Bioorganometallic Chemistry

1.31 Bioorganometallic Chemistry N Metzler-Nolte, Ruhr-Universitaet Bochum, Bochum, Germany ª 2007 Elsevier Ltd. All rights reserved. 1.31.1

Introduction

883

1.31.2

Naturally Occuring Organometallics

884

1.31.2.1

Cobalamins

884

1.31.2.2

Hydrogenases (H2ases)

887

1.31.2.3

Carbon Monoxide Dehydrogenase (COdH) and Acetyl Coenzyme A Synthase (ACS)

889

1.31.2.4

Methyl coenzyme M reductase (F430)

890

1.31.3

Medicinal Organometallic Chemistry

891

1.31.3.1

Anticancer Agents

891

1.31.3.2

Antimicrobial Agents

894

1.31.3.2.1 1.31.3.2.2

Antibacterial Antimalarial

894 895

1.31.3.3

NO/CO Drugs

895

1.31.3.4

Radiopharmaceuticals

896

Toxicology and Environment

896

1.31.3.5 1.31.4

Organometallic Compounds For Biological Studies

897

1.31.4.1

Amino Acid, Peptide, and Protein Derivatives

897

1.31.4.2

DNA, RNA, and PNA Derivatives

902

1.31.4.3

Others

904

1.31.4.3.1 1.31.4.3.2 1.31.4.3.3 1.31.4.3.4

1.31.5

Sugar derivatives Lipids Receptor ligands Peptide synthesis

904 904 906 908

Biosensors Based on Organometallics

909

1.31.5.1

Protein-based Redox Probes

909

1.31.5.2

DNA Sensors

909

1.31.5.3

Metallo-immuno Assays

911

1.31.5.4

Colorimetric Assays and Luminescent Probes

912

1.31.5.5

Heavy Metal Probes for Crystallography and Electron Microscopy

913

References

914

1.31.1 Introduction In a recent contribution to COMC (1995), Riordan pointed out that ‘the phrase ‘‘bioorganometallic chemistry’’ is rather nebulous, conjuring different visions to various communities of scientists.’ In one attempt to capture this breadth, bioorganometallic chemistry has been defined as ‘‘. . . the study of organometallic complexes with bioligands. . ., and the use of these derived complexes in a variety of applications and basic research studies. . . .’’ It is rather a pleasure to see so many different fields combined under one conceptual roof. Bioorganometallic chemistry is certainly a hot and exciting development, given that for many years organometallic compounds were, by the great majority of researchers, believed to be highly sensitive and unstable to air and moisture. Today, we see that this notion was premature. There are many exciting uses of organometallic compounds in medicine, medicinal diagnostics

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Bioorganometallic Chemistry

Figure 1

and bio-related analytics, molecular recognition of biomolecules, and bioprobes—to name just several important categories. On top of that, there is a growing number of organometallic compounds in nature. The most famous one, vitamin B12, has been isolated in pure form about 60 years ago. Being a vital cofactor for a number of enzymatic transformations, it was long thought to be the only organometallic compound in nature. There is now well-founded evidence for organometallic intermediates and reactivity in over one dozen cases, and it is safe to forecast that this number will grow. Figure 1 tries to visualize the place of ‘‘bioorganometallic chemistry.’’ Recently, the first book entitled ‘‘Bioorganometallic Chemistry’’ was published.1 It is obviously impossible to condense this book, which has over 440 pages, into this one single chapter. The author has chosen to give an overview of different aspects of what ‘‘bioorganometallics’’ means to him Several chapters in Volume 12 of this series cover selected aspects in more detail (‘‘medicinal organometallic chemistry,’’ ‘‘organometallic receptors,’’ ‘‘organometallic compounds in biosensing,’’ and ‘‘environmental and biological aspects of organometallic compounds’’). An introductory chapter for students can be found in another book.2 There are a number of reviews on the various subsections of this chapter, which are cited in the appropriate subsections. We have recently reviewed the bioorganometallic chemistry of ferrocene.3 Volume 8 of Comprehensive Coordination Chemistry II (2003) has a number of excellent chapters on the model chemistry related to organometallic enzymes.

1.31.2 Naturally Occuring Organometallics Organometallic compounds serve as cofactors, active sites, or intermediates in a number of biomolecules and biomolecular transformations. Compounds as yet identified are the cobalamins (vitamin B12 and derivatives), the hydrogenase enzyme family (H2ases), acetyl coenzyme A synthase (ACS) and carbon monoxide dehydrogenase (COdH), and the Ni-containing reaction center in methyl coenzyme M reductase (F430). These four classes are treated in more detail in the following sections. The discussion is, however, limited to the biological (organometallic) chemistry involved, and model complexes are not covered in any depth. Not treated are the other enzyme centers which exhibit reactivity that would typically be classified as ‘‘organometallic,’’ such as the nitrogenase enzymes which convert atmospheric dinitrogen into ammonia under ambient conditions.4 Iron carbene complexes may be involved in reductive dehalogenation reactions of cytochromes P450.5 Gaseous ethylene is an important signaling molecule for many plants.6 It is effective at nanomolar concentrations, suggesting the presence of high-affinity receptors. It has been proposed that a Cu(I) center is involved in ethylene binding in Arabidopsis plants.7 This is another beautiful example of bioorganometallic chemistry in nature, for which a model complex has been presented.8

1.31.2.1 Cobalamins Cobalamins are a family of cobalt-containing cofactors, also known as the vitamin B12 family (Scheme 1). It was observed in the early 1900s that raw liver extracts could cure an otherwise fatal disease, pernicious anemia. In 1948, a red crystalline compound was isolated from liver extracts (cyanocobalamin), which was structurally characterized by Dorothy Hodgkin in 1956. These discoveries were honored by Nobel prizes in 1934 (to Whipple, Minot, and

Bioorganometallic Chemistry

Scheme 1

Murphy) and 1964 (to Hodgkin). Methylcobalamin and adenosylcobalamin are the two biologically active cofactors. They are transformed into the stable cyanocobalamin during the isolation process. Cyanocobalamin or aquocobalamin is the active ingredient in vitamin B12-containing medicines, both are converted into the physiologically active forms in the body. The cobalamins constitute the first, and for a long time the only, well-characterized examples of genuine organometallics in nature. There is an immense literature on cobalamins, including books and reviews on all the aspects of the enzymology, chemistry, and model compounds.9–12 Therefore, only a few details are discussed here. Cobalamins are unique cofactors from a synthetic point of view, which make use of the special properties of the metal–carbon bond.13 They can provide carbanions (‘‘nature’s Grignard reagents’’), carbocations, or carbon radicals. In the latter group, an adenosyl radical is released from coenzyme B12 which is used to initiate a 1,2-rearrangement. A classification has been proposed depending on the nature of the migrating and receiving groups (Scheme 2).14,15 Class I enzymes are carbon-skeleton mutases (initial C–C bond breakage) and include glutamate mutase (GluM), 2-methyleneglutamate mutase (MGM), isobutyryl CoA mutase, and methylmalonyl CoA mutase (MMCM) (CoA ¼ coenzyme A). The rearrangement of (R)-methylmalonyl CoA to succinyl CoA catalyzed by MMCM, which is probably the best-studied enzyme in this field, is shown in Scheme 3. Different crystal structures of MMCM from Propionibacterium shermanii have been solved by Mancia and co-workers. The first was reconstituted with desulfoCoA,16 while three structures were solved with adenosylcobalamin cofactor and the substrate as well as two inhibitors similar to substrate and products.17 In these crystals, no adenosyl group could be detected bound to Co, as it was

Scheme 2

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Bioorganometallic Chemistry

Scheme 3

apparently lost during crystallization. A previous structure with cofactor bound, but no substrate, had the adenosyl group attached to cobalt.18 All structures were quite similar, suggesting that activation and substrate rearrangement take place without major conformational changes. It is interesting to note that MMCM is the only coenzyme B12-dependent enzyme which occurs in both mammals and bacteria. It is a necessary enzyme in the metabolism of fatty acids with an odd number of carbon atoms. Lack of a functional enzyme is the origin for the human metabolic disease called methylmalonic acidemia. Class II eliminases catalyze the migration of a hydroxyl or amino group to a carbon atom which already carries one hydroxyl group (initial C–O or C–N bond breakage), followed by an elimination step. Examples are propanediol dehydratase (DD) and glycerol dehydratase (GD). Ribonucleotide triphosphate reductase (RTPR) is also commonly counted into this class of B12-dependent enzymes.19 RTPR catalyzes the reduction of ribonucleoside triphosphates to 29-deoxyribonucleoside triphosphates, but without a rearrangement reaction. Finally, class III aminomutase enzymes catalyze the migration of an amino group (initial C–N bond breakage). Examples include -lysine-5,6-aminomutase and D-ornithine-4,5-aminomutase, both of which require pyridoxal phosphate as an additional cofactor. The above three groups differ in a number of aspects, in addition to the migrating groups involved. Class I and III bind the adenosylcobalamin cofactor in a ‘‘base-off/His-on’’ mode. The cobalamin nucleotide loop is buried in a hydrophobic pocket of the enzyme, which in turn provides a histidine group from the active site to occupy the axial ligand position on the cobalt atom. Class II, in contrast, has the nucleotide loop remaining coordinated to the metal in all structures known so far. Class I and II also differ in their reactivity and EPR spectroscopic patterns. Class I EPR spectra show a relatively strong hyperfine coupling between a Co(II) center and the organic radical. Class II enzymes exhibit only weak coupling so that the two features are well resolved. Chemically, class I enzymes do not generally tolerate any alterations in the cofactor structure, while class II enzymes are rather promiscuous toward structural variations in the coenzyme. Taking all these considerations together, it may well be that the primary step in catalysis, homolysis of the metal–carbon bond, is initiated by more than one mechanism, which is quite an intriguing thought for an organometallic chemist and should spur more research in the area in the future. Methylation reactions are the second major class of reactions that require methylcobalamin as a cofactor.9,13,15 In fact, corrinoid-dependent methyl transferases are ubiquitous and occur in all organisms except plants. More than a dozen methyl transferases from different species have been isolated and characterized. Results in conjunction with the ACS/ COdH system are described below for the synthesis of acetyl CoA. By far, the best studied example is methionine synthase (MetS).9 MetS catalyzes a methyl group transfer from N5-methyl tetrahydrofolate to homocysteine to form methionine via a methyl-cob(III)alamin intermediate (Scheme 3). The cobalamin thus serves as a shuttle for the methyl group transfer, shifting between Co(I) and Co(III) redox states. Formally, the methyl group is transferred as a carbocation. The highly reactive Co(I) intermediate gets occasionally inactivated as Co(II). Reactivation involves electron transfer from a ferridoxin and methylation. Numerous studies were carried out on MetS from various species. The MetS (650–896) cobalamine-binding domain from Escherichia coli was the first cobalamin-dependent enzyme to be structurally

Bioorganometallic Chemistry

Scheme 4

characterized.20,21 It showed the ‘‘base-off/His-on’’ binding motif already discussed above. Scheme 4 shows a schematic drawing of this enzyme fragment. More recently, a Co-corrinoid-dependent methyl transfer reaction that does not require methyl tetrahydrofolate was discovered.22 Rather, these enzymes can use O-methyl compounds such as methanol and methyl ethers as a source of the methyl group by cleavage of the O–C bond. Methylcobalamin is also involved in other biomethylation reactions. Examples include the methylation of inorganic As salts to volatile methyl arsines, as well as the biomethylation of Hg2þ salts to yield the highly toxic methyl– mercury cation [CH3–Hg]þ (see also Section 1.31.3.5). In this reaction, methylcobalamin serves as a Grignard-type reagent and the methyl group is transferred as a carbanion.

