OrganometallicâPeptide Bioconjugates: Synthetic Strategies and

Review pubs.acs.org/CR

Organometallic−Peptide Bioconjugates: Synthetic Strategies and Medicinal Applications Bauke Albada*,† and Nils Metzler-Nolte*,‡ †

Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands Inorganic Chemistry I − Bioinorganic Chemistry, Ruhr University Bochum, Universitätsstrasse 150, 44780-D Bochum, Germany

‡

ABSTRACT: Peptides are important biological molecular entities in biomedical research. They can be prepared in a large variety of shapes, with a host of chemical functions, and tailored for specific applications. Organometallic medicinal chemistry is a relatively young field that explores biomedical and bioanalytical applications of organometallic complexes, that is, metal compounds with at least one direct, covalent metal−carbon bond. The conjugation of peptides to such medicinally active organometallic moieties started only about 20 years ago, and it has been very beneficial for the development of bioorganometallic chemistry in general. Similarly, the biomedical properties of peptides have been altered by their conjugation to organometallic (OM) moieties. In this review, synthetic methods by which OM moieties can be conjugated to peptides via a carbon−metal bond are described, and selected medicinal applications of such conjugates are discussed. Inorganic coordination complexes between metal ions and peptides are excluded from this review. Also, the labeling of peptides with radiometals and applications of radiolabeled peptides will not be treated herein. First, modifications of the peptide backbone (either N- or C-terminally, or both) with organometallic moieties will be described, including the insertion of OM moieties as part of the peptide backbone. Then sidechain modifications will be reported, among them the most recent strategies for chemoselective arene metalation on peptides. Finally, approaches by which multiple metalation can be achieved are explored. In each section, selected examples of biological applications are highlighted.

CONTENTS 1. Introduction 1.1. Peptides and Organometallics in Medicinal Chemistry 1.2. Organometallic Peptide Chemistry: A Sensitivity Issue 1.3. Scope of This Review 2. Peptide Backbone Modifications 2.1. Direct Conjugation to the N-Terminus in the Solid Phase 2.1.1. Half-Sandwich Complexes 2.1.2. Sandwich Complexes 2.1.3. Cy clopentadienyl-Isolobal Tris(pyrazolyl)borate Complexes 2.1.4. NCN Pincer Complexes 2.1.5. Sensing Protein−Peptide Interactions by Use of Ferrocene 2.2. Direct Conjugation to the N-Terminus in Solution 2.3. Indirect Conjugation to the N-Terminus: Solid- and Solution-Phase Methods 2.4. Direct Conjugation to the C-Terminus 2.5. Indirect Conjugation to the C-Terminus 2.6. Incorporation of Organometallic Moieties in the Peptide Backbone 3. Side-Chain Modifications 3.1. Direct Metalation of Proteinogenic Amino Acid Residues © XXXX American Chemical Society

3.2. Direct Metalation of Unnatural Amino Acids 3.3. Indirect Metalation of Amino Acid SideChain Functionalities 4. Metalation of Aromatic Amino Acid Residues 4.1. Arene Metalation of Amino Acids, Their Residues, and Dipeptides 4.2. Arene Metalation on Biologically Active Peptides 5. Miscellaneous and Combined Metalation Strategies 6. Conclusions 7. Outlook Author Information Corresponding Authors Notes Biographies Dedication Abbreviations Proteinogenic Amino Acids Nonproteinogenic Amino Acids Human Cancer Cell Lines References

B B B C E E F H K L L L M P Q

V X Y AA AC AF AG AG AH AH AH AH AH AH AI AI AI AI

R T Received: March 4, 2016

U A

DOI: 10.1021/acs.chemrev.6b00166 Chem. Rev. XXXX, XXX, XXX−XXX

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1. INTRODUCTION The term “organic chemistry” originated from a time when it was thought that living systems were composed solely of organic molecules. However, it did not take long to realize that atoms originally classified as “inorganic” are also important for the orchestration of life. In fact, inorganic complexes were likely of key importance in early stages of the development of life on earth.1 Despite this increase in our understanding of living systems, the terms organic and inorganic remain in use, although their strict connection with life and nonlife has been lost. Nowadays, both the organic and inorganic aspects of living systems are prolifically studied, and it has become very well established that they go hand-in-hand when it comes to maintaining life.2 For example, it has been estimated that approximately 30% of all enzymes have a catalytically crucial transition metal ion bound to their protein framework.3,4 Considering the inorganic part, it was not until the identification of vitamin B12 that chemists realized that a rather exotic family of inorganic compoundsthe group of organometallic compounds, a class of compounds that is characterized by a carbon−metal bondalso plays a role in living systems.5,6 Whereas metal salts of organic medicinal compounds and even biologically active coordination complexes (like cisplatin and its derivatives) are nowadays used in the clinic on a large scale,7 this is not yet the case for organometallic (OM) complexes. At this moment, only very few organometallic complexes have entered clinical trials: for example, Cp2TiIICl2 (Cp = cyclopentadienyl) did not meet the formulation criteria after phase II, and ferroquine (a ferrocene-containing analogue of the antimalarial drug chloroquine), which will enter phase III trials in the near future.8 On the other hand, the idea that organometallic complexes can be beneficial within a biomedical context has become generally accepted: several (bi)annual conferences discuss the applications of metals in medicine, and books and book chapters have appeared. The new field of organometallic medicinal chemistry has grown rapidly, quickly reaching its current level of sophistication where various organometallic complexes can be made biocompatible, or turned into bioconjugates that target specific disease-related biomolecules.9 Their mode of action is oftentimes unique, which opens up the possibility for entirely new medications.10−13 Obviously, before organometallic medicine can be generally applied, organometallic complexes and their conjugates have to outgrow the laboratory stage and make their way to the clinic. Attempts to realize this are underway as more organometallic complexes are studied in vitro, and some are even studied in small living systems.14,15

biological targets, or they have properties that are otherwise undesired. Fortunately, a significant number of organometallic molecules have been identified that do not suffer from these drawbacks. Even more, a variety of OM fragments has become available that can be attached to biologically active compounds like peptides, proteins, saccharides, and DNA, to name a few. Of these, peptides are a particularly topical and intriguing class of compounds since peptide conjugates in general are gaining importance in biochemical and pharmaceutical studies.16−20 At this moment, a significant number of peptides are applied in the clinic,21 and conjugates of peptides with bioactive compounds like radiolabels or chemotherapeutic agents are being explored.22 A number of reviews have appeared recently that cover the wider scope of preparation and applications of transition metal bioconjugates.23−26 Although some touched on the synthesis of organometallic−peptide conjugates,27 the field has expanded significantly so that its own review is warranted. After the preparation of a few exploratory conjugates of small peptides with readily available OM moieties, the number of strategies that have been applied in this field has increased rapidly. This has resulted in a large variety of OM−peptide conjugates, highlights of which are displayed in the chronological overview below (Chart 1). As can be seen, the complexity of OM− peptides conjugates has increased. The field has quickly moved from ultrashort peptides that were used in proof-of-principle studies to application of biologically active OM−peptide conjugates that possess a variety of biologically relevant functional groups. The set of available strategies has become so diverse that today almost any conceivable organometallic− peptide conjugate can be prepared; the main limitation is the availability or accessibility of a properly functionalized OM fragment. Apart from applications in bioanalytical and medicinal areas, organometallic peptide conjugates have also been extensively applied in catalysis. These will not be treated here in depth; the reader is directed toward excellent reviews that have been composed.28−32 However, some examples of OM−peptide conjugates that have been applied only in catalysis studies will be mentioned when a unique method for preparation of the OM−peptide conjugate is employed that might be useful medicinal application as well. 1.2. Organometallic Peptide Chemistry: A Sensitivity Issue

Until recently, organometallic chemistry and peptide chemistry were separated by a chasm filled with oxygen, moisture, and protic solvents. Whereas peptides find their biological activity in aqueous environments in which oxygen and water are crucial components for the biochemical pathways that support the organism, many organometallic moieties do not tolerate these conditions. However, recent advances in both fields have allowed bridges to be formed. Many organometallic complexes have been recognized to be sufficiently air- and moisture-stable so that they can be applied in bioconjugation strategies that have emerged.33 Also, peptide chemistry allows many elegant orthogonal deprotection and conjugation strategies that are selective and mild enough to be applied to at least moderately stable OM fragments. The conjugates that are known to date have shown that the hybrid molecules possess properties that are directly related to the interplay between peptide and OM fragment.34 This has been especially studied for ferrocenederivatized peptides.35−44 In many cases, the OM moiety is used to add exotic properties onto a bioactive peptide in order

1.1. Peptides and Organometallics in Medicinal Chemistry

Living systems are governed by many types of interactions, most of which are tuned at sub-Ångstrom precision. Slight aberrations in desired molecular interactions can cause disease(s) to emerge, and the discovery of molecules that can alter disease-related undesired biomolecular interactions is a major goal of the field of medicinal chemistry. Similarly, the field of organometallic medicinal chemistry [alternatively called inorganic or (bio)organometallic medicinal chemistry] seeks to identify organometallic moieties that can invoke such responses.8 However, most organometallic compounds as such are incompatible with biological systems: many of them are poorly soluble in water or are unstable in aqueous environments, some even react violently with oxygen from the air, they may react in a nonspecific way with many B


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Chart 1. Display of Various Organometallic−Peptide Conjugatesa

a

The chart gives an impression of the type of bioconjugates published in the last three decades in relation to medicinal studies. The presence of a carbon−metal bond between peptide and organometallic fragment was the selection criterion for this review; peptidic backbones in the earlier structures are highlighted with bold lines for clarity. The oxidation state of metal ions in the complexes is omitted for clarity, as is a possible overall charge of the respective compound. A similar chronological display of arene metalations is shown in Chart 7.

OM−peptide conjugates, and we describe their biomedical and bioanalytical applications. It should be noted that we included only OM−peptide conjugates that contain a metal−carbon (M−C) bond between the organometallic fragment and the peptide. This excludes conjugates in which an organometallic moiety is attached to a peptide by a heteroatom,46,47 a strategy that usually results in metastable or even dynamic complexes that are rather governed by principles described by classical coordination chemistry. Similarly, coordination complexes of

to elucidate its mode of action or to modulate its properties with respect to the targeting of a specific disease-related molecular aberration.45 1.3. Scope of This Review

This review deals with various strategies and applications for conjugates of peptides and organometallic (OM) moieties, spanning the years 1988−2015. In view of the relatively young age of the field of peptide-based organometallic medicinal chemistry, we particularly focus on synthetic routes that yield C


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peptides with metal ions will not be treated; they have been covered extensively in the past.48 Also not included are the earliest methods that relied on the application of amino acidbased organometallic building blocks that can be incorporated into peptides,49 as these have been reviewed in detail earlier50,51 and the methods that are used along these synthetic pathways hardly change in a fundamental manner.52 Those building blocks are designed in such a fashion that they can be inserted by standard solid-phase peptide synthetic approaches. Nowadays, most peptides are obtained via Fmoc/tBu-based peptide chemistry (Fmoc = 9-fluorenylmethoxycarbonyl; tBu = tertbutyl); a large variety of amino acid building blocks can be obtained from commercial sources. Protocols that use room temperature or microwave-assisted elevated temperature during the coupling and deprotection reactions are highly optimized and are routinely applied. With these methods, bioactive peptides of intermediate lengths, ∼20 amino acid residues, can be prepared overnight with relatively high purity. Fundamentally, an OM group can be attached to the N- or C-terminus of a peptide, to its side-chain functionalities, or even to the backbone amido groups. As a fifth alternative, the OM group itself can be part of the peptide backbone, replacing the typical C(carbonyl)−C(α)−N peptide backbone (section 2.6). This review is structured according to the methods by which an OM group can be attached to a peptide structure, following the division as it is depicted in Figure 1. Attachment of the OM fragment on the N-terminus is the most straightforward route and hence was one of the first approaches explored in great depth;44,53 the N-terminal amino group was directly modified by use of an OM moiety that contains a carboxylic acid (ad) (sections 2.1 and 2.2). Alternatively, the Nterminus can be converted into another functional group, which in turn can react specifically with an appropriate functionality in the organic-ligand part of the OM fragment (aid) (section 2.3). Yet another alternative route employs conversion of the Nterminus into a different functional group that can react directly with the metal of an OM or coordination complex under formation of an OM−peptide conjugate (route eid) (also section 2.3). Here, we note the subtle difference between routes aid and eid. In route aid the OM fragment is attached to the Nterminus via its organic ligand, whereas in route eid the OM fragment is attached to the N-terminal functionality via its metal ion. An example of the first is click conjugation, whereas an example of the latter is the reaction of an N-terminally positioned acetylene with Co2(CO)8. Each of these strategies can be applied before or after the peptide has been cleaved from the resin, but this depends on the stability of the formed OM complex under (usually acidic) peptide-cleavage conditions and the absence of competing groups in the cleaved peptide, respectively. Solid-phase peptide synthesis occurs from C-to-N terminus, that is, by attaching protected amino acid residues, in which the carboxylic acid has been activated, to the amino group of resinbound protected peptides. The C-terminal carboxyl group of the peptide is constantly attached to the solid support during peptide synthesis. This makes C-terminal modification far less straightforward. In cases where a resin handle (usually referred to as linker) is used that yields a C-terminal reactive functional group (route bd), the OM complex can in principle be attached after cleavage of the peptide from the resin (section 2.4). However, this modification can suffer from epimerization of the C-terminal amino acid residue and does not tolerate the presence of a variety of functional groups like those in Asp, Glu,

Figure 1. Schematic overview of different metalation strategies presented in different sections of this review. Abbreviations: a and e = N-terminal conjugation, b and f = C-terminal conjugation, c = conjugation to functional side chains, d = conjugation to π-electroncontaining side chains; π = π-electronic side-chain residue; subscript d = direct, subscript id = indirect; FG = functional group; FGC = functional group conversion.

Lys, His, and Cys, to name the most prominent incompatible amino acid residues. Nevertheless, in certain cases a C-terminal carboxylic acid moiety can theoretically be used. More popular, however, have been indirect methods in which a functional group at the C-terminus is produced that can react with OM fragments in an orthogonal manner (routes bid and fid) (section 2.5). For this, specialized linkers have been developed. Attachment of the OM fragment to a side-chain functionality via its organic part can be realized during the peptideelongation steps that are performed on the solid support or at the end of peptide elongation (section 3). For this approach, orthogonal protecting groups are often used that can be removed under mild conditions that do not cleave the peptide from the resin nor remove other protecting groups (route cd or cid) (section 3.1).54 Alternatively, π-electron-containing amino D


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2. PEPTIDE BACKBONE MODIFICATIONS

acid side-chain functionalities can be metalated directly by suitable OM reagents in which the metal reacts with the carbon atoms that contain the π-electrons (route d). Depending on the type of OM complex employed, this strategy can be applied to still immobilized and protected peptides or to already cleaved and deprotected peptides. For this approach, one can use (i) the naturally occurring histidine residue, which is conveniently converted into a N-heterocyclic carbene (section 3.1), (ii) an unnatural amino acid residue that has been inserted in the peptide, like propargylglycine (sections 3.2 and 3.3), or (iii) one of the three aromatic amino acids (Phe, Trp, and Tyr) that can be metalated under formation of a mixed-sandwich complex (section 4). This last method has been applied on amino acids and their residues (section 4.1) but also on bioactive peptides (section 4.2). Miscellaneous approaches that were not covered in one of the earlier sections are summarized at the end, together with conjugates that contain multiple OM moieties (section 5). In each of the above cases, the harsh acidic conditions that are usually required to cleave and deprotect the peptide from the resin form a difficult-to-take hurdle for many OM complexes and thus often determine which of the routes should be followed. For example, in the case of ferrocenoylconjugated peptides it has been shown that the presence of water, which is usually added to scavenge reactive carbocations, facilitates oxidation of the ferrocene group to the ferrocinium ion, which is much less stable and rapidly loses the CpFe fragment, resulting in a purely organic cyclopentadienederivatized peptide. Thus, for the cleavage of ferrocenylconjugated peptides it has been proven constructive to apply phenol instead of water. Similarly, instead of oxidation of the metal ion by one of the applied scavengers, reduction is also possible. For example, reduction of metal fragments by applied silanes has been observed.55,56 Finally, differences in the behavior of identical functional groups are sometimes negligible but not always. For example, an amide-bond forming reaction can be performed equally well on the N-terminal amino group or internal side chains that contain an amino group (as, for example, the lysine residue). This is also the case for triazole-bond formation between an alkyne and an organic azide, direct OM complex formation on alkynes by Co2(CO)8 or Sonogashira cross coupling reactions, or N-heterocyclic carbene (NHC) complex formation. However, whereas the attachment of an amino-groupcontaining OM moiety is usually conveniently performed on side-chain or N-terminally positioned carboxylic acids, modification of a C-terminal carboxylic acid moiety is usually more cumbersome. This is mostly due to the presence of a substitution at the Cα carbon atom, but folding of the peptide is also known to contribute to this. In order to avoid duplication of content, overlapping strategies will be mentioned only briefly at the beginning of the treatment of a new site of metalation when similar strategies have been treated in depth before. Finally, common amino acids and their residues are noted according to IUPAC rules by their one-letter or threeletter codes;57 the stereochemistry of the α-carbon center is assumed to be L as occurring naturally, unless noted otherwise. For metal complexes, formal oxidation states are given in Roman numerals as superscripts after the metal atom.

