Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

Dynamic Ir(III) Photosensors for the Major Human Drug-Metabolizing Enzyme Cytochrome P450 3A4.

Probing the activity of cytochrome P450 3A4 (CYP3A4) is critical for monitoring the metabolism of pharmaceuticals and identifying drug-drug interactions. A library of Ir(III) probes that detect occupancy of the CYP3A4 active site were synthesized and characterized. These probes show selectivity for CYP3A4 inhibition, low cellular toxicity, Kd values as low as 9 nM, and are highly emissive with lifetimes up to 3.8 µs in cell growth media under aerobic conditions. These long emission lifetimes allow for time-resolved gating to distinguish probe from background autofluorescence from growth media and live cells. X-ray crystallographic analysis revealed structure-activity relationships and the preference or indifference of CYP3A4 toward resolved stereoisomers. Ir(III)-based probes show emission quenching upon CYP3A4 binding, then emission increases following displacement with CYP3A4 inhibitors or substrates. Importantly, the lead probes inhibit the activity of CYP3A4 at concentrations as low as 300 nM in CYP3A4-overexpressing HepG2 cells that accurately mimic human hepatic drug metabolism. Thus, the Ir(III)-based agents show promise as novel chemical tools for monitoring CYP3A4 active site occupancy in a high-throughput manner to gain insight into drug metabolism and drug-drug interactions.

Metalloimmunotherapy with Rhodium and Ruthenium Complexes: Targeting Tumor-Associated Macrophages.

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.

Development of a Highly Selective Ni(II) Chelator in Aqueous Solution.

The design, synthesis, and characterization of a novel Ni(II) chelator SG-20 is reported. SG-20 is selective in binding to Ni(II) versus other metal ions including Cu(II), Fe(II), Co(II), and Zn(II). At pH = 7.1, SG-20 binds Ni(II) with a Kd = 7.0 ± 0.4 µM. Job analysis indicates that SG-20 binds to both Ni(II) and Cu(II) with a 1:1 stoichiometry. Affinity of SG-20 for Ni(II) is pH dependent and decreases upon lowering to pH 4.0. A green solid was isolated from the reaction of SG-20 with NiCl2·6H2O in MeOH and characterized by X-ray photoelectron spectroscopy (XPS), electronic absorption and infrared (IR) spectroscopies, and mass spectrometry. Collectively, XPS and IR analysis revealed Ni-N and Ni-O interactions and a shift in C-O asymmetric and symmetric stretches consistent with Ni binding. Attempts to crystalize a mononuclear complex were unsuccessful, likely due to the Ni-SG-20 complex being in equilibrium with higher order species in solution. However, reaction of SG-20 with NiCl2·6H2O in water followed by slow evaporation yielded green crystals that were characterized by electronic absorption spectroscopy (λmax = 260 nm) and X-ray crystallography. These analyses revealed that SG-20 supports formation of a complex cluster containing six SG-20 ligands, 15 Ni(II), and three Na(I) centers, with two distinct types of Ni atoms in its outer and inner core. The nine Ni atoms present in the inner core were bound by oxo and carbonate bridges, whereas the six Ni atoms present in its outer shell were bound to N, O, and S donor atoms derived from SG-20. Overall, X-ray crystallographic analysis revealed that two chelator arms of SG-20 bind to one Ni(II) ion with an axial aqua ligand, whereas the third arm is free to interact with Ni ions within the central cluster, supporting the goal of Ni capture.

Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2':6',2″-terpyridine) with L = 6,6'-dimethyl-2,2'-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7-11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9-11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.

Ru(II)-Based Acetylacetonate Complexes Induce Apoptosis Selectively in Cancer Cells.

