Benzylic Hydroxylation Research Articles

Synthesis of Oxasmaragdyrin–Amino Acid Conjugates

A series of covalently linked oxasmaragdyrin‐amino acid and BF2‐oxasamaragdyrin–amino acid conjugates were synthesized by treating oxasmaragdyrin or its BF2 complex that contain a benzylhydroxyl group at one of the meso positions with an appropriate fluorenylmethyloxycarbonyl‐protected amino acid in CH2Cl2 in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide·HCl/hydroxybenzotriazole under basic conditions at room temperature. The conjugates are stable, highly soluble in all organic solvents, and confirmed by HRMS. 1D and 2D NMR spectroscopic techniques were used to characterize the oxasmaragdyrin–amino acid conjugates. Absorption, fluorescence, and electrochemical studies of conjugates indicate that the conjugates strongly absorb and emit in visible‐NIR region with decent quantum yields, singlet state lifetimes and are stable under electrochemical conditions. Furthermore, the conjugates showed minimal change in their spectral and electrochemical properties relative to that of the unconjugated oxasmaragdyrin or its BF2 complex suggest that the characteristic features of the oxasmaragdyrin and its BF2 complex is retained in the conjugates, which will be a useful feature for Near‐IR fluorescence imaging and other applications.

Regiospecific Isomerization of 2‐Benzoxazinon‐2‐yl Benzoic Acid Toward Some Nitrogen Nucleophiles as Environmental Insecticide

Based on the strategies of receptor structure‐guided benzoxazinone design, a series of nitrogen nucleophiles such as benzyl amine, sodium azide, 4,4‐bis o‐toluidine, 4‐butanolamine, glucosamine, 2‐amino pyridine, 2‐picolinyl amine, hydroxyl amine, and hydrazine derivatives, for example, hydrazine hydrate, semicarbazide, thiosemicarbazide, methylhydrazide, phenylhydrazide, could be reacted with 2‐benzoxazine‐2‐yl benzoic acid 1. According the basicity of nucleophiles, regiospecific isomerization of benzoxazinone has been considered through formation of the spiro derivatives. Organic reagents can be controlled on the course of reaction of benzoxazinonyl benzoic acid 1. Preliminary bioassays indicated that the insecticidal spectra of the synthesized compounds were ecofriendly biodegradable materials due to isomerization. Among these analogues, the quinazoline 2–4 showed 100% mortality against Nilaparvata lugens (LC50 = 0.087 mg/L). The insecticidal potency of our designed analogues was dual‐controlled by isomerization to quinazolinone and spiro derivatives that observed in vitro and shed light on the novel insecticidal mechanism. The chemical structure of the products can be confirmed by microanalytical, spectral data, optimized and stimulated by quantum chemical parameters.

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry

The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography–high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment.Graphical abstractStudies of metabolism of carbamazepine and ibuprofen in fish

P450 BM3-Catalyzed Regio- and Stereoselective Hydroxylation Aiming at the Synthesis of Phthalides and Isocoumarins.

Cytochrome P450 BM3 monooxygenases are able to catalyze the regio- and stereoselective oxygenation of a broad range of substrates, with promising potential for synthetic applications. To study the suitability of P450 BM3 variants for stereoselective benzylic hydroxylation of 2-alkylated benzoic acid esters, the biotransformation of methyl 2-ethylbenzoate, resulting in both enantiomeric forms of 3-methylphthalide, was investigated. In the case of methyl 2-propylbenzoate as a substrate the regioselectivity of the reaction was shifted towards β-hydroxylation, resulting in the synthesis of enantioenriched R- and S-configured 3-methylisochroman-1-one. The potential of P450 BM3 variants for regio- and stereoselective synthesis of phthalides and isocoumarins offers a new route to a class of compounds that are valuable synthons for a variety of natural compounds.

Control of stereoselectivity of benzylic hydroxylation catalysed by wild-type cytochrome P450BM3 using decoy molecules

The benzylic hydroxylation of non-native substrates was catalysed by cytochrome P450BM3, wherein “decoy molecules” controlled the stereoselectivity of the reactions.