2.1. Direct Conjugation to the N-Terminus in the Solid Phase

The vast majority of conjugation strategies are those based on an amide-bond-forming reaction between organometallic fragments that contain a carboxylic acid functionality (Figure 2, A)

Figure 2. Direct conjugation of OM fragments to the N-terminus of a resin-bound, side-chain-protected peptide. Examples of OM fragments that have been conjugated by this approach are shown. PG = protecting group.

and the amino group of a resin-bound peptide (Figure 2, B). After cleavage of the OM−peptide conjugate from the resin, a bioconjugate is formed that has a C-terminal carboxamide (C), ester (D), or carboxylic acid (E). As this is the most-applied approach, this method deserves to be treated before other methods are mentioned; it serves as a backdrop against which the other conjugation strategies can be appreciated. Most of these amide-bond-forming strategies are based on methods that are commonly used in peptide chemistry.58 The carboxylic acid functionalized OM complexes that have been coupled to peptides can roughly be divided in two groups: (i) those in which the carboxylic acid moiety is directly attached to the (aromatic) ligand of the organometallic fragment (group 1, Figure 2), and (ii) those in which a spacer is present between E


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the organometallic fragment and the acid (group 2, Figure 2). Fortunately, the reactivity of both these groups is comparable and similar conditions can be used for the coupling of the members of each group. In the couplings of OM fragments belonging to group 1 (of which ferrocene carboxylic acid 1 is the most prominent member), a change in the physicochemical properties of the OM moiety as a result of the acid-to-amide conversion is to be expected;59 for those that belong to group 2, such a change usually does not occur, although the spacer can have an effect on the coupling efficiency and the properties of the conjugate.60 Here it should be mentioned that coupling of an OM fragment from the first group to a resin-bound amino group that contains a neighboring bulky carbon atom might be problematic for steric reasons. Using a spacer between the OM moiety and the carboxylic acid, that is, using an OM moiety that belongs to group 2, may increase the coupling efficiency. Depending on the structural properties of the OM fragment, issues similar to those in peptide chemistry can arise, for example, inferior coupling efficiency caused by on-resin folding of highly structured peptides or incomplete Fmoc removal. Usually, OM carboxylic acids do not contain a chiral center on the Cα atom, so that epimerization, as is sometimes observed with amino acids, is not an issue.61 When it comes to strategies for these conjugation reactions, preactivation of the carboxylic acid moiety of the OM unit can be used (Figure 2). During the solid-phase synthesis of peptides, this coupling method can be conveniently applied to derivatize the N-terminus or specifically installed amino-groupcontaining side chains. Especially when the OM fragment can be obtained in large amounts or from commercial sources, like ferrocene carboxylic acid 1, an excess of OM−C(O)OH can be applied in order to achieve near-quantitative coupling yields. Coupling reagents that are applied for this conjugation reaction are the phosphonium and uronium salt-based dehydrating agents commonly applied in peptide chemistry, but other dehydrating condensation reagents were also used, like HOBt/ DIC (HOBt = N-hydroxybenzotriazole; DIC = N,N′diisopropylcarbodiimide).62,63 An alternative to in situ activated esters of the carboxylic acid functionalized OM complexes is preformed activated esters. Such compounds can be prepared beforehand, purified by methods like column chromatography, and are stable during prolonged storage. These less reactive activated esters are particularly useful for stoichiometric coupling of acids to amino groups, for example, in solution. Although these activated esters are usually coupled to amino groups, competitive labeling has been observed with hydroxyl groups (of Ser, Thr, and Tyr), sulfhydryl groups (of Cys), and even guanidinium groups (of Arg).64 Prominent preactivated esters are the fluorophenyl ester (OPfp, 2), its monosulfonated 4-sulfotetrafluorophenyl (STP, 3) derivative,65 N-hydroxysuccinimide (NHS or OSu, 4),66−68 and its sulfonated derivative (sulfo-NHS, 5) (Chart 2). When the NHS esters are used, one should be aware of the Lossen rearrangement,69−71 which primarily occurs when formation of the active ester is slow due to steric hindrance or electronic effects and which leads to introduction of an extra β-alanine residue in the OM−peptide conjugate (Scheme 1).72 Phenol and hydroxamic esters are conveniently prepared from the carboxylic acid, the phenol or N-hydroxysuccinimide, and a carbodiimide. When N,N′-dicyclohexylcarbodiimide (DCC, 6) is used, one of the products of this reaction, N,N′dicyclohexylurea (DCU, 7), precipitates easily out of the solution and the preactivated ester is usually obtained in near-

Chart 2. Activated Esters, Coupling Reagent DCC, and Related DCU Product Formed after Formation of the Peptide Bond

Scheme 1. Lossen Rearrangement That Leads to Introduction of a β-Alanine Moiety in the Peptidea

a

Dotted arrows indicate formation of the desired product (center); for the Lossen rearrangement, dominant (bold arrows) and alternative (thin arrows) pathways are indicated.

quantitative yields with a purity that is sufficient for the conjugation reaction. However, care should be taken when DCC is applied, as DCU is difficult to remove entirely, and it is known to induce strong immunogenic responses; to avoid this, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, another carbodiimide), can be used. Specific examples of the above-mentioned approach are given in the following subsections. This shows the versatility and reliability but also the limitations of these conjugation reactions. 2.1.1. Half-Sandwich Complexes. Carboxylic acid functionalized half-sandwich complexes of the type CpMI(CO)3 have been attached to peptidyl amino groups under standard dehydrating amide-bond-forming conditions. The first member of the group 7 metals of this class of η5cyclopentadienyl metallocarbonyl complexes is the manganesecontaining CpMnI(CO)3, also known as cymantrene (2, Figure 2). This robust moiety is stable in air and water and is easily functionalized with organic moieties73 that allow straightforward attachment to peptides.74−77 For example, the carboxylic acid functionalized cymantrene moiety was used by the groups of Jaouen and Neundorf to conveniently attach it to the NF


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tribution studies of the 99mTc-containing CpTcI(CO)3− octreotide conjugate revealed that tissue that contains elevated levels of somatostatin receptors, like the adrenals and pancreas, rapidly take up the conjugate and that it is slowly cleared from that tissue. Even though the uptake in muscles, fat, and organs not involved in clearance was low, substantial accumulation of radioactivity in the liver and intestines was observed at all time points of the experiment. Interestingly, changing the label from 111 In-DTPA (as used in the clinically applied OctreoScan85,86) to the more lipophilic CpTcI(CO)3 changed the clearance route from renal for the former to hepatobiliary for the latter, albeit that accumulation in the liver and intestines likely interferes with tumor imaging. An interesting approach to prepare these half-sandwich complexes was recently described in which the Diels−Alder dimerized cyclopentadienyl, that is, Thiele’s acid (HCpC(O)OH)2, was used by Alberto and co-workers87 (Scheme 2). After

terminus of resin-bound peptides by HOBt/DIC activation. Interestingly, alkylation of an aromatic ring that was directly attached to the Cp ring of the OM fragment, which occurred when TFA/water was used,60 was successfully suppressed by use of TFA/DMB (95:5 v/v) (TFA = trifluoroacetic acid; DMB = 1,3-dimethyoxybenzene). Attachment of this CpMnI(CO)3-moiety to the cell-penetrating peptide (CPP) GLRKRLRKFRNKIKEK-NH2 allowed quantification of the internalization of the OM−peptide conjugate by means of graphite furnace atomic absorption spectroscopy (GF-AAS). Treatment of HT29 and MCF7 cells with approximately 20 μM of the conjugates resulted in cellular molar concentrations of 31−108 μM for HT29 cells and 12−36 μM for MCF7 cells. The presence of a carboxyfluorescein dye indicated localization of the conjugates in endosomes and lysosomes, clearly pointing toward endosomal uptake; no uptake in the nucleus or in the mitochondria was observed. However, when the keto group in the linker was reduced to a methylene moiety, pronounced nuclear accumulation, probably in the nucleolus, was observed, which was accompanied by a slight decrease in IC50 value from 60 to 40 μM.78 When other peptides were used as carriers for the CpMn(CO)3 moiety, for example, enkephalin, neurotensin, nuclear localization sequence (NLS), and transactivator of transcription of human immunodeficiency virus (TAT), it was observed that the cellular uptake was poorly dependent on the type of peptide, that is, 0.22−0.35 nmol of Mn/mg of cell protein in HT29 cells.79 After 6 h of incubation, the concentration of Mn inside the cell was not significantly higher than that outside the cell, signaling poor uptake of the conjugates. Interestingly, whereas the CpMnI(CO)3 moiety itself and the CPP itself did not reduce the viability of MCF7 and HT29 cells, the IC50 values of the OM−peptide conjugates ranged from 40 to 70 μM against MCF7. In order to encourage the release of the toxic OM moiety from the endosomes, an enzymatically cleavable site (GFLG sequence, which is cleaved by the cathepsin B protease) was incorporated into the conjugates. Indeed, 2-fold lower IC50 values were observed when a cleavage site was present; allowing the release of two CpMnI(CO)3 moieties resulted in IC50 values of 15 μM for MCF7 and 35 μM for HT29. For human B-cell leukemia (Nalm-6) cells, significant DNA fragmentation was observed in a flow cytometric measurement when the cells were exposed to these cleavable CpMnI(CO)3 conjugates.80 The two elements below Mn, rhenium (Re) and technetium (Tc), allow for similar labeling strategies. In principle, 186Reand 188Re-containing CpReI(CO)3 can be used in radiotherapy, and 99mTc-containing CpTcI(CO)3 can be used in diagnostic imaging. It was already shown by Schatzschneider and coworkers78 that changing the Mn to Re did not significantly alter the biological properties. Via a double ligand transfer (DLT) reaction,81 CpReI(CO)3 and CpTcI(CO)3 have been prepared by Spradau and Katzenellenbogen and subsequently coupled to protein and peptide amino groups by the activated ester strategy.82 Even in the presence of a potentially competing ketone functionality in the organometallic moiety, which could form a Schiff base with the amino group, amide-bond formation was the dominant reaction (>70%),80 although 8% isolated yield was finally obtained in the case of 99mTc-containing CpTcI(CO)3−octreotide.83 Importantly, the 99mTc-containing CpTcI(CO)3−octreotide conjugate did not show decomposition when stored in human plasma at 37 °C over the course of 60 min; the chemical robustness of other CpTcI(CO)3 and CpReI(CO)3 complexes has been demonstrated.81,84 Biodis-

Scheme 2. Retro-Diels−Alder Reaction of Thiele’s Acid Leading to Labeling of the Peptide N-Terminus with Cp99mTcI(CO)387

the coupling of two peptides to this dimer, which was done on a trityl resin with the endo−endo form of Thiele’s acid and resulted in 25−30% of the dimeric peptides 8, a retro-Diels− Alder reaction was performed in the presence of [99mTcO4]−, Na[H3BC(O)OH], disodium tartrate·2H2O, and N2B4O7· 10H2O, and yields of the isolated purified OM−peptide conjugates 9 were between 25% and 90%. Interestingly, use of [99mTcO4]− in principle allows labeling of a peptide using the eluate of the [99mTcO4]− generator and performing the reaction in one vial. The peptides that were used in this conjugation reaction were shown to have low micromolar affinity for the PEPT2 receptor (a membrane protein that transports a variety of biomolecules across the biological membrane),87 with Ki values of 1.2−18 μM, in anticipation of their active transportation across biological membranes. Half-sandwich complexes of group 6 and group 8 metals also exist, and both have been successfully conjugated to amino acid residues. To be specific, both the group 8 metal containing the three-legged piano stool complex [CpC(O)OSu]FeII(CO)2Me, which is the OSu ester of 10 (Figure 2), and its group 6 metal containing the four-legged analogue [CpC(O)OSu]MoII(CO)3Me, which is the OSu ester of 11 (Figure 2), were successfully conjugated to β-alanine ethyl ester by Jaouen and G


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co-workers.88 One of the benefits of these group 6 and group 8 metal-containing half-sandwich complexes is that their metalbound alkyl group offers an additional route for derivatization. Specifically, CpFeII(CO)2(η1-CH2C(O)OSu, which is the OSu ester of 12 (Figure 2), has been conjugated to amino acid derivatives, even though the reaction was very slow (reaction time was 1 week); its Mo-containing counterpart was unreactive. This difference in reactivity was explained by the interaction of C−M bonding electrons with the carbonyl πorbital.88 2.1.2. Sandwich Complexes. Metallocenoyl derivatives like cobaltociniumoyl, ferrocenoyl, and ruthenocenoyl have been successfully attached on resin-bound peptides using their respective acids (13, 1, and 14, Figure 2, respectively) and commonly applied amide-bond forming reagents like BOP, PyBOP, HATU, and TBTU, to name a few (see Abbreviations for the chemical names of these coupling reagents). For this reaction, solvents that are usually applied in solid-phase peptide synthesis (SPPS) (e.g., DCM, DMF, NMP) can be used depending on the solubility of the components of the reaction mixture (DCM = dichloromethane; DMF = N,N-dimethylformamide; NMP = N-methylpyrrolidone). One should also be aware of side reactions that can occur when the resin-bound protected OM−peptide conjugate is exposed to the deprotection cocktail. Particularly susceptible to decomposition are ferrocenoyl derivatives, as they oxidize to ferrociniumoyl derivatives that are much less stable and hydrolytically decompose to cyclopentadiene-peptide derivatives and the other half of the sandwich complex; this instability of the ferrocenoyl group in FcC(O)−peptide derivatives is less problematic in biological studies (vide infra). To avoid this decomposition, the strongly acidic (TFA-based) cleavage mixture should contain phenol instead of water.89 Stability issues are hardly present when FcC(O)−, RcC(O)−, and Cc+C(O)−peptides are used, they can be stored for days in water and at room temperature [Fc = ferrocene, FeII(η5C5H5)2; Rc = ruthenocene, RuII(η5-C5H5)2; Cc+ = cobaltocene cation, that is, cobaltocinium, CoIII(η5-C5H5)2]. Whereas the ferrocenoyl− and ruthenocenoyl−peptide conjugates contain a neutral OM fragment, their cobaltociniumoyl counterparts have a cationic OM fragment that is reduced only at very low potentials, for example, at −1.38 V (vs Fc/Fc+) in acetonitrile. Due to the positively charged nature of the OM fragment, cobaltocinium serves as a positively charged OM fragment that is isostructural with ferrocene. As such, it has proven to be very applicable in biochemical and physiological studies. In fact, it has been shown that the ease of cellular uptake of a simian virus nuclear localization sequence (NLS, sequence PKKKRKV) was increased by the presence of a positively charged cobaltocinium or neutral ferrocenoyl moiety in the conjugate; the OM fragment was shown to be essential for active endocytosis and release of the conjugate into the cytoplasm.90 In the absence of the OM fragment, the NLS peptides remained adhered to the outer membrane and no uptake was observed. Importantly, whereas the cells actively took up larger OM−peptide conjugates, smaller versions of the conjugates only sparingly entered the cells, mainly by passive diffusion. It has been suggested that the OM fragment plays an important role when it comes to the apparently rapid escape of OM−NLS bioconjugates from the endosomes. As the antiproliferative effect of these Cc+C(O)− and FcC(O)−NLS conjugates was >1 mM, it was reasoned that the mere presence of the OM moiety inside the nucleus does not lead to toxic

effects.91 The hydrophobic nature of the ferrocenoyl group was implemented to increase the lipophilicity of neuropeptides in order to increase their permeation through the blood−brain barrier (BBB). Indeed, using an in vitro brain endothelial cell system, it was shown that a FcC(O)−enkephalin conjugate performed significantly better than the corresponding Cc+C(O) and acetylated derivatives when it comes to passage through the membrane from the apical to the basal side. It was hypothesized that the increased lipophilicity of the conjugate resulted in increased uptake, as also cell lines that do not express the enkephalin receptor also readily incorporate the conjugates. Importantly, cell viability was hampered only at very high concentrations of the conjugates, proving their low toxicity.92 However, we could demonstrate a certain level of cytotoxicity when metallocenoyl groups were attached to poly(arginine) derivatives: novel metallocene derivatives of a cell-penetrating poly(arginine) peptide [abbreviated as McC(O)− (Arg)9(Phe)2Lys-NH2, where Mc = metallocene, a generic term that stands for ferrocene, Fc, or ruthenocene, Rc] were designed and their biological activities were investigated. We proposed that such metallocene−poly(arginine) peptides induce lysosomal membrane permeabilization and thereby could be developed toward targeted anticancer drugs. The IC50 values of 50 μM were higher than anticipated, but still the lowest ones we witnessed so far for Fc−peptide conjugates.93 Cellular uptake in malignant cell lines was also shown by an FcC(O)−RGD conjugate, where the electrochemical properties of the Fc moiety were used to assess depletion of the FcC(O)− RGD conjugate from the applied solution.94 Conjugation of the Fc moiety to an RGD peptide was anticipated to lead to a potent antitumor agent, as the oxidized form of the Fc moiety (Fc+) is known to have antitumor activity.95,96 Even though reducing or Fenton conditions were required, enhanced DNA cleavage was observed for simple ferrocenoyl amino acid derivatives.97 Importantly, good reversibility of the oxidation and reduction of the Fc moiety was proven, allowing the Fc moiety to be used as an electrochemical probe. Uptake of the RGD-containing peptide in B16 melanoma cells was 5 times higher than that for a GGG control peptide, 83% versus 17%; removal of a C6 spacer between the FcC(O) moiety and the RGD peptide resulted in a significantly lower uptake of 42%. Cell viability studies supported this finding, with IC50 values of 5.2 ± 1.4 μM for FcC(O)−C6−RGD, 19.6 ± 3.3 μM for FcC(O)−RGD, and 24.8 ± 3.9 μM for FcC(O)−GGG [the IC50 values of FcC(O)OH and RGD were 49.0 ± 5.8 μM and 21.6 ± 3.8 μM in this study], as well as apoptosis rates of 3.4% for FcC(O)OH, 6.0% for RGD, 12.6% for FcC(O)−RGD, and 24.4% for FcC(O)−C6−RGD. On the basis of flow cytometry analysis, it was suggested that the ferrocenoyl RGD conjugates arrest the tumor cell cycle at several phases, followed by induced apoptosis. Apart from attaching Fc to tumor-targeting peptides, ruthenocene (Rc) has also been conjugated to peptides, mostly via ruthenocene carboxylic acid, which can be quite conveniently obtained from ruthenocene. Specifically, RcC(O)−octreotide was prepared in 75% crude yield.98 Biological activity of this conjugate and its related FcC(O) and Cc+C(O) conjugates was evaluated by determining their effect on cell viability and cellular uptake efficiency.99 One of the advantages of a stable ruthenium-containing label is that it adds intracellular detectability of the OM−peptide conjugate by atomic absorption spectroscopy (AAS).100 Cyclic voltammetry revealed half-wave potentials of 0.14 V for FcC(O)−octreotide and −1.18 V for Cc+C(O)−octreotide and an oxidative wave at H


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0.53 V (vs Fc/Fc+); the electrochemical processes are quasireversible for the Fc and Cc+ conjugates. None of the McC(O)−octreotide conjugates significantly hampered the cell viability of HeLa, PT45, and HepG2 cells, making them ideal candidates to study receptor-mediated cellular uptake. Indeed, the cellular uptake of FcC(O)−octreotide was 5-fold higher than that of its acetylated form but also than that of its ruthenocenoylated counterparts. Vesicular distribution was found in live HepG2 cells, and none of the McC(O)− octreotide conjugate was found in the nucleus nor in the cytosol; endosomal uptake was shown, which was then likely followed by storage in the lysosomes. Interestingly, a general trend with increasing receptor-specific uptake was observed for the McC(O)−octreotide conjugates in the order Rc < Fc < Cc+. Recently, the effect of electron-poor metallocenes on the antiproliferative activity of Leu-enkephalin or neurotensin was assessed.101 For this, 1′-hexafluoropropan-2-ol (HFA)-equipped carboxylic acid derivatives of both ferrocene and ruthenocene were prepared by Friedel−Crafts acylation followed by hydrolysis of the bis(aryl) ketone (Scheme 3).

