A series of structurally related mono- and bis-NHC–iridium(I) (NHC: N-heterocyclic carbene) complexes have been investigated for their suitability as potential anticancer drugs. Their spectral behaviour in aqueous buffers under physiological-like conditions and their cytotoxicity against the cancer cell lines MCF-7 and HT-29 are reported. Notably, almost all complexes exhibit significant cytotoxic effects towards both cancer cell lines. In general, the cationic bis-carbene complexes show higher stability and greater anticancer activity than their neutral mono-carbene analogues with IC₅₀ values in the high nanomolar range. Furthermore, to gain initial mechanistic insight, the interactions of these iridium(I)–NHC complexes with two model proteins, namely lysozyme and cytochrome c, were explored by HR-ESI-MS analyses. The different protein metalation patterns of the complexes can be roughly classified into two distinct groups. Those interactions give us a first idea about the possible mechanism of action of this class of compounds. Overall, our findings show that iridium(I)–NHC complexes represent very interesting candidates for further development as new metal-based anticancer drugs.

We report the synthesis of metallocene compounds Cp₂M with two different electron-withdrawing substituents on both cyclopentadienyl rings (hexafluoroacetone (HFA) and chlorobenzoyl (1–5); HFA and COOH (6 and 7), M=Fe or Ru). The COOH-containing derivatives were used to synthesize peptide bioconjugates with enkephalin (8 and 9) and neurotensin (10 and 11) as well as fluorescein-labeled neurotensin (12). All the molecules were fully characterized, including X-ray structures for 6 and 7. The physicochemical properties (lipophilicity and electrochemistry) and cytotoxicity on MCF-7, HT-29, and PT-45 cancer cells were evaluated for selected compounds. Electrochemical investigation by cyclic voltammetry revealed that all bis-substituted metallocenes are up to 300 mV harder to oxidize compared to the monosubstituted 2-ferrocenylhexafluoropropan-2-ol (FcHFA: Δ=214 mV; disubstituted derivatives: up to Δ=512 mV; both vs. FcH^0/+). For the bis-substituted compounds, log P determinations by RP-HPLC showed increased lipophilicity in comparison to the monosubstituted FcHFA and RcHFA. Cellular uptake was investigated by fluorescence microcopy, and this revealed endosomal entrapment for 12.

Peptide dendrimers are a class of molecules of high interest in the search for new antibiotics. We used microwave-assisted, copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC; “click” chemistry) for the simple and versatile synthesis of a new class of multivalent antimicrobial peptides (AMPs) containing solely arginine and tryptophan residues. To investigate the influence of multivalency on antibacterial activity, short solid-phase- synthesized azide-modified Arg-Trp-containing peptides were “clicked” to three different alkyne-modified benzene scaffolds to access scaffolds with one, two, or three peptides. The antibacterial activity of 15 new AMPs was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial strains, including a multidrug-resistant Staphylococcus aureus (MRSA) strain. With ultrashort (2–3 residues) peptides, a clear synergistic effect of the trivalent display was observed, whereas this effect was not apparent with longer peptides. The best candidates showed activities in the low-micromolar range against Gram-positive MRSA. Surprisingly, the best activity against Gram-negative Acinetobacter baumannii was observed with an ultrashort dipeptide on the trivalent scaffold (MIC: 7.5 μM). The hemolytic activity was explored for the three most active peptides. At concentrations ten times the MIC values, <1 % hemolysis of red blood cells was observed.

Thiazolium salts 2a–b derived from a dipeptide Boc-Gly-(Thz-Ala)-OMe (1b) containing the nonproteinogenic amino acid L-thiazolylalanine (Thz-Ala) can be used to generate the corresponding N,S-heterocyclic carbene (NSHC) gold(I) (3a–b) and silver(I) (4) complexes. Reaction of the NSHC-gold(I) iodide 3b with Boc-Cys-Gly-OMe gives access to the peptide bioconjugate 5, which contains a NSHC-Au-(S-Cys) unit. Compounds 3–5 constitute the first coin metal NSHC-peptide complexes. All new compounds were comprehensively characterized by ¹H, ¹³C and 2D-NMR spectroscopy, IR spectroscopy, and mass spectrometry. Their cytotoxicity was studied in vitro against three different tumor cell lines (A549, Jurkat T and MiaPaca2) and IC₅₀ values in the low micromolar range (< 25 μM), and as low as 0.4 μM in the best case were observed. All new Au complexes show good stability and promising properties and, as a result, this novel type of gold(I) carbene complex opens possibilities for the design of new metal-based drugs with promising antitumor characteristics.

