Synthesis, Spectroscopic Properties, and Photoinduced CO-Release Studies of Functionalized Ruthenium(II) Polypyridyl Complexes: Versatile Building Blocks for Development of CORM–Peptide Nucleic Acid Bioconjugates

A series of ruthenium­(II) dicarbonyl complexes of formula [RuCl₂(L)­(CO)₂] (L = bpy^CH3,CH3 = 4,4′-dimethyl-2,2′-bipyridine, bpy^CH3,CHO = 4′-methyl-2,2′-bipyridine-4-carboxyaldehyde, bpy^CH3,COOH = 4′-methyl-2,2′-bipyridine-4-carboxylic acid, CppH = 2-(pyridin-2-yl)­pyrimidine-4-carboxylic acid, dppzcH = dipyrido­[3,2-a:2′,3′-c]­phenazine-11-carboxylic acid), and [RuCl­(L)­(CO)₂]⁺ (L = tpy^COOH = 6-(2,2′:6′,2″-terpyridine-4′-yloxy)­hexanoic acid) has been synthesized. In addition, a high-yield synthesis of a peptide nucleic acid (PNA) monomer containing the 2-(pyridin-2-yl)­pyrimidine ligand was also developed, and this compound was used to prepare the first Ru­(II) dicarbonyl complex, [RuCl₂(Cpp-L-PNA)­(CO)₂],(Cpp-L-PNA = <i>tert</i>-butyl-<i>N</i>-[2-(<i>N</i>-9-fluorenylmethoxycarbonyl)­aminoethyl]-<i>N</i>-[6-(2-(pyridin-2-yl)­pyrimidine-4-carboxamido)­hexanoyl]­glycinate) attached to a PNA monomer backbone. Such metal-complex PNA–bioconjugates are attracting profound interest for biosensing and biomedical applications. Characterization of all complexes has been undertaken by IR and NMR spectroscopy, mass spectrometry, elemental analysis, and UV–vis spectroscopy. Investigation of the CO-release properties of the Ru­(II) complexes in water/dimethyl sulfoxide (49:1) using the myoglobin assay showed that they are stable under physiological conditions in the dark for at least 60 min and most of them even for up to 15 h. In contrast, photoinduced CO release was observed upon illumination at 365 nm, the low-energy shoulder of the main absorption maximum centered around 300 nm, establishing these compounds as a new class of PhotoCORMs. While the two 2,2′-bipyridine complexes release 1 equiv of CO per mole of complex, the terpyridine, 2-(2′-pyridyl)­pyrimidine, and dipyrido­[3,2-a:2′,3′-c]­phenazine complexes are less effective CO releasers. Attachment of the 2-(2′-pyridyl)­pyrimidine complex to a PNA backbone as in [RuCl₂(Cpp-L-PNA)­CO₂] did not significantly change the spectroscopic or CO-release properties compared to the parent complex. Thus, a novel class of Ru­(II)-based PhotoCORMs has been established which can be coupled to carrier delivery vectors such as PNA to facilitate cellular uptake without loss of the inherent CORM properties of the parent compound

Synthesis, Spectroscopic Properties, and Photoinduced CO-Release Studies of Functionalized Ruthenium(II) Polypyridyl Complexes: Versatile Building Blocks for Development of CORM–Peptide Nucleic Acid Bioconjugates

