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As the carrier of the inheritable information in cells, DNA has been the target of metal complexes for over 40 years. In this chapter, the focus will be on non-covalent recognition of the highly structured DNA surface by substitutionally inert metal complexes capable of either sliding in between the normal base pairs as metallointercalators or flipping out thermodynamically destabilized mispaired nucleobases as metalloinsertors. While most of the compounds discussed are based on ruthenium(II) and rhodium(III) due to their stable octahedral coordination environment and low-spin 4d6 electronic configuration, most recent developments of alternative metal complexes, based on both transition metals and main group elements, will also be highlighted. A particular focus of the coverage is on structural data from X-ray structure analysis, which now provides details of the interaction at unprecedented details and will enable development of novel DNA binding probes for fundamental studies as well as new anticancer drug candidates.

Antimicrobial resistance is a growing global concern in human and veterinary medicine, with an ever-increasing void in the arsenal of clinicians. Novel classes of compounds including carbon monoxoide-releasing molecules (CORMs), for example the light-activated metal complex [Mn(CO)3(tpa-κ3N)]Br, could be used as alternatives/to supplement traditional antibacterials. Avian pathogenic Escherichia coli (APEC) represent a large reservoir of antibiotic resistance and can cause serious clinical disease in poultry, with potential as zoonotic pathogens, due to shared serotypes and virulence factors with human pathogenic E. coli. The in vitro activity of [Mn(CO)3(tpa-κ3N)]Br against multidrug-resistant APECs was assessed via broth microtitre dilution assays and synergy testing with colistin performed using checkerboard and time-kill assays. In vivo antibacterial activity of [Mn(CO)3(tpa-κ3N)]Br alone and in combination with colistin was determined using the Galleria mellonella wax moth larvae model. Animals were monitored for life/death, melanisation and bacterial numbers enumerated from larval haemolymph. In vitro testing produced relatively high [Mn(CO)3(tpa-κ3N)]Br minimum inhibitory concentrations (MICs) of 1024 mg/L. However, its activity was significantly increased with the addition of colistin, bringing MICs down to ≤32 mg/L. This synergy was confirmed in time-kill assays. In vivo assays showed that the combination of [Mn(CO)3(tpa-κ3N)]Br with colistin produced superior bacterial killing and significantly increased larval survival. In both in vitro and in vivo assays light activation was not required for antibacterial activity. This data supports further evaluation of [Mn(CO)3(tpa-κ3N)]Br as a potential agent for treatment of systemic infections in humans and animals, when used with permeabilising agents such as colistin.

The copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was used for the first time to attach a biologically active carbon monoxide delivery agent to modified nanodiamond (ND) as a highly biocompatible carrier. The [Mn(CO)(3)(tpm)](+) photoactivatable CO-releasing molecule (PhotoCORM) on the surface retained the carbon monoxide release properties of the parent compound as shown with the myoglobin assay.

In this paper we investigate metal-organic compounds as infrared (IR) active labels by scattering scanning near-field infrared microscopy (IR s-SNOM, often also abbreviated as s-SNIM) with a lateral resolution of 90 × 90 nm(2). Tailor-made IR spectroscopic probes based on cymantrene (CpMn(CO)(3) with Cp = η(5)-C(5)H(5)) conjugated to a cysteine-modified pseudoneurotensin (pNT-Cys-OH) peptide were prepared by automated microwave-assisted solid phase peptide synthesis (SPPS) and characterized by HPLC, ESI-MS and IR. Well-defined patterned self-assembled monolayers on a gold surface were prepared by microcontact printing of 1-octadecanethiol (ODT) followed by additional incubation in ethanolic solution of the cymantrene-peptide derivative. The self-assembled monolayers have been evidenced by infrared reflection absorption spectroscopy (IRRAS) and AFM. CO laser source radiation was tuned (1944, 1900, 1798 and 1658 cm(-1)) for imaging contrast with good matching correlation between spectroscopic and topographic patterns at specific characteristic metal carbonyl and amide bands (1944 cm(-1) (λ = 5.14 μm) and 1658 cm(-1) (λ = 6.03 μm)). Cymantrene probes provide an attractive method to tag a unique spectroscopic feature on any bio(macro)molecule. Introducing such probes into super-resolution IR s-SNOM will enable molecular tracking and distribution studies even in complex biological systems.

