations, transfer hydrogenations, and alkene hydrogenations with high yield and enantioselectivity.84-88 This field of artificial metalloenzymes is not limited to peptide/protein scaffolds but has recently been expanded to the world of DNA. This approach is particularly innovative since DNA, as opposed to RNA, does not normally display any catalytic properties. As in the case of protein scaffolds, both the supramolecular and covalent anchoring approaches have been investigated for the preparation of DNAzymes. The resulting hybrids have been shown to catalyze DielsAlder and Michael reactions, together with Friedel-Crafts (81) Steinreiber, J.; Ward, T. R. Coord. Chem. Rev. 2008, 252, 751– 766. (82) Carey, J. R.; Ma, S. K.; Pfister, T. D.; Garner, D. K.; Kim, H. K.; Abramite, J. A.; Wang, Z.; Guo, Z.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 10812–10813. (83) Okrasa, K.; Kazlauskas, R. J. Chem. Eur. J. 2006, 12, 1587–1596. (84) Pierron, J.; Malan, C.; Creus, M.; Gradinaru, J.; Hafner, I.; Ivanova, A.; Sardo, A.; Ward, T. R. Angew. Chem., Int. Ed. 2008, 47, 701–705. (85) Letondor, C.; Humbert, N.; Ward, T. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4683–4887. (86) Creus, M.; Pordea, A.; Rossel, T.; Sardo, A.; Letondor, C.; Ivanova, A.; Le Trong, I.; Stenkamp, R. E.; Ward, T. R. Angew. Chem., Int. Ed. 2008, 47, 1400–1404. (87) Skander, M.; Humbert, N.; Collot, J.; Gradinaru, C.; Klein, G.; Loosli, A.; Sauser, J.; Zocchi, A.; Gilardoni, F.; Ward, T. R. J. Am. Chem. Soc. 2004, 126, 14411–14418. (88) Letondor, C.; Pordea, A.; Humbert, N.; Ivanovna, A.; Mazurek, S.; Novic, M.; Ward, T. R. J. Am. Chem. Soc. 2006, 128, 8320–8328. (89) Oltra, N. S.; Roelfes, G. Chem. Commun. 2008, 6039–6041. (90) Boersma, A. J.; Feringa, B. L.; Roelfes, G. Angew. Chem., Int. Ed. 2009, 48, 3346–3348. (91) Coquiere, D.; Feringa, B. L.; Roelfes, G. Angew. Chem., Int. Ed. 2007, 46, 9308–9311. (92) Fournier, P.; Fiammengo, R.; J€aschke, A. Angew. Chem., Int. Ed. 2009, 48, 4426–4429.
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and allylic amination reactions, with high enantioselectivity.89-92
Conclusions We hope this brief survey, based on recent examples, better illuminates the new landscape offered by bioorganometallic chemistry. Now that the initial intellectual proscriptions against organometallic compounds in biological media have been breached, an astonishing fertility is reinvigorating organometallic chemistry. There are a host of areas in the life sciences, some of which have been presented here, that can be enriched by exploiting the peculiarities of organometallic reaction chemistry, structural chemistry, and spectroscopic properties. We particularly hope this subject finds a training ground among younger chemists and all others who possess the versatility and curiosity to navigate such a highly interdisciplinary field. At this stage, the only limit to bioorganometallics seems to be the imagination.
Glossary of Biological Terms Angiogenesis: the process of blood vessel growth. In the context of cancer research, an antiangiogenic compound is one that prevents the growth of blood vessels in the tumor, thus starving the tumor cells of oxygen and nutrients. Apoptosis: programmed cell death. Unlike necrosis, apoptosis is not harmful to the organism and is not a result of trauma. Diseases such as cancer are associated with dysfunctional apoptotic pathways, allowing uncontrolled proliferation; restoring normal apoptosis is one of the targets of cancer research. ATP: adenosine-50 -triphosphate is a product of cellular respiration that transports chemical energy within for metabolism. Glioma: a brain or spine tumor arising from glial cells. GSH: glutathione, a tripeptide involved in the scavenging of reactive oxygen species and maintaining redox homeostasis in the cell. The oxidized form of GSH is GSSG, a dimer possessing a sulfur-sulfur bond. In vitro: literally “within the glass”, here generally referring to experiments with cultured cells. In vivo: literally “within the living”, here generally referring to experiments with animals. Kinase: an enzyme that transfers a phosphate group from ATP to a protein (phosphorylation). In cancer research, specific kinase inhibitors are being developed primarily to interfere with DNA repair mechanisms. Metastasis: the spread of a cancer tumor from one organ or part to another nonadjacent organ or part, often via the circulatory or lymphatic system. The original tumor is called the primary tumor, and the tumor arising from metastasis is called the secondary tumor. Reactive oxygen species: small oxygen-containing molecules such as H2O2, OH°, and ONOO- which play a role in normal cell signaling pathways but which at elevated concentrations can cause DNA and protein damage.
Acknowledgment. E.A.H. and G.J. thank Roger Alberto, Vincent Artero, Paul Dyson, Emanuela Licandro, Eric Meggers, Michele Salmain, and Wolfgang Weigand for their expert contributions to this review.
