Mechanism of hydroxylation of aromatic compounds: II. Evidence for the involvement of superoxide anions in enzymatic hydroxylations

The mechanism of hydroxylation reactions catalyzed by m-hydroxybenzoate-4-hydroxylase and anthranilate hydroxylase from Aspergillus niger was investigated using superoxide dismutase from ovine erythrocytes. Inclusion of superoxide dismutase in the assay mixtures of the two enzymes resulted in complete inhibition of the hydroxylation reaction, indicating the possible involvement of superoxide anions (O2−) in these reactions.

Investigation of regio- and sterochemistries of microbial biotransformation /

Epoxides can be hydrolyzed by fungi to produce chiral diols. The first part of this thesis presents an investigation of the microbial hydrolysis of aziridines comparable in structure to epoxide biotransformation substrates. Biotransformation of the aziridines 1 -methyl-2-phenyl aziridine, 2- phenylaziridine and N-methyl-7-aza bicyclo[4.1.0] heptane was studied using Beauveria sulfurescens, Aspergillus niger and Diplodia gossypina but no evidence for enzymic hydrolysis was obtained. The hydroxylation reaction performed by the fungus Beauveria sulfurescens ATCC 7159 has been studied for many years and several models describing the hydroxylating pattern exhibited by this fungus have been proposed. The second part of this thesis presents a test of the proposed models. The ability of Beauveria sulfurescens to hydroxylate thirty potential substrates was examined, and the data suggest that none of the earlier proposed models accounts for all of the bioconversion results. A possible explanation is proposed, suggesting that there is more than one enzyme responsible for the hydroxylation reactions performed by Beauveria sulfurescens.

Paracetamol degradation intermediates and toxicity during photo-Fenton treatment using different iron species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds (radical clocks), which can produce both unrearranged and ring opened products upon oxidation, have been key tools in these investigations. In this study, we introduce several cyclopropyl containing fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation. The probes are shown to be capable of distinguishing radical from cationic intermediates due to the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these probes by P450(BM3) and P450(Biol) gives results consistent with a radical but not a cationic intermediate in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened products gives remarkably homogeneous rates for oxygen rebound of (2-3) x 10(10) s(-1). The effects of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation are discussed.

Selective hydroxylation of small alkanes by the particulate methane monooxygenase

The particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol under ambient temperatures and pressures. Other small alkanes and alkenes are also substrates of this enzyme. We measured and compared the initial rate constants of oxidation of small alkanes (C1 to C5) catalyzed by pMMO. Both primary and secondary alcohols were formed from oxidation of n-butane and n-pentane. The alcohols produced from alkane oxidation can be further oxidized, probably by pMMO, to aldehydes and ketones. The apparent regioselectivity for n-butane and n-pentane is 100% 2-alcohols because the formation of primary alcohols is slower than further oxidation of these alcohols. The hydroxylation at the secondary carbons is highly stereoselective: (R)-alcohols are preferentially formed. The enantiomeric excess increases slightly with decreasing reaction temperature. The steric course of hydroxylation on primary carbons was also studied by using isotopically substituted ethane: (S)- or (R)-CH_3-CHDT, and (S)- or (R)-CD_3- CHDT and the reactions were found to proceed with 100% retention of configuration. A primary isotopic effect of k_H/k_D=5.0 was observed in these experiments.

Superconductor mixed oxides La2-xSrxCuO4+/-lambda for catalytic hydroxylation of phenol in the liquid-solid phase

Superconductor mixed oxides are often used as catalysts at high temperature in gas-solid phase oxidations and considered not suitable for lower temperature reactions in the liquid-solid phase; here the catalysis of La2-xSrxCuO4+/-lambda (x = 0, 0.1, 0.7, 1) mixed oxides in phenol hydroxylation at lower temperatures are studied, and we find that the value of x has a significant effect on catalytic activity: the lower its value, the higher the catalytic activity; a mechanism is proposed to explain the experimental phenomena.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

Reactions of steroidal epoxides with strong organic bases and an investigation into the syntheses of some labelled pregnane derivatives

Reactions of 5,6- and 4,5-epoxycholestane derivatives with strong bases were investigated. Epoxidation of 3a-acetoxycholest-5-ene also gave a new compound along with the anticipated epoxides. Interconversions of the latter were observed. Some possible mechanisms of its formation and rearrangements have been pIioposed. No reaction was observed with any of the 5,6- and 4,5-steroidal epoxides employed in the present study, using potassium tertiary butoxide under refluxing conditions. n-Butyllithium reacted only with 5,6-epoxycholestanes bearing a ketal moiety at the C3 carbon. Opening of the ketal group was observed with n-butyllithium in the case of a ~-epoxide. The reaction was also investigated in the absence of epoxide functionality. A possible mechanism for the opening of ketal group has been proposed. Lithium diethylamide (LDEA) was found effective in rearranging 5,6- and 4,5-epoxides to their ~orresponding allylic alcohols. These rearrangements presumably proceed via syn-eliminations, however the possibility of a corresponding anti-elimination has not been eliminated. A substituent effect of various functional groups (R = H, OH, OCH2CH20) at C3 has-been observed on product distribution in the LDEApromoted rearrangements of the corresponding epoxides. No reaction of these epoxides was observed with lithium diisopropylamide (LDA) • In the second part of the project, several attempts were made towards the sYRthesis of deoxycorticoste~one~17,2l,2l~d3' a compound desirable for the 2l-dehydroxylation studies of deoxycorticosterone. Several routes were investigated, and some deuterium labelled pregnane derivatives were prepared in this regard. Microbial 21-hydroxylation of progesteronel7,21,21,2l- d4 by ~ niger led to loss of deuterium from C21 of the product. An effort was made to hydroxylate progesterone microbially under neutral condtions.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis.

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Reactions catalyzed by bacterial cytochromes P450

The cytochromes P450 are a large family of oxidative haemoproteins that are responsible for a wide variety of oxidative transformations in a variety of organisms. This review focuses upon the reactions catalyzed specifically by bacterial enzymes, which includes aliphatic hydroxylation, alkene epoxidation, aromatic hydroxylation, oxidative phenolic coupling, heteroatom oxidation and dealkylation, and multiple oxidations including C-C bond cleavage. The potential for the practical application of the oxidizing power of these enzymes is briefly discussed.

Condensed phase combustion travelling waves with sequential exothermic or endothermic reactions

The one-dimensional propagation of a combustion wave through a premixed solid fuel for two-stage kinetics is studied. We re-examine the analysis of a single reaction travelling-wave and extend it to the case of two-stage reactions. We derive an expression for the travelling wave speed in the limit of large activation energy for both reactions. The analysis shows that when both reactions are exothermic, the wave structure is similar to the single reaction case. However, when the second reaction is endothermic, the wave structure can be significantly different from single reaction case. In particular, as might be expected, a travelling wave does not necessarily exist in this case. We establish conditions in the limiting large activation energy limit for the non-existence, and for monotonicity of the temperature profile in the travelling wave.