A series of ruthenium­(II) dicarbonyl complexes of formula [RuCl₂(L)­(CO)₂] (L = bpy^CH3,CH3 = 4,4′-dimethyl-2,2′-bipyridine, bpy^CH3,CHO = 4′-methyl-2,2′-bipyridine-4-carboxyaldehyde, bpy^CH3,COOH = 4′-methyl-2,2′-bipyridine-4-carboxylic acid, CppH = 2-(pyridin-2-yl)­pyrimidine-4-carboxylic acid, dppzcH = dipyrido­[3,2-a:2′,3′-c]­phenazine-11-carboxylic acid), and [RuCl­(L)­(CO)₂]⁺ (L = tpy^COOH = 6-(2,2′:6′,2″-terpyridine-4′-yloxy)­hexanoic acid) has been synthesized. In addition, a high-yield synthesis of a peptide nucleic acid (PNA) monomer containing the 2-(pyridin-2-yl)­pyrimidine ligand was also developed, and this compound was used to prepare the first Ru­(II) dicarbonyl complex, [RuCl₂(Cpp-L-PNA)­(CO)₂],(Cpp-L-PNA = <i>tert</i>-butyl-<i>N</i>-[2-(<i>N</i>-9-fluorenylmethoxycarbonyl)­aminoethyl]-<i>N</i>-[6-(2-(pyridin-2-yl)­pyrimidine-4-carboxamido)­hexanoyl]­glycinate) attached to a PNA monomer backbone. Such metal-complex PNA–bioconjugates are attracting profound interest for biosensing and biomedical applications. Characterization of all complexes has been undertaken by IR and NMR spectroscopy, mass spectrometry, elemental analysis, and UV–vis spectroscopy. Investigation of the CO-release properties of the Ru­(II) complexes in water/dimethyl sulfoxide (49:1) using the myoglobin assay showed that they are stable under physiological conditions in the dark for at least 60 min and most of them even for up to 15 h. In contrast, photoinduced CO release was observed upon illumination at 365 nm, the low-energy shoulder of the main absorption maximum centered around 300 nm, establishing these compounds as a new class of PhotoCORMs. While the two 2,2′-bipyridine complexes release 1 equiv of CO per mole of complex, the terpyridine, 2-(2′-pyridyl)­pyrimidine, and dipyrido­[3,2-a:2′,3′-c]­phenazine complexes are less effective CO releasers. Attachment of the 2-(2′-pyridyl)­pyrimidine complex to a PNA backbone as in [RuCl₂(Cpp-L-PNA)­CO₂] did not significantly change the spectroscopic or CO-release properties compared to the parent complex. Thus, a novel class of Ru­(II)-based PhotoCORMs has been established which can be coupled to carrier delivery vectors such as PNA to facilitate cellular uptake without loss of the inherent CORM properties of the parent compound

An Environmentally Benign and Cost-Effective Synthesis of Aminoferrocene and Aminoruthenocene

An improved synthesis of aminoferrocene has been carried out that adheres with the basic green chemistry guidelines. Amination from aqueous NH₃ as the nitrogen source, with the inexpensive CuI/Fe₂O₃ couple as cocatalyst in ethanolic solution, makes the process environmentally attractive as well as a viable alternative for all practical purposes. This procedure has also been applied to prepare aminoruthenocene, being reported for the first time

Phosphodiester Cleavage Properties of Copper(II) Complexes of 1,4,7-Triazacyclononane Ligands Bearing Single Alkyl Guanidine Pendants

Three new metal-coordinating ligands, L¹·4HCl [1-(2-guanidinoethyl)-1,4,7-triazacyclononane tetrahydrochloride], L²·4HCl [1-(3-guanidinopropyl)-1,4,7-triazacyclononane tetrahydrochloride], and L³·4HCl [1-(4-guanidinobutyl)-1,4,7-triazacyclononane tetrahydrochloride], have been prepared via the selective N-functionalization of 1,4,7-triazacyclononane (tacn) with ethylguanidine, propylguanidine, and butylguanidine pendants, respectively. Reaction of L¹·4HCl with Cu­(ClO₄)₂·6H₂O in basic aqueous solution led to the crystallization of a monohydroxo-bridged binuclear copper­(II) complex, [Cu₂L¹₂(μ-OH)]­(ClO₄)₃·H₂O (<b>C1</b>), while for L² and L³, mononuclear complexes of composition [Cu­(L²H)­Cl₂]­Cl·(MeOH)_0.5·(H₂O)_0.5 (<b>C2</b>) and [Cu­(L³H)­Cl₂]­Cl·(DMF)_0.5·(H₂O)_0.5 (<b>C3</b>) were crystallized from methanol and DMF solutions, respectively. X-ray crystallography revealed that in addition to a tacn ring from L¹ ligand, each copper­(II) center in <b>C1</b> is coordinated to a neutral guanidine pendant. In contrast, the guanidinium pendants in <b>C2</b> and <b>C3</b> are protonated and extend away from the Cu­(II)–tacn units. Complex <b>C1</b> features a single μ-hydroxo bridge between the two copper­(II) centers, which mediates strong antiferromagnetic coupling between the metal centers. Complexes <b>C2</b> and <b>C3</b> cleave two model phosphodiesters, <i>bis</i>(<i>p</i>-nitrophenyl)­phosphate (BNPP) and 2-hydroxypropyl-<i>p</i>-nitrophenylphosphate (HPNPP), more rapidly than <b>C1</b>, which displays similar reactivity to [Cu­(tacn)­(OH₂)₂]²⁺. All three complexes cleave supercoiled plasmid DNA (pBR 322) at significantly faster rates than the corresponding <i>bis</i>(alkylguanidine) complexes and [Cu­(tacn)­(OH₂)₂]²⁺. The high DNA cleavage rate for <b>C1</b> {<i>k</i>_obs = 1.30 (±0.01) × 10^–4 s^–1 vs 1.23 (±0.37) × 10^–5 s^–1 for [Cu­(tacn)­(OH₂)₂]²⁺ and 1.58 (±0.05) × 10^–5 s^–1 for the corresponding <i>bis</i>(ethylguanidine) analogue} indicates that the coordinated guanidine group in <b>C1</b> may be displaced to allow for substrate binding/activation. Comparison of the phosphate ester cleavage properties of complexes <b>C1</b>–<b>C3</b> with those of related complexes suggests some degree of cooperativity between the Cu­(II) centers and the guanidinium groups