Organometallic analogues of chloroquine show promise as new antimalarial agents capable of overcoming resistance to the parent drug chloroquine. Here, the synthesis and characterization of three new cymantrene (CpMn(CO)(3)) and cyrhetrene (CpRe(CO)(3)) 4-aminoquinoline conjugates with either an amine or amide linker are reported. The antimalarial activity of the new organometallic conjugates N-(2-(7-chloroquinolin-4-ylamino)ethyl)-4-cymantrenylbutanamide (3), N-(2-(7-chloroquinolin-4-ylamino)ethyl)-4-cyrhetrenylbutanamide (4) and N-(7-chloroquinolin-4-yl)-N'-(cymantrenylmethyl)ethane-1,2-diamine (6) was evaluated against a chloroquine-sensitive (CQS) and a chloroquine-resistant strain (CQR) of the malaria parasite Plasmodium falciparum. The cymantrene complex with an amine linker (6) showed good activity against the CQS strain but was inactive against the CQR strain. In contrast, cymantrene and cyrhetrene compounds with an amide linker were active against both the CQS and the CQR strain. In addition, the antibacterial, anti-trypanosomal and anti-leishmanial activity of the compounds was evaluated. Compound 6 showed submicromolar activity against Trypanosoma brucei at a concentration where the toxicity to normal human cells is low. No significant effect was noticed on the exchange of manganese for rhenium in the CpM(CO)(3) moiety in any of the biological assays.

Organometallic complexes conjugated to cell-penetrating peptides (CPPs) are promising systems for diagnostic imaging and therapeutic applications in human medicine. Recently, we reported on the synthesis of cymantrene(CpMn(CO)(3))-CPP conjugates with biological activity on different cancer cell lines. However, the precise mechanism of cytotoxicity remained elusive in these studies. To investigate the role of the metal center and the linker between the CpM(CO)(3) moiety and the peptide, a number of derivatives with manganese replaced by rhenium and the keto linker originally used substituted by a methylene group were prepared and fully characterized by (1)H NMR spectroscopy, infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis as well as X-ray structure determination. The organometal-peptide conjugates as well as carboxyfluorescein-labeled derivatives thereof were prepared by solid-phase peptide synthesis, purified by high-performance liquid chromatography, and analyzed by mass spectrometry. Fluorescence microscopy studies of MCF-7 human breast cancer cells revealed an efficient cellular uptake and pronounced nuclear localization of the bioconjugates with the methylene linker compared with systems with the keto group. In addition, the latter also showed a higher cytotoxicity. In contrast, the variation of the metal center from manganese to rhenium had a negligible effect. The structure-activity relationships determined in the present work will aid in the further tuning of the biological activity of organometal-peptide conjugates.

Over the past years, numerous promising new metalorganic lead structures have been developed exhibiting highly active cytostatic properties. However, the efficiency of such chemotherapeutics in the treatment of tumors is often limited by their low therapeutic index due to their short half-life, lack of tumor selectivity, and associated side effects. Furthermore, the membrane barrier often restricts their cellular uptake by passive diffusion. In this contribution, we describe the synthesis, cellular uptake, and biologic activity of a series of cymantrene-peptide conjugates. Cymantrene CpMn(CO)(3) is a robust organometallic group, which is stable in air and water and easy to functionalize. In this work, some new cymantrene derivatives with different linkers between the half-sandwich complex and the carboxylate group were attached to the cell-penetrating peptide sC18 that should act as a transporter for the metal moiety. All conjugates were characterized for their cytotoxic activity on human breast adenocarcinoma cells (MCF-7) and human colon carcinoma cells (HT-29). We found that bioconjugates bearing two cymantrene groups were more active than the monofunctionalized ones. By the introduction of a cathepsin B cleavage site next to the organometallic group, the biologic activity could be in increased even further. Fluorescence microscopy studies and apoptosis assays gave preliminary hints on the mode of action of these systems.

