Synthesis, Characterization, and Glutathionylation of Cobalamin Model Complexes [Co(N4PyCO₂Me)Cl]Cl₂ and [Co(Bn-CDPy3)Cl]Cl₂

Synthetic Co­(III) complexes containing N5 donor sets undergo glutathionylation to generate biomimetic species of glutathionylcobalamin (GSCbl), an important form of cobalamin (Cbl) found in nature. For this study, a new Co­(III) complex was synthesized derived from the polypyridyl pentadentate N5 ligand N4PyCO₂Me (<b>1</b>). The compound [Co­(N4PyCO₂Me)­Cl]­Cl₂ (<b>3</b>) was characterized by X-ray crystallography, UV–vis, IR, ¹H NMR, and ¹³C NMR spectroscopies and mass spectrometry (HRMS). Reaction of <b>3</b> with glutathione (GSH) in H₂O generates the biomimetic species [Co­(N4PyCO₂Me)­(SG)]²⁺ (<b>5</b>), which was generated <i>in situ</i> and characterized by UV–vis and ¹H NMR spectroscopies and HRMS. ¹H NMR and UV–vis spectroscopic data are consistent with ligation of the cysteine thiolate of GSH to the Co­(III) center of <b>5</b>, as occurs in GSCbl. Kinetic analysis indicated that the substitution of chloride by GS^– occurs by a second-order process [<i>k</i>₁ = (10.1 ± 0.7) × 10^–2 M^–1 s^–1]. The observed equilibrium constant for formation of <b>5</b> (<i>K</i>_obs = 870 ± 50 M^–1) is about 3 orders of magnitude smaller than for GSCbl. Reaction of the Co­(III) complex [Co­(Bn-CDPy3)­Cl]­Cl₂ (<b>4</b>) with GSH generates glutathionylated species [Co­(Bn-CDPy3)­(GS)]²⁺ (<b>6</b>), analogous to <b>5</b>. Glutathionylation of <b>4</b> occurs at a similar rate [<i>k</i>₂ = (8.4 ± 0.5) × 10^–2 M^–1 s^–1], and the observed equilibrium constant (<i>K</i>_obs = 740 ± 47 M^–1) is slightly smaller than for <b>5</b>. Glutathionylation showed a significant pH dependence, where rates increased with pH. Taken together, these results suggest that glutathionylation is a general reaction for Co­(III) complexes related to Cbl

Cumyl Ester as the C-Terminal Protecting Group in the Enantioselective Alkylation of Glycine Benzophenone Imine

Cumyl ester is an optimal C-terminal protecting group for glycine benzophenone imine in asymmetric alkylation reactions catalyzed by <i>Cinchona</i> chiral phase-transfer catalysts. High levels of enantioselectivity have been obtained (up to 94% ee) with this substrate, which provides an attractive alternative to the analogous <i>tert</i>-butyl ester. N-terminal imines and the C-terminal esters can be cleaved from alkylation products by hydrogenolysis, while maintaining acid-labile side chain protecting groups

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(<b>1</b>)]­(OTf) (<b>7</b>; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru­(bpy)₂(<b>2</b>)]­(Cl)₂ (<b>8</b>; bpy = 2,2′-bipyridine), where <b>1</b> and <b>2</b> are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds <b>7</b> and <b>8</b> were characterized using various spectroscopic techniques and elemental analysis; <b>7</b> and <b>8</b> both show exceptionally long excited state lifetimes. Re­(I)-based complex <b>7</b> inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor <b>1</b> with a second-order rate constant of 211000 ± 42000 M^–1 s^–1. Irreversible ligation of <b>7</b> to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-<b>1</b>-Re­(CO)₃(phen). Compound <b>7</b> was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with <b>7</b> 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 <b>7</b> 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

DFT Investigation of Ligand Photodissociation in [Ru^II(tpy)(bpy)(py)]²⁺ and [Ru^II(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 <i>cis</i> fashion to the terpyridine ligand

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

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)]Cl(2) (tpy = 2,2′:6′,2″-terpyridine) with L = 6,6′-dimethyl-2,2′-bipyridine (Me(2)bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (Me(2)dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (Me(2)Ph(2)dppn; 11), [Ru(tpy)(Me(2)bpy)(4)]Cl(2) (7) and [Ru(tpy)(Me(2)dppn)(4)]Cl(2) (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 (1)O(2) 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