We report the synthesis of trifluoromethylated metallocenes (M=Fe, Ru) and related metal-free compounds for comparison of their biological properties with the aim to establish structure–activity relationships toward the anti-proliferative activity of this compound class. All new compounds were comprehensively characterized by NMR spectroscopy (¹H, ¹³C, ¹⁹F), mass spectrometry, IR spectroscopy, and elemental analysis. A single-crystal X-ray structure was obtained on the Ru derivative, 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3). The cytotoxicity of all compounds was tested on MCF-7, HT-29, and PT-45 cells, and IC₅₀ values as low as 12 μM were observed. Both the metallocene moiety and the hydroxy function are crucial for cytotoxicity. In addition, the activity decreased sharply even if only one trifluoromethyl group was replaced with a methyl group. Electrochemical investigations by cyclic voltammetry revealed that all CF₃-containing compounds are harder to oxidize than the unsubstituted metallocenes. Moreover, log P determination by RP-HPLC showed the fluorinated derivatives to have higher lipophilicity, with log P values up to 4.6. At the same time, the generation of reactive oxygen species (ROS) in Jurkat cells by these compounds was investigated by flow cytometry. Strong ROS production was shown exclusively for the bis-CF₃ derivative 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) after 6 and 24 h. Also on the Jurkat cell line, only compound 1 strongly induces necrosis after 24 and 48 h, as shown by annexin V/propidium iodide staining. No induction of apoptosis was observed. We propose that compound 1 is more efficiently incorporated into cancer cells relative to all other derivatives, causing significant induction of oxidative stress within the cell, which ultimately leads to cell death.

The photophysical properties of [Re(CO)₃(L-N₃)]Br (L-N₃=2-azido-N,N-bis[(quinolin-2-yl)methyl]ethanamine), which could not be localized in cancer cells by fluorescence microscopy, have been revisited in order to evaluate its use as a luminescent probe in a biological environment. The Re^I complex displays concentration-dependent residual fluorescence besides the expected phosphorescence, and the nature of the emitting excited states have been evaluated by DFT and time-dependent (TD) DFT methods. The results show that fluorescence occurs from a ¹LC/MLCT state, whereas phosphorescence mainly stems from a ³LC state, in contrast to previous assignments. We found that our luminescent probe, [Re(CO)₃(L-N₃)]Br, exhibits an interesting cytotoxic activity in the low micromolar range in various cancer cell lines. Several biochemical assays were performed to unveil the cytotoxic mechanism of the organometallic Re^I bisquinoline complex. [Re(CO)₃(L-N₃)]Br was found to be stable in human plasma indicating that [Re(CO)₃(L-N₃)]Br itself and not a decomposition product is responsible for the observed cytotoxicity. Addition of [Re(CO)₃(L-N₃)]Br to MCF-7 breast cancer cells grown on a biosensor chip micro-bioreactor immediately led to reduced cellular respiration and increased glycolysis, indicating a large shift in cellular metabolism and inhibition of mitochondrial activity. Further analysis of respiration of isolated mitochondria clearly showed that mitochondrial respiratory activity was a direct target of [Re(CO)₃(L-N₃)]Br and involved two modes of action, namely increased respiration at lower concentrations, potentially through increased proton transport through the inner mitochondrial membrane, and efficient blocking of respiration at higher concentrations. Thus, we believe that the direct targeting of mitochondria in cells by [Re(CO)₃(L-N₃)]Br is responsible for the anticancer activity.