By combining organometallic groups and peptides, a large number of conjugates with interesting new biological properties can be prepared. Especially, attachment to cell-penetrating peptides (CPP) that act as efficient cell delivery vehicles has come to the fore. However, the presence of the metal moiety in such systems can interfere with standard conjugate synthesis procedures which therefore need to be optimized for every new compound. In this work, we report on the preparation of six new cymantrene-sC18 peptide bioconjugates that were prepared by solid phase peptide synthesis (SPPS) techniques. The cymantrene complexes were chosen for their different linker to the peptide, to study the influence of the linker group on cellular uptake and cell viability of the conjugates. Interestingly, the attachment of the metal complex leads to a non-standard cleavage of the Rink amide linker used in the SPPS protocol under trifluoroacetic acid (TFA) treatment, resulting in peptide amides that are N-alkylated at the C-terminus. Furthermore, we found that depending on the type of cymantrene moiety attached, the formation of reactive carbocations which result from decomposition of the resin linker is facilitated and can alkylate the metal complex moiety. Both effects were analyzed by MS/MS studies and cleavage mixtures for efficient elimination of this byproduct formation were identified. Moreover, initial biological testing of the cytotoxicity of one of the bioconjugates gave promising results. Concentration-dependent cell viability studies of Cym1-sC18 on human MCF-7 breast adenocarcinoma cells gave an IC(50) value of 59.8 (+/- 6.7) microM and demonstrate their potential in anticancer chemotherapy.

A series of five tris(2-pyridylmethyl)amine (tpa) ruthenium complexes [Ru(tpa)(N-N)](PF(6))(2) with N-N=bpy (2,2'-bipyridine), phen (1,10-phenanthroline), dpq (dipyrido[3,2-d:2',3'-f]quinoxaline), dppz (dipyrido[3,2-a;2',3'-c]phenazine), and dppn (4,5,9,16-tetraazadibenzo[a,c]naphthacene) was prepared and characterized by NMR, UV-Visible (UV/Vis), and fluorescence spectroscopy as well as cyclic voltammetry. Structures optimized with density functional theory methods (DFT, BP86, TZVP) without constraints show C(1) symmetry while in solution, the (1)H and (13)C NMR spectra are in accordance with an average C(s) symmetry. This is thought to be due to a low energy barrier for flipping of the equatorial pyridine ring from one side of the N-N plane to the other. The electronic structure of the compounds was studied with DFT and a change in the highest occupied molecular orbital (HOMO) character from Ru t(2g) for the bpy, phen, and dpq to N-N ligand-based for the dppz and dppn complexes was found. TDDFT calculations showed dominant N-N-based intra-ligand charge transfer (ILCT) transitions in the latter two complexes mixed with metal-to-ligand charge transfer (MLCT) bands found for all five compounds. DNA binding of the complexes was studied with UV/Vis titrations, the fluorescent ethidium bromide displacement assay, and CD spectroscopy. The affinity increases with the aromatic surface area of of the bidentate N-N ligand in the order bpy<

Cym-CO-CH2-CH2-COOH was prepared in good yield by Friedel-Crafts reaction of cymantrene (Cym, CpMn(CO)3) with succinic anhydride for the IR labelling of peptides and fully characterized, including an X-ray structure analysis (monoclinic space group P2(1)/n, a=5.727(3)A, b=19.865(9)A, c=10.518(5)A, beta=91.211(9) degrees). The compound was isolated in pure form without the need for chromatographic work-up and subsequently used for solution-phase synthesis of a bioconjugate with phenylalanine methyl ester to allow a complete spectroscopic characterization of this model system. The cymantrene keto carboxylic acid also turned out to be a very robust marker in automated microwave-assisted solid phase peptide synthesis (SPPS). [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu) was prepared on a Wang resin and labelled with the cymantrene derivative on the solid support under microwave irradiation in all steps. The metal-carbonyl marker stayed intact during cleavage from the resin with concentrated trifluoroacetic acid. After simple precipitation and lyophilization, the cymantrene-enkephalin bioconjugate could be obtained in analytically pure form without the need of HPLC purification. As required, the compound is non-cytotoxic against MCF-7 cells at up to 100 microM. This protocol thus allows one to introduce organometallic IR spectroscopic labels to peptides in a very straightforward way.