KHF2, a mild and selective desilylating agent for phenol t-butyldimethylsilyl (TBDMS) ethers.

TBDMS (t-BuMe2Si, t-butyldimethylsilyl) ethers of a variety of phenols have been deprotected with KHF2 in MeOH, at room temperature. Carboxylic ester and labile phenolic acetate were unaffected under these conditions. In competition reactions between TBDMS ethers of a phenol and two primary benzylic alcohols, the phenolic ether underwent cleavage whereas the alcohol ethers remained intact. From a substrate containing both a phenolic hydroxyl group and a secondary, doubly benzylic hydroxyl group protected as TBDMS ethers, the phenol was rapidly and selectively released. Cleavage of TBDMS, TBDPS, and TIPS ethers of a phenol was also compared. TBDMS and TBDPS ethers underwent cleavage at room temperature within 30 min, whereas removal of the TIPS ether required 2.5 hours. Ease of cleavage appears to be TBDMS ≈ TBDPS > TIPS. At 60 °C, TBDMS ethers of primary benzylic, allylic, and unactivated alcohols can be efficiently desilylated over a prolonged period (13-17 h). Thus, KHF2 proves to be a mild and effective reagent for the selective desilylation of phenol TBDMS ethers at room temperature.

Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3.

Enzymatic conversion of natural glycosides to their corresponding hydroxylated products using cytochromes P450 has significant advantages over synthetic chemistry and even enzyme-catalyzed glycosylation of chemicals. At present, the basic strategy for making glycosides of stilbenoid compounds is to use the glycosylation activity of enzymes, such as glycosyltransferases. Here, an efficient synthesis of a valuable (E)-astringin, a piceatannol glucoside, was developed using CYP102A1 via the highly regioselective C-3' hydroxylation of polydatin, a resveratrol glucoside. (E)-astringin is a high added value compound found in plants and wine. Benzylic hydroxylation of polydatin provides an attractive route to (E)-astringin, a catechol product. Thus far, chemical and enzymatic methods of producing (E)-astringin have not been developed. In the present study, a set of CYP102A1 mutants from Bacillus megaterium was found to catalyze regioselective hydroxylation of polydatin at the C-3' position to generate an (E)-astringin, a piceatannol glucoside.

Computational insights into the oxidation of mono- and 1,4 disubstituted arenes by the Toluene Dioxygenase enzymatic complex

Toluene Dioxygenase (TDO) enzymatic complex has been widely used for preparation of enantiopure cis-cyclohexadienediols for synthetic applications. Along the last 30 years, a variety of mono- and di-substituted arenes have been studied as substrates for this enzyme, and some interesting observations have been reported regarding the yield and selectivity of the biotransformation. Nevertheless, none of them has been explained considering the active site’ structural and electronic features. In this work we present the first computational model of TDO’s active site, with a description of its architecture and interactions with the substrate to understand and predict substrate orientation. Our findings indicate that in the O2-free TDO, the iron(II) is stabilized by the coordination of an oxygen atom from the neighboring Gln215 residue. Besides, the active site is comprised by four pockets with different relative affinities for the substrates’ substituents. Monosubstituted arenes adopt a pose in which the alkyl chains maximize the London dispersion interactions with minimal steric clashes, giving an explanation for the observed trend in the benzylic hydroxylation yields. Finally, the computational results allowed us to rationalize the enantiomeric excess of 1,4-disubstituted arenes and the regioselectivity of the dihydroxylation of fluorobenzene. These data were used to develop and validate an affinity index (AI), as a quantitative indicator of the orientation preference for these substrates. This practical and easy-to-use tool can be applied to successfully predict the orientation of the para-disubstituted benzenes into the TDO active site.

Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists

Aiming to discover dual-acting β2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β2 adrenergic antagonist/D2 receptor agonist with Ki values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders.

Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling

Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions.

Chemoenzymatic synthesis of enantiopure hydroxy sulfoxides derived from substituted arenes.