The effect of ferrocene groups, linked by either an amido or a sulfonamido group to β-lactam antibiotics, was one of the earliest examples of OM−peptide chemistry (17−20, Chart 3). Chart 3. Ferrocene−β-Lactam Derivatives with Moderate Antibacterial Activities102

Even though the OM−β-lactam conjugates, prepared by Vata and co-workers,102 displayed activity against Gram-positive bacteria, the activities were lower than those of nonderivatized β-lactams. In a more extensive fashion, the effect of metallocenoyl groups on the activity of antimicrobial peptides (AMPs) has also been studied. Whereas many peptides exert their activity inside a living cell, AMPs do not necessarily need to be taken up by the bacterium, as they primarily attack the outside of the bacterial cell envelope. Using a large variety of organometallic antimicrobial peptides (OM−AMPs), researchers were able to make synthetic antimicrobial peptides (synAMPs). For some derivatives, the activity approaches that of vancomycin, which has long been considered the antibiotic of last resort103 but is now facing resistant strains.104 By use of OM fragments, the groups of Metzler-Nolte and Bandow took the challenge to prepare very potent OM−AMPs that would be active against a broad range of bacteria without being toxic to human cells (vide infra).107−109 With short Arg-Trp-based AMPs as the peptide part that possesses measurable activity, it was hoped that the metal fragment could contribute to the activity of the conjugate. This was inspired by the fact that the presence of a ferrocenoyl ring in the well-known anticancer drug tamoxifen changed the pharmacodynamics profile due to redox activity of the OM fragment.105,106 Initially, organometallic tri- to pentapeptides were presented that possessed modest antibacterial activity; the most active compound was FcC(O)−WRRF-NH2, with a minimal inhibitory concentration (MIC) of 50 μM.107 In a later study, FcC(O) and the isostructural Cc+C(O) moieties were attached to resin-bound Arg-Trp-based pentapeptides, and the cleaved OM−AMPs were tested for their antibacterial activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus.108 Interestingly, the more hydrophobic FcC(O) derivatives tended to be more active than the more hydrophilic Cc+C(O)−AMPs, which was an indirect indication that the primary target of these peptides was the bacterial membrane. This indication was further strengthened in a later study, in which the ruthenocenoyl derivatives of the same WRWRW pentapeptides were identified as yet a more potent nonhemolytic and nontoxic OM−AMP.109 Despite having indications that the membrane was the primary target of these amphipathic OM− AMPs,37 the exact mode-of-action was not precisely determined at the time, nor was the contribution of the OM fragment to

Scheme 3. Synthesis of Electron-Poor Ferrocene and Ruthenocene Carboxylic Acid Derivatives101

This provided the required electron-poor HFA-derivatized ferrocenecarboxylic acids (15) and ruthenocenecarboxylic acids (16) that could be coupled to resin-bound peptide precursors. Both compounds showed higher half-wave potentials than ferrocene, 512 mV for the HFA-ferrocene and 470 mV for the HFA-ruthenocene derivative; in comparison, HFA-modified ferrocene has a potential of 214 mV (vs FcH0/+). Whereas the HFA-ruthenocene conjugates with peptides could be cleaved by the standard cleavage mixture of TFA/water/TIS (95:2.5:2.5 v/ v/v), the HFA-ferrocene counterparts were cleaved by use of TFA/phenol/TIS (85:10:5 v/w/v) (TIS = triisopropylsilane). The OM−peptide conjugates were obtained in 14−30% isolated yields. Unfortunately, the IC50 values of the peptide derivatives were >100 μM, whereas those of the organometallic building blocks were in the range 10−60 μM. This nontoxic behavior of the OM−peptide bioconjugates was attributed to endosomal entrapment. I


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Chart 4. Lead Optimization from Organic AMP to Organometallic AMP with 8−16-fold Increased Activitya109,111,122

a Replacing the N-terminal arginine residue with an organometallic metallocenoyl moiety, in combination with an L-to-D substitution scan, led to an 8−16-fold increase in antibacterial activity, allowing OM−AMPs to approach the activity of vancomycin.

aromaticity of the Cp rings in Rc and especially Oc,114 the yield of OcC(O)OH was even lower than that of RcC(O)OH (21% vs 53% overall yield, respectively, based on the metallocene). As these Fc−, Rc−, and Oc−AMPs did not show any signs of decomposition in various aqueous solutions, leakage of the metal ion from the OM fragment is not the most likely source of their high antibacterial activity. In order to determine the underlying cause for the higher activity of Rc− and Oc−AMPs when compared to Fc−AMPs, several differences between the metallocenoyl groups should be considered (Figure 3). First of all, their redox potentials differ:

the antibacterial activity assessed in great detail. This was assessed only later (vide infra). The hydrophobic nature of the uncharged ferrocenoyl derivative has been pointing toward increased hydrophobicity as being the primary cause for the enhanced activity of FcC(O)-derivatized AMPs. This was indeed confirmed by comparing the antibacterial and hemolytic properties of lipidated with those of ferrocenoylated Arg-Trp-based AMPs.110 On the basis of retention times found by HPLC analysis of a set of lipidated peptides on a C18 column (HPLC = high-pressure liquid chromatography), it was found that the lipophilicity of the FcC(O) moiety lies in between those of hexanoic and octanoic acids and is closer to the former [i.e., comparable to a C(O)C6H13 lipid]. Since the trends in antibacterial and hemolytic activity of the ferrocenoylated AMPs follow those of the lipidated peptides, it was shown that the increase in antibacterial activity of FcC(O)−AMPs was due to their increased hydrophobicity and was not significantly assisted by a redox process. This also explains the usually lower activity of the significantly more hydrophobic cobaltociniumoyl derivatives. Whereas lipophilicity could explain the increased activity of ferrocenoylated AMPs, this was not the case for ruthenocenoylated AMPs. On the basis of their retention times (tR) on a C18 column in HPLC analysis, which are almost identical, very comparable antibacterial activities would be expected. This was not observed, however. When RcC(O)− WRWRW-NH2 (21, Chart 4, tR = 20.1 min) was compared to FcC(O)−WRWRW-NH2 (22, Chart 4, tR = 20.2 min), the former is 4 times more active against Bacillus subtilis (MIC values of 2.9 vs 12 μM) and 8 times more active against methicillin-resistant S. aureus (MRSA; MIC = 5.8 vs 48 μM). Interestingly, replacing Rc with osmocene [Oc, OsII(η5-C5H5)2] did not significantly increase the antibacterial activity: the MIC value of OcC(O)−WRWRW-NH2 (23, Chart 4) against MRSA was 2.7 μM.111 The synthesis of this novel OM−AMP was facilitated by the preparation of osmocenecarboxylic acid (24, Figure 2), which was prepared according to an optimized procedure analogous to that usually applied for preparation of ferrocenecarboxylic acid112 and ruthenocenecarboxylic acid.113 Due to the lower

Figure 3. Schematic comparison of C−M distances and redox potentials between the three different metallocenoyl derivatives of group 8 metals. Distances are derived from the crystal structures (Cambridge Structural Database codes 1154857 for Fc, 665899 for Rc, and 1259660 for Oc); other values are taken from the literature as referred to in the text. The red ellipsoid of Fc and green ellipsoid of Rc and Oc indicate that the metal center of Fc does not function as an Hbond acceptor, whereas in Rc and Oc it does (see text for details).

Fc = 0.307 V, Rc = 0.693 V, and Oc = 0.633 V.115 In the presence of electron-withdrawing substituents, the ease of oxidation decreases; electron-donating substituents have the opposite effect. Apart from the redox potential, the size and chemical properties also differ significantly. Regarding size, the volume of the Fc moiety is significantly less than that of Rc and Oc: VFc = 767 Å3,116 VRc = 818 Å3, and VOc = 806 Å3.117 These differences emerge from the differences in M−C bond lengths and distances between the Cp rings. The chemical properties of these three metallocene derivatives are also very different since J


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that are formed by ferrocene derivatives have all been mentioned but not been unambiguously proven.50,110 Contrary to what one might expect, antiproliferative activity of Fc derivatives is rather independent of the redox potentials of the conjugates.50 Apart from in vivo applications, FcC(O)−peptide derivatives have also been applied as functional units for nanomaterials.124 By use of a peptide sequence that was derived from β-amyloid (Aβ), FcC(O)−KLVFF-NH2, which contains the hydrophobic core of Aβ1−42, up to 93% reduction of fibril formation of the Aβ1−42 (10 μM) sequence was observed when 40 μM Fc− peptide was applied; 50% inhibition was already observed with a molar ratio of 0.18 of the FcC(O)−KLVFF-NH2 to Aβ1−42.125 Instead of fibrils or fibrillar material, insoluble globular aggregates were observed, and a 5-month incubation period with the above-mentioned concentrations did not lead to notable fibrils. Whereas the isolated FcC(O)−KLVFF-NH2 peptide exhibits a reversible one-electron redox reaction, with a cathodic peak at 0.546 V and anodic peak at 0.624 V (vs Ag/ AgCl), the oxidative current decreases sharply when Aβ1−42 is present, indicating association of the OM−peptide with the Aβ1−42 peptide. Interestingly, the presence of the Fc moiety significantly increased the stability of the peptide against proteolysis: 82% of H-KLVFF-NH2 was degraded after 3 h and completely after 24 h, whereas 32% of the FcC(O)-KLVFFNH2 was degraded after 3 h and 52% after 24 h when exposed to chymotrypsin at 37 °C. The redox activity of the ferrocenoyl moiety was directly targeted in order to induce gelation of conjugates of ferrocenoyl with ultrashort peptides.43,126 Specifically, FcC(O)−Phe-Lys(Cbz)-OMe and its inverse sequence, which were prepared by standard methods, revealed a low tendency to undergo extensive self-organization. Heating and cooling, or the application of sonication, resulted in an increase in viscosity of the solutions of these peptides in toluene, but it remained that monosubstituted ferrocene derivatives were not capable of directed self-assembly. However, inserting a C6 spacer between the dipeptide and the Fc moiety led to formation of OM− peptide conjugates that instantly formed strong gels in aromatic solvents, with minimum gelation concentrations (mgc) < 0.1 wt %. A gradually decreasing prominent negative Cotton effect at 500 nm when the gel was slowly heated to 80 °C indicated a chiral organization of the ferrocene moiety. The presence of the Fc moiety allowed redox-responsive sol−gel transition cycles to be performed with redox-active chemicals. When Fe(ClO4)3 was applied on top of the gel, it transformed into a solution state and the color of the mixture turned green, which is attributed to the oxidation of Fc to Fc+. Upon addition of ascorbic acid, the gel was again formed. 2.1.3. Cyclopentadienyl-Isolobal Tris(pyrazolyl)borate Complexes. Whereas group 8 metals and cobalt can conveniently be attached as coordinatively saturated metallocenoyl (in the case of Co, as cobaltociniumoyl) groups and group 7 metals as half-sandwich complexes, other metals cannot be attached via either of these organic ligands. For those metals, one can use ligands that are isolobal to the cyclopentadienyl family of ligands: tris(pyrazolyl)borates. Specifically, a so-called scorpionate Tp* complex with tungsten has been attached to the N-terminus of a peptide by use of standard SPPS coupling protocols (Scheme 4) [Tp* = hydridotris(3,5dimethylpyrazolyl)borate].127 Via an η2-coordinated alkyne, the carboxylic acid of the complex (28 in Figure 2) could be coupled to a growing peptide chain. It showed good stability

both the ruthenium(II) and osmocenium(II) ions of ruthenocene and osmocene, respectively, are known to be Hbond acceptors, that is, Lewis bases;118,119 on the other hand, the d orbitals of the Fe(II) ion do not extend sufficiently from the sandwich formed by the Cp rings to allow for such an attractive interaction.120 Apart from these activity-enhancing attributes, the presence of a ruthenocenoyl moiety in the AMP allowed detailed analysis of the mode of action of these short AMPs. Specifically, the high electronic density of the ruthenium(II) ion facilitated visualization of Rc-AMP in the bacterial membrane by means of transmission electron microscopy (TEM).45 In addition, utilizing the fact that ruthenium does not naturally occur in bacteria, AAS was successfully applied to quantitatively analyze the distribution of Rc−AMP; it was shown by Bandow and coworkers45 that 89% of the AMP was located in the bacterial membrane, with the rest equally distributed between cell wall and cytoplasm. Since the proteomic profile of B. subtilis treated with RcC(O)−WRWRW-NH2 was virtually identical to that of purely organic H-RWRWRW-NH2 (25, Chart 4) it was concluded that the mode of action (MOA) of these peptides was virtually identical, although the former was faster-acting than the latter. Realizing the potential of the OM−AMPs, the same group pursued the preparation of OM−AMPs with improved activity. It was already shown that the potential therapeutic window of lipidated AMPs of the same family could significantly be improved by an L-to-D substitution scan of five of the seven amino acid residues.121 Indeed, when the same Lto-D substitution scan approach was applied on the McC(O)− WRWRW-NH2 pentapeptide, four diastereomeric OM−AMPs (26, Chart 4) with MIC values as low as 0.7−1.5 μM were identified.122 For comparison, vancomycin (27) and gramicidin S displayed MIC values of 0.6 μM and 2.8 μM, respectively, against the same MRSA strains in the same assay. Importantly, these last two well-established antibacterial peptides require more amino acids to achieve their activity: seven amino acids and two sugars for vancomycin (1447 mass units) and 10 amino acids for gramicidin S (1200 mass units) versus five amino acids and one OM fragment for the OM−AMPs (1145 mass units). Since none of the OM−AMPs were hemolytic, the four members of this special family of synAMPs now represent the most active AMPs known to date. Considering the observation that the membrane is the primary target of these OM−AMPs (vide infra), both of these factors may be important. In the tightly orchestrated supramolecular medium that is formed by the membrane, inserting a slightly larger molecule into the membrane should have a larger effect when it is compared to a smaller similar molecule. Also, at the interface between bulk water and lipophilic interior of the membrane, the presence of an H-bond acceptor can have a profound effect on the strength of the interaction, and thus on the activity. In view of the redox-active nature of ferrocenoylated peptides, their potential as superoxide dismutase (SOD) mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration has been assessed. Moderate SOD activity was determined (EC50 of 310 and 575 μM) but strong inhibition of tyrosine nitration was observed for FcC(O)−Orn-Orn-OrnNH2 (Orn = L-ornithine).123 The mechanism of the often-observed enhanced activity of ferrocenoyl−peptide conjugates is unclear. Unspecific DNA binding or damage, inhibition of topoisomerase, or even immediate attack on biomolecules by reactive oxygen species K


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Scheme 4. Synthesis of Tp*WII(I)(CO)-Metalated Leuenkephalin and Neurotensin Derivatives127

2.1.5. Sensing Protein−Peptide Interactions by Use of Ferrocene. The specific reactivity of the N-terminus of a peptide can also be used for other heterogeneous strategies. For example, Kraatz and co-workers130 showed that a gold nanoparticle-functionalized screen-printed carbon electrode (GNP-SPCE) could be modified with a reactive ferrocene unit, which in turn could be modified with a peptide in buffered solution (Figure 4, 32). The immobilized peptide (33) bound

against air and water, and the presence of both carbon monoxide (CO) and iodido (I) ligands allows potential applications in infrared tracing and 125I radiolabeling. OM− peptide conjugates 29 and 30 (Scheme 4) proved to be stable under 3% (v/v) hydrazine, which was used to remove ivDdE groups from Lys [ivDdE = 1-(4,4-dimethyl-2,6-dioxocyclohex1-ylidene)isovaleryl], and under 20% TFA in DMF, which was applied to cleave the conjugate from the resin and to remove tert-butyl-based protecting groups; isolated yields ranged from 29% to 56%. 2.1.4. NCN Pincer Complexes. In 2003, van Koten and co-workers128 described the attachment of a PtII NCN pincer complex to a resin-bound PLL-linked hexapeptide GPPFPF. For this, an earlier produced [X-PtII(NCN)]Val-OH (X = I or Br) residue was used,129 which was coupled to the resin-bound peptide by use of TBTU and NEM in DMF (NEM = Nethylmorpholine). Although not a quantitative analysis technique, from the absence of the starting material in MALDI-TOF MS analysis of a fraction of the photocleaved conjugate, it was concluded that the OM−peptide conjugate was obtained quantitatively (MALDI-TOF MS = matrixassisted laser desorption ionization time-of-flight mass spectrometry). Unfortunately, treatment of this conjugate with 95% TFA and 5% H2O resulted in approximately 30% loss of the biomarker in only 30 min. To solve this, a PtII−NCN complex that has a carboxylic acid functionality directly attached to the aromatic ring of the pincer was attached (31 in Figure 2); it was shown that this could be done on all 20 proteinogenic amino acid residues. These more robust OM−peptide conjugates were indeed more stable to acidic treatment, and it was shown that they could be used to visualize the presence of KI3 in water or the presence of SO2 in a carrier gas by a colorimetric reaction.

