Aromatic Hydroxylation Reactions Research Articles

Hydrogen-Bond-Assisted Catalysis: Hydroxylation of Paclitaxel by Human CYP2C8.

Paclitaxel (PTX, or Taxol), a chemotherapeutic agent widely employed in the treatment of various cancers, undergoes metabolic transformations through the cytochrome P450 enzymes CYP3A4 and CYP2C8. CYP3A4 catalyzes the aromatic hydroxylation reaction of PTX, whereas CYP2C8 demonstrates a distinct reactivity pattern, producing 6α-hydroxypaclitaxel via alkane hydroxylation. Despite the significant impact of PTX metabolism on its anticancer efficacy, the detailed mechanisms underlying these transformations have remained largely unclear. In this study, we employed hybrid quantum mechanics and molecular mechanics (QM/MM) calculations to elucidate the mechanism of PTX metabolism by human CYP2C8. Our QM/MM results reveal that the hydroxylation of PTX by CYP2C8 follows an atypical rebound mechanism. Either of the two hydrogen atoms at the C6 position of PTX can be abstracted, leading to a common radical intermediate. Although the subsequent rebound barrier is unusually high, stereochemical scrambling is unlikely, as the rebound barrier for the formation of the 6α-hydroxylated PTX─the actual product─is significantly lower than that for the 6β-hydroxylated metabolite. Thus, product selectivity is determined by the non-rate-determining rebound step. Furthermore, the hydroxyl group at the C7 position of PTX plays a catalytic role by facilitating the hydrogen abstraction and rebound steps. Our study also confirms a pronounced stability of the transition state in the high-spin sextet spin state, enabled by the enzyme's specific substrate positioning.

Proton-assisted activation of a MnIII-OOH for aromatic C-H hydroxylation through a putative [MnVO] species.

Adding HClO4 to [(BnTPEN)MnIII-OO]+ in MeOH generates a short-lived MnIII-OOH species, which converts to a putative MnVO species. The potent MnVO species in MeCN oxidizes the pendant phenyl ring of the ligand in an intramolecular fashion. The addition of benzene causes the formation of (BnTPEN)MnIII-phenolate. These findings suggest that high valent Mn species have the potential to catalyze challenging aromatic hydroxylation reactions.

In Situ Fluorescence Imaging Reveals Contribution of Cerebral Hydroxyl Radicals in Hyperhomocysteinemia-Induced Alzheimer-like Dementia.

Elevated plasma level of homocysteine, also termed as hyperhomocysteinemia, is acknowledged as a significant and independent risk factor of Alzheimer's disease. However, the mechanistic insight has not been thoroughly elucidated yet. In this work, 3,5-dihydroxybenzyloxy was explored as the unique reaction trigger and integrated into the naphthalimide fluorophore via a carbamate linker to afford a new probe for •OH imaging. •OH treatment induced aromatic hydroxylation and subsequent elimination reaction to release the caged fluorophore, accompanied with a highly specific and sensitive turn-on fluorescence response. Cell imaging results revealed that excess homocysteine triggered overwhelming •OH production, which was mediated by N-methyl-d-aspartate receptor and NADPH oxidase, and the resultant •OH stress further initiated neuronal ferroptosis, also confirmed by western blot analyses. Additionally, hyperhomocysteinemic mouse models were established, and Alzheimer-like dementia of the mice was observed from behavioral tests. Most importantly, with this probe, cerebral •OH fluctuation was in situ visualized in live mice, which positively correlated with the severity of Alzheimer-like dementia induced by hyperhomocysteinemia. These results reveal that cerebral •OH stress may be the critical nexus linking hyperhomocysteinemia and Alzheimer's disease. This work provides a robust fluorescence probe for in situ visualizing the cerebral •OH fluctuations and illuminating critical insights into •OH contributions in brain disorders.

Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase.