The synthesis, chemical and biological characterization of seven Ru(II) polypyridyl complexes containing acetylacetonate (acac) ligands are reported. Electronic absorption spectra were determined and electrochemical potentials consistent with Ru(III/II) couples ranging from +0.60 to +0.73 V vs Ag/AgCl were measured. A series of complexes were screened against MDA-MB-231, DU-145, and MCF-10A cell lines to evaluate their cytotoxicities in cancer and normal cell lines. Although most complexes were either nontoxic or equipotent in cancer cells and normal cell lines, compound 1, [Ru(dpqy)(acac)(py)](PF6), where dqpy is 2,6-di(quinolin-2-yl)pyridine, showed up to 2.5:1.0 selectivity for cancer as compared to normal cells, along with nanomolar EC50 values in MDA-MB-231 cells. Lipophilicity, determined as the octanol/water partition coefficient, log Po/w, ranged from -0.33 (0.06) to 1.15 (0.10) for the complexes. Although cytotoxicity was not correlated with electrochemical potentials, a moderate linear correlation between lipophilicity and toxicities was observed. Cell death mechanism studies indicated that several of the Ru-acac compounds, including 1, induce apoptosis in MDA-MB-231 cells.

Dual-Action Ru(II) Complexes with Bulky π-Expansive Ligands: Phototoxicity without DNA Intercalation.

We report the synthesis and photochemical and biological characterization of Ru(II) complexes containing π-expansive ligands derived from dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn) adorned with flanking aryl substituents. Late-stage Suzuki couplings produced Me2dppn ligands substituted at the 10 and 15 positions with phenyl (5), 2,4-dimethylphenyl (6), and 2,4-dimethoxyphenyl (7) groups. Complexes of the general formula [Ru(tpy)(L)(py)](PF6)2 (8-10), where L = 4-7, were characterized and shown to have dual photochemotherapeutic (PCT) and photodynamic therapy (PDT) behavior. Quantum yields for photodissociation of monodentate pyridines from 8-10 were about 3 times higher than that of parent complex [Ru(tpy)(Me2dppn)(py)](PF6)2 (1), whereas quantum yields for singlet oxygen (1O2) production were ∼10% lower than that of 1. Transient absorption spectroscopy indicates that 8-10 possess long excited state lifetimes (τ = 46-50 µs), consistent with efficient 1O2 production through population and subsequent decay of ligand-centered 3ππ* excited states. Complexes 8-10 displayed greater lipophilicity relative to 1 and association to DNA but do not intercalate between the duplex base pairs. Complexes 1 and 8-10 showed photoactivated toxicity in breast and prostate cancer cell lines with phototherapeutic indexes, PIs, as high as >56, where the majority of cell death was achieved 4 h after treatment with Ru(II) complexes and light. Flow cytometric data and rescue experiments were consistent with necrotic cell death mediated by the production of reactive oxygen species, especially 1O2. Collectively, this study confirms that DNA intercalation by Ru(II) complexes with π-expansive ligands is not required to achieve photoactivated cell death.

Ru(II) Polypyridyl Complexes Derived from Tetradentate Ancillary Ligands for Effective Photocaging.