1.31.2.2 Hydrogenases (H2ases) H2ases are a family of enzymes which catalyze the reversible conversion of dihydrogen into protons and electrons. They are found in archaebacteria and bacteria, as well as in green algae. These organisms use dihydrogen gas as their energy source; they are now often found in anaerobic environments.23 In the H2ase enzyme clusters, electrons are passed along a chain of FeS clusters to finally generate chemical reductants like NADH. H2ases are commonly classified by their active site (Scheme 5).24,25 NiFe-H2ase contains a bimetallic Ni–Fe center. In the NiFeSe subclass, the rare amino acid selenocysteine replaces one cysteine coordinated to Ni. The FeFe (also called Fe-only) class contains two Fe atoms that are held in place by a sulfur atom from a nearby FeS cluster. Finally, an H2ase exists that was long thought to be metal free. It has recently been shown that even this H2ase contains one Fe atom in an Fe(CO)2 group, which is most probably functional.26–28 It does not, however, contain any FeS clusters.28,29 According to its biological function, this iron–sulfur cluster-free hydrogenase from methanogenic archaea is termed H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd). In all H2ases, the Fe center is coordinated to carbonyl and cyanide ligands. Both ligands are ubiquitous in inorganic chemistry. Equally well, they are highly toxic and in fact strong enzyme inhibitors, for example, for Fe-containing

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Bioorganometallic Chemistry

Scheme 5

Scheme 6

heme oxygenases. Although we now understand the origin of these ligands in the H2ase enzymes, it is still a miracle why nature uses the Fe(CO)x(CN)y fragment. One characteristic of H2ases is the ability to split dihydrogen heterolytically, that is, there must be a hydride intermediate. This was shown as early as 1954 by isotopic exchange between D2O and H2, to yield HOD and HD, being catalyzed by H2ase enzymes (Scheme 6). The most common enzymes for the H2 uptake are the NiFe H2ases.25 They were also the first to be structurally characterized.30,31 The organometallic Fe(CO)(CN)2 core, which has a low-spin Fe(II) center, is linked to the Ni atom by two bridging cysteine residues (Scheme 5). The Ni center is furthermore coordinated by two more cysteine residues. One of them may be substituted by selenocysteine in the NiFeSe enzymes. Bridging and terminal cysteine residues are linked pairwise by a Cys–X–X–Cys motif. In the inactive form, the two metal centers are furthermore bridged by an oxide or hydroxide ion (Scheme 5). In addition to the atomic coordinates, the crystal structures reveal exit channels for protons (via hydrogen donor–acceptor groups such as carboxyl, amino hydroxyl, etc.) and electrons (a line of FeS clusters characteristically ˚ from the bimetallic core in opposite directions. Furthermore, a hydrophobic channel filled with Xe spaced by about 12 A) atoms in one crystal structure looks like a plausible H2 access route.32 A similar channel is identified in all other H2ases. Obviously, the dihydrogen splitting reaction is reversible, and this is the case in fermentative bacteria that generate dihydrogen. It should be noted that although isolated enzymes catalyze reaction (1) in Scheme 6 completely reversibly, it will be typically committed to only one direction in any given organism or metabolic path. Thus, the Fe-only H2ases are often used for H2 production.33,34 They are more efficient than the NiFe class, but also far more sensitive to dioxygen.25 The active site is shown in Scheme 5 with two Fe(CO)(CN) centers, bridged by a dithiol with three light atoms in the chain.35–37 Although the ambiguity (X ¼ CH2, NH, or O) could not be resolved by X-ray crystallography, the bridge is now generally assumed to be bis(methylthiol)amine (X ¼ NH).25,38 The bimetallic site is linked to the protein via a cysteine residue of an adjacent Fe4S4 cluster in addition to the hydrogen bonds to the cyanide ligands. The activation of dihydrogen is a major topic in organometallic chemistry, and numerous structural as well as functional model systems for the H2ase enzymes were proposed, which have been the topic of many books and reviews. A detailed discussion of all those is found in a recent book chapter.25 The thoughts particularly intriguing to an organometallic chemist are as follows. (i) H2ases constitute a very old class of enzymes. Likewise, mixed Fe(CO)x(CN)y compounds have been known for over a century. There is now a renewed interest in this class of compounds. Compounds like [Fe(CN)5(CO)]3

Bioorganometallic Chemistry

and trans-[Fe(CN)4(CO)2]2 were recently studied in great detail, in particular, with respect to their spectroscopic properties by Koch and co-workers.39,40 Hieber and co-workers prepared the first iron carbonyl sulfide complex Fe2S2(CO)6 and the iron hydride FeH2(CO)4 in the 1930s. Seyferth and co-workers examined iron carbonyl sulfides in more detail in 1980,41 but the field really took off following the structural characterization of an Fe-only H2ase.42–44 (ii) Several different redox states have been characterized for the H2ase enzymes, including activated forms in the presence of H2 or CO-inhibited forms. IR spectroscopy of the CO and CN ligands is a great help in identifying and characterizing these different states, as well as to distinguish between terminal and bridging carbonyls. It is not at all surprising for the organometallic chemist (but indeed, an uncommon experiment for a biologist) that the exact nature of the CO and CN ligands in the first crystal structure of NiFe H2ase from Desulfovibrio gigas could only be resolved by subsequent IR spectroscopic investigations!45–47 Another long-known and simple compound serves as an excellent spectroscopic model for the Fe(CO)(CN)2 core in NiFe H2ases, namely, [CpFe(CO)(CN)2].48,49 (iii) It is surprising that the exact mechanism of dihydrogen activation in H2ases still remains unclear. In fact, even the site of activation is not identified beyond doubt in the NiFe family. Dihydrogen complexes were first isolated and fully characterized by Kubas in 1984,50,51 and their bonding situation is now well understood.52 Stable Fe(II) dihydrogen complexes were later synthesized by Morris and co-workers.53 This, together with the fact that the above-mentioned hydrophobic access channel points toward the Fe atom in NiFe H2ase, seems to favor the Fe center as the primary point of H2 binding. Prior to the structural results, well-founded spectrosocpic and chemical evidence led Lindahl to suggest Ni(Cys)–hydride intermediates.54 A number of theoretical calculations have tackled the problem, invoking also bridging hydrides.

1.31.2.3 Carbon Monoxide Dehydrogenase (COdH) and Acetyl Coenzyme A Synthase (ACS) The interconversion of CO and CO2 is carried out by carbon monoxide dehydrogenases (COdH’s). These enzymes are major components in the global carbon cycle. They are often, but not exclusively, found in bifunctional enzymes together with ACS, which synthesizes acetyl coenzyme A from CO, a methyl group, and the thiol coenzyme A. Scheme 7 demonstrates the relevance of COdH and ACS in the early stages of carbon assimilation of anaerobic organisms.55 Monofunctional COdH includes two distinct classes, namely, an aerobic protein with a molybdopterin cofactor, and anaerobic phototrophs. Only the second class is relevant to this review, and all known anaerobic COdHs contain an NiFeS active site (called the C cluster), at which the CO-to-CO2 transformation takes place. Spectroscopic and structural studies from different species reveal some diversity in the C cluster. The two most extensively studied proteins are from Rhodospirillum rubrum (monofunctional COdH) and Moorella thermoacetica (formerly Clostridium thermoaceticum, bifunctional COdH and ACS).10 X-ray structural results are available for R. rubrum,56 and another monofunctional COdH from Carboxydothermus hydrogenoformans (Scheme 8).57 Both consist of a cuboidal NiFe3S4 cluster with an additional exo-Fe atom. All mechanistic proposals so far involve bimetallic pathways, but despite some effort, the exact nature of the catalytic species remains in question. One suggestion is the attack of an Fe–OH to the Ni-bound CO, followed by CO2 liberation from the metallocarboxylic acid.58 A very similar proposal reverses the two metals, that is, Ni–OH and Fe–CO

Scheme 7

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Bioorganometallic Chemistry

Scheme 8

Scheme 9

form the intermediates.59 Finally, Ludden an co-workers suggested a different pathway with Ni hydride intermediates.60,61 From an organometallic point of view, there is a striking similarity to the industrial water-gas shift reaction. A notable difference is that the latter generates dihydrogen, rather than protons and electrons (Scheme 8, bottom). All known ACS enzymes are bifunctional in that they possess a C cluster with COdH activity in addition to an A cluster (the ACS active site, Scheme 9). In the enzymes, a ‘‘CO tunnel’’ is described through which CO can pass directly from the C cluster, where it is generated from CO2, to the A cluster, where acetyl CoA synthesis takes place.62–64 Again, two mechanisms were proposed that differ in the order of binding events and redox states involved. In essence, however, CO binds to an Ni–CH3 species, followed by insertion and generation of an Ni–acetyl species, which upon reaction with CoA liberates the acetyl CoA product. It is interesting to note that methylation of Ni occurs by reaction with methyl cobalamin (Scheme 7). In M. thermoacetica, the cobalamin is the cofactor for a rather unique protein called the corrinoid iron sulfur protein (CFeSP). The above process, even if mechanistic details still remain in question, resembles the industrial Monsanto acetic acid synthesis process (Scheme 9, bottom). In this case, however, the reaction is catalyzed by a low-valent Rh catalyst. Numerous structural and functional models for both COdH and ACS were published, many of these results were recently reviewed.10,65

