Quinol-containing ligands enable high superoxide dismutase activity by modulating coordination number,charge, oxidation states and stability of manganese complexes throughout redox cycling

Reactivity assays previously suggested that two quinol-containing MRI contrast agent sensors for H₂O₂, [Mn(H2qp1)(MeCN)]²⁺ and [Mn(H4qp2)Br₂], could also catalytically degrade superoxide. Subsequently, [Zn(H2qp1)(OTf)]⁺ was found to use the redox activity of the H2qp1 ligand to catalyze the conversion of O₂˙⁻ to O₂ and H₂O₂, raising the possibility that the organic ligand, rather than the metal, could serve as the redox partner for O₂˙⁻ in the manganese chemistry. Here, we use stopped-flow kinetics and cryospray-ionization mass spectrometry (CSI-MS) analysis of the direct reactions between the manganese-containing contrast agents and O₂˙⁻ to confirm the activity and elucidate the catalytic mechanism. The obtained data are consistent with the operation of multiple parallel catalytic cycles, with both the quinol groups and manganese cycling through different oxidation states during the reactions with superoxide. The choice of ligand impacts the overall charges of the intermediates and allows us to visualize complementary sets of intermediates within the catalytic cycles using CSI-MS. With the diquinolic H4qp2, we detect Mn(iii)-superoxo intermediates with both reduced and oxidized forms of the ligand, a Mn(iii)-hydroperoxo compound, and what is formally a Mn(iv)-oxo species with the monoquinolate/mono-para-quinone form of H4qp2. With the monoquinolic H2qp1, we observe a Mn(ii)-superoxo ↔ Mn(iii)-peroxo intermediate with the oxidized para-quinone form of the ligand. The observation of these species suggests inner-sphere mechanisms for O₂˙⁻ oxidation and reduction that include both the ligand and manganese as redox partners. The higher positive charges of the complexes with the reduced and oxidized forms of H2qp1 compared to those with related forms of H4qp2 result in higher catalytic activity (k_cat ∼ 10⁸ M⁻¹ s⁻¹ at pH 7.4) that rivals those of the most active superoxide dismutase (SOD) mimics. The manganese complex with H2qp1 is markedly more stable in water than other highly active non-porphyrin-based and even some Mn(ii) porphyrin-based SOD mimics.

Manganese complexes with polydentate quinol-containing ligands are found to catalyze the degradation of superoxide through inner-sphere mechanisms. The redox activity of the ligand stabilizes higher-valent manganese species.     

Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450cam‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide

The hydroperoxo iron(III) intermediate P450_camFe^III–OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450_cam, could be transiently observed in the peroxo‐shunt oxidation of the substrate‐free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450_camFe^III–OOH species as a function of varying reaction conditions, such as pH, and concentration of H₂O₂, camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate‐free form of P450_cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid–base catalyst and is mainly governed by the ability of H₂O₂ to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$ . Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic O?O bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)‐camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe^III–OOH intermediate in the oxidation of the natural substrate of P450_cam.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min^?1. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL^?1 for isomer I and 0.40–2.40 µg mL^?1 for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL^?1 for isomer I and 0.0356 and 0.1078 µg mL^?1 for isomer II, respectively.     

An Approach to Transfer Methods from HPLC to UHPLC Techniques in Some Carbapenems

Stability-indicating LC methods were developed and validated for the quantitative determination of doripenem, meropenem and tebipenem in the presence of their degradation products formed during forced degradation studies. Isocratic HPLC and UHPLC separations were performed with a core–shell Kinetex 1.7, 2.6 and 5 µm, all C18, 100A, 100 × 2.1 mm columns and the mobile phase composed of acetonitrile and 12 mmol L⁻¹ ammonium acetate in different ratios. The flow rates of the mobile phase were: 0.5 mL min⁻¹ for 1.7 µm column, and 1.0 mL min⁻¹ for 2.6 and 5 µm ones. Detection wavelength was 298 nm and temperature was set at 30 °C. All analysed drugs were exposed to stress conditions which caused their hydrolysis and thermal degradation. The methods were validated by evaluation of linearity, accuracy, precision, selectivity and robustness. Proposed methods were successfully applied for the determination of investigated antibiotics during kinetic studies in aqueous solutions and in the solid state. The advantages of chromatographic procedures which are based on the use of C18 stationary phases with different particle sizes in the analysis of selected carbapenems were discussed.

     

Liquid chromatography tandem mass spectrometry with ion trap and triple quadrupole analyzers for determination of thyreostatic drugs in urine and muscle tissue

Liquid chromatography tandem mass spectrometry methods were developed and validated to screen for and confirm residues of the thyreostatic drugs: tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil in bovine and porcine urine and muscle tissues using dimethylthiouracil as internal standard. Thyreostats were extracted from urine samples with diethyl ether after derivatisation with 3-iodobenzylbromide in basic medium (pH 8.0) and analyzed by gradient elution on a Nucleosil C18 column with ion trap mass spectrometry detection using an electrospray source and triple quadrupole MS detection with turbo spray source. Thyreostats were extracted from muscle tissue with methanol, the denaturation of matrix protein was performed and then the same steps as for the urine samples were carried out. The methods were validated in accordance with the Commission Decision 2002/657/EC. Good thyreostats recoveries were obtained (from 82% to 117%) as well as acceptable within-lab reproducibility. The values of the decision limit CCα and the detection capability CCβ of five thyreostatic drugs are found to be below the recommended concentration set at 10 μg L(-1) (kg(-1)). The results of the validation demonstrate that liquid chromatography mass spectrometry with ion trap detection does not meet the criteria for confirmation for some thyreostats and therefore was applied for screening purpose only.     

The effect of lipid oxidation on the water permeability of phospholipids bilayers

The effect of lipid oxidation on water permeability of phosphatidylcholine membranes was investigated by means of both scattering stopped flow experiments and atomistic molecular dynamics simulations. Formation of water pores followed by a significant enhancement of water permeability was observed. The molecules of oxidized phospholipids facilitate pore formation and subsequently stabilize water in the membrane interior. A wide range of oxidation ratios, from 15 to 100 mol%, was considered. The degree of oxidation was found to strongly influence the time needed for the opening of a pore. In simulations, the oxidation ratio of 75 mol% was found to be a threshold for spontaneous pore formation in the tens of nanosecond timescale, whereas 15 mol% of oxidation led to significant water permeation in the timescale of seconds. Once a pore was formed, the water permeability was found to be virtually independent of the oxidation ratio.     

Preorganization in highly enantioselective diaza-Cope rearrangement reaction

Crystal structure and activation entropy data indicate that H-bond directed diaza-Cope rearrangement of chiral diimines takes place with a high degree of preorganization. CD spectroscopy and HPLC data show that there is inversion of stereochemistry for the reaction with excellent enantioselectivity.