Metal complexes have many proven applications in the caging and photochemical release of biologically active compounds. Photocaging groups derived from Ru(II) traditionally have been composed of ancillary ligands that are planar and bi- or tridentate, such as 2,2'-bipyridine (bpy), 2,2':6',2″-terpyridine (tpy), and 1,10-phenanthroline (phen). Complexes bearing ancillary ligands with denticities higher than three represent a new class of Ru(II)-based photocaging groups that are grossly underdeveloped. Because high-denticity ancillary ligands provide the ability to increase the structural rigidity and control the stereochemistry, our groups initiated a research program to explore the applications of such ligands in Ru(II)-based photocaging. Ru(TPA), bearing the tetradentate ancillary ligand tris(2-pyridylmethyl)amine (TPA), has been successfully utilized to effectively cage nitriles and aromatic heterocycles. Nitriles and aromatic heterocycles caged by the Ru(TPA) group show excellent stability in aqueous solutions in the dark, and the complexes can selectively release the caged molecules upon irradiation with light. Ru(TPA) is applicable as a photochemical agent to offer precise spatiotemporal control over biological activity without undesired toxicity. In addition, Ru(II) polypyridyl complexes with desired photochemical properties can be synthesized and identified by solid-phase synthesis, and the resulting complexes show properties to similar to those of complexes obtained by solution-phase synthesis. Density functional theory (DFT) calculations reveal that orbital mixing between the π* orbitals of the ancillary ligand and the Ru-N dσ* orbital is essential for ligand photodissociation in these complexes. Furthermore, the introduction of steric bulk enhances the photoliability of the caged molecules, validating that steric effects can largely influence the quantum efficiency of photoinduced ligand exchange in Ru(II) polypyridyl complexes. Recently, two new photocaging groups, Ru(cyTPA) and Ru(1-isocyTPQA), have been designed and synthesized for caging of nitriles and aromatic heterocycles, and these complexes exhibit unique photochemical properties distinct from those derived from Ru(TPA). Notably, the unusually greater quantum efficiency for the ligand exchange in [Ru(1-isocyTPQA)(MeCN)2](PF6)2, Φ400 = 0.033(3), uncovers a trans-type effect in the triplet metal-to-ligand charge transfer (3MLCT) state that enhances photoinduced ligand exchange in a new manner. DFT calculations and ultrafast transient spectroscopy reveal that the lowest-energy triplet state in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is a highly mixed 3MLCT/3ππ* excited state rather than a triplet metal-centered ligand-field (3LF) excited state; the latter is generally accepted for ligand photodissociation. In addition, Mulliken spin density calculations indicate that a majority of the spin density in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is localized on the isoquinoline arm, which is opposite to the cis MeCN, rather than on the ruthenium center. This significantly weakens the Ru-N6 ( cis MeCN) bond, which then promotes the ligand photodissociation. This newly discovered effect gives a clearer perception of the interplay between the 3MLCT and 3LF excited states of Ru(II) polypyridyl complexes, which may be useful in the design and applications of ruthenium complexes in the areas of photoactivated drug delivery and photosensitizers.

Catch and Release Photosensitizers: Combining Dual-Action Ruthenium Complexes with Protease Inactivation for Targeting Invasive Cancers.

Dual action agents containing a cysteine protease inhibitor and Ru-based photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Ru-based PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Ru-based complexes were synthesized with the formula [Ru(bpy)2(1)](O2CCF3)2 (3), where bpy = 2,2'-bipyridine and 1 = a bipyridine-based epoxysuccinyl inhibitor; [Ru(tpy)(NN)(2)](PF6)2, where tpy = terpiridine, 2 = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2'-bipyridine (4); 6,6'-dimethyl-2,2'-bipyridine (5); benzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (6); and 3,6-dimethylbenzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (7). Compound 3 contains a [Ru(bpy)3]2+ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds 4-7 were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes 5 and 7 undergo photoactivated ligand dissociation, whereas 6 and 7 generate singlet oxygen. Inhibitors 1-7 all potently and irreversibly inhibit CTSB. Compounds 4-7 were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that 4-7 potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound 7 causes efficient cell death under light conditions, consistent with its ability to release a Ru(II)-based photosensitizer and to also generate 1O2.

DFT Investigation of Ligand Photodissociation in [RuII(tpy)(bpy)(py)]2+ and [RuII(tpy)(Me2bpy)(py)]2+ Complexes.

Photoinduced ligand dissociation of pyridine occurs much more readily in [Ru(tpy)(Me2bpy)(py)]2+ than in [Ru(tpy)(bpy)(py)]2+ (tpy = 2,2':6',2″-terpyridine; bpy = 2,2'-bipyridine, Me2bpy = 6,6'-dimethyl-2,2'-bipyridine; py = pyridine). The S0 ground state and the 3MLCT and 3MC excited states of these complexes have been studied using BP86 density functional theory with the SDD basis set and effective core potential on Ru and the 6-31G(d) basis set for the rest of the atoms. In both complexes, excitation by visible light and intersystem crossing leads to a 3MLCT state in which an electron from a Ru d orbital has been promoted to a π* orbital of terpyridine, followed by pyridine release after internal conversion to a dissociative 3MC state. Interaction between the methyl groups and the other ligands causes significantly more strain in [Ru(tpy)(Me2bpy)(py)]2+ than in [Ru(tpy)(bpy)(py)]2+, in both the S0 and 3MLCT states. Transition to the dissociative 3MC states releases this strain, resulting in lower barriers for ligand dissociation from [Ru(tpy)(Me2bpy)(py)]2+ than from [Ru(tpy)(bpy)(py)]2+. Analysis of the molecular orbitals along relaxed scans for stretching the Ru-N bonds reveals that ligand photodissociation is promoted by orbital mixing between the ligand π* orbital of tpy in the 3MLCT state and the dσ* orbitals that characterize the dissociative 3MC states. Good overlap and strong mixing occur when the Ru-N bond of the leaving ligand is perpendicular to the π* orbital of terpyridine, favoring the release of pyridine positioned in a cis fashion to the terpyridine ligand.

Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of the Cysteine Protease Cathepsin L.

Two new Re(I)- and Ru(II)-based inhibitors were synthesized with the formulas [Re(phen)(CO)3(1)](OTf) (7; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru(bpy)2(2)](Cl)2 (8; bpy = 2,2'-bipyridine), where 1 and 2 are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds 7 and 8 were characterized using various spectroscopic techniques and elemental analysis; 7 and 8 both show exceptionally long excited state lifetimes. Re(I)-based complex 7 inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor 1 with a second-order rate constant of 211000 ± 42000 M-1 s-1. Irreversible ligation of 7 to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-1-Re(CO)3(phen). Compound 7 was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with 7 followed by imaging via confocal microscopy showed substantial intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of 7 to label CTSL in living cells. Overall this study reveals that a Re(I) complex can be attached to an enzyme inhibitor and enhance potency and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due to long excited state lifetimes and non-native element composition.

Unusual Role of Excited State Mixing in the Enhancement of Photoinduced Ligand Exchange in Ru(II) Complexes.

Four Ru(II) complexes were prepared bearing two new tetradentate ligands, cyTPA and 1-isocyTPQA, which feature a piperidine ring that provides a structurally rigid backbone and facilitates the installation of other donors as the fourth chelating arm, while avoiding the formation of stereoisomers. The photophysical properties and photochemistry of [Ru(cyTPA)(CH3CN)2]2+ (1), [Ru(1-isocyTPQA)(CH3CN)2]2+ (2), [Ru(cyTPA)(py)2]2+ (3), and [Ru(1-isocyTPQA)(py)2]2+ (4) were compared. The quantum yield for the CH3CN/H2O ligand exchange of 2 was measured to be Φ400 = 0.033(3), 5-fold greater than that of 1, Φ400 = 0.0066(3). The quantum yields for the py/H2O ligand exchange of 3 and 4 were lower, 0.0012(1) and 0.0013(1), respectively. DFT and related calculations show the presence of a highly mixed 3MLCT/3ππ* excited state as the lowest triplet state in 2, whereas the lowest energy triplet states in 1, 3, and 4 were calculated to be 3LF in nature. The mixed 3MLCT/3ππ* excited state places significant spin density on the quinoline moiety of the 1-isocyTPQA ligand positioned trans to the photolabile CH3CN ligand in 2, suggesting the presence of a trans-type influence in the excited state that enhances ligand exchange. Ultrafast spectroscopy was used to probe the excited states of 1-4, which confirmed that the mixed 3MLCT/3ππ* excited state in 2 promotes ligand dissociation, representing a new manner to effect photoinduced ligand exchange. The findings from this work can be used to design improved complexes for applications that require efficient ligand dissociation, as well as for those that require minimal deactivation of the 3MLCT state through low-lying metal-centered states.

Photoactivated inhibition of cathepsin K in a 3D tumor model.