Crystals of MIL-88B-Fe and NH₂-MIL-88B-Fe were prepared by a new rapid microwave-assisted solvothermal method. High-purity, spindle-shaped crystals of MIL-88B-Fe with a length of about 2 μm and a diameter of 1 μm and needle-shaped crystals of NH₂-MIL-88B-Fe with a length of about 1.5 μm and a diameter of 300 nm were produced with uniform size and excellent crystallinity. The possibility to reduce the as-prepared frameworks and the chemical capture of carbon monoxide in these materials was studied by in situ ultrahigh vacuum Fourier-transform infrared (UHV-FTIR) spectroscopy and Mössbauer spectroscopy. CO binding occurs to unsaturated coordination sites (CUS). The release of CO from the as-prepared materials was studied by a myoglobin assay in physiological buffer. The release of CO from crystals of MIL-88B-Fe with t_1/2=38 min and from crystals of NH₂-MIL-88B-Fe with t_1/2=76 min were found to be controlled by the degradation of the MIL materials under physiological conditions. These MIL-88B-Fe and NH₂-MIL-88B-Fe materials show good biocompatibility and have the potential to be used in pharmacological and therapeutic applications as carriers and delivery vehicles for the gasotransmitter carbon monoxide.

We report the facile synthesis of four different trifluoromethylated metallocene triazoles and their biological evaluation (M = Fe and Ru). The cytotoxicity of all compounds was evaluated using MCF-7, HT-29, PT-45 and GM5657 cells and IC₅₀ values as low as 33 μM were found. It was shown that the metallocene moiety is crucial for the cytotoxic effect. The electrochemical behavior of triazoles 3–6 was investigated by cyclic voltammetry. These electrochemical measurements revealed that all triazoles are less prone to oxidation than ferrocene. Moreover, Log P determination by RP-HPLC showed increased lipophilicity for the fluorinated derivatives with Log P values up to 3.8. Furthermore, successful bioconjugation could be achieved by coupling triazole 7 to the amino acid L-leucine.

Concurrently with the emergence of purely organic derivatives of the naturally occurring antibiotic platensimycin (1a), herein, we describe the design, synthesis and biological evaluation of both the enantiomers of a C6–C7 ferrocene-fused platensimycin derivative 2b. (S,S_P)- and (R,R_P)-2b were prepared in nine steps starting from commercially available 4-ferrocenyl-4-oxobutyric acid via highly diastereoselective Michael additions of optically active planar-chiral ferrocene-fused cyclohexanone derivatives (5) with acrylate ester as the key step. Manual superimposition of (S,S_P)-2b on platensimycin bound to the active site of its target enzyme FabF suggests that the former fits nicely in the active site and the C6–C7-fused ferrocene occupies a pocket similarly to the C8–C9-fused tetracyclic cage of 1a. Antimicrobial activities of (S,S_P)- and (R,R_P)-2b were tested against various Gram-positive and Gram-negative bacterial strains.

The mixed-ligand tris(pyrazolyl)borate sandwich compound [TpmRuTp′]Cl (2) was prepared by microwave-assisted synthesis from Tp′Ru(COD)Cl (1) [Tp = tris(pyrazolyl)borate; Tp′ = p-bromophenyltris(pyrazolyl)borate; Tpm = tris(pyrazolyl)methane; COD = 1,4-cyclooctadiene]. Subsequently, 2 was converted to the azide-functionalized [TpmRu(p-N₃C₆H₄Tp)] (3), which can be readily coupled to biomolecules by Cu-catalyzed azide–alkyne cycloaddition (Cu-AAC) in solution, as exemplified by the covalent attachment to pentyne-functionalized HC(CH₂)₂–CO–Val–OtBu (4), and an alkyne derivative of the neuropeptide enkephaline HC(CH₂)₂–CO–ENK–OH (ENK = enkephaline, Tyr–Gly–Gly–Phe–Leu). The resulting triazole compounds [TpmRu({p-C₂N₃H–(CH₂)₂–CO–Val–OtBu}Tp)]Cl (5) and [TpmRu({p-C₂N₃H–(CH₂)₂–CO–ENK–OH}Tp)]Cl (6), represent the first [3+2] cycloaddition products of azide-functionalized Tp compounds. They were characterized by NMR spectroscopy and mass spectrometry. Furthermore, 1, 2, and 3 as well as the reaction intermediate [(κ²-N,N′-Tpm)RuCl(Tp′)] (2a) were characterized in the solid state by single-crystal X-ray diffraction.