Electrochemiluminescent Monomers for Solid Support Syntheses of Ru(II)-PNA Bioconjugates: Multimodal Biosensing Tools with Enhanced Duplex Stability

The feasibility of devising a solid support mediated approach to multimodal Ru­(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru­(II)-PNA-like monomers, [Ru­(bpy)₂(Cpp-L-PNA-OH)]²⁺ (<b>M1</b>), [Ru­(phen)₂(Cpp-L-PNA-OH)]²⁺ (<b>M2</b>), and [Ru­(dppz)₂(Cpp-L-PNA-OH)]²⁺ (<b>M3</b>) (bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine, Cpp-L-PNA-OH = [2-(<i>N</i>-9-fluorenylmethoxycarbonyl)­aminoethyl]-<i>N</i>-[6-(2-(pyridin-2yl)­pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru­(II)-PNA oligomers and characterized by IR and ¹H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, <b>M1</b> was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH₂ (<b>PNA1</b>) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH₂ (<b>PNA4</b>) to give <b>PNA2</b> (H-g-c-a-a-t-a-a-a-a-<i><b>M1</b></i>-lys-NH₂) and <b>PNA3</b> (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-<i><b>M1</b></i>-lys-NH₂), respectively. The two Ru­(II)-PNA oligomers, <b>PNA2</b> and <b>PNA3</b>, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (<b>DNA</b>: 5′-T-T-T-<b>T-T-T-T-A-T-T-G-C</b>-T-T-T-3′) showed no major variations, suggesting that the electronic properties of the Ru­(II) complexes are largely unaffected by hybridization. The thermal stability of the <b>PNA·DNA</b> duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (<i>T</i>_m) was almost 8 °C higher for <b>PNA2·DNA</b> duplex, and 4 °C for <b>PNA3·DNA</b> duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru­(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, <b>PNA2</b>, <b>PNA3</b>, <b>PNA2·DNA</b> and <b>PNA3·DNA</b> displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru­(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools

Molecular and Cellular Characterization of the Biological Effects of Ruthenium(II) Complexes Incorporating 2‑Pyridyl-2-pyrimidine-4-carboxylic Acid

A great majority of the Ru complexes currently studied in anticancer research exert their antiproliferative activity, at least partially, through ligand exchange. In recent years, however, coordinatively saturated and substitutionally inert polypyridyl Ru­(II) compounds have emerged as potential anticancer drug candidates. In this work, we present the synthesis and detailed characterization of two novel inert Ru­(II) complexes, namely, [Ru­(bipy)₂(Cpp-NH-Hex-COOH)]²⁺ (<b>2</b>) and [Ru­(dppz)₂(CppH)]²⁺ (<b>3</b>) (bipy = 2,2′-bipyridine; CppH = 2-(2′-pyridyl)­pyrimidine-4-carboxylic acid; Cpp-NH-Hex-COOH = 6-(2-(pyridin-2-yl)­pyrimidine-4-carboxamido)­hexanoic acid; dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine). <b>3</b> is of particular interest as it was found to have IC₅₀ values comparable to cisplatin, a benchmark standard in the field, on three cancer cell lines and a better activity on one cisplatin-resistant cell line than cisplatin itself. The mechanism of action of <b>3</b> was then investigated in detail and it could be demonstrated that, although <b>3</b> binds to calf-thymus DNA by intercalation, the biological effects that it induces did not involve a nuclear DNA related mode of action. On the contrary, confocal microscopy colocalization studies in HeLa cells showed that <b>3</b> specifically targeted mitochondria. This was further correlated by ruthenium quantification using High-resolution atomic absorption spectrometry. Furthermore, as determined by two independent assays, <b>3</b> induced apoptosis at a relatively late stage of treatment. The generation of reactive oxygen species could be excluded as the cause of the observed cytotoxicity. It was demonstrated that the mitochondrial membrane potential in HeLa was impaired by <b>3</b> as early as 2 h after its introduction and even more with increasing time

Phosphodiester Cleavage Properties of Copper(II) Complexes of 1,4,7-Triazacyclononane Ligands Bearing Single Alkyl Guanidine Pendants