Figure 4. Attachment of a pentadecapeptide on a preactivated ferrocenecarboxylic acid that was immobilized on a solid surface. The peptide contained a C-terminal carboxylic acid moiety and several functional side-chain groups, and reaction occurred at the N-terminus. The presence of a C-terminal histidine (His) residue did not interfere with this conjugation strategy.130

to the reverse transcriptase (RT) of human immunodeficiency virus 1 (HIV-1), which changed the characteristics of the redoxactive label, allowing electrochemical detection of the virus by square-wave voltammetry. By use of human serum that was spiked with RT, linear quantification of the enzyme was possible in the range 1−500 pg/mL (0.9−427 fM), with a lower detection limit of 0.8 pg/mL (0.7 fM). Importantly, a clear distinction was observed between the response of the system to RT and to integrase, an enzyme that is known for its interaction with the applied peptide sequence, and to protease. 2.2. Direct Conjugation to the N-Terminus in Solution

OM−peptide conjugates can also be prepared in solution. This is an appealing strategy when larger amounts of the conjugate L


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need to be obtained,43 when amino-containing peptidederivatives need to be conjugated with acid-labile ferrocene groups for bioanalytic purposes,131 or when the OM−peptide conjugate disintegrates under the harsh conditions that are usually required to cleave the OM−peptide from the resin.89 Solution-phase coupling of an OM fragment with a peptide is usually performed with (close to) stoichiometric ratios of the reagents, whereas solid-phase-based methods rely on use of an excess of the acylating agent. Carboxylic acid functionalized organometallic moieties can be conveniently coupled to peptides by use of isobutylchloroformate, N-methylmorpholine, and triethylamine, which produce volatile side products and organic salts that can easily be removed by standard washing procedures. In particular, sandwich and half-sandwich complexes have been attached by this method. Amido-linked OM− peptide conjugates can also be prepared with ferrocenecarboxylic acid chloride (FAC), and the sulfonamide-linked counterparts can be prepared with ferrocenesulfonyl chloride (FSC),102,132 although the latter usually provides lower yields.131 Both approaches are limited to organic solvents and peptide conjugates that do not contain competing reactive functional groups.133,134 Coupling reagents such as DCC/ HOBt/Et3N or EDCI/NMM/DMAP are also applied in certain occasions [NMM = N-methylmorpholine, DMAP = 4(dimethylamino)pyridine].135−137 Alternatively, stable cobalt and molybdenum carbonyl complexes of OSu-activated hex-4ynoic acid were prepared and reacted with mono- and dipeptides to give formation of the OM conjugates in respectable yields of 30−53%.138 OM moieties that are functionalized with (an) aldehyde(s) are conveniently conjugated to the amino functionality of peptides by formation of a Schiff base or imine. Since the imine is readily hydrolyzed, leading to the initial reaction components, these moieties were reduced to secondary amino groups by use of NaBH4 (at 0 °C).139 One drawback of solution-phase conjugation chemistry, however, is the more delicate purification methods that may need to be used and the fact that the process of solution-phase elongation of the peptide that leads up to the final conjugation reaction is more timeconsuming than solid-phase peptide synthesis. Nevertheless, solution-phase conjugation chemistry offers an attractive alternative to solid-phase based approaches, and the technique has been applied in several interesting cases. Ferrocene−peptide conjugates in which the ferrocene moiety has a different redox chemistry than the often-applied amidated ferrocenoyl moiety are sometimes desired. In order to achieve this, arylferrocene OM moieties can be used, which also offer additional tuning abilities by means of ortho, meta, and para positioning of the Fc and C(O)OH moieties (34−36, Figure 5). Notable examples of these derivatives were provided by Kenny and co-workers.140,141 N-(Ferrocenyl)benzoyl−peptide conjugates were prepared in solution by coupling various protected dipeptide esters (A, Figure 5) with the appropriate ferrocenebenzoic acid (B, Figure 5) by use of DCC or EDCI, HOBt, and TEA in DCM at 0 °C, producing the OM−peptide conjugates in 25−73% yields (TEA = Et3N). Longer peptides were also attached by the same procedure (accompanied by 29−55% yield), but the resulting OM−peptide conjugates were less active than the smaller counterparts.142 The related but significantly larger N-(6-ferrocenyl-2-naphthoyl) dipeptide ethyl esters were prepared by use of the reagents mentioned, accompanied by 23−89% yields.143 Especially the ferrocenyl− naphthoyl conjugates proved to be very active against H1299

Figure 5. Direct conjugation of OM−C(O)OH fragments at the Nterminal amino group of peptides in solution. The C-terminus of the peptide is protected as an ester or amide, and competing side-chain functionalities are omitted or protected to ensure that only the desired reaction can take place.

lung cancer cells (IC50 1.3 ± 0.1 μM), showing that a lower redox potential and higher lipophilicity can lead to OM− peptide conjugates with more potential anticancer activities.144 Vibrational analysis revealed a high level of conjugation between the aromatic ring and the ferrocene unit; subsequent B3LYP/6-31G(d,p) calculations revealed localization of the highest occupied molecular orbital (HOMO) on the Fc unit and of the lowest unoccupied molecular orbital (LUMO) on the conjugate phenyl ring.145 In addition to these examples, solution-phase methods were used to construct peptide−OM− peptide conjugates, that is, constructs in which the OM fragments forms an integral part of the peptide backbone (vide infra). OM fragments other than sandwich and half-sandwich derivatives have also been attached. In 2000, van Koten and co-workers146 prepared attached PtII NCN pincer complexes to the N-terminal amino group of an amino acid residue via Schiff base formation followed by reduction with NaB(CN)3H. Later on, this method was extended to the solid phase, using the amino acid conjugates prepared in the just-mentioned study to attach the PtII complex to a resin-bound peptide (vide supra, section 2.1.4.). 2.3. Indirect Conjugation to the N-Terminus: Solid- and Solution-Phase Methods

Not all OM fragments contain a reactive moiety that is suitable for direct covalent linkage to the N-terminal amino group of the peptide. To allow for the attachment of such fragments, the N-terminus is derivatized with a specific functional group that can react with the OM fragment via its intrinsic reactivity or a functional group that is inherently part of the OM moiety. This approach has been equally well utilized with soluble OM moieties and peptides that were attached to the resin as well as peptides that were in solution (Scheme 5). Carbon−carbon triple bonds have proven to be very useful in the formation of organometallic−peptide conjugates. Their reactivity is virtually orthogonal to all biologically relevant functional groups,147 with the exception of reactive thiols/ thiolates like activated cysteine residues,148,149 making them stable during many manipulations and modifiable on demand. In those derivatizations, the carbon−carbon core of the alkyne M


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zebrafish embryos was severely compromised, probably by inhibition of the cyclooxygenase 2 (COX-2) enzyme.155 Although not explored in great detail, evidence emerges for a promising application of this OM fragment as a carbon monoxide releasing molecule (CORM): this is a very interesting class of compounds that can locally deliver trace amounts of CO in order to inhibit O2-dependent pathways.156 For example, octreotide, which contains an N-terminal dicobalt hexacarbonyl alkyne moiety and was obtained in good yield (73%) by reacting the alkyne-containing octreotide with Co2(CO)8 under N2 atmosphere, was active on HeLa cells (IC50 ≈ 10 μM) and moderately active on PT45 (IC50 ≈ 30 μM) and HepG2 (IC50 ≈ 65 μM) cells, whereas Co2(CO)8 was only moderately active against HeLa cells (IC50 ≈ 20−40 μM).99 Apart from using cobalt to react with an acetylene group to form an OM−peptide conjugate, complexes of the group 6 metals molybdenum (Mo) and tungsten (W) can also react with acetylene groups. 157 [The previously mentioned Tp*WII(CO)(I)−alkyne η2 complex, in section 2.1.3, by Metzler-Nolte and co-workers,127 was not prepared via this indirect method.] Application of this interesting chemistry has been developed by Curran et al.158−162 in the past 15 years (see Figure 1 for a representative structure). The first systems studied were amino ester residues that contained acetylene groups on the amino group. When 2 equiv of acetylene derivative reacted with 1 equiv of WII(CO)3(dmtc)2 in refluxing methanol under inert atmosphere, the orange color of the methanolic solution of WII(CO)3(dmtc)2 changed to a deep green color, which is indicative of the monoalkyne coordinating complex (dmtc = N,N-dimethyldithiocarbamate).158 During the course of several hours, the color changed again, now from lemon to yellow, signifying the formation of a bis(alkyne) coordinating complex, which was typically isolated in 32−54% yield.159 The chemical shifts of the acetylene protons were used to probe the conformational flexibility of the formed complexes; no less than three conformations were observed, which corresponds to the theoretically and experimentally expected number. As the molecules can occupy all of these different conformations in a dynamic fashion, which was evidenced in the 1H NMR spectra, this tungsten bridge between two acetylene moieties offers a promising approach to preorganize biologically active peptides. Indeed, this approach was successfully applied to cyclize peptides;160 even mimics of β- and γ-turns were prepared with isolated yields of 24− 28%.161,162 Interconversion between the different conformations of the metallacyclic peptides was dependent on the size of the ring. Now that the fundamentals of this approach to cyclize peptides have been discovered, it is expected that the method will also become applicable to peptides with functionalized side chains and biological targets. An alternative to this direct conversion of the acetylene moiety to an OM unit is its orthogonal reaction with an OM fragment via a cross-coupling reaction. The discovery of Pdcatalyzed cross-coupling reactions, which was rewarded with the Nobel Prize in Chemistry 2001, has attracted the attention of synthetic chemists across a wide chemical landscape.163 Hoffmanns and Metzler-Nolte166 described the first Sonogashira cross-coupling reaction164,165 between an OM fragment and a peptide in 2006 (Scheme 6B). By use of either an Nterminally or an internally positioned iodoarene (4-iodobenzoic acid or 4-iodophenylalanine), conjugation of the peptide to two different acetylene-derivatized ferrocene moieties was realized.

Scheme 5. General Strategies for Indirect Metalation of Peptides on Their N-Termini by Orthogonal Conjugation Chemistrya

a

X = NH2 or OH; Y and Z are functional groups that display crossreactivity with each other.

becomes incorporated into the bridge that connects the peptide and the OM fragment. The acetylene moiety can be converted into an organometallic complex by use of Co2(CO)8, which leads to the formation of a metal−carbonyl complex (Scheme 6A). Even Scheme 6. Examples of Indirect Metalation of Peptides on Their N-Termini by Orthogonal Conjugation Chemistrya

a

(A) Cobalthexacarbonyl−acetylene complex formation on acetylenepeptide; (B) Sonogashira cross-coupling reaction between iodoarylpeptide and acetylene-OM moiety; (C) CuAAC of azido-OM to acetylene-peptide; (D) CuAAC of acetylene-OM to azidopeptide.

though Co2(CO)8 has been applied as a protecting group for alkynes as early as 1971,150 the role of the formed acetylene− dicobalt hexacarbonyl complex as a functional OM complex in (bio)active peptides was only studied much later, most notably by the work of Gust and Ott and co-workers.151,152 and by Metzler-Nolte and co-workers.153,154 Important insight into the possible mode of action of these compounds, when it comes to their anticancer activity, has been provided recently by use of an OM−aspirin conjugate: it was shown that vascularization in N


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(37) moiety was decorated with an acetylene-functionalized Leu-enkephalin peptide (38) (Scheme 7). Interesting differ-

For transformation of dipeptides, a dried and degassed mixture of tetrahydrofuran (THF) and Et3N in a Schlenck tube was mixed with the peptide, 1.1 equiv of OM-acetylene, and 2 mol % PdII(PPh3)2Cl2 and 3 mol % CuI; precipitation of the protonated base salt as side-product was used to estimate completion of the reaction (6 h). A typical yield of 70−91% was achieved for internally metalated dipeptides. For metalation of Leu-enkephalin, a modified procedure needed to be used; the peptide had a 4-iodophenylalanine residue at the 4-position that could be metalated in degassed DMF/Et3N (1:1 v/v) with 1.1 equiv of Fc-acetylene, 30% PdII(PPh3)2Cl2, and 50% CuI. After 12 h of reaction time, 48% of the OM−peptide conjugate was obtained after purification by HPLC. Metalation of an Nterminally positioned 4-iodoarene by the same procedure resulted in 23% purified product. This example illustrates how conditions that can be applicable in metalation of one peptide might not be applicable in the metalation of another peptide. Another prominent method for derivatization of acetylene moieties is the copper(I)-catalyzed acetylene−azide cycloaddition (CuAAC) reaction,167−169 a reaction that was simultaneously shown to be applicable on peptides170 and small molecules.171 Instead of reacting the acetylene moiety with an iodoarene by use of a Pd catalyst, the CuAAC reaction clicks an acetylene to an organic azide by use of a Cu(I) catalyst. A detailed mechanism for this important reaction was recently established.172 In principle, there is no preference for the positioning of the two reactive sites: both the peptide and the OM fragment can be derivatized with either the azido or acetylene group (Scheme 6C,D).173 In the last step of the synthetic procedure, before cleavage of the peptide from the resin, it is sometimes desired to apply a chelating agent that helps to remove metal salts from the polymeric core of the resin. As was the case in the initial studies of amide-linked OM− peptide conjugates, ferrocene derivatives also dominate the assembly of triazole-linked OM−peptide constructs. This has been facilitated by the fact that both azido- and acetylenefunctionalized Fc groups are readily available. As acetylene or azido groups can be installed in peptides on demand,174 their conjugation reaction is limited only by the ease of the reaction itself. In cases where the azido or acetylene moiety is directly attached to the Fc complex, its CuAAC conjugation affects the redox potential of the OM fragment. For example, conversion of the Fc−CCH moiety into its 1,2,3-triazole unit results in a shift of +97 mV of the redox potential (against Fc/Fc+).59 In this specific case, where various linkers were used between the Fc unit and the peptide nucleic acid chain, four different redox potentials were obtained, each of which corresponded to one of the four nucleobases of DNA. The applicability of CuAAC for conjugation of ruthenocene to peptides and derivatives like peptide nucleic acid (PNA) has been shown by Patra and Metzler-Nolte,175 and for titanocene, by Tacke and coworkers176 (see Figure 1 for an exemplary structure). One of the most appealing features of the CuAAC reaction is its wide tolerance for functional groups, at least for those that are commonly found in peptides. This tolerance opens up the possibility to conjugate the peptides in a post-SPPS fashion. Whereas this can be seen as an option that is additional to SPPS-based CuAAC conjugation for derivatization of one peptide, CuAAC is virtually the only option if one wants to conjugate two larger peptides with one OM fragment. The facility of this approach was first shown in 2008 by MetzlerNolte et al.:177 in one synthetic step, a 1,1′-diazidoferrocene

Scheme 7. Indirect Metalation of Two N-Terminally Modified Peptides with a Bis(azido)ferrocene Moiety by CuAAC Chemistry177

ences in redox chemistry were observed: whereas the ferrocenyl monotriazole conjugate showed reversible redox chemistry, the 1,1′-ditriazole conjugated ferrocenyl construct (39) was irreversibly oxidized. An alternative multistep strategy for the labeling of peptides with 99mTc or Re coordination complexes uses an N-terminally positioned electron-rich carbon atom as a donor group, resulting in organometallic−peptide conjugates. Using an isocyanide-functionalized N-terminus, Pietzsch and Kozminski and co-workers178−182 showed that a metal−carbon bond is formed between the coordination complex and the peptide, resulting in a lipophilic 4 + 1 (or κ1 on the side of the peptide) mixed-ligand complex (Chart 5, generic structure 40).183 In recent years, these OM−peptide conjugates have been successfully applied in tumor-imaging strategies184 and have proven to be significantly stable to allow biological tests.179 Using the mixed-ligand framework of these OM−peptide conjugates, hydrophilic coligands were introduced (41 and 42, Chart 5), which shifted the excretion of these conjugates from hepatobiliary to an equal rate of renal and hepatobiliary pathways, thus improving the in vivo properties.185 Unfortunately, target-background ratios in the tumor xenographs were low, demanding further optimizations. Nevertheless, the labeling procedure requires mild reaction conditions, and a variety of bioactive peptides were reacted with the coordination complex by simply mixing the two components (Chart 5).186 Importantly, in view of the instability of isocyanides under acidic conditions that are often applied in HPLC-based peptide purification, a basic mobile phase has to be used, for example, buffers that contain 0.01 M NH3. Also, the labeling reaction was negatively affected by the presence of oxygen in the system. Nevertheless, with a specific activity of ∼20 GBq/μmol, the procedure has comparable labeling efficiency to the clinically approved 99mTc-Apcitide and 99mTc-Depreotide, which bind 99m Tc by use of an N3S chelator (specific activity ∼20 GBq/ μmol). Half-sandwich complex derivatives based on CpMnI(CO)3 and CpReI(CO)3 have been attached by classical solution-phase coupling strategies like the preparation and isolation of O


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Chart 5. Indirect OM−Peptide Conjugate Formation by Means of an N-Terminal Isocyanide Groupa

a

The basic core of the OM−peptide conjugate is shown, as well as two examples in which the lipophilic nature of the 4 + 1 complex was suppressed. These OM−peptide conjugates can be prepared in the presence of several peptidyl functional groups, as is apparent from the structures of the peptides that have been successfully used.178−186. M = 99mTcI or ReI.

preactivated OSu esters;187 this route was also applied on proteins.188 Specifically, proteins can be labeled with an organometallic−peptide bioconjugate that contains an additional reactive functionality. For example, Eppinger and coworkers189 showed that an epoxide-containing OPfp esteractivated amino acid derivative (43) was conjugated with an amino-group-containing half-sandwich complex CyRuII (44) or Cp*′RhIII (45) (Cy = p-cymene; Cp*′ = substituted pentamethylcyclopentadienyl), with formation of an OM− peptide conjugate in excellent yields (Scheme 8, 46 and 47). These complexes were subsequently conjugated to the reactive cysteine moiety in papain, a very powerful nucleophile that reacts with the epoxide moiety. The resulting OM−protein conjugates were applied as artificial metalloenzymes in asymmetric catalysis.190 Similar approaches that target reactive serine residues in proteases are also available.191,192

by incorporating a C-terminal glycine or proline residue or by using another amino acid spacer. In view of these difficulties for obtaining C-terminally labeled peptides and the ease by which N-terminally labeled peptide can be obtained (vide supra), only a few alternative approaches have been described for direct derivatization of the C-terminus of peptides. In 1999, Metzler-Nolte and co-workers196 explored the ability to introduce an organometallic moiety early on in the synthetic route toward OM bioconjugates. For this, ferrocene amines, obtained from ferrocene aldehyde (48) and a primary amine,195 were coupled in solution to N-protected amino acid residues by use of DCC/DhbtOH [DhbtOH = 3-hydroxy1,2,3-benzotriazin-4(3H)one], with good yields of 54−71% (Scheme 9). Interestingly, the change of potential of about 50 mV when the amino-group-containing CpFeIICp′CH2N(H)R (49) was converted into the amido-group-containing CpFeIICp′CH2N(R1)C(O)R2 (50) allowed them to follow the coupling reaction electrochemically (Cp′ = monosubstituted cyclopentadienyl). Extension of the C-terminally modified amino acid residue in solution was performed by HBTU/Et3N-mediated amide-bond formation. As a result, one of the very few peptides was prepared in which the OM unit was attached on the nitrogen atom of the C-terminal backbone amido group of the peptide. Interestingly, this method allowed for preparation of one of the first heterobimetallic OM−peptide conjugates (51 and 52 in Scheme 9): benzoic acid chromium tricarbonyl was attached to the N-terminus of the dipeptide in 64% yield.197 Cyclic and square-wave voltammetry on a tripeptide that was derivatized with ferrocenyl groups on both the N- and C-termini did not show an electronic interaction

2.4. Direct Conjugation to the C-Terminus

In addition to the N-terminus of the peptide backbone, the Cterminus is also an attractive anchor point for an OM moiety. However, due to the C-to-N nature of solid-phase synthesis of peptides, derivatization of the C-terminal end of the peptide is usually less straightforward. One way of avoiding this is the application of linkers that are sensitive toward acid, so that the attached peptide can be cleaved under dilute acidic conditions, such as 1% TFA or hexafluoropropanol. Specifically, trityl-based linkers61 allow modification of the C-terminal carboxylic acid of the peptide after SPPS has been completed and the (purified) side-chain-protected peptide has been obtained. The main disadvantage of this route would be the risk of epimerization of the C-terminal amino acid residue,193,194 which can be avoided P


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electronically decouples the effect of the amide on the redox potential of Fc, and is complemented by a method that attaches aminoferrocene (Fc-NH2) to the C-terminus of peptides.198,199 In a more recent example, azidomethylferrocene was clicked to 1,1,1-trifluoro-2-(trifluoromethyl)-3-butyn-2-ol, after which the OM fragment was conjugated to Boc-Leu-OH by use of EDCI and DMAP. This coupling was performed at room temperature for 1 day, in 61% yield. Unfortunately, this conjugate was very susceptible to hydrolysis even in slightly alkaline conditions, preventing further biological testing.200 Importantly, in this case, chain elongation proceeds from Cto N-terminus, as in solid-phase peptide synthesis, so epimerization of the C-terminus is negligible and stereochemically pure compounds are usually obtained. Although this method should also be applicable for on-resin OM−peptide synthesis with the backbone amide linker (BAL),201−203 so far, no examples have emerged in the literature.