Enantiopure β-hydroxy sulfoxides and catechol sulfoxides were obtained, by chemoenzymatic synthesis, involving dioxygenase-catalysed benzylic hydroxylation or arene cis-dihydroxylation and cis-diol dehydrogenase-catalysed dehydrogenation. Absolute configurations of chiral hydroxy sulfoxides were determined by X-ray crystallography, ECD spectroscopy and stereochemical correlation. The application of a new range of β-hydroxy sulfoxides as chiral ligands was examined.

Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

11C]MADAM Used as a Model for Understanding the Radiometabolism of Diphenyl Sulfide Radioligands for Positron Emission Tomography (PET).

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [11C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [11C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 μM. Urine samples after perfusion of [11C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [11C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [11C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.

Green oxidation of alcohols with hydrogen peroxide catalyzed by a tetra-cobalt polyoxometalate in water

In this work it was demonstrated that the sodium and potassium salts of a sandwich-type tetra-cobalt tungstophosphate, [Co4(H2O)2(PW9O34)2]10−, are efficient catalysts for the selective oxidation of alcohols into corresponding aldehydes with hydrogen peroxide in water. Among these two salts, Na10[Co4(H2O)2(PW9O34)2]·27H2O complex showed better catalytic activity for the alcohols’ oxidation. Using trace amounts of catalyst, different benzylic and aliphatic hydroxyl groups were converted into the corresponding carbonyl compounds in high to excellent yields. In this work green and efficient catalytic oxidation of various alcohols to aldehydes and ketons with a sandwich-type tetra-cobalt tungstophosphate, [Co4(H2O)2(PW9O34)2]10−, is developed. High-to-excellent yields for oxidation of different alcohols by hydrogen peroxide in water are some advantageous of this catalytic system.

Sterically controlled functionalization of carbon surfaces with -C6H4CH2X (X = OSO2Me or N3) groups for surface attachment of redox-active molecules.

Glassy carbon electrodes were modified by electrochemical reduction of a diazonium molecule ((i)Pr3SiOCH2C6H4N2(+)BF4(-)) featuring a triisopropylsilyl-protected benzylic hydroxyl group. This electrochemical process introduced a monolayer of (i)Pr3SiOCH2C6H4- groups onto the surface of the electrode. The bulky -Si(i)Pr3 protecting group not only prevents the uncontrolled growth of structurally ill-defined and electronically blocking polyphenylene multilayers, but also separates the phenyl groups in the monolayer. Thus, the void spaces between these aryl units should allow a better accommodation of sizable molecules. Removal of the -Si(i)Pr3 protecting groups by (n)Bu4NF exposed the reactive benzylic hydroxyl functionalities that can undergo further transformations to anchor functional molecules. As an example, redox-active ferrocene molecules were grafted onto the modified electrode via a sequence of mesylation, azidation, and copper-catalyzed [3 + 2] cycloaddition reactions. The presence of ferrocenyl groups on the surface was confirmed by X-ray photoelectron spectroscopic and electrochemical studies. The resulting ferrocene-modified glassy carbon electrode exhibits cyclic voltammograms typical of surface-bound redox active species and remarkable electrochemical stability in an acidic aqueous environment.

ChemInform Abstract: Asymmetric Epoxidation of Alkenes and Benzylic Hydroxylation with P450tol Monooxygenase from Rhodococcus coprophilus TC‐2.

P450tol monooxygenase was discovered as a unique and highly enantioselective enzyme for asymmetric epoxidation of some terminal alkenes containing electron-withdrawing groups and benzylic hydroxylation of several ethylbenzenes giving the corresponding useful and valuable products, such as (R)-2- and 3-substituted styrene oxides, (S)-4-substituted styrene oxides, and (S)-benzylic alcohols, in high ee.

Cytochrome P450-mediated bioactivation of mefenamic acid to quinoneimine intermediates and inactivation by human glutathione S-transferases.