Three new metal-coordinating ligands, L¹·4HCl [1-(2-guanidinoethyl)-1,4,7-triazacyclononane tetrahydrochloride], L²·4HCl [1-(3-guanidinopropyl)-1,4,7-triazacyclononane tetrahydrochloride], and L³·4HCl [1-(4-guanidinobutyl)-1,4,7-triazacyclononane tetrahydrochloride], have been prepared via the selective N-functionalization of 1,4,7-triazacyclononane (tacn) with ethylguanidine, propylguanidine, and butylguanidine pendants, respectively. Reaction of L¹·4HCl with Cu­(ClO₄)₂·6H₂O in basic aqueous solution led to the crystallization of a monohydroxo-bridged binuclear copper­(II) complex, [Cu₂L¹₂(μ-OH)]­(ClO₄)₃·H₂O (<b>C1</b>), while for L² and L³, mononuclear complexes of composition [Cu­(L²H)­Cl₂]­Cl·(MeOH)_0.5·(H₂O)_0.5 (<b>C2</b>) and [Cu­(L³H)­Cl₂]­Cl·(DMF)_0.5·(H₂O)_0.5 (<b>C3</b>) were crystallized from methanol and DMF solutions, respectively. X-ray crystallography revealed that in addition to a tacn ring from L¹ ligand, each copper­(II) center in <b>C1</b> is coordinated to a neutral guanidine pendant. In contrast, the guanidinium pendants in <b>C2</b> and <b>C3</b> are protonated and extend away from the Cu­(II)–tacn units. Complex <b>C1</b> features a single μ-hydroxo bridge between the two copper­(II) centers, which mediates strong antiferromagnetic coupling between the metal centers. Complexes <b>C2</b> and <b>C3</b> cleave two model phosphodiesters, <i>bis</i>(<i>p</i>-nitrophenyl)­phosphate (BNPP) and 2-hydroxypropyl-<i>p</i>-nitrophenylphosphate (HPNPP), more rapidly than <b>C1</b>, which displays similar reactivity to [Cu­(tacn)­(OH₂)₂]²⁺. All three complexes cleave supercoiled plasmid DNA (pBR 322) at significantly faster rates than the corresponding <i>bis</i>(alkylguanidine) complexes and [Cu­(tacn)­(OH₂)₂]²⁺. The high DNA cleavage rate for <b>C1</b> {<i>k</i>_obs = 1.30 (±0.01) × 10^–4 s^–1 vs 1.23 (±0.37) × 10^–5 s^–1 for [Cu­(tacn)­(OH₂)₂]²⁺ and 1.58 (±0.05) × 10^–5 s^–1 for the corresponding <i>bis</i>(ethylguanidine) analogue} indicates that the coordinated guanidine group in <b>C1</b> may be displaced to allow for substrate binding/activation. Comparison of the phosphate ester cleavage properties of complexes <b>C1</b>–<b>C3</b> with those of related complexes suggests some degree of cooperativity between the Cu­(II) centers and the guanidinium groups

Molecular and Cellular Characterization of the Biological Effects of Ruthenium(II) Complexes Incorporating 2‑Pyridyl-2-pyrimidine-4-carboxylic Acid

A great majority of the Ru complexes currently studied in anticancer research exert their antiproliferative activity, at least partially, through ligand exchange. In recent years, however, coordinatively saturated and substitutionally inert polypyridyl Ru­(II) compounds have emerged as potential anticancer drug candidates. In this work, we present the synthesis and detailed characterization of two novel inert Ru­(II) complexes, namely, [Ru­(bipy)₂(Cpp-NH-Hex-COOH)]²⁺ (<b>2</b>) and [Ru­(dppz)₂(CppH)]²⁺ (<b>3</b>) (bipy = 2,2′-bipyridine; CppH = 2-(2′-pyridyl)­pyrimidine-4-carboxylic acid; Cpp-NH-Hex-COOH = 6-(2-(pyridin-2-yl)­pyrimidine-4-carboxamido)­hexanoic acid; dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine). <b>3</b> is of particular interest as it was found to have IC₅₀ values comparable to cisplatin, a benchmark standard in the field, on three cancer cell lines and a better activity on one cisplatin-resistant cell line than cisplatin itself. The mechanism of action of <b>3</b> was then investigated in detail and it could be demonstrated that, although <b>3</b> binds to calf-thymus DNA by intercalation, the biological effects that it induces did not involve a nuclear DNA related mode of action. On the contrary, confocal microscopy colocalization studies in HeLa cells showed that <b>3</b> specifically targeted mitochondria. This was further correlated by ruthenium quantification using High-resolution atomic absorption spectrometry. Furthermore, as determined by two independent assays, <b>3</b> induced apoptosis at a relatively late stage of treatment. The generation of reactive oxygen species could be excluded as the cause of the observed cytotoxicity. It was demonstrated that the mitochondrial membrane potential in HeLa was impaired by <b>3</b> as early as 2 h after its introduction and even more with increasing time