Scheme 8. Synthesis of Epoxide-Containing OM−Amino Acid Conjugates Applicable in Site-Selective Conjugation Reactions with the Uniquely Reactive Cysteine Residue in Papain190

2.5. Indirect Conjugation to the C-Terminus

Employing the versatility of linkers nowadays available for preparation of a variety of C-terminally modified peptides, indirect modifications of the C-terminus of peptides have attracted much attention in recent years. This is mostly because, for an indirect conjugation strategy, the reactive intermediate can be generated from (cheap) commercially available reagents and can be obtained in pure form before the OM conjugation reaction is performed. One of the first examples of this approach was described by Metzler-Nolte and co-workers in 2009 (Scheme 10).154 By use of Ellman’s version of Kenner’s safety catch linker, a Leuenkephalin derivative was prepared that had an alkylated Cterminal carboxamide (53). One drawback of this approach is the usually low yield of the cleaved peptide; in the example mentioned, the C-terminally alkylated Leu-enkephalin derivative was obtained in only 22% yield. Fortunately, subsequent metalation of the triple bond with Co2(CO)8, leading to formation of a dicobalthexacarbonyl−alkyne complex (54), proceeded quantitatively. Stretching vibrations of the carbonyl groups attached to Co ions were observed at 2095, 2053, and 2022 cm−1. As an alternative for synthesis of the C-terminal acetylene-functionalized peptide, the BAL linker (55) can also be used;204 however, to our knowledge, this approach has not been used in organometallic−peptide conjugation reactions. In order to counter the low yield of the C-terminal acetylenemodified peptide, Albada and co-workers205 described a new type of linker that relies on a silyl group as connecting unit between peptide-acetylene and resin (Scheme 11). Silyl groups are particularly suited for the protection of acetylene moieties, and the Si−C bond to the more electronegative sp-hybridized acetylene carbon atom is more readily cleaved than the remaining Si−C(sp3) bonds. The first generation of this new type of linker, the SAM1 linker (56, SAM = silyl-based alkynemodifying), proved to be stable during manual SPPS at room temperature and during automated microwave-assisted SPPS, during which coupling reactions were performed at 75 °C. Importantly, a one-pot cleavage-click procedure was described to yield triazolylferrocene peptide conjugate 57 directly from the new SAM1 linker and ferrocene azide. The yield of this one-pot procedure was significantly higher (59%) than that of the alternative route that consisted of two individual steps (16%) (Scheme 11). Unfortunately, the SAM1 linker was not compatible with acid-mediated removal of the peptide protecting groups, as the arylacetylene moiety hydrates upon

Scheme 9. Direct Metalation of a C-Terminal Carboxamide Group of Small Peptides via Schiff Base Formation, Reduction, and Acylationa 196

a

Two peptides prepared by means of solution-phase conjugation reactions are shown..

between the two ferrocenyl moieties, and the distance between the two iron(II) ions was estimated to be ∼10 Å. This method results in a Fc-derivatized peptide that contains a methylene moiety between the carboxamide and Fc fragment, which Q


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Scheme 10. Indirect Metalation of Peptidyl Carboxamide Groups by Use of Safety Catch or BAL Linkersa

a

After assembly of the peptide, the acylsulfonamide linker group is alkylated with iodoacetonitrile in the presence of DIPEA, after which the resinbound acyl group of the peptide is converted into a C-terminal acetylene-functionalized carboxamide.154 The BAL linker can also be applied to generate C-terminal acetylene-derivatized peptides.204.

Scheme 11. First Two Members of the Family of Silyl-Based Alkyne-Modifying Linkersa

a

In this type of linker, the peptide is assembled on a linker molecule that contains a silyl−alkyne bond. Whereas the SAM1 linker provides sidechain-protected arylacetylene-functionalized peptides,205 the SAM2 linker provides protected and deprotected acetylene-functionalized peptides;208 both linkers are compatible with a one-pot cleavage-click conjugation reaction with an organometallic moiety, in this case azidomethylferrocene.

exposure to strong acids that are usually needed for this deprotection step.206,207 One year later, Albada and coworkers208 therefore described the acid-compatible SAM2 linker (58); in fact, the resin-bound peptide was susceptible to acid-mediated deprotection of the side chain without removal of the peptide from the resin. The very convenient one-pot cleavage-click reaction was shown to be applicable also for this SAM2 linker, affording C-terminally triazole-linked OM−peptide conjugates 59 in 26−53% yield (Scheme 11). The additional chemistry offered by this linker makes it a very promising candidate for bioconjugation reactions, as was recently shown by a one-pot copper-free cleavage-Sonogashira conjugation reaction.209,210

Scheme 12. Incorporation of a 1,1′-Dicarboxylic Acid Ferrocene Moiety in a Parallel β-Sheet Mimica42

2.6. Incorporation of Organometallic Moieties in the Peptide Backbone

a

This approach is limited to soluble peptides or resins that contain a high loading of linker molecules, and thus densely packed peptides.

Apart from modification of one of the two terminal groups of the peptide backbone, an organometallic moiety can also be incorporated into the peptide backbone. This is facilitated by the possibility that many organic moieties of organometallic complexes allow suitable modifications. When identical functional groups are introduced, such as two carboxylic acid functionalities, the peptide backbone alters directionality: instead of having a backbone with one C- and one N-terminus, it now consists of two C- or two N-termini (see also Scheme 12 for a OM−peptide conjugate with two C-termini). Alternatively, when both an amino and a carboxylic acid moiety are

present on the OM moiety, the directionality of the peptide backbone is not altered. Which of the two options is applied has major consequences for the applications. Meldal and co-workers211,212 used unsymmetrically substituted imidazolium salts in which one side contains a carboxylic acid and the other side an amino group (Chart 6). By incorporation of two NHC precursor fragments, coordination of a proximate enolate (in 60) was suppressed and a bisNHC-coordinated Pd complex was formed (in 61). The resinR


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orientation of the backbone: 1′-aminoferrocene-1-carboxylic acid214−217 (64) was incorporated into small peptides (65, 66).218−221 In an elaborate attempt to study the presence of Fc units in the peptidic backbone, even “peptides” were prepared that contain only Fc units; by use of Fmoc-Fc-C(O)OH (67) and SPPS methods that are common for Fmoc-based SPPS, tri-, tetra-, and penta-“peptides” (68) were obtained in 12−18% isolated yield (Scheme 13).222 The prepared oligo-Fc “peptides” displayed a stepwise oxidation process, suggesting electronic and electrostatic interactions in these oligomers. The first antiparallel β-sheet-mimicking Fc−peptide hybrid appeared from a collaborative effort by Kraatz, Kirin, MetzlerNolte, and Rapic and co-workers in 2006.41 Together with the necessary monosubstituted control compounds, the helical properties of ferrocene−peptide hybrid structures that contained the 1′-aminoferrocene-1-carboxylic acid unit as a turn inducer were studied. It was found that the amino acid residue attached to the amino group of the Fc unit determines the helical chirality of the Fc−peptide hybrid. In a study that appeared later that year,223 the incorporation of an Fc-based amino acid moiety in longer peptides was presented. It was shown that the Fc unit was also able to support a helical structure supported by an antiparallel β-sheet-like alignment of the strands. These examples revealed the influence of an OM moiety in the peptide backbone on the structure of peptides. As a result, it became possible to design specific structural features into peptides by use of organometallic moieties. For a more indepth treatment of these studies, the reader is referred to an excellent review that appeared on this topic.44 A novel approach for the incorporation of ferrocene moiety in the backbone of bioactive peptides was recently reported by Neundorf and co-workers.224 By use of a 1,2-ferroceno-fused cyclopentene backbone moiety in which the amino group was protected with Fmoc, an OM amino acid derivative was prepared that was compatible with SPPS. This building block was applied in a double coupling reaction on a resin-bound Cterminal fragment of the human peptide hormone calcitonin (hCT), the hCT(24−32) unit. After manual coupling of FmocGln(Trt)-OH, the full OM-modified sequence of hCT(9−32) (69) was prepared by automated SPPS procedures (Figure 6)

Chart 6. N-Heterocyclic Carbene Complexes Incorporated into the Peptide Backbone211,212

bound versions of these OM−peptide conjugates were applied in heterogeneous cross-coupling reactions. When it comes to metallocene groups, the functionalities needed for the conjugation reaction can be positioned either on the same (1,2- or 1,3- substitution) or on different Cp rings. Using a 1,1′-dicarboxylic acid ferrocene unit (62), several groups prepared parallel β-sheet mimics in which the Fc unit composed the turn part of the peptide (63) (Scheme 12).42 These studies have been reviewed extensively elsewhere44 after Herrick et al. launched the concept in 1996.213 Importantly, the authors did not observe notable differences between peptide turns induced by neutral ferrocene (Fc) and by positively charged cobaltocinium (Cc+), which indicates that alignment of the peptide strands is determined by structural features of the sandwich complex and the peptide, not by the charge of the complex. Clearly, such incorporation of an OM unit in the peptide backbone has a significant impact on the physicochemical properties of the peptide. A more advanced building block allowed incorporation into peptides without affecting the native

Scheme 13. Incorporation of Ferrocene in the Backbone of Peptides218−221

S


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Scheme 14. Synthesis of (A) Fem-modified195 and (B) Fedmodified226 Peptide Backbone

Figure 6. Ferrocene-modified human peptide hormone calcitonin.225

(Trt = triphenylmethane). Interestingly, even though the peptide was cleaved with TFA/water/TIS, oxidation of the Fc moiety was not described. Internalization studies showed that the OM−peptide conjugate is taken up to a similar extent as the native peptide, and no cytotoxic effect was observed up to 250 μM for the OM-hCT(9−32) conjugate.225 Following their initial work on tungsten−bis(alkynylpeptide) complexes, Curran et al. prepared alkyne-bridged tripeptides and reacted the triple bond with WII(CO)3(dmtc)2 under formation of a diastereomeric WII(CO) (dmtc)2(alkynylpeptide) complex 70 (dmtc = N,N-dimethyldithiocarbamate) (Figure 7).158 An interesting observation was made that such

Figure 7. Symmetrical alkyne-bridged bis(peptide) in which the triple bond is metalated with a WII(dmtc)2 complex.158

alkyne−tungsten complexes show fast rotation of the alkyne about the tungsten center, even at 23 °C. Another important conclusion, at least from a biological perspective, is that the peptides do not seem to be preorganized in a particular fashion under influence of tungsten complexation. As these complexes have not been applied in biomedical settings, the reader is referred to the cited papers for more details.158 Apart from incorporation of an OM moiety in the peptide backbone, or the incorporation of a nonpeptidelike molecular fragment that allows coordination of an OM fragment, some OM moieties can be attached to the amide backbone of the peptide (Scheme 14). An early example emerged in 1986,195 when the chromophoric (yellow) ferrocenylmethyl (Fem) group was attached to a peptide bond in order to manipulate the secondary structure of peptide derivatives or, additionally, to solubilize the peptides by making them more lipophilic (Scheme 14A). The conjugation reaction proceeded by a reductive alkylation reaction of amino acids or amino acid esters with ferrocene aldehyde 48.195 Subsequent extension of the peptide chain on the amino group (of, for example, derivative 71) proceeded without complications; extension on the C-terminus of Boc-Gly-(Fem)Ala-OH with H-Leu-OtBu gave the OM−tripeptide conjugate in 92% yield, although 27% D-alanine was also formed (for comparison, coupling of BocGly-Ala-OH with H-Leu-OtBu under identical conditions gave 84% yield and 12% D-alanine). Epimerization of the C-terminal amino acid residue, generally observed when the amino group is acylated and the carboxylic acid is activated, is not avoided by the Fem group. However, when Tcboc-(Fem)Ala-OH was coupled with H-Leu-OtBu, the OM-dipeptide was obtained in 85% yield with 80% under optimized conditions. The histidinyl imidazole ring can be converted into an Nheterocyclic carbene (NHC), a heterocyclic moiety in which the carbene functionality on the C2 atom has a high affinity for metal ions.241 This approach was initially explored by Erker and co-workers242 and was later extended to peptides by Albrecht and co-workers.243−246 Starting from the unprotected amino acid histidine, Erker and co-workers242 prepared a backboneprotected moiety in which both imidazole nitrogen atoms can be alkylated by use of Meerwein’s reagent, [Et3O+·BF4−]. This procedure, however, which was exemplified by PhC(O)-HisOEt, resulted in epimerization of the chiral Cα atom and Oalkylation of the PhC(O) group. With n-propyl bromide and NaHCO3, a much cleaner reaction took place, and the desired histidinium salt was obtained with >70% yield, even when the reaction was performed on a 150 g scale. Similar results were obtained with isopropyl iodide (95% yield) and also with BocHis-OMe (yields were 92% for the n-propyl and 69% for the isopropyl alkylated imidazole, respectively). The resulting bisalkylated imidazole ring was subsequently deprotonated at the C2 atom with Ag2O, resulting in the Arduengo carbene in nearquantitative yields. These bis(carbene) AgI complexes can directly be converted into late transition metal histidylidene complexes by reacting them with a suitable metal precursor in a noncoordinating solvent. For example, transmetalation with PdII(CH3CN)2Cl2 resulted in bis(carbene) PdII complexes (in 82% or 86% yield), and reaction with [RhI(cod)Cl]2 resulted in diastereomeric monocarbene RhI complexes (in 65% or 73% yield) (cod = cyclooctene). Similar procedures were applied by Albrecht and co-workers, first on histidine residues243,244 and later on small peptides.245,246 After solution-phase synthesis of the peptide, for which Boc-His(εMe)-OMe was the starting material, the histidinium peptide was obtained by methylation of the pyridine-like nitrogen atom in the monoalkylated imidazole ring (Scheme 15). The bis-alkylated imidazole ring in peptide 77 could be converted into the organometallic NHC complex via a one-pot procedure with Ag2O and either [RuII(Cy)Cl2]2, [RhI(cod)Cl]2, or [IrIII(Cp*)Cl2]2 in DCM at room temperature. Yields were 50% for RuII(η6-Cy)(η1-NHC), about 70% for RhI(cod) (78), and 81% for IrIIICp OM−peptide conjugates. A minor drawback of this system is the use of Ag2O: this light-sensitive material works best if it is freshly prepared and provides a clean reaction only when used in the dark, as was noted by the authors. Due to the hydrophobic nature of the prepared OM−peptide conjugates, purification by flash chromatography was feasible. The resulting Rh I monochloride complex was converted into a catalytically active

3.1. Direct Metalation of Proteinogenic Amino Acid Residues

Importantly, most functional groups that are present in the set of proteinogenic amino acid residues do not allow direct formation of carbon−metal bonds; only a few methods are known in which aromatic amino acid side-chain functionalities are metalated (vide infra). Other methods rely on rather classical modifications with σ-bond formation of a few residues like lysine amino groups, cysteine thiol groups, or carboxylic acid groups of aspartic and glutamic acid. The proteinogenic amino acid residues lysine (Lys, K), aspartic acid (Asp, D), and glutamic acid (Glu, E)229 can be subjected to metalation, similarly to the N-terminal amino group and C-terminal carboxylic acid moiety (vide supra). Apart from the necessary orthogonal deprotection of side-chain functional groups of these amino acid residues, for which detailed procedures have been established,230 the strategies mentioned can be applied relatively straightforwardly. For example, amino groups in proteins can be metalated with organometallic pyrylium salts, which provide chromophoric organometallic conjugates with absorption maxima in the range 390−450 nm.231,232 The cylindrical shape of sandwich complexes of the ferrocene type has inspired applications of such molecular pillars to bridge between two topologically neighboring groups in peptides. This has been described as an alternative to disulfide-bridged,233 thioether-bridged,234 or carbon−carbonbridged235 approaches. Specifically, a 1,1′-difunctionalized ferrocene moiety can bridge two remote parts of a peptide, thereby restraining the conformation of the peptide (Figure 8, 75). For example, two lysine residues at positions i and i + 3 in a peptide were linked together by use of 1,1′-ferrocenedicar-

Figure 8. (Left) A 310-helix induced by 1,1′-ferrocene diacid-bridged lysine residues in the i and i + 3 positions.38 (Right) Ability to fix a peptide backbone was also shown in an Fc Leu-enkephalin derivative; a stabilizing hydrogen bond is indicated by the dashed line.236 U


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3.2. Direct Metalation of Unnatural Amino Acids

Scheme 15. Metalation of Bis-alkylated Histidine Residues by Means of Their Conversion into N-Heterocyclic Carbenesa