1.31.2.4 Methyl coenzyme M reductase (F430) It is worth noting that the toxicity of one of the simplest organometallic Ni compounds, Ni(CO)4, was discovered almost as soon as the compound itself. Consequently, nickel was long considered a toxic metal with no function in biology. Therefore, it came as a big surprise when Thauer discovered that nickel was absolutely required for the growth of

Bioorganometallic Chemistry

Scheme 10

methane-producing archaebacteria. These bacteria are strict anaerobes, they die quickly when exposed to dioxygen.66 Several C1 donors are used by the rather diverse class of methanogens, in particular, acetate and CO2. Dihydrogen or formate serve as reducing equivalents. As a unifying theme for all different pathways, a methyl equivalent is reduced to methane in the final, energy-conserving step of the catalytic cycle.67,68 This observation led to the isolation of Nicontaining enzymes from these bacteria and to their characterization as Ni-containing macrocycles with a reduced porphyrinoid ring, which is known as cofactor F430 (Scheme 10).69,70 In the activated state, the enzyme is believed to contain Ni(I), to which methyl groups can be oxidatively added. Methane is finally released upon addition of a proton and reductive elimination, and the Ni(I) state is regenerated.10 Although this is certainly an intriguing and convincing mechanism for an organometallic chemist, it is not without question. Based on density functional theory (DFT) studies, Sigbahn and Crabtree have suggested a mechanism for methane production that does not involve any Ni–methyl intermediates.71

1.31.3 Medicinal Organometallic Chemistry Medicinal aspects have always played a major role in the development of bioorganometallic chemistry. In fact, Salvarsan, the first cure for syphilis developed by Ehrlich in the early 1900s, is an organometallic compound. Today, we have a more detailed understanding of the molecular basis of diseases, and refined synthetic methods as well as structure–activity relationships (SAR). Given the thermodynamic as well as kinetic stability of organometallic compounds, the multitude of structural possibilities, and the additional properties of metal complexes such as redox activity, it is likely that the importance of medicinal organometallic chemistry is going to grow.72 In this introductory chapter, only a few examples are highlighted. A more comprehensive chapter on ‘‘medicinal organometallic chemistry’’ follows in Volume 12 of this series.

1.31.3.1 Anticancer Agents Anticancer agents have been a focus for the drug development for many years. Following the success of Cisplatin, Pt(NH3)2Cl2, which is one of the three most prescribed anticancer drugs, numerous other metal compounds have been tested, among them many organometallics. To the present day, however, none of these compounds has successfully passed clinical trials. An anti-proliferative effect has been demonstrated for metallocenes by Ko¨pf and Ko¨pf-Maier. Even simple ferrocenium salts were shown to have an anti-proliferative effect on certain types of cancer cells. The mechanism of action has not yet been elucidated and several targets including nuclear DNA, the cell wall, and the enzyme topoisomerase were proposed. Osella et al. showed that ferrocenium salts may generate hydroxyl radicals in physiological solutions.73,74 Whether these radicals damage the DNA or other targets, as for example, the cell wall, is unclear. In addition, there are conflicting reports on whether or not the redox state of the iron atoms is crucial for cytotoxicity. Neuse and co-workers found significantly enhanced cytotoxicity when ferrocenes were bound to polymeric supports.75–77 For the bent metallocene dihalides, structure–activity relationships were established for the halides and substitution of the Cp rings.78–81 Also, hydrolysis reactions were studied in detail with a view on aqueous stability. Model studies with amino acids, nucleic acids, proteins, and blood plasma provided more insight into the mechanism of action.82 Titanium compounds were most active, and titanocene dichloride has entered clinical trials.83 Although very promising

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Bioorganometallic Chemistry

in animal models, the clinical response was not encouraging enough to justify continuing trials, which were recently abandoned for titanocene dichloride. Due to its decomposition and low solubility in water, there were also problems with the formulation of the drug. Mainly because titanocene dichloride seems superficially similar to Cisplatin with two halide ligands in a cis-position, a related mode of action was assumed, that is, binding to DNA and eventually apoptosis of the cancer cell.84–86 Despite much effort, at no point was clear evidence for such a mode of action obtained. Instead, Ti binding to transferrin following hydrolysis was proposed,87 and even a stimulatory effect of aqueous Ti species on hormone-dependent breast cancer cells was observed.88 To circumvent some of these problems, modifications have recently been proposed. Titanocenes with amino groups were synthesized to increase aqueous solubility, and ansatitanocenes exhibit much greater hydrolytic stability.89–91 Both groups of compounds show promising biological activity. Research has also concentrated on molybdocene derivatives. Several X-ray structures with the Cp2Mo fragment coordinated to nucleobases were obtained.92–94 In addition, extensive spectroscopic studies, mainly by 1H and 31P NMR, were carried out in solution.93,95–97 Although Cp2MoCl2 was originally less active than Cp2TiCl2, it may in the long run be a more successful lead structure.97 Harding and co-workers investigated cellular uptake and intracellular localization of different bent metallocene dihalides by X-ray fluorescence.98,99 Only low levels of Ti and V were detected inside cells, and only Mo seemed to accumulate in significant amounts in the cellular nuclei (Figure 2). These findings agree well with the notion that all metallocenes have a different biological profile. Interestingly, molybdocene dichloride was also shown to hydrolyze phosphate esters and is thus a rare case of an organometallic nuclease.100,101 Ruthenium arenes are another interesting class of organometallics with proven anticancer activity.102 The most active complex [(6-biphenyl)Ru(ethylene–diamine)Cl]þ 1 (Scheme 11) had an activity comparable to Carboplatin against a human ovarian cancer cell line.103,104 The interaction of this compound with different biomolecules has been studied, and again, DNA has been suggested as the primary target.105,106 It is, however, unclear at present which events following the initial binding of the drug lead to the cell death. Sadler and co-workers solved the co-crystal structure of [(cymene)RuCl2] with lysozyme in order to shed more light on the possible interactions of this class of organometallics with proteins.107 As can be seen in Scheme 12, the organometallic compound occupies a pocket of the protein, but both chloride ions remain coordinated to Ru under the conditions of crystallization and one imidazole ring from a histidine binds to the Ru atom. More recently, another approach to organometallic anticancer agents was proposed. Organometallic fragments were mainly seen as large lipophilic groups that can replace phenyl rings in drugs. This approach has led to a ferrocene derivative (‘‘ferrocifen,’’ 2) of tamoxifen 3 (Scheme 13).108,109 Tamoxifen, a so-called selective estrogen receptor modulator (SERM), is the first-line drug for patients with hormone-dependent breast cancer. It works by competitive binding to the estrogen receptor (ER), thus repressing estradiol-mediated DNA transcription in the tumor tissue.110 Although tamoxifen is a highly active drug, it does not work on hormone-independent cancers, which constitute about one-third of all patients. In addition, expression of the ER may become down-regulated under tamoxifen treatment, turning the drug ineffective. Ferrocifen is a tamoxifen derivative, in which one of the phenyl rings has been replaced by a ferrocenyl group (Scheme 13). It is as active as tamoxifen on hormone-dependent cancer cell lines. Surprisingly, it is also active against hormone-independent cancer cell lines.109 Other organometallic fragments in place of the ferrocenyl group were also

Cp2 TiCl2

(a)

Cp MoCl 2 2

(b)

K

(c)

Figure 2 Distribution of metal compounds inside a single cell as studied by X-ray fluorescence: (a) Cp2TiCl2 is barely found inside the cell, (b) Cp2MoCl2 is well taken up and partly accumulated in the nucleus, and (c) K serves as a reference.

Scheme 11

Bioorganometallic Chemistry

Scheme 12

Scheme 13

tested, but found to be inactive in the later test.88,109,111 This suggests two different modes of action for ferrocifen. In addition to tamoxifen-like binding to the ER receptor, which was independently shown,109 a second pathway must exist which is critically dependent on the metal. In an elegant study, redox activation has been proposed as the second mode of action.112 The active metabolite hydroxyferrocifen is readily oxidized yielding a chinone methide intermediate which is activated for nucleophilic attack by nucleophiles. Extensive structure–activity relationship studies in correlation with electrochemical properties support this hypothesis. It is particularly noteworthy, and highly encouraging for the organometallic chemist, that redox activity of the metallocene is the key for additional biological activity that exceeds that of a purely organic analog. This idea, which is related to the concept of ‘‘oxidative stress’’ in connection with reactive oxygen species (ROS), has in fact been suggested previously113 and is gaining new popularity recently.72

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Co2(CO)6(alkyne) complexes represent another class of molecules with anti-proliferative properties in cancer cells. Derivatives of well-known inhibitors of cyclooxygenase (COX) enzymes were particularly active.114–117 Many analgetics and anti-inflammatory drugs are COX inhibitors. This class is commonly known as non-steroidal antiinflammatory drugs (NSAID). The link between inflammation and cancer has been pointed out118 and organic irreversible COX-2 inhibitors were published.119 The most active metal derivative today is the dicobalt hexacarbonyl complex of (2-propyn-1-yl)acetylsalicylate (Co-ASS, 4; Scheme 13). which is derived from the drug acetylsalicylic acid (Aspirin).120 This compound is a potent inhibitor of COX. Its anti-proliferative effect is greater than that of Cisplatin, and Co complexes were generally more active than the metal-free derivatives. Other cellular targets were also evaluated, and the cellular uptake of Co was quantified by atomic absorption spectroscopy (AAS).117,120 As for ferrocifen, an additional, metal-specific mode of action seems to be involved. In this context, but seemingly unrelated, reactive intermediates derived from Co2(CO)6(alkynes) have been investigated computationally.121 Finally, yet another class of metal carbonyls with anti-neoplastic properties was discovered recently by Schmalz and co-workers.122 They tested iron carbonyl derivatives of nucleosides, such as 5 (Scheme 13). For this series of compounds, a clear structure–activity relationship emerges.123 The most active derivatives have IC90 values in the low mM range against BJAB cell lines. In addition, selected derivatives of this class of compounds showed good in vitro activity against leukemia cells from patients with acquired resistance against common anticancer drugs. The mechanism of cell death was also investigated. Although the BJAB cells were finally apoptotic, apoptosis did not seem to be initiated by the regular signaling cascades.122,123 Further investigation is in progress,124 and it is certainly interesting to note that organometallic compounds may invoke a new mechanism of action.

