Protein-surfactant film voltammetry of wild-type and mutant cytochrome P450 BM3

We are investigating the redox chemistry of wild-type (WT) and mutant (1-12G) cytochrome P450 BM3. Absorption spectra in solution feature the Fe(III) Soret at 418 nm for WT and a split Soret for 1-12G at 390 and 418 nm. Voltammetry of the proteins within DDAPSS films on the surface of carbon electrodes reveal nearly identical Fe(III/II) potentials (approximately -200 mV vs Ag/AgCl), but significant differences in k degrees , 250 vs 30 s(-)(1), and Fe(III/II)-CO potentials, -140 vs -115 mV, for WT vs 1-12G. Catalytic reduction of dioxygen by the proteins on rotating-disk electrodes was analyzed using Levich and Koutecky-Levich treatments. The data reveal 1-12G n and k(obs) values that are, respectively, 1.7 and 0.07 times those of WT, suggesting that the two proteins differ strikingly in their reactions with dioxygen.     

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry

The interest in therapeutic drug monitoring has increased over the last few years. Inter‐ and intra‐patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield^TM C₁₈ column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease. Copyright © 2015 John Wiley & Sons, Ltd.     

The arene oxede-oxepine valence isomerization; a dynamic nmr investigation using a prochiral substituent

The geminal -methytene protons in 3-ethylnaphthalene-l,2-oxide show chemical shift nonequivalence in ¹H NMR spectra recorded at ambient temperature consistent with a high barrier to enantiomerization via the orepine tautomer. The methylene protons in -benzoyloxytoluene-2,3-oxide which are a singlet at ambient temperature split into an AB system at -135 °C and there is evidence of ca. 5% of the oxepine isomer. Lineshape analysis of the coalescing arene oxide AB Signals provides the activation parameters for the valence isomerization/degenerate racemization process: ΔGXXX = 7.6 kcal mol^-1, ΔHXXX = 8.6 kcal mol^-l, ΔSXXX= 5.8 cal K^-l mol^-1.     

Reactions of phosphorus halides with urethane

By the reaction of phosphorus trichloride, thiophosphoryl chloride, phosphorus oxychloride and phosphorus pentachloride with urethane in stoichiometric ratio 1:1 or 1:2 in benzene, compounds of the type

and

(X = S, O or Cl; n = 1 or 2) have been synthesized. A probable mechanism for their formation has also been suggested.     

Base-Catalyzed Dehydration of 3-Substituted Benzene cis-1,2-Dihydrodiols: Stabilization of a Cyclohexadienide Anion Intermediate by Negative Aromatic Hyperconjugation

Evidence that a 1,2-dihydroxycyclohexadienide anion is stabilized by aromatic "negative hyperconjugation" is described. It complements an earlier inference of "positive" hyperconjugative aromaticity for the cyclohexadienyl cation. The anion is a reactive intermediate in the dehydration of benzene cis-1,2-dihydrodiol to phenol. Rate constants for 3-substituted benzene cis-dihydrodiols are correlated by σ(-) values with ρ = 3.2. Solvent isotope effects for the reactions are k(H(2)O)/k(D(2)O) = 1.2-1.8. These measurements are consistent with reaction via a carbanion intermediate or a concerted reaction with a "carbanion-like" transition state. These and other experimental results confirm that the reaction proceeds by a stepwise mechanism, with a change in rate-determining step from proton transfer to the loss of hydroxide ion from the intermediate. Hydrogen isotope exchange accompanying dehydration of the parent benzene cis-1,2-dihydrodiol was not found, and thus, the proton transfer step is subject to internal return. A rate constant of ～10(11) s(-1), corresponding to rotational relaxation of the aqueous solvent, is assigned to loss of hydroxide ion from the intermediate. The rate constant for internal return therefore falls in the range 10(11)-10(12) s(-1). From these limiting values and the measured rate constant for hydroxide-catalyzed dehydration, a pK(a) of 30.8 ± 0.5 was determined for formation of the anion. Although loss of hydroxide ion is hugely exothermic, a concerted reaction is not enforced by the instability of the intermediate. Stabilization by negative hyperconjugation is proposed for 1,2-dihydroxycyclohexadienide and similar anions, and this proposal is supported by additional experimental evidence and by computational results, including evidence for a diatropic ("aromatic") ring current in 3,3-difluorocyclohexadienyl anion.     

Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and benzo[b]furans: synthesis of benzo[b]thiophene 2,3-oxide

Enzymatic cis-dihydroxylation of benzo[b]thiophene, benzo[b]furan and several methyl substituted derivatives was found to occur in both the carbocyclic and heterocyclic rings. Relative and absolute configurations and enantiopurities of the resulting dihydrodiols were determined. Hydrogenation of the alkene bond in carbocyclic cis-dihydrodiols and ring-opening epimerization/reduction reactions of heterocyclic cis/trans-dihydrodiols were also studied. The relatively stable heterocyclic dihydrodiols of benzo[b]thiophene and benzo[b]furan showed a strong preference for the trans configuration in aqueous solutions. The 2,3-dihydrodiol metabolite of benzo[b]thiophene was utilized as a precursor in the chemoenzymatic synthesis of the unstable arene oxide, benzo[b]thiophene 2,3-oxide.