NIH Shift Research Articles

Synthetic Copper-(Di)oxygen Complex Generation and Reactivity Relevant to Copper Protein O2-Processing.

Synthetic copper-dioxygen complex design, generation and characterization, play a crucial role in elucidating the structure/function of copper-based metalloenzymes, including dopamine β-monooxygenase, lytic polysaccharide monooxygenases, particulate methane monooxygenase, tyrosinase, hemocyanin, and catechol oxidase. Designing suitable ligands to closely mimic the variable active sites found in these enzymes poses a challenging task for synthetic bioinorganic chemists. In this review, we have highlighted a few representative ligand systems capable of stabilizing various copper-dioxygen species such as CuII-(O2 •-)(superoxide), Cu2 II-(μ-η 1:η 1-O2 2-) (trans/cis-peroxide), Cu2 II-(μ-η 2:η 2-O2 2-)(side-on peroxide) and Cun II--OOH (hydroperoxide) species. Here, we discuss the ligand type utilized, syntheses, and spectroscopic characterization of these species. We also delineate reactivity patterns, particularly electrophilic arene hydroxylation by a side-on peroxo species which occurs via a "NIH shift" mechanism and thermodynamic-kinetic relationships among Cu2-(O2 •-)/O2 2-/-OOH moieties.

Elusive Active Intermediates and Reaction Mechanisms of ortho-/ipso-Hydroxylation of Benzoic Acid by Hydrogen Peroxide Mediated by Bioinspired Iron(II) Catalysts.

Aromatic hydroxylation of benzoic acids (BzOH) to salicylates and phenolates is fundamentally interesting in industrial chemistry. However, key mechanistic uncertainties and dichotomies remain after decades of effort. Herein, the elusive mechanism of the competitive ortho-/ipso-hydroxylation of BzOH by H2O2 mediated by a nonheme iron(II) catalyst was comprehensively investigated using density functional theory calculations. Results revealed that the long-postulated FeV(O)(anti-BzO) oxidant is an FeIV(O)(anti-BzO•) species 2 (anti- and syn- are defined by the orientation of the carboxyl oxygen of BzO to the oxo), which rules out the noted two-oxidant mechanism proposed previously. We propose a new mechanism in which, following the formation of an FeV(O)(syn-BzO) species (3) and its electromer FeIV(O)(syn-BzO•) (3'), 3/3' either converts to salicylate and phenolate via intramolecular self-hydroxylation (route A) or acts as an oxidant to oxygenate another BzOH to generate the same products (route B). In route A, the rotation of the BzO group along the C-O bond forms 2, in which the BzO group is orientated by π-π stacking interactions. An electrophilic ipso-addition forms a phenolate by concomitant decarboxylation or an ortho-attack forms a cationic complex, which readily undergoes an NIH shift and a BzOH-assisted proton shift to form a salicylate. In route B, 3 oxidizes an additional BzOH molecule directed by hydrogen bonding and π-π stacking interactions. In both routes, selectivity is determined by the chemical property of the BzO ring. These mechanistic findings provide a clear mechanistic scenario and enrich the knowledge of hydroxylation of aromatic acids.

Regioselective oxidation of heterocyclic aromatic hydrocarbons catalyzed by cytochrome P450: A case study of carbazole

Recently there are increasing interests in accurately evaluating the health effects of heterocyclic PAHs. However, the activation mechanism and possible metabolites of heterocyclic PAHs catalyzed by human CYP1A1 is still elusive to a great extent. Here, leveraged to high level QM/MM calculations, the corresponding activation pathways of a representative heterocyclic PAHs, carbazole, were systematically explored. The first stage is electrophilic addition or hydrogen abstraction from N-H group. Electrophilic addition was evidenced to be more feasible and regioselectivity at C3 and C4 sites were identified. Correlations between energy barriers and key structural/electrostatic parameters reveal that O-Cα distance and Fe-O-Cα angle are the main origin for the catalytic regioselectivity. Electrophilic addition was determined as the rate-determining step and the subsequent possible reactions include epoxidation, NIH shift (the hydrogen migration from the site of hydroxylation to the adjacent carbon) and proton shuttle. The corresponding products are epoxides, ketones and hydroxylated carbazoles, respectively. The main metabolites (hydroxylated carbazoles) are estimated to be more toxic than carbazole. The regioselectivity of carbazole activated by CYP1A1 is different from the environmental processes (gas and aqueous phase). Collectively, these results will inform the in-depth understanding the metabolic processes of heterocyclic PAHs and aid the accurate evaluation of their health effects.