1.31.3.2 Antimicrobial Agents 1.31.3.2.1

Antibacterial

The first drug which was discovered by systematic screening was, in fact, an organometallic one. Compound number 606 in Paul Ehrlich’s laboratory proved to be particularly effective against syphilis. It was marketed as Salvarsan in Europe (arsphenamine in the USA, 6, Scheme 14) and its structure was originally thought to be an analog of azobenzene with an AsTAs double bond. Later studies made an oligomeric structure more likely, and arsenobenzene is a cyclic hexamer. In the absence of a crystal structure for Salvarsan, a recent mass spectrometric study provides the best structural data today.125 Interestingly, the hydrochloride of Salvarsan is too toxic for humans, so the compound had to be administered in basic solution, in which it is only poorly soluble. The more soluble NeoSalvarsan 7 was later introduced to the market for the same disease. Both compounds are easily oxidized in air and in the body, and indeed the phenarsine oxide is the active metabolite. This compound, Mapharsen, which probably also has an oligomeric structure, was also marketed in the 1920s. The As-containing antibiotics were later gradually replaced by penicillins and sulfonamides. There is, however, renewed interest due to the growing resistance in bacteria to many of the common antibiotics. Also, organic mercury compounds have been in use as mild antiseptics until recently. Concerns have been raised126 (and subsequently debated)127 that some cases of autism and Asperger’s syndrome in children might be related to mercury poisoning. The design concept for the anticancer-active ferrocifen mentioned above is replacement of a phenyl ring by a ferrocene substituent. The very same principle has previously been applied to a number of antimicrobial agents. The first ferrocene derivatives of penicillins and cephalosporins have been synthesized by Edwards and co-workers.128–130 Further derivatives were later investigated.131–136 Unlike the anti-malarials discussed below, there has been no real breakthrough for organometallic antibacterials. Small peptides are a promising class of antibacterial compounds. They are mostly comprised of cationic and lipophilic amino acids, and minimal motifs containing only Arg and Trp have been suggested. Presumably, interaction of these peptides with the bacterial cell membrane contributes to their activity. Assuming that metallocenes are bulky, lipophilic groups, which may (cobaltocenium) or may not (ferrocene) possess additional charges, we have

Scheme 14

Bioorganometallic Chemistry

Scheme 15

prepared a number of metallocene peptide conjugates and tested their antibacterial activity on different Grampositive and Gram-negative bacteria (Scheme 15).137 In these compounds, the metal complex did not increase the overall activity, but could switch selectivity. For example, the ferrocene–peptide conjugate 8Fe is about 5 times more active against the Gram-positive Staphylococcus aureus than against Gram-negative E. coli, whereas the activity is reversed for the cobaltocenium–peptide conjugate 8Co.138 For both compounds, the minimum inhibitory concentration (MIC) is comparable to the natural antibiotic peptide Pilosulin 2, which is also more active against E. coli.

1.31.3.2.2

Antimalarial

Antimalarial drugs is another area of medicinal chemistry which is successfully investigated. The malaria parasite Plasmodium falciparum is increasingly developing strains which are resistant against common antimalarial drugs such as chloroquine. Brocard and co-workers have synthesized a ferrocene analog of chloroquine,139 which is active even against chloroquineresistant Plasmodium strains with IC50 values in the low nM range.140–142 Ferroquine 9 was able to protect mice from infection and is now in clinical phase I trials (Scheme 16). Trapping of the compound in the food vacuole of the parasite and inhibition of hemozoin formation is the primary mechanism of action.141,143,144 In this respect, ferroquine is similar to chloroquine 10, but clearly, the lipophilic metallocene is needed for enhanced bioactivity. Structure–activity relationships have been established.145 At present, it appears that subtle changes such as increased lipophilicity and differences in geometric and electronic structure suffice to account for the activity even against chloroquine-resistant strains.144,146 It remains to be established, however, whether there is an additional, as yet undiscovered, mode of action, similar to the case of ferrocifen. It is worth noting that the two optical isomers of ferroquine exist due to the planar chirality of the unsymmetrically 1,2substituted ferrocene moiety. Both enantiomers were prepared by enzymatic resolution of an ester intermediate in >98% optical purity. Both isomers display similar activity in vitro.147 Although both enantiomers are less active than the racemate in vivo, the (þ)-enantiomer displays better curative effects than the optical antipode. This different behavior indicates different pharmacokinetics of the two enantiomers. Ferrocene derivatives of other antimalarial drugs like artemisinine, quinine, and mefloquine have also been tested,141,148,149 as well as various other chloroquine-derived organometallics.150 Moss and coworkers synthesized and tested chloroquine and ferroquine derivatives with other organometallic groups.151–153

1.31.3.3 NO/CO Drugs Carbon monoxide, although toxic in higher concentrations, appears to be an important signaling molecule in the body, particularly with relation to the cardiovascular system. Metal carbonyls are among the oldest and best investigated classes of organometallic compounds. However, it has only recently been discovered that many simple metal carbonyls like Fe(CO)5, Mn2(CO)10, and [RuCl2(CO)3]2 may release CO under physiological conditions (possibly with irradiation) and will thus effect vasodilation and reduce acute hypertension in vivo.154 More compounds were tested, and [RuCl(CO)3(glycinate)] 11 appears to be the most promising candidate so far. The compound had remarkable protective effects in animal experiments. A condition of induced ischemia was survived with no damage

Scheme 16

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Bioorganometallic Chemistry

in isolated rat hearts when 50% of the tissue was damaged without addition of 11. Correspondingly, survival rates of mice with a heart transplant were much higher when 11 was given during the transplant. These effects may partly be attributed to the inhibition of blood platelet aggregation of the compound.155 As yet, the exact mechanism of action is unclear. Inhibition of heme enzymes has been suggested as well as CO acting as a reductant. There may be some relation to the ACS/COdH reactivity discussed above. Although it is still early in the investigation of metal carbonyls as CO-releasing drugs, there seems to be much promise in the area.154 Actually, neither the notion of CO being an important signaling molecule, nor the fact that CO-containing metal complexes have potential as drugs comes as a surprise. Carbon monoxide is isoelectronic with NOþ. NO, on the other hand, has been known to be a major second messenger with a variety of functions. This discovery was awarded a Nobel prize recently, and Science magazine declared NO the molecule of the year in 1992. Nitroprusside sodium Na[Fe(CN)5NO], which is an organometallic compound by definition (although this volume deliberately excludes cyanide ligands), has been an important drug for over a century.

1.31.3.4 Radiopharmaceuticals Metal carbonyls and the isoelectronic isonitriles do also play an important role as radiopharmaceuticals. 99mTc is probably the most frequently used metal isotope for radioimaging. 99mTc (t1/2 ¼ 6 h) is by far the most widely used metal isotope in radiopharmaceuticals. This pre-eminent position is due to its very favorable physical properties that allow chemical synthesis and radioimaging on a reasonable timescale without extended exposition of the patient. The low-valent Tc isonitrile complex [Tc(CN–CH2–C(CH3)2–OCH3)]þ (Sestamibi, 12) was reported by Davison in 1983,156 and has found widespread use in radioimaging of the heart (Cardiolite by DuPont). The d 6-low-spin configuration makes this compound particularly stable, even under physiological conditions. Its selectivity for cardiac tissue has been attributed to its size, lipophilicity, and positive charge. 99m Tc radiopharmaceuticals must be prepared and used ‘‘on the spot.’’ Ideally a ‘‘kit’’ formulation without the need for subsequent purification should be provided for maximum ease and safety of use by the technical staff in hospitals. Alberto, Schubiger and co-workers have made a significant step forward towards this goal by the preparation of the [99mTc(CO)3(H2O)3]þ cation 13.157,158 This compound can be readily synthesized in one step from TcO4 by BH4 reduction in the presence of CO. It has been used, inter alia, for the labeling of serotonergic receptors in the brain and Histagged antibodies.159,160 A more recent and also more convenient preparation for 13 uses the long-known boranocarbonate [BH3CO2H] (Scheme 17).161 A ‘‘kit’’ preparation using this chemistry is marketed by the Mallinkrodt company (Isolink). The Tc(CO)3 fragment has quickly found widespread use, for instance, for the labeling of peptides by various groups,162–168 vitamin B12,167 and a number of other biomolecules.169 A simple route has been proposed for the synthesis of CpTc(CO)3 compounds with an acetyl-substituted Cp ring.170 The labeling of biomolecules with CpTc(CO)3 by double ligand transfer (DLT) reactions from ferrocene derivatives was used by Katzenellenbogen and co-workers.163,171,172 DLT is, in fact, quite an old technique that was originally explored by Wenzel and co-workers in the 1960s.173

1.31.3.5 Toxicology and Environment Environmental issues related to the toxicology of organometallic compounds have played an immense role in the past, and they may likely continue to do so. Two books have been published on the subject.174,175 Volume 12 of this series has a full chapter on ‘‘environmental and biological aspects of organometallic compounds.’’ Therefore, this section only superficially touches a few examples involving As, Hg, Sn, and Pb. While the term ‘‘bioorganometallic’’ is certainly appropriate to describe CO- or CN-inhibited forms (and thus poisoning) of heme proteins, such species will not be discussed here. On the other hand, the toxicology of organometallic compounds is inevitably related to their reactions with biomolecules, especially proteins.176 Organoarsenic compounds have a history not just for good (see Salvarsan above), but also for use as poison gas in World War I. Lewisite (ClCHTCHAsCl2) was tested but luckily never used. Research into possible antidotes led to the development of 2,3-dimercaptopropanol, known as mercaprol or British anti-Lewisite (BAL). This compound to