Two peptide nucleic acid (PNA) oligomers containing two different organometallic moieties, namely derivatives of azidoferrocene (Fc-N₃) and ferrocene carboxylic acid (Fc-COOH) for PNA5 and derivatives of β-cymantrenoylpropionic acid [Cym-CO(CH₂)₂COOH] and the rhenium bisquinoline tricarbonyl complex [Re(BQ-N₃)(CO)₃]Br for PNA6, have been prepared on a solid support by using two different synthetic methods: Peptide coupling and click chemistry. PNA5 and PNA6 were unambiguously characterized by ESI-MS or MALDI-TOF MS and their purity checked by LC–MS. As expected, the bioconjugate PNA6 presents two broad and strong absorption bands at 1933 and 2032 cm^–1 in its IR spectrum due to the presence of six metal–carbonyl bonds. It has also been demonstrated that the presence of the Mn complex in PNA6 did not significantly alter the fluorescence properties of the Re complex in aqueous solution compared with a previously reported Re–PNA bioconjugate (PNA7). Photoexcitation of PNA6 at 350 nm reveals two distinct emission bands at about 434 and 595 nm in aqueous solutions. PNA6 was successfully electroporated into HeLa cells, as shown by high-resolution continuum-source atomic absorption spectroscopy, which was used to measure the concentrations of both Re and Mn. However, no fluorescence of the Re complex in PNA6 was observed in living cells, even at a concentration 20 times higher than that previously reported for PNA7.

Organometallic complexes possess great potential for imaging applications in biology, due to their kinetic stability and often favourable intrinsic properties. In this work we present a new class of Re^I-tricarbonyl complexes with a substituted bis(phenanthridinylmethyl)amine (bpm) ligand. The complex Re(CO)₃(R-bpm) could be conveniently prepared by microwave synthesis from [Re(CO)₃(H₂O)₃]Br and a suitably substituted bis(phenanthridinylmethyl)amine (R-bpm). Complex 5, with R=CH₂-CO₂-CH₃, was characterized by a single-crystal X-ray structure. Complex 6 (R=CH₂-C₆H₄-CO₂H) was used in solid-phase peptide synthesis (SPPS) to label the neurotensin(8–13) (NT) fragment N-terminally. The complexes show luminescence emission with large Stokes shifts (λ_ex=350 nm, λ_em=570 nm). Cellular uptake and intracellular localization studies in several cell lines demonstrate the utility of the new Re(CO)₃(R-bpm) complexes for fluorescence imaging and reveal significant differences between the simple methyl ester 5 and the NT bioconjugate 7.

In the course of finding ligands suitable for application in bioorganometallic systems, “third-generation” Tp-transfer agents p-BrC₆H₄TpM [M = Na (1), K (2), Rb (3), Cs (4), Tl (5); Tp = tris(pyrazolyl)borate], p-BrC₆H₄Tp*Tl (6) [Tp* = tris(3,5-dimethylpyrazolyl)borate], (p-BrC₆H₄Tp)₂Mg (7), p-BrC₆H₄Tp^MeK (8) [Tp^Me = tris(3-methylpyrazolyl)borate] and p-BrC₆H₄Tp*K (9) bearing a functionalizable 4-bromophenyl group on the boron atom were synthesized and characterized. Monovalent 6 and magnesium-sandwich compound 7 were structurally characterized in the solid state by X-ray diffraction. Compound 6 forms a dimer by κ²-coordination of Tl to one Tp ligand and to the bromine atom of a neighbouring 4-bromophenyl group.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