A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year’s history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C–C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

Endocrine-disrupting metabolic activation of 2-nitrofluorene catalyzed by human cytochrome P450 1A1: A QM/MM approach.

Nitropolycyclic aromatic hydrocarbons (NPAHs) present one of the most important airborne pollutants. Recent studies have shown that one of the most abundant NPAHs, 2-Nitrofluorene (NF), was supposed to be converted to endocrine-disrupting metabolites by cytochrome P450 1A1 (CYP1A1) in human cells. However, the mechanism is still largely unexplored. Here the metabolic activation and transformation mechanism of NF catalyzed by CYP1A1 were systematically studied with the aid of Molecular Dynamics, Density Functional Theory and Quantum Mechanics/Molecular Mechanics techniques. We evidence that CYP1A1 can activate NF through two elementary processes: (i) electrophilic addition (12.4kcal·mol-1) or hydrogen abstraction (38.2kcal·mol-1) and (ii) epoxidation (5.9 and 8.7kcal·mol-1) or NIH shift (12.5 and 14.9kcal·mol-1) or proton shuttle (12.1kcal·mol-1). Electrophilic addition was found to be the rate-determining step while epoxidation rather than NIH shift or proton shuttle is the more feasible pathway after electrophilic addition. Metabolites 6,7-epoxide-2-nitrofluorene and 7,8-epoxide-2-nitrofluorene were identified as the major epoxidation products. Epoxides are unstable and easy to react with hydrated hydrogen ions and hydroxyls to produce endocrine disrupter 7-hydroxy-2-nitrofluorene. Toxic analysis shows that some of the metabolites are more toxic to model aquatic organisms (e.g. Green algea) than NF. Binding affinity analysis to human sex hormone binding globulin reveals that NF metabolites all have endocrine-disrupting potential. This study provides a comprehensive understanding on the biotransformation process of NF and may aid future studies on various NPAHs activation catalyzed by human P450 enzyme.

Biomimetic synthesis of L-DOPA inspired by tyrosine hydroxylase

L-3,4-dihydroxyphenylalanine (L-DOPA) is in high demand as the cornerstone for treatment of Parkinson's disease. The current production of L-DOPA is associated with poor productivity and long production period. Biomimetic system inspired from tyrosine hydroxylase was developed to achieve the production of L-DOPA from tyrosine with high reactivity, efficiency, and specificity. The biomimetic system owned close resemblance of component and structure in comparison with tyrosine hydroxylase, consisting of tyrosine as substrate, a redox complex of Fe2+ and EDTA as the catalyst to simulate the active center of the natural tyrosine hydroxylase, hydrogen peroxide as the oxidant, and ascorbic acid as the reductant. HPLC, HPLC-MS/MS, 1H NMR, and specific rotation identified L-DOPA was generated. The system showed high catalytic activity and regioselectivity for hydroxylation of tyrosine as equal to tyrosine hydroxylase. FeIVO2+ was formed as the major active species, and NIH shift was observed. EDTA accelerated the reaction by reducing the redox potential of Fe3+/Fe2+ couple. Density functional theory calculation suggested formation of FeIVO2+ was more thermodynamically favorable. The biomimetic system shared analogous catalytic mechanism with TyrH. Process parameters was optimized for maximum production of L-DOPA, namely 6.4 mM tyrosine, 1.6 mM Fe2+, 1.92 mM EDTA, 150 mM H2O2, and 35 mM ascorbic acid in 0.2 M glycine-HCl buffer at pH 4.5 and 60 °C. The yield, titer, and productivity were obtained as 52.01%, 3.22 mM, and 48,210.68 mg L−1 h−1, respectively. The proposed method exhibited an amazing productivity, might provide a promising strategy to industrialize L-DOPA production.