As an alternative to the histidine residue, a suitable side-chain functionality can be converted into a NHC carbene precursor. This concept has been applied by Xu and Gilbertson250 for the preparation of a peptide conjugate of the second-generation Grubbs catalyst (Scheme 16A). By use of an alkyl−zinc amino Scheme 16. Synthesis of (A) Peptide Conjugate with Second-Generation Grubbs Catalyst250 and (B) 4Hydroxyproline-based N-Heterocyclic Carbene Complexed with RhI(cod)Cl252

a

According to Albrecht and co-workers.245,246

species by a ligand-substitution reaction in which chloride was exchanged for phosphine. RhI complexes of peptides with functional groups, such as Tyr or Met, were also prepared, and for the latter, coordination of the thioether sulfur atom to the RhI center was observed after the NHC OM−peptide conjugate was treated with KPF6 (79). Peptide-based NHC−metal conjugates have been primarily studied in catalytic applications and much less in biomedical settings, although a few examples emerged recently. In line with the application of simple metal NHC complexes in the treatment of cancer and infectious diseases,247 the antiproliferative activities of NHC−gold amino acid conjugates were studied.248 Very acceptable antitumor activities were obtained against HeLa, HepG2, and HT-29 that were comparable to that of cisplatin. It was concluded that the [NHC−AuI]+ fragment alone was not responsible for the toxicity of the compounds, and it was suggested that differential uptake contributed to the observed activities. Interestingly, before or after this coordination of the methionine residue to the RhI ion, the cod ligand could be replaced with carbon monoxide (CO), which provided peptide 80, by bubbling CO(g) through the solution for 10 min, followed by stirring overnight under a CO atmosphere. For a peptide in which Met was substituted for Tyr, which could potentially be an O ligand, coordination of the phenol oxygen atom was not observed. Clearly, the softer sulfur atom facilitated coordination to RhI and the harder hydroxyl group did not. Alkylation of the Cys sulfhydryl moiety provides another entry into the preparation of organometallic−peptide conjugates. However, this approach is mostly applied for protein modifications, as shown by several examples.249 These will not be treated in depth here.

acid residue (81) in combination with iodoarylimidazolium salts (82), an amino acid building block was prepared that could be incorporated into a peptide and subsequently converted into an OM−peptide conjugate (83). In order to obtain a phosphine-coordinated complex (i.e. the peptidic version of the second-generation Grubbs catalyst), the firstgeneration Grubbs catalyst had to be used. The second generation of the Grubbs catalyst contains both an NHC and a P(CyHex)3 ligand, which would lead to a bis-NHC complex. Very recently, hydroxyproline, a natural building block that has invited various applications in biomimetic studies,251 was modified with NHC−RhI complexes by Zhao and Gilbertson252 (Scheme 16B). After synthesis of the suitable NHC precursor building block, which was obtained in its protected form in ∼85% yield in two steps from 4-trans-hydroxyproline, it was incorporated into tripeptides (like 84) by solution-phase methods in 61% yield. This was then followed by complexation V


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to rhodium, which gave 41−53% yields of the OM−peptide conjugate (85). Alternatively, 4-trans-hydroxy-L-proline was converted into 4-cis-azido-L-proline, which was then converted into an organometallic−peptide conjugate by use of acetyleneferrocene and CuAAC. This led to a new class of HIV-1 antagonists having an IC50 value of 0.2 μM for the inhibition of HIV-1 cell infection.253 Another alternative to the histidine-based NHC-coordinating unit is the commercially available thiazolylalanine residue (as in peptide 86).254 This moiety is conveniently converted into a thiazolyl-2-ylidene N,S-heterocyclic carbene (NSHC) after alkylation of the nitrogen atom, with formation of a thiazolium salt, and deprotonation of the C2 atom. This carbene can then be converted into an organometallic complex by reaction with CyRuII and Cp*RhIII precursor complexes (Scheme 17, 87 and

Scheme 18. Synthesis of NSHC−Gold(I) Peptide Complexesa and Gold(I)-bridged Bis(peptide) Complexb 255

Scheme 17. Application of L-Thiazolylalanine in Formation of NSHC-Complexed CyRuIICl2 and Cp*RhIIICl2254

88, respectively). Due to the OM reagents used in this reaction, which dictate the use of dry DCM under inert atmosphere, only protected amino acids and di- and tripeptides could be used. Yields of 24−28% of the mixed-sandwich complexes were obtained. Alternatively, the NSHC obtained from the alkylated thiazolylalanine peptide 89 can be metalated with various gold(I) species, such as AuI(Cl) (DMS), AuI{N(SiMe3)2} (tht), or even AuI−cysteine derivatives (DMS = dimethyl sulfide; tht = tetrahydrothiophene) (Scheme 18). Deprotonation of the C2 atom by Ag2O can be applied in the synthesis of NHC−metal complexes, even though the higher acidity of the H2 proton in the thiazolium moiety results in a relatively weaker nucleophile when compared to those obtained from imidazolium salts. The subsequent transmetalation reaction of the AgI−NSHC complex, which could be isolated by HPLC, with AuI(Cl)(DMS) was successful and provided OM conjugate 90, but reaction with AuI(I)(DMS) gave a mixture of products. This complication was avoided by a one-pot procedure in which the carbene was generated by deprotonation with the strongly nonnucleophilic gold(I)-containing base AuI{N(SiMe3)2}(tht), which was obtained by an in situ metathesis reaction between K[N(SiMe3)2] and AuI(Cl) (tht). The thus-obtained NSHC− AuI−iodido complex 91 could be reacted with Boc-Cys-GlyOMe 92 via formation of a bis(peptide) Au I-bridged bioconjugate 93. Whereas the gold(I)-bridged bis(peptide) conjugate did have an IC50 value of ∼25 μM or higher against

a By use of Ag2O and AuI(Cl)(DMS) or AuI{N(SiMe3)2}(tht). bBy use of NSHC−gold(I) iodide complex and Boc-Cys-Gly-OMe.

three human tumor cell lines (lung carcinoma A549, pancreatic carcinoma MiaPaca2, and T-dell leukemia Jurkat) after 24 h of incubation, the NSHC−gold(I)−iodido complex displayed IC50 values of 0.4 ± 0.01 μM against A549, 6.2 ± 0.1 μM against Jurkat, and 16.6 ± 0.2 μM against MiaPaca2.255 Clearly, promising activities are obtained for these NSHC−gold(I) complexes are observed,247 and it would be interesting to study their complexes with peptides that specifically target cancer cells.256 The orthogonal chemistry of the acetylene moiety is also useful for modification of internal amino acid residues.257 This has been mentioned before, in the description of metalation of modified N-terminus (section 2.1), and similar strategies apply for an amino acid residue that contains this moiety in its side chain. In principle, any amino acid building block that possesses an alkyne functionality can be used for this.258 For example, lysine residues that have a variety of orthogonally removable protecting groups are available; the liberated side-chain amino group can be acylated with molecules that contain an acetylene and carboxylic acid, like propargyl acid or 5-hexynoic acid. Considering the application of propargylglycine, Gasser et al.154 reacted an internally positioned propargylglycine derivative W


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with Co2(CO)8, with formation of Co2(alkyne) (CO)6containing peptide 94 (Figure 9). Application of the tumor-

Scheme 19. 4-Acetylphenylalanine-Derived C,NCoordinating Organometallic Amino Acid Residue Incorporated at the N-Terminus of a β-Turn Peptide259

Figure 9. Structural formulas of Co2(alkyne)(CO)6 (94) and Tp*WII(I)(CO) (95) metalated Leu-enkephalin derivatives.154

targeting peptide octreotide that was functionalized with this OM fragment was recently described.99 Another example by Metzler-Nolte and co-workers127 used this amino acid residue to attach a Tp*WII(I)(CO) complex via η2-coordination to the alkyne 95, Figure 9). The unnatural amino acid p- or m-acetylphenylalanine is an alternative suitable precursor to prepare C,N-ligands for OM− peptide conjugates.259 Two strategies have been developed: (1) a full protection, metalation, and deprotection cycle to obtain metalated amino acids that, in principle, can be incorporated into proteins in vivo, for example, by use of Escherichia coli; or (2) a monoprotection and metalation cycle to obtain monoprotected metallo amino acids that can be used in synthesis of peptides (Scheme 19). For synthesis of the OM− amino acid conjugate via strategy 1, the amino acid 96 was simultaneously protected with a 9-borabicyclononanyl (9-BBN) group in order to avoid the otherwise favorable coordination of the amino acid moiety to the metal synthon. After the metalation reaction, the 9-BBN protecting group is removed by ethylenediamine, which concomitantly replaces the chloride counterions for the metal ion (97 and 98). Concerning the second strategy, monoprotected metalated amino acid residues were obtained that could be incorporated into peptides by standard solution-phase Boc-based peptide synthesis procedures; depending on the OM synthon, a monomeric (for Cp*IrIIICl, 99) or dimeric (for PdIICl, 100) species of the building block was obtained. Unfortunately, removal of the Nterminal Boc group with TFA was accompanied by decomposition of the metallopeptides, which limited the application of these peptides to asymmetric catalysis in aprotic solvents. Importantly, however, metallopeptides 101 and 102 remained intact during the synthesis, indicating potential applicability in medicinal or biology-centered studies.

incorporation of previously synthesized organometallic amino acid building blocks51 but only modifications on already (partly) assembled peptides. For this indirect kind of metalation, Lys, Asp, Glu, and Cys are also available, as well as modified versions of (natural) amino acid residues that allow orthogonal conjugation strategies. Lysine has one of the most-addressed side-chain groups in bioconjugation chemistry. The amino acid residue itself is available with orthogonal protecting groups on the amino groups, allowing selective deprotection of either the side-chain or backbone amino group. This opportunity has been harvested substantially for indirect introduction of OM groups in cases where direct attachment of an OM−carboxylic acid was not applicable. For example, the side-chain amino group was converted into an isocyanide by reacting with an isocyanocontaining acid; the resulting moiety was applied in a 4 + 1 labeling reaction with a 99mTc complex (as described in section 2.3 and Chart 5).260 Very often, substituted phenylalanine residues are applied; these allow for several types of conjugation strategies that can also be used to attach an OM group. In 1998, Beck and co-

3.3. Indirect Metalation of Amino Acid Side-Chain Functionalities

As mentioned earlier, the spectrum of available metalation sites in a protein can be expanded significantly by incorporation of nonproteinogenic amino acid residues. At this point, the reader should be reminded that this review does not cover the X


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workers261 showed that backbone-protected p-ethynylphenylalanine derivative 103 provided Au-containing (104) or Ptcontaining (105) OM−peptide in good yields when treated with Ph3PAuICl or trans-(Ph3P)2PtIICl2 (Scheme 20).

Scheme 21. Spontaneous Gold(I)−Azide Click Conjugation of an Arylacetylene Moietya 263

Scheme 20. Synthesis of AuI− and PtII−Phosphine− Acetylene Amino Acid Conjugates261

a

The arylacetylene moiety was installed on a 4-iodophenylalanine residue after peptide assembly on the resin was completed.

Apart from direct metalation of the acetylene moiety, OM reagents with an organic moiety that specifically reacts with the acetylene group can be applied. For example, iodophenylalanine is used in cross-coupling reactions, as it can be conveniently introduced in a peptide during SPPS. MetzlerNolte and co-workers229 successfully applied a Sonogashira cross-coupling reaction for the functionalization of Leuenkephalin with a ferrocene moiety; this method is very similar to the C-terminal, N-terminal, or side-chain variations discussed earlier. It should be noted, however, that this reaction is preferably performed under an inert atmosphere due to potential inactivation of the catalytically active Pd species.166 Alternatively, the Sonogashira reaction can be employed to introduce a TMS-protected acetylene moiety whose unique reactivity is temporarily shielded. After removal of the trimethylsilyl protecting group from resin-bound peptide 106, the arylacetylene moiety can be reacted with a “spring-loaded” gold−azido complex, resulting in organometallic−peptide conjugate 107 (Scheme 21). This typical click reaction, which does not require a metal salt as catalyst, was initially discovered by Gray and co-workers,262 who inferred that during the click reaction, an N-to-C shift of the AuI ion takes place with formation of an OM complex. Even though this organometallic complex is more stable than the initially formed N-coordination complex, disintegration of the OM bioconjugate under acidic conditions can occur during the final cleavage and purification of the peptide construct. Yields of the gold(I)−triazole-linked peptides are 16−18%. Importantly, the AuI−peptide conjugates

showed high stability in biological buffers at neutral pH over the course of 72 h, making them applicable in biomedical experiments, and some of these peptides showed potent antiproliferative effects on HT29 and MCF7 cancer cells.263 Targeting of mitochondria was shown by AAS, where a strong and selective inhibition of the enzyme thioredoxin reductase (TrxR), a regulator of cellular redox processes, was shown. This results in elevated levels of reactive oxygen species and strong effects on the respiration of mitochondria. Although p53 mutant MDA-MB231 breast cancer cells were resistant to cisplatin, proliferation of these cells was hampered by the AuI− peptide conjugates.

4. METALATION OF AROMATIC AMINO ACID RESIDUES Transition metal ions can have significant affinity for aromatic moieties. Many of them bind strongly to cyclopentadienyl (Cp) groups, but they also have affinity for benzene-like aromatic rings. Therefore, by use of the appropriate organometallic precursors, mixed-sandwich complexes of arene groups and half-sandwich organometallic synthons can be prepared.264 This was initially explored with amino acids and simple derivatives thereof (like dipeptides) but has recently been successfully applied to the metalation of a variety of biologically active peptides (Chart 7). Y


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Chart 7. Chronological Summary of Mixed-Sandwich Complexes Prepared from Aromatic Naturally Occurring Amino Acid Residuesa

a

While the formal oxidation state of the metal ions in the complexes is indicated, the compounds’ overall charge is omitted for clarity and simplicity. Z


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Scheme 22. Example of Application of Reactivity of CpRuIIMetalated Chloroarenes for Synthesis of the Vancomycin Carboxylate-Binding Pocket269−277

4.1. Arene Metalation of Amino Acids, Their Residues, and Dipeptides

All amino acid residues that contain an aromatic side-chain functionalityphenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W)have been metalated with suitable organometallic reagents. In this reaction an η6 complex is formed by π-coordination of the metal ion to the carbon atoms of the aromatic ring. Strategies of this kind of arene-selective derivatization of amino acids, simple peptides, and also of larger peptides and proteins have been reviewed by Beck and coworkers51 and Grotjahn,265 and recently by Monney and Albrecht.23 Here we include the most recent contributions to this field and show their relationship to previously reviewed strategies. Emphasis on synthesis of the complexes is given, and the structural consequences of metalation are discussed in a few selected cases. Initial metalation studies on aromatic amino acid side-chain groups were performed with a [RuII(η5-Cp)(η1-MeCN)3](PF6) complex. In 1987, Gill and co-workers266 used this reagent to metalate backbone-protected Ac-AAAr-OEt (AAAr = Phe, Tyr, or Trp) via a thermal ligand exchange reaction in 1,2dichloroethane at 40−50 °C (see 108, Chart 7, for an example). About seven years later, Sheldrick and Gleichmann267 applied [RuIII(η5-Cp*)(μ2-Cl)2]2 in MeOH and NaOMe under reflux conditions (3 h) to metalate a variety of amino acid residues: RuII(η5-Cp*)(η6-Phe), 109 in Chart 7, in 88% yield; RuII(η5-Cp*)(η6-Phe4Cl) in 38% yield (Phe4Cl = 4chlorophenylalanine); RuII(η5-Cp*)(η6-Tyr) in 90% yield; RuII(η5-Cp*)(η6-DOPA) in 90% yield (DOPA = 3,4-dihydroxyphenylalanine); RuII(η5-Cp*)(η6-Phser) in 38% yield (Phser = threo-β-phenylserine); RuII(η5-Cp*)(η6-Trp) in 44% yield; and RuII(η5-Cp*)(η6-Trp5‑OH) in 45% yield (Trp5‑OH = 5-hydroxytryptophan). Importantly, all reactions were performed under argon atmosphere by standard Schlenk techniques, and MeOH was distilled under argon before use. It was found that the two Phe complexes were stable in aqueous solutions and under exposure to air, whereas the Tyr and DOPA complexes disintegrated into their starting amino acid derivatives and an OM fragment of undefined composition. That the Cp*RuII fragment was bound to the six-membered ring of the Trp indole side chain, leading to the two stereoisomers, was inferred from chemical shifts of the four protons of that ring. Coordination to the C6 ring was concluded from the observation that the pyrrole CH group was not affected by coordination. The lower yield of the chlorinated Phe residue was attributed to the electron-withdrawing nature of the halogen and the resulting lower tendency of the phenyl ring to form η6 complexes. Interestingly, metalation of the chlorinated aromatic ring resulted in an activation of this ring toward nucleophilic attack. This was shown by Rich and Pearson and co-workers on slightly different systems, where in a mixed-sandwich complex of chlorinated phenylalanine derivatives and CpRuII species (110, Scheme 22),268 the chlorinated carbon atom became susceptible to a nucleophilic aromatic substitution (SNAr) reaction with phenol nucleophiles (Scheme 22).269−276 Those CpRuII complexes were prepared by mixing Boc-(4-Cl)PheOH, its methyl ester, or a Boc-Asn-(4-Cl)Phe-OMe dipeptide with [RuII(η5-Cp*)(η1-MeCN)3](PF6), which gave 85−100% yields.277 As result of metalation of the aromatic group, the 1H NMR signal of protons attached to the aromatic ring shifted upfield from 7.1−7.3 ppm to 5.7−5.9 ppm in the 1H NMR spectrum. After formation of the bis(aryl) ether, the CpRuII

fragment could be detached from the aromatic ring in 111 by irradiation with a 300 W sunlamp. Instead of the dimeric metalating species [RuIII(η5-Cp*)(μ2Cl)2]2, the monomeric species [RuII(η5-Cp*)(η1-MeCN)3](OTf) could also be used, as was shown by Sheldrick and coworkers278 Stoichiometric ratios of amino acid and OM reagent were mixed in THF and subjected to reflux conditions; depending on the substrate, the mixture was refluxed between 3 h and 3 days. The following phenylalanine-containing complexes were formed: amino acid RuII(η5-Cp*)(η6-Phe), 109 in Chart 7, in 37% yield; dipeptide H-[RuII(η5-Cp*)(η6Phe)][RuII(η5-Cp*)(η6-Phe)]-OH, 112 in Chart 7, in 37% yield; and cyclic dipeptide cyclo[RuII(η5-Cp*)(η6-Phe)][RuII(η5-Cp*)(η6-Phe)] in 40% yield. The cyclic diketopiperazine complex was used to study photochemical stability of the sandwich complex: irradiation of a solution of the bis-metalated dipeptide with an Hg lamp (254−380 nm) for 3 h resulted in complete removal of both OM moieties. Synthesis of the tryptophan complexes gave results similar to those of the phenylalanine complexes: amino acid RuII(η5-Cp*)(η6-Trp) in 36% yield; dipeptide H-[RuII(η5-Cp*)(η6-Trp)][RuII(η5-Cp*)(η6-Trp)]-OH in 46% yield; and cyclic dipeptide cyclo[RuII(η5Cp*)(η6-Trp)][RuII(η5-Cp*)(η6-Trp)]-OH in 33% yield. Importantly, the same paper presented the first intramolecular competition reaction between two arene amino acid side-chain groups: Trp versus Phe. By use of H-Phe-Trp-OH and H-TrpPhe-OH, selective metalation of the Trp side chain by reaction with [RuII(η5-Cp*)(μ2-Cl,μ2-Cl)2]2 was observed. It was hypothesized that the selectivity of the Cp*RuII fragment is dominated by its preference for the partially localized arene πsystems in indole over the more delocalized arene π-systems in AA