Mefenamic acid (MFA) has been associated with rare but severe cases of hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, and hypersensitivity reactions that are believed to result from the formation of reactive metabolites. Although formation of protein-reactive acylating metabolites by phase II metabolism has been well-studied and proposed to be the cause of these toxic side effects, the oxidative bioactivation of MFA has not yet been competely characterized. In the present study, the oxidative bioactivation of MFA was studied using human liver microsomes (HLM) and recombinant human P450 enzymes. In addition to the major metabolite 3'-OH-methyl-MFA, resulting from the benzylic hydroxylation by CYP2C9, 4'-hydroxy-MFA and 5-hydroxy-MFA were identified as metabolites resulting from oxidative metabolism of both aromatic rings of MFA. In the presence of GSH, three GSH conjugates were formed that appeared to result from GSH conjugation of the two quinoneimines formed by further oxidation of 4'-hydroxy-MFA and 5-hydroxy-MFA. The major GSH conjugate was identified as 4'-OH-5'-glutathionyl-MFA and was formed at the highest activity by CYP1A2 and to a lesser extent by CYP2C9 and CYP3A4. Two minor GSH conjugates resulted from secondary oxidation of 5-hydroxy-MFA and were formed at the highest activity by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability of seven human glutathione S-transferases (hGSTs) to catalyze the GSH conjugation of the quinoneimines formed by P450s was also investigated. The highest increase of total GSH conjugation was observed with hGSTP1-1, followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this study show that, next to phase II metabolites, reactive quinoneimines formed by oxidative bioactivation might also contribute to the idiosyncratic toxicity of MFA.

Regio- and stereoselective benzylic hydroxylation to synthesize chiral tetrahydroquinolin-4-ol and tetrahydro-1H-benzo[b]azepin-5-ol with Pseudomonas plecoglossicidas

Two whole-cell biocatalysts were found to have C–H oxidation activity with high R-enantioselectivity on 1,2,3,4-tetrahydroquinoline (THQ) and 2,3,4,5-tetrahydro-1H-benzo[b]azepine (TBA). R-1,2,3,4-tetrahydroquinolin-4-ol ((R)-THQL) and R-2,3,4,5-tetrahydro-1H-benzo[b]azepin-5-ol ((R)-TBAL) were smoothly obtained with 82% yield in 99% e.e. and 99% yield in 98% e.e., respectively.

Genotoxicity of 1-methylpyrene and 1-hydroxymethylpyrene in Chinese hamster V79-derived cells expressing both human CYP2E1 and SULT1A1.

1-Methylpyrene (1-MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1-MP, catalyzed by multiple CYP isoforms, gives rise to 1-hydroxymethylpyrene (1-HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1-MP and 1-HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1-MP and 1-HMP to induce cytotoxicity (using the CCK-8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co-exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5-4 μM and 5-20 μM, under conditions where no reduction in cell viability/growth occurred, 1-HMP and 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79-hCYP2E1-hSULT1A1 cells in these concentration ranges, with 1-MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1-HMP in V79-hCYP2E1-hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1-MP was significantly reduced by either 1-aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1-MP and cause genotoxicity in mammalian cells.

Role of substrate positioning in the catalytic reaction of 4-hydroxyphenylpyruvate dioxygenase-A QM/MM Study.

Ring hydroxylation and coupled rearrangement reactions catalyzed by 4-hydroxyphenylpyruvate dioxygenase were studied with the QM/MM method ONIOM(B3LYP:AMBER). For electrophilic attack of the ferryl species on the aromatic ring, five channels were considered: attacks on the three ring atoms closest to the oxo ligand (C1, C2, C6) and insertion of oxygen across two bonds formed by them (C1-C2, C1-C6). For the subsequent migration of the carboxymethyl substituent, two possible directions were tested (C1→C2, C1→C6), and two different mechanisms were sought (stepwise radical, single-step heterolytic). In addition, formation of an epoxide (side)product and benzylic hydroxylation, as catalyzed by the closely related hydroxymandelate synthase, were investigated. From the computed reaction free energy profiles it follows that the most likely mechanism of 4-hydroxyphenylpyruvate dioxygenase involves electrophilic attack on the C1 carbon of the ring and subsequent single-step heterolytic migration of the substituent. Computed values of the kinetic isotope effect for this step are inverse, consistent with available experimental data. Electronic structure arguments for the preferred mechanism of attack on the ring are also presented.