Natural diversity of FAD-dependent 4-hydroxybenzoate hydroxylases

4-Hydroxybenzoate 3-hydroxylase (PHBH) is the most extensively studied group A flavoprotein monooxygenase (FPMO). PHBH is almost exclusively found in prokaryotes, where its induction, usually as a consequence of lignin degradation, results in the regioselective formation of protocatechuate, one of the central intermediates in the global carbon cycle. In this contribution we introduce several less known FAD-dependent 4-hydroxybenzoate hydroxylases. Phylogenetic analysis showed that the enzymes discussed here reside in distinct clades of the group A FPMO family, indicating their separate divergence from a common ancestor. Protein homology modelling revealed that the fungal 4-hydroxybenzoate 3-hydroxylase PhhA is structurally related to phenol hydroxylase (PHHY) and 3-hydroxybenzoate 4-hydroxylase (3HB4H). 4-Hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Genome mining suggests that the 4HB1H activity is widespread in the fungal kingdom and might be responsible for the oxidative decarboxylation of vanillate, an import intermediate in lignin degradation. 4-Hydroxybenzoyl-CoA 1-hydroxylase (PhgA) catalyzes an intramolecular migration reaction (NIH shift) during the three-step conversion of 4-hydroxybenzoate to gentisate in certain Bacillus species. PhgA is phylogenetically related to 4-hydroxyphenylacetate 1-hydroxylase (4HPA1H). In summary, this paper shines light on the natural diversity of group A FPMOs that are involved in the aerobic microbial catabolism of 4-hydroxybenzoate.

Frequent occurrence of triclosan hydroxylation in mammals: A combined theoretical and experimental investigation

Triclosan (TCS) is a widespread antimicrobial agent with many adverse health risks. Its hepatoxicity invariably points to the activation of constitutive androstane receptor (CAR), which regulates cytochrome P450 (CYP) genes that are critical for oxidative metabolism. Here, we provide the theoretical and experimental evidences showing that metabolic activation of TCS frequently occurs through aromatic hydroxylation in mammals. CYP-mediated oxidation was predicted to take place at each aromatic C‒H bond. Molecular docking and in vitro approaches reveal oxidative reaction could be efficiently catalyzed by CAR-regulated CYP2B6 enzyme. Parallel reaction monitoring (PRM) high-resolution mass spectrometry was utilized to identify and profile TCS oxidative metabolites in paired mouse liver, bile, feces, plasma and urine. We found multiple hydroxylated isomers including the products generated via the NIH shift of chlorine, as well as their subsequent conjugates. These metabolites showed isomer-specific retention in mice. Glucuronide conjugates are more readily excreted than the sulfates. Moreover, for the first time, isomeric hydroxylated metabolites were detected in the urine and stool of human subjects used TCS-contained household and personal care products. Collectively, these findings suggest that hydroxylation is an important, yet often underestimated element that worth considering to fully evaluate the biological fates and health risks of TCS.

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para-hydroxybenzoate catabolism.

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.

Biodegradation of sulfamethoxazole by a bacterial consortium of Achromobacter denitrificans PR1 and Leucobacter sp. GP.

In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6-96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer-Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.

Mechanistic Studies of TiO2 Photocatalysis and Fenton Degradation of Hydrophobic Aromatic Pollutants in Water.

HO-adduct radicals have been investigated and confirmed as the common initial intermediates in TiO2 photocatalysis and Fenton degradations of water-insoluble aromatics. However, the evolution of HO-adduct radicals to phenols has not been completely clarified. When 4-d-toluene and p-xylene were degraded by TiO2 photocatalysis and Fenton reactions, respectively, a portion of the 4-deuterium or 4-CH3 group (18-100 %) at the attacked ipso position shifted to the adjacent position of the ring in the formed phenols (NIH shift; NIH is short for the National Institutes of Health, to honor the place where this phenomenon was first discovered). The results, combined with the observation of a key dienyl cationic intermediate by in situ attenuated total reflectance FTIR spectroscopy, indicate that, for the evolution of HO-adduct radicals, a mixed mechanism of both the carbocation intermediate pathway and O2 -capturing pathway occurs in both aqueous TiO2 photocatalysis and aqueous Fenton reactions.

μ-Nitrido Diiron Macrocyclic Platform: Particular Structure for Particular Catalysis.

