Inhibitory effects and spectral interactions of isomeric methoxyindazoles on recombinant nitric oxide synthases.

A series of isomeric methoxyindazoles has been evaluated as inhibitors of purified recombinant neuronal, inducible, and endothelial nitric oxide synthases (NOS). 7-Methoxyindazole (7-MI) was the most active compound of this series and displayed selectivity toward the constitutive neuronal (NOS I) and endothelial (NOS III) NOS isoforms, the inducible NOS II being almost insensitive to this inhibitor. 6-, 5-, and 4-Methoxyindazoles were almost inactive against all three NOS isoforms. Inhibition of NO and citrulline formation catalyzed by neuronal NOS in the presence of 7-MI appeared to be competitive versus both substrate L-arginine (L-arg) and (6R)-5,6,7,8-tetrahydrobiopterin (BH(4)) cofactor. 7-MI only slightly inhibited NADPH oxidase activity and was inactive against the cytochrome c (cyt c) reductase activity of neuronal NOS at concentrations up to 100-fold higher than its IC(50) value for inhibition of citrulline formation. UV/Vis and EPR studies indicated that 7-MI interacts with the oxygenase domain of neuronal NOS (NOS I(oxy)) in an identical manner but with a much lower affinity than 7-nitroindazole (7-NI). These results demonstrate that an indazole derivative bearing an electron-rich substituent in the 7-position is also a NOS I inhibitor and that such a compound presents strong similarities with the mechanism of inhibition of 7-NI.

Enantioselective Sulfoxidation Catalyzed by Vanadium Haloperoxidases.

Vanadium haloperoxidases catalyze the oxidation of halides by hydrogen peroxide to produce hypohalous acid. We demonstrate that these enzymes also slowly mediate the enantioselective oxidation of organic sulfides (methyl phenyl sulfide, methyl p-tolyl sulfide, and 1-methoxy-4 (methylthio)benzene) to the corresponding sulfoxides (turnover frequency 1 min(-)(1)). The vanadium bromoperoxidase from the brown seaweed Ascophyllum nodosum converts methyl phenyl sulfide to the (R)-enantiomer of the sulfoxide (55% yield and 85% enantiomeric excess (ee)). At low peroxide concentrations a selectivity of 91% can be attained. The enzyme catalyzes the selective sulfoxidation reaction over a broad pH range with an optimum around pH 5-6 and remains completely functional during the reaction. When the vanadium bromoperoxidase from the red seaweed Corallina pilulifera is used the (S)-enantiomer (18% yield and 55% ee) is formed. In contrast, the vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the production of a racemic mixture (54% yield), which seems to be an intrinsic characteristic of this enzyme.