The conjugation of cymantrene CpMn(CO)(3) to cell-penetrating peptide hCT(18-32)-k7 alters the intracellular distribution in MCF-7 cells compared to the unmodified peptide, as visualized by fluorescence microscopy, and leads to an increased nuclear accumulation; the peptide and cymantrene compound themselves are not toxic, but the bioconjugate shows a significant cytotoxicity with an IC(50) value of 36 micromol l(-1).

A series of five ruthenium(II) polypyridyl complexes [Ru(bpy)2(N--N)]Cl2 was tested against human HT-29 and MCF-7 cancer cell lines. Cellular uptake efficiency and cytotoxicity were found to increase with the size of the aromatic surface area of the N--N ligand. The most active compound carrying the dppn ligand exhibits a low micromolar IC(50) value against both cell lines comparable to that of cisplatin under similar conditions. Continuous measurement of oxygen consumption, extracellular acidification rate, and impedance of the cell layer with a chip-based sensor system upon exposure to the complexes showed only small changes for the first two parameters throughout the series. A significant and irreversible decrease in impedance was, however, found for the dppn compound. This suggests that its biological activity is related to modifications in cell morphology or cell-cell and cell-matrix contacts.

With the established chemistry of bridged [(porphyrinate)FeIII-X-CuII(ligand)]n+ [X = O2- (oxo), OH- (hydroxo), O22- (peroxo)] complexes, we investigated the effect of cobalt ion substitution for copper or copper and iron. Thus, in this report, the generation and characterization of new mu-oxo, micro-hydroxo, and micro-peroxo (micro-X) assemblies of [(porphyrinate)MIII-X-CoII/III(TMPA)]n+ assemblies is described, where M = FeIII or CoIII and TMPA = tris(2-pyridylmethyl)amine. The mu-oxo complex [(F8TPP)FeIII-O-CoII(TMPA)]+ (1, F8TPP = tetrakis(2,6-difluorphenyl)porphyrinate) was isolated by an acid-base self-assembly reaction of a 1:1 mixture of (F8TPP)FeIII-OH and [CoII(TMPA)(MeCN)]2+ upon addition of triethylamine. The crystal structure of 1.2C4H10O proved the presence of an unsupported Fe-O-Co moiety; angleFe-O-Co = 171.6 degrees and d(Fe...Co) = 3.58 A. Complex 1 was further characterized by UV-vis (lambdamax = 437 (Soret) and 557 nm), 1H NMR [delta 40.6 (pyrrole-H), 8.8 and 8.7 (m-phenyl-H), 8.0 (p-phenyl-H), 4.4 (PY-4H), 2.6 (PY-3H), 1.0 (PY-5H), -1.1 (PY-6H), and -2.7 (TMPA-CH2-) ppm], electrospray ionization (ESI) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric methods, Evans method NMR (microeff = 3.1), and superconducting quantum interference device (SQUID) susceptometry (J = -114 cm-1, S = 1). The micro-hydroxo analogue [(F8TPP)FeIII-(OH)-CoII(TMPA)]+ (2) [UV-vis lambdamax = 567 nm; delta 78 ppm (pyrrole-H); Evans NMR microeff = 3.7] was generated by addition of 1 equiv of triflic acid to 1. The protonation is completely reversible, and 1 is regenerated from 2 by addition of triethylamine. While (F8TPP)FeII/[CoII(TMPA)(MeCN)]2+/O2 chemistry does not lead to a stable micro-peroxo species, a dicobalt micro-peroxo complex [(TPP)CoIII-(O22-)-CoIII(TMPA)]2+ (3, TPP = meso-tetraphenylporphyrinate) forms from a reaction of O2 with a 1:1 mixture of the CoII precursor components at -80 degrees C [UV-vis lambdamax = 435 (Soret), 548, and 583 (weak) nm; silent EPR spectrum; diamagnetic NMR spectrum]. The oxygenation/deoxygenation equilibrium is reversible; warming solutions of 3 releases approximately 1 equiv of O2 and the reduced complexes are reformed.

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covalent modification was established on the basis of the enhanced depurination associated with N-alkylation. The site-selective alkylation at mismatched DNA renders these conjugates useful tools for the covalent tagging of DNA base pair mismatches and new chemotherapeutic design.