Collagenolytic activity of cathepsin K is important for many physiological and pathological processes including osteoclast-mediated bone degradation, macrophage function and fibroblast-mediated matrix remodeling. Here, we report application of a light-activated inhibitor for controlling activity of cathepsin K in a 3D functional imaging assay. Using prostate carcinoma cell line engineered to overexpress cathepsin K, we demonstrate the utility of the proteolytic assay in living tumor spheroids for the evaluation and quantification of the inhibitor effects on cathepsin K-mediated collagen I degradation. Importantly, we also show that utilizing the ruthenium-caged version of a potent nitrile cathepsin K inhibitor (4), cis-[Ru(bpy)2(4)2](BF4)2 (5), offers significant advantage in terms of effective concentration of the inhibitor and especially its light-activated control in the 3D assay. Our results suggest that light activation provides a suitable, attractive approach for spatial and temporal control of proteolytic activity, which remains a critical, unmet need in treatment of human diseases, especially cancer.

New Ru(II) Complex for Dual Activity: Photoinduced Ligand Release and (1)O2 Production.

The new complex [Ru(pydppn)(biq)(py)](2+) (1) undergoes both py photodissociation in CH3CN with Φ500 =0.0070(4) and (1)O2 production with ΦΔ =0.75(7) in CH3 OH from a long-lived (3) ππ* state centered on the pydppn ligand (pydppn=3-(pyrid-2-yl)benzo[i]dipyrido[3,2-a:2',3'-c]phenazine; biq = 2,2'-biquinoline; py=pyridine). This represents an order of magnitude decrease in the Φ500 compared to the previously reported model compound [Ru(tpy)(biq)(py)](2+) (3) (tpy=2,2':6',2''-terpyridine) that undergoes only ligand exchange. The effect on the quantum yields by the addition of a second deactivation pathway through the low-lying (3) ππ* state necessary for dual reactivity was investigated using ultrafast and nanosecond transient absorption spectroscopy, revealing a significantly shorter (3) MLCT lifetime in 1 relative to that of the model complex 3. Due to the structural similarities between the two compounds, the lower values of Φ500 and ΦΔ compared to that of [Ru(pydppn)(bpy)(py)](2+) (2) (bpy=2,2'-bipyridine) are attributed to a competitive excited state population between the (3) LF states involved in ligand dissociation and the long-lived (3) ππ* state in 1. Complex 1 represents a model compound for dual activity that may be applied to photochemotherapy.

Effects of Methyl Substitution in Ruthenium Tris(2-pyridylmethyl)amine Photocaging Groups for Nitriles.

Four complexes of the general formula [Ru(L)(CH3CN)2](PF6)2, [L = TPA (5), MeTPA (6), Me2TPA (7), and Me3TPA (8)] [TPA = tris[(pyridin-2-yl)methyl]amine, where methyl groups were introduced consecutively onto the 6-position of py donors of TPA, were prepared and characterized by various spectroscopic techniques and mass spectrometry. While 5 and 8 were isolated as single stereoisomers, 6 and 7 were isolated as mixtures of stereoisomers in 2:1 and 1.5:1 ratios, respectively. Steric effects on ground state stability and thermal and photochemical reactivities were studied for all four complexes using (1)H NMR and electronic absorption spectroscopies and computational studies. These studies confirmed that the addition of steric bulk accelerates photochemical and thermal nitrile release.

Selective Release of Aromatic Heterocycles from Ruthenium Tris(2-pyridylmethyl)amine with Visible Light.

Three complexes of the general formula [Ru(TPA)L2](PF6)2 [TPA = tris(2-pyridylmethyl)amine], where L = pyridine (1), nicotinamide (2), and imidazole (3), were prepared and characterized spectroscopically. X-ray crystallographic data were obtained for 1 and 3. Complexes 1-3 show strong absorption in the visible region and selective release of heterocycles upon irradiation with visible light. Time-dependent density functional theory calculations are consistent with the presence of singlet metal-to-ligand charge-transfer bands in the visible region in 1-3. Caged heterocycles 1-3 are highly stable in solution in the dark, including in cell growth media. Cell viability data show no signs of toxicity of 1-3 against PC-3 cells at concentrations up to 100 µM under light and dark conditions, consistent with Ru(TPA) acting as a nontoxic and effective photocaging group for aromatic heterocycles.

Selective Photodissociation of Acetonitrile Ligands in Ruthenium Polypyridyl Complexes Studied by Density Functional Theory.