The hydroxylase component (S5HH) of salicylate-5-hydroxylase catalyzes C5 ring hydroxylation of salicylate but switches to methyl hydroxylation when a C5 methyl substituent is present. The use of 18O2 reveals that both aromatic and aryl-methyl hydroxylations result from monooxygenase chemistry. The functional unit of S5HH comprises a nonheme Fe(II) site located 12 Å across a subunit boundary from a one-electron reduced Rieske-type iron-sulfur cluster. Past studies determined that substrates bind near the Fe(II), followed by O2 binding to the iron to initiate catalysis. Stopped-flow-single-turnover reactions (STOs) demonstrated that the Rieske cluster transfers an electron to the iron site during catalysis. It is shown here that fluorine ring substituents decrease the rate constant for Rieske electron transfer, implying a prior reaction of an Fe(III)-superoxo intermediate with a substrate. We propose that the iron becomes fully oxidized in the resulting Fe(III)-peroxo-substrate-radical intermediate, allowing Rieske electron transfer to occur. STO using 5-CD3-salicylate-d8 occurs with an inverse kinetic isotope effect (KIE). In contrast, STO of a 1:1 mixture of unlabeled and 5-CD3-salicylate-d8 yields a normal product isotope effect. It is proposed that aromatic and aryl-methyl hydroxylation reactions both begin with the Fe(III)-superoxo reaction with a ring carbon, yielding the inverse KIE due to sp2 → sp3 carbon hybridization. After Rieske electron transfer, the resulting Fe(III)-peroxo-salicylate intermediate can continue to aromatic hydroxylation, whereas the equivalent aryl-methyl intermediate formation must be reversible to allow the substrate exchange necessary to yield a normal product isotope effect. The resulting Fe(III)-(hydro)peroxo intermediate may be reactive or evolve through a high-valent iron intermediate to complete the aryl-methyl hydroxylation.

HIF1 inhibition of the biflavonoids against pancreas cancer: drug-likeness, bioavailability, ADMET, PASS, molecular docking, molecular dynamics, and MM/GBSA calculations

Pancreatic cancer is an aggressive disease with a high death rate and is difficult to treat. This disease, in the most cases, is asymptomatic until it progresses to an advanced stage. Therefore, the search for bioactive molecules is urgent to combat pancreatic cancer. Then, this work analyzed the interaction potential of agathisflavone and amentoflavone molecules against the HIF1 target using the ADMET, molecular docking, and molecular dynamics simulations. More recent drug-likeness filters that combine physicochemical and physiological parameters have shown that high polar surface area (TPSA > 75 Å2) drives biflavonoids out of the toxic drug space of Pfizer dataset. Regarding the pharmacokinetic descriptors, it was possible to notice that Amentoflavone showed a better order of passive cell permeability (Papp = 8 × 10−6 cm/s) and better metabolic stability, biotransformed by aromatic hydroxylation reactions by the CYP3A4 isoenzyme on the human liver, that favor its hepatic clearance. The molecular docking and molecular dynamics simulations indicated the high interaction potential and stability between the ligands analyzed (highlighted the amentoflavone molecule), respectively. The MM/GBSA calculations showed that the amentoflavone ligand registered the highest ΔG binding value of −32.6957 kcal/mol with the HIF1 target. Then, this molecule may be used as a potential inhibitor of pancreatic cancer. In this perspective, the present work represents an initial step in the virtual bioprospecting a pharmacological tool for treating of pancreatic cancer. Communicated by Ramaswamy H. Sarma

QM/MM Calculations Suggest Concerted O-O Bond Cleavage and Substrate Oxidation by Nonheme Diiron Toluene/o-Xylene Monooxygenase.

The nonheme diiron toluene/o-xylene monooxygenase (ToMO) is the most studied toluene monooxygenase that mediates an aromatic hydroxylation reaction. In this work, QM/MM calculations were performed to understand the reaction mechanism. It is revealed that the μ-η2 :η2 peroxodiferric species is the reactive intermediate after the binding of the O2 molecule to the reduced diferrous center. Subsequently, both a stepwise and a concerted mechanism involving the critical O-O bond cleavage and C-O bond formation were considered. The latter was calculated to be more favorable, suggesting that the formation of a high-valent diferryl Q intermediate is not needed. The isomeric formation of the phenol product was found very facile. The first step was calculated to be rate-limiting, with a barrier of 17.6 kcal/mol for the ortho-hydroxylation.

Rate-Limiting Step of Epoxidation Reaction of the Oxoiron(IV) Porphyrin π-Cation Radical Complex: Electron Transfer Coupled Bond Formation Mechanism.