Scheme 17

Bioorganometallic Chemistry

date is a potent and rather versatile antidote against metal poisoning. It works best for those soft heavy metal ions which form strong metal–sulfur bonds (Hg, Pb, As, etc.). The water-soluble complex is then excreted. Of historical interest is the fact that wallpapers in the nineteenth century were frequently painted with metal salts, for instance, the so-called Scheele’s green (cupric arsenite). In the damp and ill-ventilated rooms at the time, these wallpapers gave off volatile As compounds, mainly consisting of trimethylarsine. Methylcobalamin is evidently a vital cofactor for the molds that produce these arsines.177 They were, for a long time, thought to be responsible for illnesses and deaths among people living in these rooms. The famous Napoleon Bonaparte was long thought to be a victim. However, this rather compelling story has little scientific credibility as trimethylarsine lacks the toxicity to be a poison gas.178 Arsine, AsH3, on the other hand, is acutely hemotoxic, resulting in almost certain death if inhaled in larger quantities.179 It has therefore nowadays mostly been banned from undergraduate teaching laboratories, where it had long been used in a famous (and reliable) analytical test for As (Marsh’s Test). Another fateful event involving organomercury compounds makes methylmercury chloride one of the most intensely studied of all organometallics. In the coastal town of Minamata, Japan, thousand of victims died from mercury poisoning in 1953.180 It turned out that the mercury originated from a factory preparing acetaldehyde, from which contaminated waste water was released into the ocean. Mercury was then accumulated in shellfish, bio-toxified by methylation, so that the highly toxic methylmercury–methylthioether CH3Hg–SCH3 was ingested by consumption of the shellfish by the local population. Organic mercury compounds, and in particular methylated mercury species, are potent neurotoxins.181 The tragic case of a colleague is well documented. She spilled a few drops of dimethylmercury on her hands when preparing an NMR sample, went home untreated (because the spill seemed minor and she was wearing vinyl gloves), and died almost one year later, after months of agony, from incurable mercury poisoning. The use of tetraethyl lead (PbEt4), which was first prepared by Lo¨wig as early as 1853, as a fuel additive has raised major environmental concerns to the effect that it is banned in most countries today.182 Diethyltin iodide was widely distributed in 1954 in France as a cure for staphylococcal infections. The sample, which was contaminated with the much more toxic triethyltin iodide, caused over 100 deaths. Tributyl- or even trioctyltin compounds, which are far more lipophilic, have been used as preservants in plastics, clothes, and as antifouling paintings on ships. They are, however, seen with increasing skepticism and have been mostly replaced by hopefully less toxic compounds. Nonetheless, these examples underscore the necessity for constant and critical evaluation of all the chemicals in common usage.

1.31.4 Organometallic Compounds For Biological Studies A large number of organometallic compounds have been covalently attached to biomolecules like amino acids, proteins, sugars, nucleobases and nucleotides, lipids, hormones, and others. Inclusion of these conjugates into the definition ‘‘bioorganometallics’’ certainly constitutes a very large subgroup. To even try to cover the field comprehensively is beyond the scope of this chapter. The discussion is hence limited to such conjugates that were used in biological studies. An excellent review, published by Severin et al. in 1998, covers organometallic amino acid and peptide derivatives.183 A Russian review deals with the ferrocene conjugates of DNA and its constituents.184 All these are also contained in our more extensive review on the bioorganometallic chemistry of ferrocene.3 Two recent book chapters cover organometallic peptide and peptide nucleic acid (PNA) conjugates185 and the labeling of proteins186 in detail.

1.31.4.1 Amino Acid, Peptide, and Protein Derivatives In 1957, Schlo¨gl reported the synthesis and characterization of several ferrocene amino acids, including ferrocenylalanine (Fer, 14; Scheme 18).187 Other organometallic amino acids include alanine, phenylalanine (Phe), glycine derivatives, as well as further ferrocene-based amino acids, which are discussed below. The Schlo¨gl paper also describes the reaction of ferrocene carboxylic acid and ferrocene carbaldehyde with amino acids. The chemistry was later picked up in numerous publications, and ferrocene carboxylic acid amides or ferrocene imines with basically all amino acids and many dipeptides were reported.3,185 Such conjugates may serve as ligands for other transition metal ions.188–194 Kraatz has prepared ferrocene carboxylic acid conjugates with short peptides that serve as enzyme inhibitors.195–197 Improved syntheses for 14 were reported, also for enantiomerically pure derivatives.198–201 Asymmetric hydrogenation was used for this purpose, as well as Pd-catalyzed coupling of iodoferrocene with a serine-derived organozinc reagent. In the 1980s, this amino acid was incorporated into small peptides, including biologically relevant peptides in which Fer replaces Phe. Examples are the neuropeptide [Fer4]-enkephalin202–204 and the peptide hormones Substance P ([Fer7]-SP, [Fer8]-SP),205 bradykinin ([Fer5]-BK, [Fer8]-BK),205 and sarcosine derivatives of angiotensin II

897

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Bioorganometallic Chemistry

Scheme 18

([Sar1,Fer8]-AT).206,207 Other organometallic peptide conjugates were based on enkephalin,204 Substance P,208 neurokinin A,209 angiotensin II,206,207 gonadotropin-releasing hormone,210 secretin,211,212 and glutathion.213,214 Some of these peptide conjugates were tested for their biological activity, for instance, receptor-binding affinity. The syntheses of these conjugates were carried out by the traditional Merrifield solid-phase peptide synthesis (SPPS) technique. The harsh conditions for deprotection and cleavage caused at least partial decomposition of the conjugates. Characterization of these early conjugates is indeed scarce. Therefore, the biological results have to be treated with caution. We have recently studied cellular uptake and subcellular localization of the metallocene peptide conjugates with ferrocenoyl or cobaltocenium groups (Scheme 18).215,216 The conjugates were prepared by SPPS, purified, and comprehensively characterized. To enable visualization inside living cells, an additional fluorescence tag was added on an orthogonally protected lysine residue. TAT–metallocene conjugates 15 were prepared to study how the lipophilic metallocene will influence cellular uptake.216 Nuclear localization was studied by binding the metallocene to the simian virus SV40 nuclear localization sequence (NLS, 16).215 Representative results are shown in Figure 3. The ferrocene–TAT conjugate 15Fe is readily taken up by HepG2 cells. It is mainly localized in the cytoplasm. Both the ferrocene-NLS and cobaltocenium-NLS conjugates 16, on the other hand, are readily taken up and also localized in the nucleus of the cells. Radioactive (99mTc(CO)3-labeled) NLS conjugates were later used by Alberto and co-workers to induce radiation damage to nuclear DNA in B16F1 mouse melanoma cells.217 Our group has initiated a project to search for milder methods for the preparation of organometallic peptide conjugates by SPPS. By using a base-labile linker to the resin and suitable side-chain protecting groups, we were able to prepare acid-sensitive organometallics amendable to SPPS techniques. As an example, enkephalin derivatives with the covalently bound Mo(allyl)(CO)2 moiety such as 17 were successfully prepared (Scheme 19).218–221 An attractive alternative is the ‘‘two-step labeling procedure.’’ In this case, a robust anchoring group is placed in the peptide at the

(a)

(b)

(c)

Figure 3 Cellular uptake and intracellular distribution of metallocene–peptide conjugates (see text): (a) cobaltocenium-NLS (16Co), (b) ferrocene-NLS (16Fe) and (c) ferrocene-TAT (15Fe). Nuclear localization is seen for the two NLS conjugates, while the ferrocene–TAT conjugate accumulates almost exclusively in the cytoplasm and does not enter the nucleus.

Bioorganometallic Chemistry

Scheme 19

desired position. In a second step, the organometallic group is bound to this anchoring group after cleavage from the resin and purification of the anchor peptide. Labeling reagents for cysteine and lysine residues are well established for this purpose in bioorganic chemistry. We have used Sonogashira coupling to link organometallics selectively to peptides in such a two-step procedure (Scheme 19). 222–224 For this purpose, p-iodo-phenylalanine (PheI) serves as the anchoring group and replaces Phe. Following studies on the model dipeptides, a modified enkephalin (H-TyrGly-Gly-PheI-Leu-OH) 18 was prepared by SPPS, purified, and reacted in solution with ferrocene alkynes to yield structurally novel organometallic peptide bioconjugates 19 (Scheme 19).224 Metallocenes, in particular ferrocene, may also serve as peptide mimetics. Herrick and co-workers were the first to recognize that 1,19-disubstituted ferrocene may serve as a peptide turn mimetic, since it holds the two peptide strands in a geometry similar to turn structures found in proteins.225,226 Further work by several groups unraveled the rules which govern those structures.196,227–241 In addition to X-ray crystallography, variable-temperature NMR, IR, and CD spectroscopy were employed to provide structural insights in solution. Very stable structures result if at least two interstrand hydrogen bonds form and it appears that out of many possibilities, only very few types of structures are actually realized. It is interesting to note that helical chirality of the metallocene is possible. Due to the low energy of rotation about the metal–Cp bonds (in the order of a few kJ mol1), M and P isomers interconvert readily in solution. They can, however, be ‘‘locked’’ by hydrogen bonds between peptide substituents on the two rings, as indicated in Scheme 20. The structural results have recently been summarized and a systematic nomenclature has been proposed.242 Depending on the metallocene backbone, three different peptide orientations are conceivable (Scheme 21). Ferrocene 1,19-dicarboxylic acid (class-I) has been predominantly studied, but examples of ferrocene1,19-diamine (class-III) have recently been published. Both ferrocene derivatives will orient the peptides in a parallel fashion. Peptide derivatives of 19-aminoferrocene-1-carboxylic acid (ferrocene amino acid, Fca, class-II) are most similar to natural turn structures as they orient the pendant peptide strands in an antiparallel fashion. Fca was

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Bioorganometallic Chemistry

Scheme 20

Scheme 21

prepared independently by two groups in 1998.243,244 Rapic and co-workers reported several differently protected Fca derivatives.245 An improved synthesis was reported by Heinze and co-workers,246 who used Fca dimers as electrochemical anion sensors. This group also contributed computational studies for structural dynamics of Fca– peptide conjugates in solution. In collaboration with Rapic’s group, we have prepared the first Fca peptide conjugates by SPPS 247 and reported the X-ray single crystal structure of the tetrapeptide Boc–Ala–Fca–Ala–Ala–OMe.248 A comprehensive paper investigates the solution and solid-state properties of Fca peptides.249 Very recently, Kraatz’s group has shown that Fca-containing peptides may serve as collagen mimics.250 The same group has also structurally characterized the first synthetic model for -barrel structures, which also incorporates Fca.251 It is highly interesting that 1,19-disubstituted ferrocenes were used as haptens to generate antibodies for the stereoselective Diels–Alder reaction.252,253 In this context, the idea of helical chirality (and the ease of interconversion between the two enantiomers) was implicitly mentioned and used by Janda and co-workers. They also reported the first synthesis of Fca derivatives (similar to the Rapic route) and the X-ray single crystal structure of an antibody, which was co-crystallized with a ferrocene derivative.254 Scheme 22 shows the helical chirality of this protein-embedded ferrocene derivative, which is only held in place by non-covalent interactions and hydrogen bonds. A number of other ferrocene-based amino acids were reported, for example, 1,2-ferrocenylbisalanine,255 ferrocenyl--alanine and 1,19-ferrocenylbis--alanine,256 1,19-ferrocenylbisglycine,257 and ferrocenylenebisvaline.258