Metal complexes that release ligands upon photoexcitation are important tools for biological research and show great potential as highly specific therapeutics. Upon excitation with visible light, [Ru(TQA)(MeCN)2](2+) [TQA = tris(2-quinolinylmethyl)amine] exchanges one of the two acetonitriles (MeCNs), whereas [Ru(DPAbpy)MeCN](2+) [DPAbpy = N-(2,2'-bipyridin-6-yl)-N,N-bis(pyridin-2-ylmethyl)amine] does not release MeCN. Furthermore, [Ru(TQA)(MeCN)2](2+) is highly selective for release of the MeCN that is perpendicular to the plane of the two axial quinolines. Density functional theory calculations provide a clear explanation for the photodissociation behavior of these two complexes. Excitation by visible light and intersystem crossing leads to a six-coordinate (3)MLCT state. Dissociation of acetonitrile can occur after internal conversion to a dissociative (3)MC state, which has an occupied dσ* orbital that interacts in an antibonding fashion with acetonitrile. For [Ru(TQA)(MeCN)2](2+), the dissociative (3)MC state is lower than the (3)MLCT state. In contrast, the (3)MC state of [Ru(DPAbpy)MeCN](2+) that releases acetonitrile has an energy higher than that of the (3)MLCT state, indicating dissociation is unfavorable. These results are consistent with the experimental observations that efficient photodissociation of acetonitrile occurs for [Ru(TQA)(MeCN)2](2+) but not for [Ru(DPAbpy)MeCN](2+). For the release of the MeCN ligand in [Ru(TQA)(MeCN)2](2+) that is perpendicular to the axial quinoline rings, the (3)MLCT state has an occupied quinoline π* orbital that can interact with a dσ* Ru-NCCH3 antibonding orbital as the Ru-NCCH3 bond is stretched and the quinolines bend toward the departing acetonitrile. This reduces the barrier for the formation of the dissociative (3)MC state, leading to the selective photodissociation of this acetonitrile. By contrast, when the acetonitrile is in the plane of the quinolines or bpy, no interaction occurs between the ligand π* orbital and the dσ* Ru-NCCH3 orbital, resulting in high barriers for conversion to the corresponding (3)MC structures and no release of acetonitrile.

Solid-phase synthesis as a platform for the discovery of new ruthenium complexes for efficient release of photocaged ligands with visible light.

Ruthenium-based photocaging groups have important applications as biological tools and show great potential as therapeutics. A method was developed to rapidly synthesize, screen, and identify ruthenium-based caging groups that release nitriles upon irradiation with visible light. A diverse library of tetra- and pentadentate ligands was synthesized on polystyrene resin. Ruthenium complexes of the general formula [Ru(L)(MeCN)n](m+) (n = 1-3, m = 1-2) were generated from these ligands on solid phase and then cleaved from resin for photochemical analysis. Data indicate a wide range of spectral tuning and reactivity with visible light. Three complexes that showed strong absorbance in the visible range were synthesized by solution phase for comparison. Photochemical behavior of solution- and solid-phase complexes was in good agreement, confirming that the library approach is useful in identifying candidates with desired photoreactivity in short order, avoiding time-consuming chromatography and compound purification.

Ruthenium tris(2-pyridylmethyl)amine as an effective photocaging group for nitriles.

Ruthenium(II) tris(2-pyridylmethyl)amine (TPA) is an effective caging group for nitriles that provides high levels of control over the enzyme activity with light. Two caged nitriles were prepared, [Ru(TPA)(MeCN)2](PF6)2 (1) and [Ru(TPA)(3)2](PF6)2 (2), where 3 is the cathepsin K inhibitor Cbz-Leu-NHCH2CN, and characterized by various spectroscopic techniques and mass spectrometry. Both 1 and 2 show the release of a single nitrile within 20 min of irradiation with 365 nm light. Complex 2 acts as a potent, photoactivated inhibitor of human cathepsin K. IC50 values were determined for 2 and 3. Enzyme inhibition for 2 was enhanced by a factor of 89 upon exposure to light, with IC50 values of 63 nM (light) and 5.6 µM (dark).