Henry C. Kelly

Heme models of peroxidase enzymes: deuteroferriheme-catalyzed chlormation of monochlorodimedone by sodium chlorite

The iron(III) complex of deuteroporphyrin(IX), deuteroferriheme, catalyzes the chlorination, by sodium chlorite, of the active methylene compound monochlorodimedone (MCD) to dichlorodimedone. Rate studies, carried out on a stopped-flow spectrophotometric time scale, show the chlorination to be zero-order in MCD, first-order in ClO2- and to display a complex dependence on heme. The active chlorinating agent is believed to be hypochlorite, OCl-, formed as a result of the initial two-electron oxidation of heme to peroxidatic intermediate by chlorite ion. This scheme is supported by the fact that the normal (4:1) heme:ClO2- molar stoichiometry is reduced in the presence of MCD to values approaching 2:1. This suggests that MCD is an effective scavenger of OCl-, which, in the absence of active methylene compound, serves as a two-electron oxidant of heme. The zero-order dependence of rate on MCD is attributed to the slow formation of OCl-, consequent to a mechanism in which the rate-limiting step is viewed to be the regeneration of free heme from peroxidatic intermediate, probably via a catalatic pathway. Support for such a mechanism is provided by the fact that addition of ascorbate greatly enhances the rate of MCD chlorination, presumably by accelerating the rate of heme regeneration via perioxidation reduction of the heme intermediate.

Nitrogen Base Donor-Cyanoborane Addition Compounds

Four amine–BH2CN addition compounds have been prepared by the addition of the respective nitrogen base to acidified tetrahydrofuran solutions of the cyanotrihydroborate ion, and this reaction appears generally useful for the synthesis of donor–cyanoborane complexes.

Some Derivatives of the Cyanotrihydroborate Ion (BH3CN

Abstract A general method for the preparation of salts of the type R4N+ BH3CN− involving metathesis of the corresponding quaternary ammonium hydroxides with NaBH3CN is illustrated with the synthesis of tetramethyl-, tetraethyl-, and benzyltrimethylammonium cyanotrihydrobo-rates. Trimethylamine-dibromocyanoborane is obtained by direct bromination of trimethylamine-cyanoborane in aqueous solution.

Effects of amine-borane structure on the stoichiometry, rate and mechanism of reaction with hypochlorous acid : hydride oxidation versus B-chlorination

Abstract Various ring substituted benzylamine-boranes as well as α-methyl- and N -methylbenzylamine-borane undergo hydride oxidation and N-chlorination on reaction with NaOCl whereas N , N -dimethylbenzylamine-borane is subject to B-chlorination producing a mixture of amine chloroboranes. Results are consistent with previously established patterns of reactivity with alkyl- and non-aromatic heterocyclic secondary amine-boranes being subject to oxidation and their tertiary counterparts to B-chlorination. Current studies reveal the borane adducts of tertiary amines in the quinoline series to react via oxidative pathways. Ring or α-methyl substitution has a negligible effect on the rate of benzylamine-borane oxidation whereas N-methyl substitution causes a three-fold decrease. The benzylamine-borane reaction displays a modest substrate isotope effect ( k BH3 / k BD3 ≅1.3), a pronounced inverse solvent isotope effect ( k D2O / k H 2 O 3) and a pH dependence of rate that indicates saturation kinetics below pH 7.5. It is suggested that both oxidation and B-chlorination involve bimolecular rate-limiting attack of HOCl on amine-borane with different intimate mechanisms arising from correspondingly different activated complexes. A low reactivity of quinoline-monochloroborane with hypochlorite precludes the involvement of a chloroborane as a common intermediate in both processes. The activation free-energy for chlorination of trimethylamine-borane is higher than δ G ≠ values obtained for oxidation of isoquinoline-borane and for a series of benzylamine-boranes. Activation enthalpies are relatively low for both processes. Unlike the chlorination reaction where the entropy term is negligible, δ S ≠ for oxidation accounts for about 20–30% of the activation barrier. It is proposed that details of amine structure determine the mode of attack of amine-borane by HOCl with steric factors playing a dominant role.

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

Abstract The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H2O2 reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (Km = 8.1 × 10−5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k3 = 7.4 sec−1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions

SYNTHESIS AND COMPARATIVE REACTIVITY OF THIOMORPHOLINE-BORANE : AQUEOUS HYDROLYSIS AND OXIDATION BY HYPOCHLORITE

Abstract Thiomorpholine-N-borane was synthesized via amine displacement of BH3 from tetrahydrofuran–borane which had been prepared from NaBH4 and BF3·Et2O in tetrahydrofuran. Acid-catalyzed hydrolysis occurs only slightly faster than for morpholine–borane, the difference being attributed to a small difference in the electronic inductive effects of sulfur and oxygen in the 4-position of the respective adducts. Reaction with NaOCl exhibits a stoichiometric [OCl−]:[S(CH2)4NHBH3] ratio of 5:1. This is attributed to consumption of 3 mol of hypochlorite for the oxidation of hydridic hydrogen in BH3, one for the chlorination of nitrogen and another in attack at sulfur presumably resulting in sulfoxide formation. At pH 9.1–10.4, the initial reaction of hypochlorite with thiomorpholine–borane is several times faster than with morpholine–borane and, unlike the reaction with morpholine–borane, relatively insensitive to pH. Whereas hypochlorite oxidation of morpholine–borane has been proposed to occur primarily through reaction with HOCl, it is speculated that thiomorpholine–borane is also susceptible to attack by hypochlorite ion.

Mesoferriheme regeneration from its hemoprotein enzyme compound I analog: absorption and kinetic properties of one- and two-electron oxidation products

Abstract Spectral and kinetic data obtained in studies of the biphasic in situ regeneration of mesoferriheme (mfh) from oxidized species formed through the NaOCl oxidation of heme are utilized to calculate extinction coefficients of dinuclear iron species which have been proposed as models of peroxidase Compounds I and II (A and B below). For the proposed regeneration mechanism, involving consecutive one-electron redox processes, the calculated values of ϵA and ϵB are 4x104 and 5x104 M−1 cm−1, respectively. These values are somewhat greater than those previously calculated for analogous derivatives of deuteroferriheme. Both k1 and k2 are independent of pH over the range 6.9–9.1 and of total phosphate buffer concentration from 8 to 50 mM at pH 6.9 suggesting that, under these conditions, there is an absence of significant dissociation of the proposed dinuclear iron species.

Polymerization of difunctional ions containing the –N–B–N– linkage: a polymeric boron(n+) ion

A novel polymeric boron(n+) ion has been prepared by vinyl polymerization of salts of the trimethylamine-(4-vinylpyridine)dihydroboron(1+) ion.