Epoxidation reactions catalyzed by high-valent metal-oxo species are key reactions in various biological and chemical processes. Because the redox potentials of alkenes are higher than those of most high-valent metal-oxo species, the electron transfer (ET) from the alkene to the high-valent metal-oxo species in the epoxidation reaction is endergonic and must be coupled with another exergonic process. To reveal the mechanism of the ET, we performed a Marcus plot analysis for the epoxidation reaction of the oxoiron(IV) porphyrin π-cation radical complex (compound I) with alkene. The Marcus plots can be simulated with a linear line with the gradient of 0.50 when the redox potential of compound I varies and 0.07 when the redox potential of alkene varies. These results indicate that the ET process is involved in the rate-limiting step and coupled with the following O-C bond formation process: ET coupled bond formation mechanism. The DFT calculations support this conclusion and disclose the details of the mechanism. As the alkene comes close to the oxo ligand, the energy of the highest occupied molecular orbital (HOMO) of the alkene increases and the energy for the ET becomes small enough to allow the ET. Finally, the ET occurs from the HOMO of the alkene to the porphyrin π-radical orbital. The shift of one electron from the HOMO of the alkene by the ET simultaneously results in the O-C half bond formation between the oxo ligand and the alkene. The ET process itself is still endergonic and reversible, but the bond formation coupled with the ET changes the overall process to exergonic and irreversible. We also discuss the similarity with the aromatic hydroxylation reaction and the relevance to the epoxidation reactions of other metal-oxo complexes and peracid.

Biocatalytic Aromaticity-Breaking Epoxidation of Naphthalene and Nucleophilic Ring-Opening Reactions.

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral trans-disubstituted cyclohexadiene synthons. Here, we show that naphthalene epoxides generated by fungal peroxygenases can be subjected to nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. This approach may represent a promising shortcut for the synthesis of natural products and APIs.

Dinuclear μ‐Phenoxo and μ‐Hydroxo Bridged Copper Complexes Exhibiting Oxidation of Phenols and Isoelectronic Compounds: Cytotoxicity and Evidences for Cellular Apoptosis

AbstractThe compartmental pentadentate ligands HLR (where R=Me, OMe, tBu) have been utilized for the synthesis of dinuclear hydroxo and phenolato bridged copper complexes viz. [Cu2(LMe)(μ‐OPh)(μ‐OH](ClO4)2]CH3CN; (1), [Cu2(LOMe)(μ‐OPh)(μ‐OH)(μ‐OH2)](ClO4)2.CH3CN; (2) and [Cu2(LtBu)(μ‐OPh)(μ‐OH)(OH2)](ClO4)2; (3). All the three complexes were characterized by various spectroscopic techniques. Tyrosinase activity was studied by insitu reaction of Cu(I) and ligands exploiting 2,4‐di‐tert‐butyl phenol as substrate. The phenolic substrate (2,4‐DTBP) underwent radical mediated C−C coupling reaction with no insertion of molecular oxygen in contrast to the oxidative aromatic hydroxylation reaction. The catalytic activities of complexes 1–3 on diphenols and related substrate molecules were investigated and all three complexes efficiently oxidize 3,5‐di‐tert‐butylcatechol. Treatment of copper complexes on o‐aminophenol and o‐phenylene diamine resulted in the oxidative coupling of substrate molecules, leading to the formation of phenoxazinone and diaminophenazine respectively. Furthermore, oxidative properties of reported copper complexes were studied in relevance to their ability in inducing apoptotic cell death. Remarkably, all three copper complexes (1, 2 and 3) exhibited excellent cytotoxic activity towards MCF‐7 cell lines (human breast cancer cell lines) with IC50 values of 1.6 μM, 2.0 μM and 0.65 μM respectively and carried out self activated DNA cleavage of plasmid pUC18 DNA.

Mechanism of Oxidative Activation of Fluorinated Aromatic Compounds by N-Bridged Diiron-Phthalocyanine: What Determines the Reactivity?

