Inhibition screening method of microsomal UGTs using the cocktail approach.

A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45-min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7µm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400µl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Chemo-genetic optimization of DNA recognition by metallodrugs using a presenter-protein strategy.

The mode of action of precious metal anticancer metallodrugs is generally believed to involve DNA as a target. However, the poor specificity of such drugs often requires high doses and leads to undesirable side-effects. With the aim of improving the specificity of a ruthenium piano-stool complex towards DNA, we employed a presenter protein strategy based on the biotin-avidin technology. Guided by the X-ray structure of the assembly of streptavidin and a biotinylated piano-stool, we explored the formation of metallodrug-mediated ternary complexes with the presenter protein and DNA. The assemblies bound more strongly to telomere G-quadruplexes than to double-stranded DNA; chemo-genetic modifications (varying the complex or mutating the protein) modulated binding to these targets. We suggest that rational targeting of small molecules by presenter proteins could be exploited to bind metallodrugs to preferred macromolecular targets.

Site-dependent excited-state dynamics of a fluorescent probe bound to avidin and streptavidin.

The excited-state dynamics of biotin-spacer-Lucifer-Yellow (LY) constructs bound to avidin (Avi) and streptavidin (Sav) was investigated using femtosecond spectroscopy. Two different locations in the proteins, identified by molecular dynamics simulations of Sav, namely the entrance of the binding pocket and the protein surface, were probed by varying the length of the spacer. A reduction of the excited-state lifetime, stronger in Sav than in Avi, was observed with the long spacer construct. Transient absorption measurements show that this effect originates from an electron transfer quenching of LY, most probably by a nearby tryptophan residue. The local environment of the LY chromophore could be probed by measuring the time-dependent polarisation anisotropy and Stokes shift of the fluorescence. Substantial differences in both dynamics were observed. The fluorescence anisotropy decays analysed by using the wobbling-in-a-cone model reveal a much more constrained environment of the chromophore with the short spacer. Moreover, the dynamic Stokes shift is multiphasic in all cases, with a approximately 1 ps component that can be ascribed to diffusive motion of bulk-like water molecules, and with slower components with time constants varying not only with the spacer, but with the protein as well. These slow components, which depend strongly on the local environment of the probe, are ascribed to the motion of the hydration layer coupled to the conformational dynamics of the protein.

Structural, spectral, electric-field-induced second harmonic, and theoretical study of Ni(II), Cu(II), Zn(II), and VO(II) complexes with [N2O2] unsymmetrical schiff bases of S-methylisothiosemicarbazide derivatives.

New unsymmetrical [N2O2] tetradentate Schiff base complexes of Ni(II), Cu(II), Zn(II), and VO(II) were synthesized by template condensation of the tetradentate precursor 1-phenylbutane-1,3-dione mono-S-methylisothiosemicarbazone with o-hydroxybenzaldehyde or its 5-phenylazo derivative. They were characterized by elemental analysis, IR, UV-vis, electron spin resonance, and NMR spectroscopy, mass spectrometry, and magnetic measurements. The crystal structures of five of them have been determined by X-ray diffraction using, in some cases, synchrotron radiation. These compounds are characterized by a large thermal stability; their decomposition temperatures range from 240 up to 310 degrees C. Complexes with the phenylazo substituent were found to possess a large second-order nonlinear optical (NLO) response, as determined both by measurements of solution-phase direct current electric-field-induced second harmonic generation and by theoretical time-dependent density functional theory (TDDFT) calculations. The molecular hyperpolarizability was found to decrease in the order Zn(II) > Cu(II) > Ni(II) approximately VO(II). The active role of the metal in determining the NLO properties of the complexes was shown through an analysis of their UV-vis spectra, which revealed the presence of metal-to-ligand (in closed-shell complexes) and ligand-to-metal (in open-shell complexes) charge-transfer bands together with intra-ligand charge-transfer transitions. Assignment of the bands was based on the analysis of the TDDFT computed spectra.

(Strept)avidin as host for biotinylated coordination complexes: stability, chiral discrimination, and cooperativity.