The ultimate objective of bioinspired catalysis is the development of efficient and clean chemical processes. Cytochrome P450 and soluble methane monooxygenase enzymes efficiently catalyze many challenging reactions. Extensive research has been performed to mimic their exciting chemistry, aiming to create efficient chemical catalysts for functionalization of strong C-H bonds. Two current biomimetic approaches are based on (i) mononuclear metal porphyrin-like complexes and (ii) iron and diiron non-heme complexes. However, biomimetic catalysts capable of oxidizing CH4 are still to be created. In the search for powerful oxidizing catalysts, we have recently proposed a new bioinspired strategy using N-bridged diiron phthalocyanine and porphyrin complexes. This platform is particularly suitable for stabilization of Fe(IV)Fe(IV) complexes and can be useful to generate high-valent oxidizing active species. Indeed, the possibility of charge delocalization on two iron centers, two macrocyclic ligands, and the nitrogen bridge makes possible the activation of H2O2 and peracids. The ultrahigh-valent diiron-oxo species (L)Fe(IV)-N-Fe(IV)(L(+•))═O (L = porphyrin or phthalocyanine) have been prepared at low temperatures and characterized by cryospray MS, UV-vis, EPR, and Mössbauer techniques. The highly electrophilic (L)Fe(IV)-N-Fe(IV)(L(+•))═O species exhibit remarkable reactivity. In this Account, we describe the catalytic applications of μ-nitrido diiron complexes in the oxidation of methane and benzene, in the transformation of aromatic C-F bonds under oxidative conditions, in oxidative dechlorination, and in the formation of C-C bonds. Importantly, all of these reactions can be performed under mild and clean conditions with high conversions and turnover numbers. μ-Nitrido diiron species retain their binuclear structure during catalysis and show the same mechanistic features (e.g., (18)O labeling, formation of benzene epoxide, and NIH shift in aromatic oxidation) as the enzymes operating via high-valent iron-oxo species. μ-Nitrido diiron complexes can react with perfluorinated aromatics under oxidative conditions, while the strongest oxidizing enzymes cannot. Advanced spectroscopic, labeling, and reactivity studies have confirmed the involvement of high-valent diiron-oxo species in these catalytic reactions. Computational studies have shed light on the origin of the remarkable catalytic properties, distinguishing the Fe-N-Fe scaffold from Fe-C-Fe and Fe-O-Fe analogues. X-ray absorption and emission spectroscopies assisted with DFT calculations allow deeper insight into the electronic structure of these particular complexes. Besides the novel chemistry involved, iron phthalocyanines are cheap and readily available in bulk quantities, suggesting high application potential. A variety of macrocyclic ligands can be used in combination with different transition metals to accommodate M-N-M platform and to tune their electronic and catalytic properties. The structural simplicity and flexibility of μ-nitrido dimers make them promising catalysts for many challenging reactions.

Intermediate Partitioning Kinetic Isotope Effects for the NIH Shift of 4-Hydroxyphenylpyruvate Dioxygenase and the Hydroxylation Reaction of Hydroxymandelate Synthase Reveal Mechanistic Complexity

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3'-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3'-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3'-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp(3) hybridization in a HPPD variant and sp(2) hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.

Substrate Hydroxylation by the Oxido–Iron Intermediate in Aromatic Amino Acid Hydroxylases: A DFT Mechanistic Study

AbstractSubstrate hydroxylation by an FeIV=O cluster model of the active center in aromatic amino acid hydroxylases (AAHs) has been investigated by means of DFT calculations. Whereas benzene was used as a model for the aromatic amino acid substrate, the water‐free FeIV=O cluster model has been used in previous studies of enzyme activation and formation of the hydroxylating intermediate. This cluster model also has the pterin cofactor placed in the first coordination sphere of the iron atom and differs substantially from models used in previous computational studies of AAH‐catalyzed hydroxylations. The formation of the (Fe)O–C(benzene) bond is associated with a free‐energy barrier (12.6 kcal mol–1) that is slightly lower than that calculated previously for the formation of the FeIV=O species. The subsequent steps, the NIH shift and the tautomerization leading to the phenol product are both associated with lower energy barriers. The substrate hydroxylation is followed by protonation of the oxidized iron‐bound cofactor to give the pterin‐4a‐carbinolamine product. The latter is subsequently dissociated upon rebinding of water molecules to produce the hexacoordinate iron complex of the enzyme resting state. Consequently, no product is released before the oxidation of both the substrate and the cofactor has been completed. Finally, the current study completes the catalytic cycle and regenerates the catalyst, with a barrier energy comparable to that of the (Fe)O–C(benzene) bond formation.