The recent discovery of the natural product platensimycin as a new antibiotic lead structure has triggered the synthesis of numerous organic derivatives for structure–activity relationship studies. Herein, we describe the synthesis, characterization and biological evaluation of the first organometallic antibiotic inspired by platensimycin. Two bioorganometallic compounds containing (η⁶-pentamethylbenzene)Cr(CO)₃ (2) and (η⁶-benzene)Cr(CO)₃ (3), linked by an amide bond to the aromatic part of platensimycin, were synthesized. Their antibiotic activities were tested against B. subtilis 168 (Gram positive) and E. coli W3110 (Gram negative) bacterial strains. Both compounds were found to be inactive against E. coli but derivative 2 inhibits B. subtilis growth at a moderate MIC value of 0.15 mM. To test the intrinsic toxicity of chromium, several chromium salts along with {η⁶-(3-pentamethylphenyl propionic acid)}Cr(CO)₃ (5) and {η⁶-(3-phenyl propionic acid)}Cr(CO)₃ (6) were tested against both bacterial strains. No activity was observed against E. coli for any of the compounds; B. subtilis growth was not inhibited by Cr(NO₃)₃ and only very weakly by 5, K₂Cr₂O₇ and Na₂CrO₄ at MIC values of 0.5, 0.68 and 1.24 mM, respectively. Compounds 2, 3, 5 and 4 (the pure organic analogue of 2) show similar cytotoxicity against HeLa, HepG2 and HT-29 mammalian cell lines. Furthermore, the cellular uptake and the intracellular distribution of compounds 2, 3 and Cr(NO₃)₃ in B. subtilis were studied using atomic absorption spectroscopy to gain insight in to the possible cellular targets. Compound 2 was found to be readily taken up and distributed almost equally among cytosol, cell debris and cell membrane in B. subtilis.

While N-heterocyclic carbenes (NHCs) are ubiquitous ligands in catalysts for organic or industrial synthesis, their potential to form stable transition metal complexes has hardly been exploited in metal bioconjugates. In this work, we describe a straightforward synthesis of carboxylato-functionalized imidazolium salts for covalent binding to peptides. Carbene complexes of Ru and Rh were prepared from these imidazolium salts using Ag₂O, followed by transmetallation. The neuropeptide [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was chosen as a model peptide. Exploratory NMR experiments identified the Ru(p-cymene)Cl₂ complex of the asymmetrically substituted imidazol-2-ylidene 3b as the most suitable metal carbene precursor for solid phase peptide synthesis (SPPS). After optimization of the conditions for SPPS, a ruthenium-NHC pseudoenkephalin (dichloro(η⁶-p-cymene)[1-methyl-3-(methyl-p-benzoyl-Gly-Gly-Phe-Leu-OH)imidazol-2-ylidene]ruthenium(II), 12) was synthesized from 3b on solid phase using the 2-Cl-Trt resin and cleavage by 2 % TFA to yield the free carboxylic acid. Peptide 12 was fully characterized by HPLC, ¹H and ¹³C NMR and ESI-MS. Characteristic NMR signals, as well as the isotope pattern of Ru in the ESI-MS, unequivocally confirm the formation of this metal-carbene peptide bioconjugate.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

This work introduces a bis(cysteine) ligand to build a small peptidic model system of hydrogenase enzymes. Fe(C₅H₄CO–Cys–OMe)₂ (LH₂) has been employed as a chelate for an iron–carbonyl complex, which mimics two essential structural properties of the hydrogenase class of enzymes, namely the coordination of the iron–carbonyl core to peptide ligands and the presence of an electrochemical relay in spatial proximity. The treatment of LH₂ with Fe₃(CO)₁₂ yields LFe₂(CO)₆ (3a), which is the first peptide-coordinated iron hydrogenase active-site model complex. Compound 3a was fully characterized spectroscopically (¹H NMR, ¹³C NMR, IR and Mössbauer spectroscopy, mass spectrometry and elemental analysis). A single-crystal X-ray analysis confirms the proposed structure and reveals a staggered conformation of the Fe₂(CO)₆S₂ core. Fourier transform infrared (FTIR) spectroelectrochemistry reveals an electronic interaction between the peptide backbone and the iron–carbonyl cluster, but not with the ferrocene subsite. The introduction of this peptidic cysteine-based ligand into hydrogenase model chemistry helps to confirm the proposed cofactor biosynthesis and understand the electronic interplay between the metal–carbonyl active site and the protein environment in this important class of enzymes.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)