Incorporation of a biotinylated ruthenium tris(bipyridine) [Ru(bpy)(2)(Biot-bpy)](2+) (1) in either avidin or streptavidin-(strept)avidin-can be conveniently followed by circular dichroism spectroscopy. To determine the stepwise association constants, cooperativity, and chiral discrimination properties, diastereopure (Lambda and Delta)-1 species were synthesized and incorporated in tetrameric (strept)avidin to afford (Delta-[Ru(bpy)(2)(Biot-bpy)](2+))(x)() subsetavidin, (Lambda-[Ru(bpy)(2)(Biot-bpy)](2+))(x)() subsetavidin, (Delta-[Ru(bpy)(2)(Biot-bpy)](2+))(x)() subsetstreptavidin, and (Lambda-[Ru(bpy)(2)(Biot-bpy)](2+))(x)() subsetstreptavidin (x = 1-4) For these four systems, the overall stability constants are log beta(4) = 28.6, 30.3, 36.2, and 36.4, respectively. Critical analysis of the CD titrations data suggests a strong cooperativity between the first and the second binding event (x = 1, 2) and a pronounced difference in affinity between avidin and streptavidin for the dicationic guest 1 as well as modest enantiodiscrimination properties with avidin as host.

Artificial metalloenzymes: (strept)avidin as host for enantioselective hydrogenation by achiral biotinylated rhodium-diphosphine complexes.

We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium-diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium-diphosphine catalyst precursor and the host proteins was determined at neutral pH: log K(a) = 7.7 for avidin (pI = 10.4) and log K(a) = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 degrees C with a 1% catalyst loading.

Alkane hydroperoxidation with peroxides catalysed by copper complexes.

Various copper(I) and copper(II) derivatives, both "simple" ones (copper acetate, perchlorate and a complex with CH3CN) and compounds containing N,O-chelating ligands, catalyse very efficient (turnover numbers attain 2200) oxidation of saturated hydrocarbons with peroxyacetic acid (PAA) or tert-butyl hydroperoxide (TBHP) in acetonitrile solution at 60 degrees C. Alkyl hydroperoxide, alcohol and ketone are formed, the main product being an alkyl hydroperoxide in the oxidation with PAA and an alcohol for the case of TBHP. It has been proposed that the oxidation with PAA is induced via the attack of species r* [HO* or CH3C(=O)O*] on the alkane, RH. A competitive attack of r* on the solvent, CH3CN, also occurs. It has been assumed that in the case of the reaction catalysed by complex Cu(CH3CN)4BF4, copper is present mainly in the form of Cu+ cation, and the rate-limiting step of the oxidation process is the formation of r* via reaction (1): CH3C(=O)OOH + Cu+ --> CH3C(=O)O* + HO- + Cu2+ or/and CH3C(=O)OOH + Cu+ --> CH3C(=O)O- + HO* + Cu2+ with initial rate W1 = k1[PAA][Cu(CH3CN)4BF4] and k1 = 1.7 mol(-1) dm3 s(-1) at 60 degrees C. The activity of the Cu-catalyst is dramatically changed on a small modification of N,O-chelating ligands in the catalyst.

Artificial metalloenzymes for enantioselective catalysis based on biotin-avidin.

Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. Incorporation of achiral biotinylated rhodium-diphosphine complexes into (strept)avidin yields artificial metalloenzymes for the hydrogenation of N-protected dehydroamino acids. A chemogenetic optimization procedure allows one to produce (R)-acetamidoalanine with 96% enantioselectivity. These hybrid catalysts display features reminiscent both of enzymatic and of homogeneous systems.

Conformational study of S-alkylated isothiosemicarbazones.

A theoretical investigation of the conformational preferences of S-alkylated isothiosemicarbazones was performed. The structures of such compounds cluster in two groups, according to the different orientation of the -SR group with respect to the hydrazine N atom of the thiosemicarbazide. While the trans arrangement may be more stable for the isolated molecules, owing to N-H...N intramolecular interactions, the cis form is preferred by most compounds in the solid state, as the result of interplay between intra- and intermolecular effects.

1-(1-Benzoylpropen-2-yl)-3-methylisothiosemicarbazide.

The structure of the title S-alkylated isothiosemicarbazide, C(12)H(15)N(3)OS, was determined by single-crystal diffractometry and compared with the structures of other compounds containing the S-alkylthiosemicarbazide moiety. Such structures cluster into two groups, according to the different orientation of the [bond]SR group with respect to the hydrazine N atom of the thiosemicarbazide. The cis arrangement is preferred by most molecules in the solid state, in spite of the possibility of intramolecular N[bond]H...N interactions in the opposite orientation.