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phenyl.279 Furthermore, the applicability of the formed RuII(η5Cp*)(η6-Phe) sandwich complex in peptide synthesis was demonstrated by some straightforward peptide-synthesis manipulations: the sandwich complex of 113 remained intact during the peptide coupling reaction, as was concluded from the high yield of monometalated dipeptide 114 in the subsequent reaction (Scheme 23). By this approach, a selectively monometalated version, 115, of an otherwise symmetrical cyclic dipeptide was obtained.

metals, Cp*IrIII and Cp*RhIII. The three aromatic amino acid residues (Phe, Tyr, and Trp) could be metalated with these OM moieties by standard Schlenk techniques (Tables 1 and 2). Table 1. Metalation of Aromatic Side-Chain Groups of Amino Acid Residues with Cp*IrIII by Use of [IrIII(η5Cp*)(η1-L)3](OTf)2

Scheme 23. Direct Metalation of Phenylalanine Residue with a Cp*RuII Moietya 278

amino acid residue

L

Ac-Tyr-OEt

THF

H-Tyr-OMe

acetone

H-Tyr-OH

THF

Ac-Trp-OMe

THF

H-Trp-OH

THF

Ac-Phe-OMe

THF

H-Phe-OMe

acetone

conditionsa

product

THF, Δ, 18 h Ac-[IrIII(η5-Cp*)(η6Tyr)]-OEt TFA, 50 °C, H-[IrIII(η5-Cp*)(η6Tyr)]-OMe 24 h TFA, 50 °C, H-[IrIII(η5-Cp*)(η6Tyr)]-OH 24 h THF, 45 °C, Ac-[IrIII(η5-Cp*)(η6Trp)]-OMe 2h TFA, 65 °C, H-[IrIII(η5-Cp*)(η648 h Trp)]-OH THF, Δ, 18 h Ac-[IrIII(η5-Cp*)(η6Phe)]-OMe TFA, rt, 24 h H-[IrIII(η5-Cp*)(η6Phe)]-OMe

yield (%) 86 85 68 72 69 77 76

a

THF = tetrahydrofuran, TFA = trifluoroacetic acid, rt = room temperature, Δ = reflux.

Table 2. Metalation of Aromatic Side-Chain Groups of Amino Acid Residues with Cp*RhIII by Use of [RhIII(η5Cp*)(η1-acetone)3](OTf)2

a

Stability of the metalated mixed-sandwich complex during several standard amino acid manipulations was shown.

After these successful approaches for synthesis of CpRuII and Cp*RuII mixed-sandwich complexes of aromatic amino acid residues, Wolff and Sheldrick280 pursued similar metalation reactions with CyRuII (Cy = cymene, 4-methylisopropylbenzene). Depending on the amino acid substrate, metalation of the aromatic side-chain functionality with [RuII(η6-Cy)(η1acetone)3](OTf)2 was performed in DCM or TFA. Importantly, the selective formation of η6 complexes was realized only for (partially) protected amino acid residues. Without this protection, κ2-N,O-coordination complexes were usually formed, except when a pendant carboxylate was present in the organic ligand of the OM fragment (resulting in 116, Chart 7).281 Alternatively, when TFA was used as a solvent, this coordination was suppressed and complex 117 (Chart 7) was formed. In the presence of stoichiometric ratios of the reagents, Ac-Phe-OMe was metalated with 87% yield (DCM, reflux, 4 h), metalation of H-Phe-OMe·HOTf proceeded with 79% yield (DCM, reflux, 48 h), Ac-Phe-OH was metalated with 80% yield (TFA, 50 °C, 8 h), and metalation of H-Phe-OH afforded 83% yield (TFA, 50 °C, 12 h). Alkaline hydrolysis of the CyRuIImetalated H-Phe-OMe material resulted in removal of the CyRuII metal fragment from the amino acid residue. Cyclic dipeptide cyclo[Phe-Phe] was bis-metalated with 73% yield (DCM, reflux, 24 h). Coordination of the Phe aromatic ring to CyRuII leads to a 0.2 ppm upfield shift of the aromatic protons and a 20−30 ppm upfield shift for the coordinated carbon atoms. Similar results were claimed for Trp and Tyr. Sheldrick and co-workers282 further extended arene-metalation reactions to organometallic complexes of two group 9

amino acid residue

conditionsa

product

Ac-Tyr-OEt

THF, rt, 2 days

Ac-Tyr-OH

THF, Δ, 16 h

H-Tyr-OMe Ac-Trp-OMe

TFA, 50 °C, 24 h THF, rt, 48 h

Ac-Phe-OMe

THF, Δ, 20 h

Ac-[RhIII(η5-Cp*)(η6-Tyr)]OEt H-[RhIII(η5-Cp*)(η6-Tyr)]OMe H-[RhIII(η5-Cp*)(η6-Tyr)]OH Ac-[RhIII(η5-Cp*)(η6-Trp)]OMe Ac-[RhIII(η5-Cp*)(η6-Phe)]OMe

yield (%) 84 78 81 68 72

a

THF = tetrahydrofuran, TFA = trifluoroacetic acid, rt = room temperature, Δ = reflux.

These tables showcase the development of metalation reactions from strictly organometallic conditions (i.e., strongly acidic or aprotic dry solvents, reflux conditions, long reaction times) to biocompatible conditions (i.e., aqueous solutions, neutral pH and room temperature, short reaction times). Formation of mixed-sandwich complexes was witnessed by the 20−40 ppm upfield shift of signals corresponding to carbon atoms of the aromatic side chains of amino acid residues upon η 6 coordination of the metal fragment. Interestingly, chemical shifts of methyl protons on the Cp*IrIII fragment depended on the type of side chain to which they were coordinated: 1.94− 2.08 ppm for the indole group of Trp, 2.25−2.28 ppm for the phenol ring of Tyr, and 2.40−2.44 ppm for the benzene ring of Phe. A similar but smaller difference was seen for Cp*RhIIImetalated residues: 1.96 ppm for indole (Trp) and 2.09−2.20 ppm for phenol (Tyr) (vide infra). Importantly, by using these two OM fragments, the authors were able to assess the influence of relativistic effects and hard−soft acid−base preferences on the stability of mixed-sandwich complexes. It was shown that these two Cp*MIII complexes stabilize the ketone form of the phenol group of Tyr, resulting in AB


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Table 3. Overview of Arene−M−Amino Acid Distancesa ligand−metal distance (Å)

a

complex

C−M

ring−M

RuII(η5-Cp)(η6-Tyr) RuII(η5-Cp*)(η6-Phe) RuII(η6-Cy)(η6-Phe) RuII(η6-Cy)(η6-HOTrp)

2.164−2.193 2.167−2.195 2.198−2.222 2.172−2.262

1.812 1.693 1.718 1.729

RhIII(η5-Cp*)(η6-Tyr)

2.158−2.193

1.776

IrIII(η5-Cp*)(η6-Tyr) IrIII(η5-Cp*)(η6-Phe)

2.145−2.210 2.155−2.215

1.758 1.781

metal−amino acid distance (Å) M−C M = Ru 2.198−2.214 (Ru−Cγ/Cδ/Cε), 2.268 (Ru−Cζ) 2.172−2.219 2.203−2.237 2.219−2.343 M = Rh 2.242−2.260 (Rh−Cγ/Cδ/Cε), 2.338 (Rh−Cζ) M = Ir 2.208−2.276 (Ir−Cγ/Cδ/Cε), 2.367 (Ir−Cζ) 2.209−2.276

M−ring

ref

1.703 1.813 1.730 1.753

285 278 280 283

1.809

282

1.817 1.744

282 282

Distances were extracted from the reported X-ray crystal structures of mixed-sandwich complexes.

metalation of the aromatic side chains of Phe (see 120, Chart 7), Trp, and Tyr was achieved by use of an RuII(η5-Cp*) complex by heating the samples for 10−30 min at 130 °C in a microwave. Also, a method was developed in which the metalation reaction could be performed at room temperature, albeit with much prolonged reaction times of 8−20 h. Although not yet performed on peptides, the use of water as a solvent (and, if needed, some THF or MeCN as cosolvent) and the ability to perform the metalation reaction at room temperature in principle allows this method to be applied on peptides.

acidification of the 4-hydroxyl group. A pronounced doublebond character was observed for Cp*IrIII (dC−O = 1.317 Å) and Cp*RhIII (dC−O = 1.312 Å); double-bond character of the C4OH group was also observed for complexes of tyrosine model compounds with CyRuII283 but not with Cp*RuII (dC−O = 1.333 Å) (for comparison, dC−O = 1.364 ± 0.016 Å in phenols and dCO = 1.222 ± 0.013 Å in benzoquinones;284 see insert in Chart 7). Whereas the IrIII(η5-Cp*)(η6-Tyr) complexes (118, Chart 7) were stable in aqueous (D2O) solutions for 14 days, this was not the case for highly similar RhIII(η5-Cp*)(η6-Tyr) complexes (119, Chart 7), which showed 50% decay over 24 h at room temperature. Surprisingly, by changing the counterion of the complex from PF6− to BF4−, a much more stable RhIII(η5-Cp*)(η6-Tyr) complex was obtained that degraded by only 20% during 7 days. Clearly, very subtle factors are at play that determine the stability of mixed-sandwich complexes of Cp*RhIII and Tyr. This detailed study has revealed several features, for example, the metal−ligand distances (Table 3) that are of importance for the application of these complexes in biological systems (vide infra). In order to appreciate the distances that are at play in these mixed-sandwich complexes, we refer the reader back to Figure 3 (in section 2.1.2), where the M−ligand distances for group 8 metallocenes (Fc, Rc, and Oc) are given. As can be inferred from this comparison, the M−ring distances of all but one of the here-described mixedsandwich complexes lie within the range bordered by Fc (dFe−ring = 1.661 Å) and Rc (dRu−ring = 1.813 Å); the exception is the Ir−Tyr ring distance for the IrIII(η5-Cp*)(η6-Tyr) complex. Even though a variety of conditions were established that allow high-yielding metalation of aromatic amino acid residues, the chemoselectivity of the reactions was hardly addressed. For this assessment, metalation of dipeptides of type H-AA1-AA2OH (where AA1 ≠ AA2 = Tyr, Phe, Trp) was studied.286 With this study, the intrinsic preference of the organometallic synthon for a specific aromatic residue could be determined. Due to the instability of Cp*RhIII and Cp*RuII complexes, these studies focused on the formation of CyRuII and Cp*IrIII complexes. For both reactions, a pronounced preference of Trp > Tyr > Phe was observed, although this was less prominent for Cp*IrIII than for CyRuII. Using the tetrameric compound [RuII(η5-Cp*)(μ3-Cl)]4, Fairchild and Holman287 introduced an important improvement in the synthetic methodology: instead of applying rigorous Schlenck techniques throughout, degassed aqueous solutions were now shown to be compatible with the metalation reactions. It was shown that near-quantitative

4.2. Arene Metalation on Biologically Active Peptides

The previously mentioned studies on amino acids, their residues, and dipeptides paved the way for preparation of mixed-sandwich complexes of peptidic aromatic amino acid side chains. This then would allow scientists to study the consequences of that metalation on physico- and biochemical properties of the mixed-sandwich complexes. Importantly, the stability of the complexes under a variety of conditions has been studied, which resulted in a better understanding of the underlying factors that caused the observed behavior. This information will be crucial for the ultimate biological applications of similar metalated peptides. However, before such applications could have been studied, which was already possible for more straightforward classically conjugated OM− peptides constructs as discussed in the first part of this review, the metalation strategies had to make one last step in their development and be made compatible with biologically interesting peptides that contain a plethora of functional groups. Ideally, a reaction would involve mixing of the two components, the peptide and the OM synthon, which would spontaneously react under biologically benign conditions (i.e. in aqueous solvents, at neutral pH, and at room temperature). Also, the reaction should be fast and chemoselective, thus limiting the need for tedious purifications. The first reported metalation of an aromatic side chain of an oligopeptide was performed by Grotjahn et al. in 1998.288,289 By use of a η5-Cp′{(CH2)2NH2}RuII(η1-MeCN)2 complex, the only arene group of secretin was metalated (121, Figure 10, also in Chart 7). This synthetic mini-protein contains 27 amino acid residues, of which the N-terminal histidine, the free Nterminus, and the three carboxylate-functionalized residues were potent competitor sites for the metalation reaction on the targeted Phe residue; in particular, the N-terminal histidine residue was a prominent competitor as it offers an N,Nbidentate ligand that usually forms very stable five-membered coordination complexes. Nevertheless, 70−100% conversion AC


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Figure 11. Metalation of aromatic side-chain functionalities of amino acid residues of soluble peptides according to a light-induced areneexchange reaction.286,290

also be performed in the presence of 0.5% HBF4 in water, which helps to suppress the reactivity of basic amino acid residues, especially histidine. After these initial experiments, the method was applied to the metalation of two small biologically active peptides, angiotensin I and II (Figure 11 and Chart 7). By use of 4 equiv of metalating agent, global metalation of all aromatic residues was observed after 8 h. The progress of this metalation reaction was visualized by 1H NMR analysis of the reaction mixture. The chemical shift of Cp ring protons in the unreacted CpRuII−naphthalene complex are found around 4.9 ppm, while when bound to Tyr or Phe, the protons are shifted downfield to about 5.1 and 5.3 ppm, respectively. Concomitantly, the Tyr protons shift upfield from 6.6 and 6.9 ppm to 5.5 and 5.7 ppm, and the Phe protons shift from 7.2 to 6.0 ppm. (These values are indicative only, as the exact values are not reported in the paper.285) Metalation of these two aromatic residues shifts their protons into a poorly occupied region of the NMR spectrum, between 5.0 and 6.5 ppm, making metalation a valuable tool to study the behavior of the metalated peptide in a biological environment. Attempts to metalate lysozyme, a 129 amino acid, 14.3 kDa protein that contains three Tyr, four Phe, and five Trp residues, by this method were not successful, which was attributed to the fact that these aromatic residues were not accessible. Although it is a significant improvement over the other two methods, for which degassed solvents and an inert atmosphere were required, the extensive reaction times and poor chemoselectivity of this method may limit its general applicability. Also, since the peptide was most likely obtained as a TFA salt of the basic groups, that is, histidinyl imidazole ring and N-terminal secondary nitrogen atom, the reactivity of these groups was automatically suppressed from participating in the metalation reaction; under neutral conditions, it is to be expected that the imidazole ring would coordinate to the CpRuII fragment, resulting in the kinetically preferred N−Ru coordination complex. The sole aromatic ring of a larger bioactive peptide, the tryptophan residue of melittin, could also be metalated by this procedure. This powerful stimulator of phospholipase A2 is the active component of bee venom. The resulting CpRuII− melittin conjugate 124 (Chart 7) showed a dramatic reduction

Figure 10. Structure of the metalated peptide secretin. The organometallic complex that was used to perform this metalation reaction is shown in the box, and competing coordination sites are highlighted by the bold traced bonds and bold atoms.288

was inferred from the 1H NMR spectrum, and the metalated mini-protein could be analyzed by techniques commonly used in peptide and protein chemistry (i.e., HPLC, MALDI-MS, ESIMS, and Edman degradation). The yields that accompanied these transformations depended on the type of degassed solvent: in CD3OD, conversion was complete in 5 h but needed heating at 60 °C, whereas in D2O, complete conversion was observed after 8 h but now at room temperature. This last finding suggests possible applications in the metalation of proteins. Importantly, formation of η1-S- and η1-N-coordination complexes was shown to be kinetically favored, but over time the organometallic synthon relocated to the thermodynamically favored arene complex. Finally, removal of the organometallic synthon from the mixed-sandwich complex could be effected by lengthy irradiation (32 h) with a 450 W Hg lamp. Along similar lines, Kudinov and co-workers285,290 recently performed a metalation of aromatic amino acids and small peptides using a CpRuII synthon (122 in Chart 7 and 123 in Figure 11). This synthon could be generated in situ from the [RuII(η5-Cp)(η6-C10H8)]+ complex by irradiation of the mixture with visible light. Importantly, this method was applicable in water alone and occurred at room temperature. Interestingly, performing this reaction with a 1:1:1 mixture of Phe/Tyr/Trp resulted in a 0.1:0.1:0.8 ratio of the metalated products, which indicates higher reactivity of the indole ring when compared to the phenol and benzene ring; this was also seen for CpRuII (vide supra, section 4.1). These reactions can AD


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of hemolytic activity: 50% hemolysis was caused by 28.4 μM CpRuII−melittin, whereas only 2.5 μM native peptide was needed to elicit the same effect. Similarly, the metalated peptide was 2-fold less active against cancer cell lines SKOV3 and MDA-MB-231 than the native peptide. These biological results suggest that metalation hampers the interaction of the bioactive peptide with the biological cell membrane. In 2012, we described a procedure in which tyrosine residues were reacted with Cp*RhIII fragment in a chemoselective manner.296 Tyrosine residues were targeted due to their importance in many cell-signaling pathways,291 aberrations of which have been directly linked to severe diseases like cancer.292 We were particularly interested in establishing conditions that would be friendly for many biomolecules: near neutral pH, room temperature, and avoiding organic solvents. Also, fast reaction times were desired in order to avoid unwanted side reactions. By use of a Cp*RhIII(H2O)3 complex, which was conveniently prepared according to a literature procedure,293 tyrosine residues in a variety of peptides were selectively metalated under biologically friendly conditions. Initial studies on Leu-enkephalin and neurotensin-targeting peptide294 showed that the reaction proceeded sufficiently fast just below neutral pH. Interestingly, the pH of the solution dropped, indicating release of a proton as a result of the metalation reaction.295 Metalation of the Phe residue was not observed, and we also noted that the reaction rate of an Nterminal tyrosine residue in which the amino group was not blocked was significantly lower than for an acetylated Nterminus. This was attributed to the proximate N-terminal amino group, which is mostly protonated at the pH value where the metalation reaction was performed. Despite this lower reactivity, the Cp*RhIII-metalated peptide 125 (Chart 7) was obtained in 65% isolated yield, allowing several biological studies. Binding affinity of the metalated peptide 125 to two of the natural receptors for enkephalins, the δ- and μ-opiate receptors (DOR and MOR, respectively), were determined. Significant reduction in the affinity constant was observed: for DOR, the reduction in Kd was 3.5 fold (15.6 nM for Cp*RhIIImetalated peptide vs 4.4 nM for native peptide), while for MOR, this reduction was 6.5 fold (93.3 nM for Cp*RhIIImetalated peptide vs 14.3 nM for normally applied nonmetalated reference peptide [DTPA,DPhe1]-octreotide).296 Nevertheless, the natural tendency of Leu-enkephalin to bind preferably to DOR was maintained, although this preference was slightly lowered as a result of metalation. Also, the OM− peptide conjugate acted as an agonist, similar to Leuenkephalin. We probed the effect of addition of the Cp*RhIII fragment to the tyrosine side-chain functionality on its three-dimensional (3D) features. The presence of this hydrophobic bulky group as a side chain of the peptide backbone reduced the number of conformations of the entire peptide, allowing determination of the structure of [RhIII(η5-Cp*)(η6-Tyr1)]Leu-enkephalin (125, Chart 7) by use of contact information derived from its twodimensional (2D) 1H ROESY spectrum (ROESY = rotatingframe nuclear Overhauser effect correlation spectroscopy).297 By use of the X-ray crystal structures of DOR and MOR receptors, reported by Kobilka and co-workers,298,299 the binding mechanism of the metalated peptide to the receptor was assessed by docking simulations. This study showed that the Cp*RhIII-metalated N-terminal tyrosine pharmacophore was able to enter the binding pocket of the opiate receptors

without distorting the fold of the protein to a significant extent.300 Compatibility of the metalation reaction on a more complex peptide was studied for [Tyr3]octreotide (126, Scheme 24). Scheme 24. Chemoselective Metalation of [Tyr3]Octreotide with [RhIII(η5-Cp*)(η1-H2O)3]X2a 296

a

X = undefined counterion.