Molecular and Cellular Characterization of the Biological Effects of Ruthenium(II) Complexes Incorporating 2‑Pyridyl-2-pyrimidine-4-carboxylic Acid

A great majority of the Ru complexes currently studied in anticancer research exert their antiproliferative activity, at least partially, through ligand exchange. In recent years, however, coordinatively saturated and substitutionally inert polypyridyl Ru­(II) compounds have emerged as potential anticancer drug candidates. In this work, we present the synthesis and detailed characterization of two novel inert Ru­(II) complexes, namely, [Ru­(bipy)₂(Cpp-NH-Hex-COOH)]²⁺ (<b>2</b>) and [Ru­(dppz)₂(CppH)]²⁺ (<b>3</b>) (bipy = 2,2′-bipyridine; CppH = 2-(2′-pyridyl)­pyrimidine-4-carboxylic acid; Cpp-NH-Hex-COOH = 6-(2-(pyridin-2-yl)­pyrimidine-4-carboxamido)­hexanoic acid; dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine). <b>3</b> is of particular interest as it was found to have IC₅₀ values comparable to cisplatin, a benchmark standard in the field, on three cancer cell lines and a better activity on one cisplatin-resistant cell line than cisplatin itself. The mechanism of action of <b>3</b> was then investigated in detail and it could be demonstrated that, although <b>3</b> binds to calf-thymus DNA by intercalation, the biological effects that it induces did not involve a nuclear DNA related mode of action. On the contrary, confocal microscopy colocalization studies in HeLa cells showed that <b>3</b> specifically targeted mitochondria. This was further correlated by ruthenium quantification using High-resolution atomic absorption spectrometry. Furthermore, as determined by two independent assays, <b>3</b> induced apoptosis at a relatively late stage of treatment. The generation of reactive oxygen species could be excluded as the cause of the observed cytotoxicity. It was demonstrated that the mitochondrial membrane potential in HeLa was impaired by <b>3</b> as early as 2 h after its introduction and even more with increasing time

EGF Receptor-Targeting Peptide Conjugate Incorporating a Near-IR Fluorescent Dye and a Novel 1,4,7-Triazacyclononane-Based ⁶⁴Cu(II) Chelator Assembled via Click Chemistry

A new Boc-protected 1,4,7-triazacyclononane (TACN)-based pro-chelator compound featuring a “clickable” azidomethylpyridine pendant has been developed as a building block for the construction of multimodal imaging agents. Conjugation to a model alkyne (propargyl alcohol), followed by deprotection, generates a pentadentate ligand, as confirmed by X-ray crystallographic analysis of the corresponding distorted square-pyramidal Cu­(II) complex. The ligand exhibits rapid ⁶⁴Cu­(II)-binding kinetics (>95% radiochemical yield in <5 min) and a high resistance to demetalation. It may thus prove suitable for use in ⁶⁴Cu­(II)-based <i>in vivo</i> positron emission tomography (PET). The new chelating building block has been applied to the construction of a bimodal (PET/fluorescence) peptide-based imaging probe targeting the epidermal growth factor (EGF) receptor, which is highly overexpressed on the surface of several types of cancer cells. The probe consists of a hexapeptide sequence, Leu-Ala-Arg-Leu-Leu-Thr (designated “D4”), followed by a Cys-β-Ala-β-Ala spacer, then a β-homopropargylglycine residue with the TACN-based chelator “clicked” to its side chain. A sulfonated near-infrared (NIR) fluorescent cyanine dye (sulfo-Cy5) was introduced at the N-terminus to study the EGF receptor-binding ability of the probe by laser-fluorescence spectroscopy. Binding was also confirmed by coimmunoprecipitation methods, and an apparent dissociation constant (<i>K</i>_d) of ca. 10 nM was determined from radioactivity-based measurements of probe binding to two EGF receptor-expressing cell lines (FaDu and A431). The probe is shown to be a biased or partial allosteric agonist of the EGF receptor, inducing phosphorylation of Thr669 and Tyr992, but not the Tyr845, Tyr998, Tyr1045, Tyr1068, or Tyr1148 residues of the receptor, in the absence of the orthosteric EGF ligand. Additionally, the probe was found to suppress the EGF-stimulated autophosphorylation of these latter residues, indicating that it is also a noncompetitive antagonist