Bioorganometallic Chemistry

Scheme 22

1,19-Ferrocenylbisalanine 20 has been prepared in an enantiomerically pure form. Via a sophisticated synthetic route, Frejd and co-workers prepared an enantiomerically pure derivative of 1,19-ferrocenylbisalanine with orthogonal protecting groups (21, Scheme 23).259–262 After lactamization, the resulting 1,19-ferrocenophane was incorporated into several peptides as a substitute for two aromatic amino acids (Phe or Tyr).260,261 Many proteins have been labeled with organometallic complexes, mostly for analytical purposes. Some of those are mentioned in Section 1.31.5 of this chapter, and the topic has been comprehensively reviewed by Salmain.186 Ryabov published an earlier review on the topic.176 The labeling techniques are mostly the same as for organic derivatives, that is, cysteine-selective reactions (maleiimides, acetic acid halogenides), activated acids, aldehydes, or thiocyanates that react with lysines, biotin-(strept)avidine labeling, and others. The use of alkoxycarbene complexes is probably most interesting for an organometallic chemist. Reaction of the tungsten methoxycarbene 22 with primary amino groups in peptides (such as lysine side chains or the amino terminus) gives the aminocarbene 23 (Scheme 24). In a reaction with the protein bovine serum albumin (BSA), four out of six lysine residues were shown to be available for reaction with the tungsten complex.263 Tungsten

Scheme 23

Scheme 24

901

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Bioorganometallic Chemistry

Scheme 25

aminocarbene adducts of lysozyme were also investigated.264 Similar results were obtained with organometallic pyrylium salts of (Cr(CO)3(arene) and ruthenocene.265,266 In two papers, Hill et al. studied ferrocenyl derivatives of the enzyme cytochrome P450cam (CyP450cam).267,268 This enzyme catalyzes the regioselective oxidation of camphor to 5-exo-hydroxycamphor. Like all cytochromes, CyP450cam has a heme group in its active center. The highlight of these papers is the X-ray single crystal structure at 2.2 A˚ resolution of a CyP450cam(C334A) mutant with the two ferrocenyl maleimides covalently bound to the enzyme (Scheme 25).268 While the Cys136 is on the periphery of the enzyme, the Cys85-bound ferrocenyl moiety is very close to the active center, and indeed, camphor is displaced by the ferrocenyl maleimide which is an irreversible inhibitor of the enzyme.

1.31.4.2 DNA, RNA, and PNA Derivatives In contrast to the amino acid, peptide, and protein conjugates, where a number of different organometallic complexes were used for labeling, DNA derivatives are by and large limited to ferrocene. This is very likely due to the attractive electrochemical properties of ferrocene. Indeed, electrochemical DNA sensors are covered in more detail in Section 1.31.5.2. Two recent reviews cover the ferrocene conjugates with nucleobases, nucleosides and nucleotides, DNA and RNA, as well as their applications.3,184 In most of the work on oligo-deoxynucleotides (ODNs) to date, very similar chemistry has been used for covalent binding of organometallics to the ODNs (Schiff base, activated acids, metal-catalyzed couplings). Anne et al. reported an interesting enzymatic reaction to extend the 39terminus of an ODN.269 They use a ferrocenylated di-deoxynucleotide uridine triphosphate (Fc-ddUTP) as the substrate, which is accepted as an enzyme substrate and terminates the oligomer. Other Fc-dUTP derivatives were also tested as substrates for common DNA polymerases.270 This approach was later extended to ferrocene-labeled RNA, and electrochemical RNA detection on Au-DNA electrodes could be achieved.271 It is interesting to note that early work on ferrocene derivatives of nucleosides uses Sonogashira coupling of ethynylferrocene to 5-iodouridine or 8-bromoadenosine.272 This reaction was later used to synthesize ferrocenylated ODNs by solid-phase synthesis techniques. Depending on the conditions, a cyclization reaction occurs with uracil derivatives.273–275 Instead of ethynylferrocene, Grinstaff et al. used ferrocene carboxylic acid propargyl amide.276–278 The same compound was used by our group before in peptide chemistry and a crystal structure has been reported.223 The stability of metallated ODNs with complementary DNA or RNA has been investigated by UV melting studies.3 PNAs are a class of DNA analogs with very promising properties for applications in molecular biotechnology or medicine. Compared to DNA, the ribose phosphate ester backbone is replaced by a pseudo-peptide backbone in

Bioorganometallic Chemistry

Scheme 26

PNA (Scheme 26). The nucleobases are linked to this backbone via a carboxymethylene linker.279,280 PNA binds to complementary DNA or RNA oligomers according to Watson–Crick rules with high stability.281–284 An increase in stability, as measured by the melting temperature (UV Tm), between 1 and 3  C per base pair in a PNA?DNA duplex has been observed compared to a homologous ds-DNA. In addition, PNA shows a higher mismatch sensitivity toward non-complementary DNA than is commonly observed in ds-DNA.285,286 These favorable properties led to numerous applications for PNA oligomers in biotechnology and as anti-sense agents.287–291 Furthermore, a large number of structural variations of the original PNA have been published.292 Our group published the first organometallic derivatives of PNA monomers.293 Ferrocene carboxylic acid and benzoic acid chromium tricarbonyl were coupled to the amino group of PNA monomers with different nucleobases in solution using HBTU as the coupling agent to give compounds 24 and 25, respectively (Scheme 27). C-terminally labeled organometallic PNA monomers and dimers were also obtained.185 Maiorana’s group have used the Ugi fourcomponent reaction (Ugi-4CR, for 26)294,295 and olefin metathesis (for 27)296 for the preparation of other metal–PNA derivatives (Scheme 28, the different colors indicate individual components in the respective reactions).297 PNA oligomers with covalently linked organometallics were also first reported by our group. Originally, ferrocene carboxylic acid and chromium tricarbonyl benzoic acid were incorporated into PNA heptamers with the sequence H-tgg atc g-Gly by solid-phase peptide synthesis techniques.298 (According to common convention, the same fourletter code is used for PNA as for DNA; small letters, however, indicate PNA oligomers. The PNA sequence is written in peptide convention from N- to C-terminus.) Whereas the conjugate Fc-CO-tgg atc g-Gly-NH2 was purified by preparative high-performance liquid chromatography (HPLC) and shown to have the correct mass by mass spectrometry (MS), the chromium tricarbonyl derivative could not be obtained in a pure form. In order to increase stability and solubility in water, we have incorporated cobaltocenium carboxylic acid into PNA oligomers by solidphase synthesis.299 Following initial studies on smaller oligomers to optimize coupling and deprotection conditions, the decamer CpCoþC5H4-CO-acc ctg tta t-Lys-OH 28 was synthesized by solid-phase synthesis techniques, purified by preparative HPLC, characterized by MALDI-TOF mass spectrometry. A square wave voltammogram was clearly indicative for the presence of the cobaltocenium group. This conjugate was also studied in the interaction with complementary DNA. Compared to acetylated PNA of the same length and sequence, a slight stabilization of about

Scheme 27

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Bioorganometallic Chemistry

Scheme 28

1  C is observed for the cobaltocenium conjugate 28, which might be attributed to an attractive force between the positively charged cobaltocenium group and the negatively charged DNA strand.299 Metallocene PNA oligomers were deposited on Au microelectrodes, and their electrochemical properties were found to be very attractive for electrochemical DNA sensors.299,300 Also, novel Re(CO)3 conjugates with PNA decamers were reported by our group.301 These conjugates have interesting spectroscopic properties.

1.31.4.3 Others Numerous other organometallic conjugates with all kinds of biomolecules were prepared. There is obviously a tremendous potential in the use of chiral biomolecules as synthons or ligands in organometallic catalysis. Furthermore, modern natural product synthesis almost inevitably involves organometallic reactions. None of these topics is touched in here, although undoubtedly bioorganometallic intermediates are involved which may even be isolable. In the following, the focus is on the sugar derivatives, lipids, receptor ligands, and the use of organometallics in peptide synthesis.

1.31.4.3.1

Sugar derivatives

Starting in 1961, a large number of ferrocene sugar conjugates were prepared. Originally, the interest was mostly in synthesis and characterization, and ester, thioester, or amide derivatives of ferrocene carboxylic acid were the main objectives. Scheme 29 summarizes many of the 1,19-ferrocene dicarboxylic acid compounds that are known. The X-ray single crystal structure of 29 was reported by Keppler’s group.302 This compound had poor cytotoxic activity. All other compounds in Scheme 29 had similar EC50 values around 20 mM against a mouse mammary tumor cell line. The same compounds had far lower antimalarial activity than quinine as shown by Itoh’s group.303 Metal carbene derivatives of sugars were extensively investigated by Do¨tz and co-workers.304,305 These compounds can be prepared by nucleophilic addition to the metal-coordinated carbene atom, by conjugate addition to the vinylogous position in alkenyl or alkynyl carbene ligands, or by stoichiometric olefin metathesis.306 Highly complex molecules with several chiral centers form in cycloaddition reactions with sugar-derived metal carbenes in excellent diastereoselectivity, as exemplified in Scheme 30. While the fully protected sugar metal carbenes are lipophilic organometallics that are soluble in organic solvents, O-deprotection will increase aqueous solubility as a result of the free hydroxy groups. Upon addition of the long alkyl chains to such molecules, organometallic gelators with interesting properties form.307

1.31.4.3.2

Lipids

Compared to many other classes of biomolecules, organometallic derivatives of lipids are a largely under-developed area of research. An early paper reports the use of ferrocene conjugates for the electrochemical HPLC detection of

Bioorganometallic Chemistry

Scheme 29

Scheme 30

fatty acids.308 Conjugates with glycerol and cholin were also prepared.309 A ferrocenyl cholin conjugate was shown to be a competitive inhibitor of the enzyme butyrylcholinesterase with a Ki value in the mM range.310 Very recently, a significant influence of the redox state of the metallocene in ferrocene-containing cationic lipids was demonstrated.311 In the neutral ferrocene state, high levels of transfection with DNA coding for enhanced green fluorescent protein (EGFP) were observed similar to standard transfection reagents. In the cationic ferrocenium state,

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Bioorganometallic Chemistry

however, transfection was almost completely shut down and only very little green fluorescence was observed. Clearly, the difference in charge and concomitantly different lipophilicity directly influenced transfection efficiency of the system.