Also with this peptide, metalation of the tyrosine residue proceeded smoothly and rapidly under formation of [RhIII(η5Cp*)(η6-Tyr3)]octreotide 127. Following the course of the reaction by HPLC revealed the formation of three side products; ESI-MS analysis revealed two products with the same m/z value, indicating the presence of conformational isomers, and a minor product that also contained a metalated D Trp residue (i.e., 128 in Scheme 24). Purification of the formed products by preparative HPLC provided 4% and 27% of the monometalated peptide and 3% dimetalated peptide. Again, using 2D 1H ROESY NMR techniques, we were able to determine the structure of most members of the molecular population. Comparison of the structure of metalated peptide AE


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127 with that of [Tyr3]octreotide 126 revealed a pronounced effect of metalation on the positioning of side chains (Figure 12), even though CD spectroscopy confirmed that the shape of

5. MISCELLANEOUS AND COMBINED METALATION STRATEGIES Apart from the more classical peptide chemistry based approaches that apply an organometallic building block and a peptide, multicomponent reactions (MCRs) allow the formation of relatively small organometallic−peptide conjugates in a single reaction step. In one approach, a kind of Ugi fourcomponent reaction (4CR) involving an amino acid, an aldehyde, an isocyano moiety, and a cyanide-stabilized tungsten complex was mixed to form a tungsten pentacarbonyl NHC complex 129 (Scheme 25A).301 A more traditional Ugi 4CR Scheme 25. Multicomponent Reactions for One-Step Synthesis of OM−Peptide Conjugates301,302

was performed by applying a chromium tricarbonyl complex (130) of benzaldehyde, an amine, a carboxylic acid, and an isonitrile (Scheme 25B). Mixing these components in warm MeOH afforded OM−peptide conjugate 131 in up to 52% isolated yields.302 As the number of available methods that are sufficiently orthogonal in their applicability increased, multiply metalated peptides have emerged. A combination of several routes that were described above was recently shown by Metzler-Nolte and co-workers.303 On a peptide nucleic acid (PNA) monomer, the N-terminus was metalated with a CpMnI(CO)3 moiety, the Cterminus was coupled to a ReI(CO)3 coordination complex via Pd-catalyzed Sonogashira cross-coupling reaction, and the sidechain acetylene moiety was coupled to azidomethylferrocene by CuAAC chemistry (132, Figure 13).304 Interestingly, the obtained compounds were potent antibacterial agents, which disordered the membrane architecture, resulting in disturbance of respiration and cell wall biosynthesis; the ferrocenecontaining derivative induced oxidative stress.305 Similar

Figure 12. Effect of metalation of the tyrosine residue in [Tyr3]octreotide with Cp*RhIII on the structure of the peptide: (top) [Tyr3]octreotide 126 and (bottom) [RhIII(η5-Cp*)(η6-Tyr3)]octreotide 127, with RhIII ion depicted as a pink sphere. Reprinted with permission from ref 296. Copyright 2012 American Chemical Society.

the backbone was not significantly affected. This was further supported by the observation that both binding affinity and in vitro activity of the metalated and nonmetalated peptides were less different than was the case for Leu-enkephalin. This is most likely because, in the case of [RhIII(η5-Cp*)(η6-Tyr3)]octreotide, the pharmacophore itself is not metalated. AF


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Scheme 27. RuII/Co0 Bis-Metalated Neurotensin Derivative 135307

Figure 13. Antibacterial trimetallic PNA unit 132.303

strategies were shown on larger peptide nucleic acids and peptides. For example, the N-terminus was metalated via an amide-bond-forming reaction, and an internal acetylene moiety was coupled with azidoferrocene by CuAAC (133, Scheme 26).306 Around the same time, the same group published a bisScheme 26. Generic Bis-Metalated PNA Conjugate 133306

shira protocol, which would allow conjugation of the OM unit via an aryliodide group. For the introduction of OM groups on the side chains of amino acid residues, a plethora of options is available; the obvious candidates are Lys or Asp/Glu in combination with metalation strategies that are common for N-terminal amino or C-terminal carboxylic acid groups, respectively. On top of that, histidine residues can be converted into N-heterocyclic carbene moieties, or unnatural amino acid residues can be introduced that allow orthogonal metalation chemistry. The affinity of late transition metal ions for aromatic rings has opened up a variety of approaches in which naturally occurring aromatic amino acid residues can be metalated in a chemoselective manner and under biologically compatible conditions. We expect that this last approach will even be applicable on proteins in due course. The application of creative chemistry, and particularly of innovative methods that make use of metal-specific reactivity, has made metal−peptide bioconjugates with unique properties available. In this review, we have highlighted some applications of such bioconjugates in studies of cell uptake and intracellular localization of peptide conjugates, in studies into the mode of action of metal-based drugs and peptides, and as biosensors and probes for structural biology.

metalated peptide that contained a ruthenocene moiety attached to the N-terminus and an N-terminal propargylglycine (134) that was converted into its dicobalt hexacarbonyl complex (135, Scheme 27).307

6. CONCLUSIONS The field of organometallic−peptide conjugates has matured over the past two decades from approaches in which amino groups and carboxylic acid functionalities of organometallic moieties and peptides were coupled by use of standard “aminoacid-like” coupling protocols to metal-specific chemoselective metalation reactions that can be performed under biologically friendly conditions. At this moment, a sufficient number of Nterminal modifications is now available: in case straightforward amide-bond-forming reactions are not tolerated or available, other routes like copper-catalyzed alkyne−azide cycloaddition reactions (CuAAC), Sonogashira cross-coupling reactions, or the introduction of C-coordinating groups like isocyanide can be considered. For C-terminal modifications, CuAAC introduction of the OM moiety on the C-terminus of carboxamideterminated peptides is very convenient, although it demands the presence of an azido group on the organometallic fragment. The most recent development uncovered a cleavage-Sonoga-

7. OUTLOOK Although new synthetic approaches for the metalation of peptides are still likely to emerge, it is expected, now that many metalation strategies have become established, that the research focus will shift even more toward the behavior of OM−peptides in biological environments. For example, even though the attachment of a ferrocenoyl moiety on the lysine side-chain amino group of short Arg-Trp-based antimicrobial peptides seemed to function merely as a lipophilic unit,110 replacing the iron(II) ion with the similar ruthenium(II) ion not only enhanced the activity of similar antibacterial peptides109 but also helped to confirm the mode of action of such short AG


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antibacterial peptides.45 The significant differences in activity of RcC(O)−AMPs versus their FcC(O) counterparts prove that differences in size, redox potential, and H-bond acceptor capabilities of the metal ion118 can have a pronounced effect on the biological activity of the conjugates. A recent study showed that an L-to-D substitution scan of all five proteinogenic amino acid residues in RcC(O)−WRWRW-NH2 revealed nonhemolytic OM−AMPs whose activities match, or even surpass, that of vancomycin, one of the most potent antibiotics that currently forms one of the last lines of defense against pathogenic bacteria.122 Chemically, strategies that allow the chemoselective metalation of specific residues in peptides under biologically friendly conditions are expected to emerge. This would open up the way to perform selective conjugation chemistry between organometallic fragments and peptides or proteins inside a living cell or on tissue. Applications of such reactions would be marking and identifying particular cells, monitoring metabolic pathways, or staining tissue samples for diagnostic purposes. At present, most studies to explore biological applications of metal−peptide conjugates are indeed of a fundamental nature. The authors merely seek to explore and extend the boundaries of the available chemical space. While this is a hallmark of a young, emerging field, there is clearly promise for translation into clinically meaningful applications in the field of targeted therapeutics. A major drawback of the best-established metalbased drug, cisplatin, is its poor selectivity for cancer cells, causing severe side effects and limiting the maximum dose. This evident clinical limitation has indeed led to the common perception that all metal-based drugs are “dirty” and nonselective in that sense. On the other hand, the concept of targeted therapy is well-established in medicinal chemistry and chemical biology. With the recent development of metal− peptide conjugates of selective, tumor-targeting peptides, the perception of metal-based drugs could be changed from within the logic of targeted therapy. Some first animal and toxicology studies are currently performed in academic laboratories, but it is probably too early to expect clinical trials within the next few years. However, we would predict that this scene is likely to change when promising data from animal testing lead to one or two candidates being pushed forward to translational development.

as an assistant professor at Wageningen University, in the Laboratory of Organic Chemistry, where he develops novel approaches for siteselective protein modifications and novel antibacterial agents. Nils Metzler-Nolte obtained his Ph.D. from LMU Munich (Germany) in 1994, did a postdoc with Professor M. L. H. Green at Oxford (U.K.), and started his independent research on bioorganometallic chemistry at the Max-Planck-Institut für Strahlenchemie (nowadays MPI for Chemical Energy Conversion) in Mülheim, Germany. He was appointed associate professor for pharmaceutical and bioinorganic chemistry at the University of Heidelberg in 2000 and full professor of bioinorganic chemistry at the Ruhr University Bochum in 2006. He served as dean of the university-wide graduate school from 2009−2012 and was vice president for early career researchers and international affairs of his university between 2010−2012. Nils was speaker of the DFG-funded research unit “Biological Function of Organometallic Compounds” and Council Member of the Society of Biological Inorganic Chemistry. His work was recognized by several fellowships and awards, the most recent being the Julius von Haast award of the Royal Society of New Zealand. He was Chair of the Gordon Research Conference “Metals in Medicine” in 2016. Nils serves on the international advisory board of several journals and is an associate editor for Dalton Transactions. With research interests in medicinal organometallic chemistry and functional metal bioconjugates, the group is running a full program from inorganic synthesis to cell biology.

DEDICATION This review is dedicated to William S. Sheldrick, an inspirational pioneer in bioorganometallic chemistry, who passed away on January 16, 2015. ABBREVIATIONS AAS atomic absorption spectroscopy Aβ β-amyloid AMPs antimicrobial peptides BAL backbone amide linker 9-BBN 9-borabicyclononanyl Boc tert-butyloxycarbonyl BOP benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate Cbz carboxybenzyl Cc+ cobaltocinium, Cp2CoIII CD circular dichroism cod cyclooctene CORM carbon monoxide releasing molecule Cp cyclopentadienyl Cp′ monosubstituted cyclopentadienyl Cp* pentamethylcyclopentadienyl CPP cell-penetrating peptide CuAAC copper(I)-catalyzed acetylene−azide cycloaddition Cy p-cymene DCC dicyclohexylcarbodiimide DCM dichloromethane DCU N,N′-dicyclohexylurea DhbtOH 3-hydroxy-1,2,3-benzotriazin-4(3H)-one DIC diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP 4-dimethylaminopyridine DMB 1,3-dimethyoxybenzene dmtc dimethyl dithiocarbamate DMF N,N-dimethylformamide

AUTHOR INFORMATION Corresponding Authors

*(B.A.) E-mail [email protected]. *(N.M.-N.) E-mail [email protected]. Notes

The authors declare no competing financial interest. Biographies Bauke Albada obtained his Ph.D. from Utrecht University (The Netherlands) in 2009, under the supervision of Professor Dr. Rob M. J. Liskamp. During his postdoctoral studies in the group of Professor Dr. Nils Metzler-Nolte in Bochum (Ruhr University Bochum, Germany), he studied novel antibacterial organometallic peptides and developed several novel methods for conjugation of bioactive peptides and organometallic moieties. His most recent stay in the laboratory of Professor Dr. Itamar Willner in Jerusalem (The Hebrew University of Jerusalem, Israel) were focused on the development and rational design of hemin-containing catalytic DNA systems that displayed enzymelike saturation kinetic behavior. Since 2016, he works AH


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δ-opiate receptor 1-ethyl-3-(3-dimethylaminopropyl)EDCI carbodiimide ESI-MS electrospray ionization mass spectrometry Fc ferrocene, Cp2FeII Fmoc 9-fluorenylmethoxycarbonyl HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HFA 1′-hexafluoropropan-2-ol HOBt N-hydroxybenzotriazole HPLC high-pressure liquid chromatography IUPAC International Union of Pure and Applied Chemistry ivDdE 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)isovaleryl M metal element MALDI-TOF matrix-assisted laser desorption ionization timeof-flight Mc metallocene, Cp2MII (M = Fe, Ru, Os, Co, or Mn) mgc minimal gelation concentration MIC minimal inhibitory concentration MOR μ-opiate receptor MRSA methicillin-resistant Staphylococcus aureus NEM N-ethylmorpholine NMM N-methylmorpholine NLS nuclear localization sequence NHC N-heterocyclic carbene NHS N-hydroxysuccinimide (also OSu) Oc osmocene, Cp2OsII OM organometallic OM−AMPs organometallic antimicrobial peptides OPfp fluorophenyl PG protecting group PNA peptide nucleic acid PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate Rc ruthenocene, Cp2RuII ROESY rotating-frame nuclear Overhauser effect correlation spectroscopy RT reverse transcriptase SAM-linker silyl-based alkyne modifying linker STP 4-sulfotetrafluorophenyl synAMPs synthetic antimicrobial peptides SOD superoxide dismutase SPPS solid-phase peptide synthesis TAT transactivator of transcription of human immunodeficiency virus TBTU N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl) uranium tetrafluoroborate tBu tert-butyl TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TIS triisopropylsilane Trt trityl, triphenylmethane

E, Glu F, Phe G, Gly H, His I, Ile K, Lys L, Leu M, Met N, Asn P, Pro Q, Gln R, Arg S, Ser T, Thr V, Val W, Trp Y, Tyr

DOR

glutamic acid phenylalanine glycine histidine isoleucine lysine leucine methionine asparagine proline glutamine arginine serine threonine valine tryptophan tyrosine

Nonproteinogenic Amino Acids

Aph DOPA Iph Orn Pgl Tal

4-acetylphenylalanine 3,4-dihydroxyphenylalanine 4-iodophenylalanine ornithine propargylglycine thiazolylalanine

Human Cancer Cell Lines

H1299 HeLa HepG2 HT29 Jurkat MCF7 MDA-MB-231 MiaPaca2 Nalm-6 PT45 SKOV3

non-small-cell lung carcinoma cervical cancer hepatocellular carcinoma colorectal adenocarcinoma T lymphocyte breast adenocarcinoma breast adenocarcinoma pancreatic adenocarcinoma B-cell precursor leukemia pancreatic carcinoma ovarian carcinoma

REFERENCES (1) See, for example, Biological Inorganic Chemistry: Structure and Reactivity, 1st ed.; Bertini, I., Gray, H. B., Stiefel, E. I., Valentine, J. S., Eds.; University Science Books: Sausalito, CA, 2007. (2) Inorganic Chemical Biology: Principles, Techniques and Applications, 1st ed.; Gasser, G., Ed.; Wiley−VCH: Weinheim, Germany, 2014; DOI: 10.1002/9781118682975. (3) Shriver, D. F.; Atkins, P. W. Inorganic Chemistry, 3rd ed.; Oxford University Press: Oxford, U.K., 1999. (4) Concepts and Models in Bioinorganic Chemistry, 1st ed.; Kraatz, H.B., Metzler-Nolte, N., Eds.; Wiley−VCH: Weinheim, Germany, 2006. (5) Hodgkin, D. C.; Pickworth, J.; Robertson, J. H.; Trueblood, K. N.; Prosen, R. J.; White, J. G. Structure of Vitamin B12: The Crystal Structure of the Hexacarboxylic Acid Derived From B12 and the Molecular Structure of the Vitamin. Nature 1955, 176, 325−328. (6) Brink, C.; Hodgkin, D. C.; Lindsey, J.; Pickworth, J.; Robertson, J. H.; White, J. G. Structure of Vitamin B12: X-ray Crystallographic Evidence on the Structure of Vitamin B12. Nature 1954, 174, 1169− 1171. (7) Bioinorganic Medicinal Chemistry, 1st ed.; Alessio, E., Ed.; Wiley− VCH: Weinheim, Germany, 2011; DOI: 10.1002/9783527633104. (8) Bioorganometallics: Biomolecules, Labeling, Medicine, 1st ed.; Jaouen, G., Ed.; Wiley−VCH: Weinheim, Germany, 2006; DOI: 10.1002/3527607692. (9) Fish, R. H. A Bioorganometallic Chemistry Perspective: Organometallic Chemistry at the Interface with Biology. J. Organomet. Chem. 2015, 782, 3−16.

ProteinogenicAmino Acids

A, Ala alanine C, Cys cysteine D, Asp aspartic acid AI


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