John W. Daly: The Early Years. The NIH Shift and Cyclic-AMP Assays: Early Pharmacological Breakthroughs

John W. Daly: The Early Years. The NIH Shift and Cyclic-AMP Assays: Early Pharmacological Breakthroughs

Diiron N-bridged species bearing phthalocyanine ligand catalyzes oxidation of methane, propane and benzene under mild conditions

Transformation of methane, the most abundant and the least reactive compound of natural gas to valuable products is one of the most difficult chemical problems of great practical importance. In Nature, methane monooxygenase enzymes transform methane to methanol in water under physiological conditions. However, chemical analogs for such a transformation are unknown. Here, we show the mild and efficient aqueous oxidation of methane by hydrogen peroxide, an ecologically and biologically relevant oxidant catalyzed by supported μ-nitrido diiron phthalocyanine dimer, (FePctBu4)2N . This bio-inspired complex containing a stable Fe – N – Fe motif catalyzes the oxidation of methane to methanol which is further transformed to formaldehyde and formic acid as is demonstrated using13CH4and18O labelling. (FePctBu4)2N - H2O2system shows a high activity in the oxidation of benzene to phenol which occurs via formation of benzene oxide and exhibits NIH shift typically accociated with biological oxidation. Mechanistic features of oxidation of methane and benzene as well as detected intermediate hydroperoxo- and high valent oxo diiron complexes support an O-atom transfer reaction mechanism relevant to bio-oxidation.

N‐Bridged Diiron Phthalocyanine Catalyzes Oxidation of Benzene with H2O2 via Benzene Oxide with NIH Shift Evidenced by Using 1,3,5‐[D3]Benzene as a Probe

Kinetic isotope effect: Incorporation of 18O from H218O2 in phenol in the presence of H216O and 16O2 and detection of benzene oxide as well as NIH shift in the course of the oxidation of PhH indicates that μ-nitrido diiron phthalocyanine operates by a biooxidation-like mechanism (see figure).

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.

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4'-hydroxylation of (S)-mephenytoin labeled with tritium in the 4' position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3'- and 5'-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC(50) values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4'-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4'-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.

Synthesis and reactivity of (μ-η 2:η 2-peroxo)dicopper(II) complexes with dinucleating ligands: Hydroxylation of xylyl linker with a NIH shift

New hexadentate dinucleating ligands having a xylyl linker, X–L–R, were synthesized, where X–L–R = 1,3-bis[bis(6-methyl-2-pyridylmethyl)aminomethyl]-2,4,6-trimethybenzene (Me 2–L–Me) and 1,3-bis[bis(6-methyl-2-pyridylmethyl)aminomethyl]-2-fluorobenzene (H–L–F). They form dinuclear copper(I) complexes, [Cu 2(X–L–R)] 2+ (Me 2–L–Me ( 1) and H–L–F ( 2)). The copper(I) complexes in acetone at −78 °C react with O 2 to produce intra- and intermolecular (μ-η 2:η 2-peroxo)dicopper(II) species depending on the concentrations of the complexes: both complexes generate intramolecular (μ-η 2:η 2-peroxo)dicopper(II) species [Cu 2(O 2)(X–L–R)] 2+ ( 1-O 2 and 2-O 2) at the concentrations below ∼5 mM, whereas at ∼60 mM, both complexes produce intermolecular (μ-η 2:η 2-peroxo)dicopper(II) species, which were confirmed by the electronic and resonance Raman spectroscopies. The electronic spectrum of 1-O 2 in acetone at concentrations below ∼5 mM showed an absorption band at ( λ max = 442 nm, ε = 5600 M −1 cm −1) assignable to the π σ ∗ (O–O)-to-Cu(II) ( ( d x 2 - y 2 + d x 2 - y 2 ) component) LMCT transition in addition to an intense band attributable to the π σ ∗ (O–O)-to-Cu(II) ( ( d x 2 - y 2 - d x 2 - y 2 ) component) LMCT transition ( λ max = 359 nm, ε = 21000 M −1 cm −1), indicating that the (μ-η 2:η 2-peroxo)Cu(II) 2 core of 1-O 2 takes a butterfly structure. Decomposition of 1-O 2 resulted in hydroxylation of the 2-position of the xylyl linker with 1,2-methyl migration (NIH shift), suggesting that the hydroxylation reaction proceeds via a cationic intermediate as proposed for closely related (μ-η 2:η 2-peroxo)Cu(II) 2 complexes having a xylyl linker. Kinetic study of the decomposition (hydroxylation of the xylyl linker) of 1-O 2 suggests that a stereochemical effect of the methyl group in the 2-position of the xylyl linker has a significant influence on a transition state for decomposition (hydroxylation of the xylyl linker).