1.31.4.3.3

Receptor ligands

Organometallic derivatives of steroid hormones have been used to study ligand–receptor interactions for quite some time. SERMs with anti-tumor activity have already been discussed in Section 1.31.3.1. Other applications were developed for metallo-immuno assays and are discussed in Section 1.31.5.3. For such applications, binding of the organometallic compound to a receptor is necessarily implied but not always proved or quantified. The steroid hormone estradiol 30 has been the subject of labeling with a number of different organometallic compounds. The structure of 30 (R ¼ H) and its derivatives is shown in Scheme 31, Table 1 summarizes the relative binding affinities of organometallic estradiol derivatives to the ER. Most derivatives so far are 17-substituted estradiols, that is, the organometallic group R is pointing backward on C17. Top and co-workers carried out molecular modeling studies to understand binding affinities of organometallic selective estrogen receptor modulators (SERMs) to the ER based on its crystal structure.312 Very recently, Gmeiner and co-workers prepared metallocene-derived receptor ligands for G-protein-coupled receptors (GPCRs) such as dopamine and serotonin receptor subtypes. They used ruthenocene and ferrocene derivatives, in which the metallocenes replaced cyclophanes as so-called ‘‘fancy bioisosters.’’319 In particular, compound 31 (Scheme 32) showed sub-nanomolar affinity and high specificity for the dopamine D4 and serotonin HT1A receptor subtypes, and may thus be a suitable lead structure for the further development of selective organometallic GPCR ligands. In the above example, as in the ferrocifen series and many others above, the organometallic group (e.g., ferrocene) simply serves as an inert, bulky, lipophilic residue. Indeed, it often replaces benzene in an organic structure. In a slightly different approach, Meggers’ group has developed organometallic compounds in which the metal’s role is to correctly place other (organic) groups in three-dimensional (3-D) space. Their target was to design analogs of protein kinase (PK) inhibitors based on the structure of the purely organic inhibitor (þ)-staurosporin.320–322 Scheme 33 shows

Scheme 31

Table 1 Relative binding affinity (RBA) of some mononuclear organometallic estradiol derivatives to the ERa,b,c R

RBAa

Referencesb

Fc (-C5H4)Ru(Cp) -U-Fc (-C6H5)Cr(CO)3 -U-(-C6H5)Cr(CO)3 -U-(-C5H4)Mn(CO)3 -U-(-C5H4)Re(CO)3 CH2-(-C5H4)Mn(CO)3 CH2-(-C5H4)Re(CO)3 [(-C5H4)Ru(Cp* )]OTf

8 2 28, 37e 11 24 15 16 2.5 0.8 0

313d 313d 314d 315 315 316 316 316 316 315

a

RBA values determined in a competitive radioreceptor binding assay at 0  C. For examples of estradiol conjugates with dinuclear organometallic complexes, see Refs: 317, 318. c See also Scheme 31. d X-ray crystal structure reported. e RBA to the ER. b

Bioorganometallic Chemistry

Scheme 32

Scheme 33

the structure of (þ)-staurosporin 32 and a CpRu analog thereof. In addition to a similar molecular shape and surface, Scheme 34 also shows that the electrostatic potential surfaces of staurosporin and its organometallic analog 33 are very comparable.72 On the basis of a co-crystal structure of enantiomerically pure (S)-33 and the PK Pim-1, it has been suggested that electrostatic interactions indeed play a crucial role to explain the very high affinity of 33 to Pim-1.72,322 Indeed, the affinity of (S)-33 to Pim-1 is far higher than that of (þ)-staurosporin. As expected, the affinity of the enantiomer (R)-33 to Pim-1 is also several orders of magnitude smaller. More than 60 PKs are known today, which are all quite similar. This makes the design of specific inhibitors for one single PK a very difficult task. It is thus remarkable that 33 is highly selective for Pim-1. Pim-1 is especially interesting since it is overexpressed in human prostate cancer cells. (S)-33 or derivatives might therefore find application as novel chemotherapeutic agents against prostate cancer. PKs in general are key players in the modulation of enzyme activity by transfer of the -phosphate group of the adenosinetriphosphate (ATP) co-substrate to hydroxy groups of either serine, threonine, or tyrosine in the target protein. This phosphorylation is especially important in the control of cellular signal transduction pathways, and disregulated kinase activity is a frequent cause of diseases. Organometallic compounds similar to 33 are stable in vitro and in vivo. One such derivative (which specifically inhibited glykogen synthase kinase 3)320 was utilized as a tool to switch on the signal transduction pathways in Xenopus frog embryos, which could even be visualized by changes in the phenotype.321 This results from the ATP-binding site being blocked by the organoruthenium compound.

32 Scheme 34

33

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Bioorganometallic Chemistry

1.31.4.3.4

Peptide synthesis

Many interesting applications of organometallic compounds for amino acid or peptide synthesis were published, for example, to obtain unnatural amino acids. In here, mention is made only of some of those in which distinct organometallic peptide intermediates were isolated and characterized. Weiß and Fischer have used aminocarbenes of chromium or tungsten as N-terminal protecting groups in conventional solution-phase peptide synthesis up to tetrapeptides.323,324 The metal carbene group is finally removed by trifluoroacetic acid (TFA) under mild conditions. Similarly, the ferrocenylmethyl (Fem) group may serve as a tag to the amino groups in peptides which enhances lipophilicity325,326 and enable electrochemical detection of the peptides.327,328 The Fem group is also removed by dilute acid. The [Fe(CO)3(C6H7)]þ cation (Fed) is an alternative to the Fem group.329–331 Compound 34 is a potent chiral auxiliary in Ugi four-component reactions (Ugi-4CR) for the synthesis of tripeptides (Scheme 35).332 Using 34 and related ferrocenylamines, numerous peptides were prepared.333–336 The ferrocenylamine can be readily regenerated for further usage.337,338 Stoichiometric olefin metathesis can be used to introduce metal carbonyl markers into peptides. Both Ugi-4CR and olefin metathesis have also been used to introduce organometallic markers into PNA, as mentioned in Section 1.31.4.2. The ruthenium-mediated coupling of aryl ethers is another attractive route to linear peptoids339 and cyclic peptides.340–343 Hegedus and co-workers developed the metal carbene chemistry mentioned in the previous paragraph into a unique route to unnatural amino acids, as shown in Scheme 36.344 Photolysis of the Cr(CO)5 amino carbene 35 produces a metal-complexed amino ketene, which can be trapped with amino acid esters to yield a dipeptide 36. Use of an appropriate chiral auxiliary on the carbene amino atom generally gives high enantiomeric purity of the newly formed stereocenter. Although the chemistry can be performed on the resin as part of an SPPS scheme, it is most successfully carried out in solution as part of a fragment condensation scheme for peptide synthesis.344 The octapeptide Boc-Gly-Ala-D-homoPhe-D-Ala-Phe-Val-Leu-Gly-OMe (homoPhe: homo-phenylalanine) was successfully prepared by this method.345 In this peptide, the central tripeptide D-homoPhe-D-Ala-Phe (marked boldface) was synthesized in solution by chromium carbene photochemistry. The tripeptide fragment was added to the tripeptide Val-Leu-Gly on the resin, and the synthesis was completed by addition of the two N-terminal amino acids by SPPS. The crude product was obtained in 72% yield based on the chromium carbene tripeptide and could be readily purified and characterized. An alternative approach to metal-catalyzed peptide synthesis was proposed by Beck and co-workers. Their method uses half-sandwich complexes of Rh, Ir, or Ru.346–351 In this reaction, N,N9-coordinated peptides are elongated at the N-terminus by condensation with amino acid esters. Scheme 37 shows the postulated reaction sequence.348 It is worth noting that formation of the peptide bond does not require activating reagents or protecting groups. The key step is nucleophilic attack of the deprotonated terminal amino group at the ester carbonyl group. Repeated addition of amino acid esters yields a growing peptide in the coordination sphere of the metal complex. All reactions proceed under relatively mild conditions in the coordination sphere of the chiral metal half-sandwich fragment and no racemization has been observed. The peptide is finally released from the metal template by methanolic HCl.

Scheme 35

Scheme 36

Bioorganometallic Chemistry

Scheme 37

Several intermediates have been isolated and characterized spectroscopically as well as by X-ray crystallography.347,348 In one paper, the catalytic formation of up to a nonapeptide H-(Gly)9-OMe on a {(p-cymene)RuCl} fragment is described.349 The formation of cyclic tetrapeptides on a metal template is also reported.350

1.31.5 Biosensors Based on Organometallics In a very general sense, Stephenson has defined the term ‘‘bioprobes’’ as ‘‘. . . functional molecules or devices that provide information about biological systems. . . .’’ The high kinetic and thermodynamic stability of many organometallic complexes, in addition to their electronic and spectroscopic properties, have spurred their use in numerous sensor applications. Among those are sensors which involve biomolecules, or which detect biomolecules. In this chapter, only a few selected examples are presented as an introduction to the field. Organometallic biosensors are comprehensively summarized in four chapters in a recent book on bioorganometallic chemistry.186,352–354 A more detailed treatment is also found in a chapter in Volume 12 of this series.

1.31.5.1 Protein-based Redox Probes Most work related to the covalent labeling of proteins with organometallic is related to the development of enzyme or antibody amperometric biosensors.186 For the majority of redox enzymes, the active center (or redox-active cofactors) are buried inside the protein and are therefore electrically inaccessible for direct electron transfer to the electrode surface of an amperometric biosensor. This problem has been resolved by (i) addition of a diffusional redox-active mediator, (ii) covalent tethering of the mediator to the protein, or (iii) immobilization of the protein in a redox-active polymer.355 Ferrocenyl derivatives have frequently been used in all three formats as mediators because of their almost ideal electrochemical properties. The redox enzyme glucose oxidase (GOD) has been a primary target of investigation. It forms the basis of amperometric glucose biosensors which are used to monitor the level of glucose in patients suffering from diabetes. In the first marketed hand-held device for patient’s use (the ExacTECHTM pen), the 1,19-dimethylferrocene/ 1,19-dimethylferrocenium couple served as a diffusional redox mediator.356,357 Subsequently, many covalent modifications of GOD were explored and more advanced devices have reached the market. Methods of ferrocene derivatization of GOD were tabulated in two recent reviews.3,186 Ferrocene labeling of all kinds of proteins and enzymes (both redox-active and non-redox-active) is comprehensively treated in one of those articles.3

1.31.5.2 DNA Sensors Similar to the labeling of enzymes with redox mediators, genosensors were developed for sequence-specific DNA detection by making use of the electrochemical properties of organometallic compounds, mainly ferrocene and its derivatives.354 Electrochemical genosensors are particularly attractive because they are highly sensitive and robust, cheap compared to other detection modes such as fluorescence, and they can be easily miniaturized.358 The potential in this technique has attracted commercial interest.359,360 DNA oligomers can be labeled with ferrocene derivatives in a number of ways (see also Section 1.31.4.2). Upon binding to complementary DNA or RNA (target) strands, an electrochemical signal can be detected, for example, after separation by HPLC361,362 or capillary gel electrophoresis (CGE).363,364 Electrochemical detection is also possible after triplex formation with a ferrocenylated ODN.365,366 The sensitivity of the system may be enhanced after DNA amplification by PCR,367 or through amplification of the current by coupling to an enzymatic reaction, for example, the oxidation of glucose by GOD.368 Ferrocene can be readily modified chemically. Depending on the

909

910

Bioorganometallic Chemistry

substituents of both rings, a different redox potential of the Fe(II)/Fe(III) couple results. The four differently substituted ferrocenes 37–40 (Scheme 38) were used as covalent labels on the 59 end of four PCR primers which differ in one base only. Thus, each redox potential ‘‘codes’’ for one specific nucleobase in the position of interest.363 Using sinusoidal voltammetry, the different potentials could be reliably differentiated, and a novel system for single nucleotide polymorphism was established.364 This strategy is, in principle, analogous to the so-called ‘‘four-color DNA sequencing.’’ Ferrocenylated ODNs were first immobilized in a self-assembled redox-active monolayer on Au electrodes by Letsinger and co-workers.369 Upon hybridization of a complementary strand, the electrochemical potential of the ferrocene changes. In addition to applications as electrochemical DNA sensors, such self-assembled DNA monolayers with electro-active groups may provide information on the mechanism of electron transfer through DNA, and indirectly also on molecular mobility within short stretches of DNA.370–374 We have recently extended this idea by the use of immobilized metallocene-labeled PNA on Au electrodes.299,300 Because PNA is an uncharged molecule, a surface with improved properties forms, and electrochemical detection, also of single mismatches, is facilitated. A slightly different approach is used in a so-called ‘‘sandwich assay.’’375,376 First, a capture probe is immobilized on an Au electrode. The target DNA binds to this capture probe if a complementary sequence is present. In addition, the target DNA carries a binding sequence (for example poly-A). The ferrocenylated poly-T signaling probe hybridizes with the poly-A sequence of the target ODN. If the target also binds to the capture probe, the ferrocene comes close to the electrode surface and a signal is measured. The system has later been characterized in detail377 and was refined.359,378 Finally, attempts have been made to perform electrochemical DNA detection without having to use metalmodified ODNs.354,376 This can be achieved by immobilizing the capture ODN on an electrode, allowing it to hybridize with the target DNA, and then adding an electro-active compound that will interact only with ds-DNA. Quite a number of compounds have been used for detection, including Co complexes, ethidium bromide, and

Scheme 38

Bioorganometallic Chemistry

Scheme 39

Scheme 40

intercalating drugs such as daunorubicin. Takenaka’s group has successfully used the ferrocene-modified naphthalenediimide 41 as threading intercalator (Scheme 39).368,379,380 This system is chemically very robust and highly sensitive, reaching a detection limit of 10 zmol DNA under favorable conditions.380 The sensitivity of the system could be enhanced by coupling it to an enzymatic reaction like the glucose oxidation using GOD.368 Furthermore, the system has the potential of direct mismatch detection by determining the number of intercalated molecules or the rate of electron transfer.379 Scheme 40 compares a perfect match situation with a case where the target strand has a single mismatch. It is assumed that less electrons will be transferred less fast if the local geometry is perturbed by a single base mismatch. Evidently, this system needs careful calibration and probably optimization for every single application. On the other hand, it is versatile in the sense that one and the same simple electro-active probe 41 is used for every ODN sequence to be investigated. An application that enables the rapid analysis of heterozygous deficiency of the human lipoprotein lipase gene has been reported.381

1.31.5.3 Metallo-immuno Assays One prominent use of organometallic complexes is in metallo-immuno assays. The traditional radio-linked immuno assay (RIA) is highly sensitive but has obvious disadvantages related to the use of radioactivity. Modern alternatives use colorimetric, fluorescence, or enzyme-linked detection schemes (ELISA). The idea of using non-radioactive metals for specific, highly sensitive detection in immuno assays was first mentioned by Cais, who used steroid

911

912

Bioorganometallic Chemistry

Scheme 41

hormones as haptens and atomic absorption spectroscopy (AAS) for detection.382,383 The idea was later modified by Jaouen’s group, who used infrared spectroscopy of metal carbonyl compounds for detection. The metal C–O stretching vibrations around 2,000 cm1 are generally quite strong. In addition, few organic molecules have absorptions in this region, which is thus rather blank and well suited for detection. A first use of this technique was published in 1985,384 and the method was described as carbonyl metallo immuno assay (CMIA) in 1992.385 Several reviews have been published.158,352,386,387 Initial work has concentrated on steroid hormones and their receptor interaction.388–390 More recently, the method was extended as an analytical tool for the quantification of antiepileptic drugs in patients. By careful choice of the organometallic complex, it has been possible to quantify two391 or even three different drugs simultaneously and independently.392 Scheme 41 shows three such organometallic conjugates of different anti-epileptic drugs as an example. The IR bands used for detection are indicated. Also, interesting applications for environmental analytics such as pesticide analysis were reported mainly by Salmain and co-workers.

1.31.5.4 Colorimetric Assays and Luminescent Probes Colorimetric, fluorescence, and luminescent assays are arguably by far the most important detection methods in bioanalytics. In relation, the importance of metal-based complexes in this field is negligible. Nevertheless, some metal complexes have very favorable properties, for example, long fluorescence or luminescence lifetimes and emission at very long wavelengths. It is probably fair to say that the potential of organometallic complexes in this area is greater than the number of workers advocating their use. There are, however, some unique uses of organometallic complexes in bioanalytics. Since the mid-1960s, lithium salts are among the most frequently used drugs for patients suffering from bipolar disorder. Severin and co-workers could show that organometallic receptors are an interesting alternative to the commonly used organic ionophores for the selective sequestering and sensing of lithium ions. A trinuclear metallamacrocycle was obtained from a dihydroxypyridine derivative and a commercially available ruthenium complex in buffered aqueous solution (Scheme 42). The macrocycle acts as a potent and selective receptor for lithium ions.393 With the help of a subsequent redox reaction, the binding event can be transduced into a color change, which allows to detect lithium ions in the pharmacologically relevant concentration range of 1 mM by the ‘‘naked eye.’’

Bioorganometallic Chemistry

Scheme 42

A synthetic receptor, which is bound via non-covalent interactions to a dye, is able to function as a chemosensor. The basic requirement is that the displacement of the dye by an analyte results in a change of its optical properties.394 Recently, it was shown that the combination of an organometallic Cp* RhIII complex with the dye azophloxine allows to selectively detect histidine- and methionine-containing peptides in water at neutral pH.395 Due to the high binding constants of the organometallic Rh complex, peptides could be detected at concentrations as low as 300 nM. In direct extension of this work, it was shown that the selectivity of such bioorganometallic assays can be increased significantly when they are performed in an array format.396 Thus, it was possible to identify all the 20 natural amino acids with a fidelity of 97% using UV–VIS spectroscopy in combination with a multivariate analysis. Closely related analytes such as leucine and isoleucine were clearly distinguishable, a result which would be very difficult to achieve with a classical ‘‘one sensor–one analyte’’ approach.

1.31.5.5 Heavy Metal Probes for Crystallography and Electron Microscopy Heavy atoms are often essential for solving the ‘‘phase problem’’ in the X-ray crystallography of biomacromolecules. In many cases, crystals are soaked with a solution of the compound, and it is hoped that the heavy atoms will occupy well-defined sites in the crystal with a high occupancy factor. Organomercury compounds are frequently used for this purpose. The ideal reagent will react efficiently with one single, well-defined site of the biomolecule, for example, a protein. It should not disturb the crystallization process, better even facilitate crystallization. Electron-dense transition metal complexes could serve such a purpose in principle. [Pt(CN)4]2 and [Au(CN)4] have been suggested, but are rather unselective, possibly reacting with multiple sites of the protein.397,398 Polynuclear Ir, Ru, or Os carbonyl clusters have been proposed for this purpose, and they can be monofunctionalized with activated esters or maleiimide groups, as exemplified for the first such monofunctional cluster 42 prepared by Jahn (Scheme 43).399 Other kinetically inert half-sandwich complexes (W, Re, Ir) might also be suitable, and in principle many of the ideas and compounds discussed in Section 1.31.4.1 are directly applicable.400–403 For instance, tungsten alkoxycarbene complexes have been used for the labeling of the enzyme lysozyme in an analogous manner to the one depicted in Scheme 24.264 However, multiple adducts were found, and it has not been shown that there was a benefit for crystallization.186 The tetrairidium cluster 42 has been instrumental in the structure elucidation of a ribosomal particle.399,404,405 There is even more potential for the site-specific covalent labeling with heavy atom derivatives in cryo-electron microscopy.186 Very large particles, such as viruses or protein aggregates, can be visualized. Because the

Scheme 43

913

914

Bioorganometallic Chemistry

electron-dense metals could be ‘‘seen’’ directly over the mostly carbon-containing protein background, site-selective labeling would be enormously helpful for the construction of images with molecular resolution. Clearly, this is an area with a lot of potential. To develop the right molecules, reagents and conditions will require a close collaboration of synthetic organometallic chemists with molecular and structural biologists.

Acknowledgments The author is most grateful to all the co-workers who have contributed to the success of this chapter. Special thanks go to Bernie Kraatz for an excellent collaboration and many stimulating discussions and to Karin Weiß for her equally excellent and ever-friendly support of the group in the Heidelberg years. Margaret Harding provided the pictures for Figure 2, the Milan group (Maiorana, Licandro, and Baldoli) freely shared its enthusiasm for organometallic PNAs, Karl Heinz Do¨tz provided valuable literature, and Dr. Fozia Noor made valuable suggestions on the final draft of the chapter.

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