The biodegradation of compounds with C-F bonds is challenging due to the fact that these bonds are stronger than the C-H bond in methane. In this work, results on the unprecedented reactivity of a biomimetic model complex that contains an N-bridged diiron-phthalocyanine are presented; this model complex is shown to react with perfluorinated arenes under addition of H2 O2 effectively. To get mechanistic insight into this unusual reactivity, detailed density functional theory calculations on the mechanism of C6 F6 activation by an iron(IV)-oxo active species of the N-bridged diiron phthalocyanine system were performed. Our studies show that the reaction proceeds through a rate-determining electrophilic C-O addition reaction followed by a 1,2-fluoride shift to give the ketone product, which can further rearrange to the phenol. A thermochemical analysis shows that the weakest C-F bond is the aliphatic C-F bond in the ketone intermediate. The oxidative defluorination of perfluoroaromatics is demonstrated to proceed through a completely different mechanism compared to that of aromatic C-H hydroxylation by iron(IV)-oxo intermediates such as cytochrome P450 Compound I.

Mechanistic insights into the biomimetic catalytic hydroxylation of arenes by a molecular Fe(NHC) complex

The mechanism of the hydroxylation of benzene by an iron(II) bis(NHC) complex, [FeII(NCCN)(CH3CN)2](PF6)2 (1; NCCN=bis(o-imidazol-2-ylidenepyridine)methane), in the presence of hydrogen peroxide was investigated. The invariance of the catalytic system towards the presence of radical scavengers and an inverse intermolecular kinetic isotope effect (KIE) allow to rule out a hydrogen abstraction pathway. Additionally, using 1,3,5-[D3]benzene as substrate, NIH-shift products were detected, resembling the reactivity of naturally occurring enzymes and some molecular biomimetic iron complexes. Supported by density functional theory calculations, the aromatic hydroxylation reaction is proposed to proceed via an iron-arene σ-complex as intermediate species.

Mechanism of aromatic hydroxylation of lidocaine at a Pt electrode under acidic conditions

Aromatic hydroxylation reactions, which are mainly catalyzed by cytochrome P450 (CYP) enzymes in vivo, are some of the most important reactions of Phase I metabolism, because insertion of a hydroxyl group into a lipophilic drug compound increases its hydrophilicity and prepares it for subsequent Phase II metabolic conjugation reactions as a prerequisite to excretion. Aromatic hydroxylation metabolites of pharmaceuticals may be obtained through various synthetic and enzymatic methods Electrochemical oxidation is an alternative with advantages in terms of mild reaction conditions and less hazardous chemicals. In the present study, we report that aromatic hydroxylation metabolites of lidocaine can be readily obtained electrochemically under aqueous acidic conditions at platinum electrodes. Our results show that the dominant N-dealkylation reaction can be suppressed by decreasing the solution pH below 0.5 resulting in selective 3-hydroxylidocaine, which is an in vivo metabolite of lidocaine. Experiments in 18O labelled water indicated that water is the primary source of oxygen, while dissolved molecular oxygen contributes to a minor extent to the hydroxylation reaction.

A New Domain of Reactivity for High-Valent Dinuclear [M(μ-O)2 M'] Complexes in Oxidation Reactions.

The strikingly different reactivity of a series of homo- and heterodinuclear [(MIII )(μ-O)2 (MIII )']2+ (M=Ni; M'=Fe, Co, Ni and M=M'=Co) complexes with β-diketiminate ligands in electrophilic and nucleophilic oxidation reactions is reported, and can be correlated to the spectroscopic features of the [(MIII )(μ-O)2 (MIII )']2+ core. In particular, the unprecedented nucleophilic reactivity of the symmetric [NiIII (μ-O)2 NiIII ]2+ complex and the decay of the asymmetric [NiIII (μ-O)2 CoIII ]2+ core through aromatic hydroxylation reactions represent a new domain for high-valent bis(μ-oxido)dimetal reactivity.

A New Domain of Reactivity for High‐Valent Dinuclear [M(μ‐O)2M′] Complexes in Oxidation Reactions

AbstractThe strikingly different reactivity of a series of homo‐ and heterodinuclear [(MIII)(μ‐O)2(MIII)′]2+ (M=Ni; M′=Fe, Co, Ni and M=M′=Co) complexes with β‐diketiminate ligands in electrophilic and nucleophilic oxidation reactions is reported, and can be correlated to the spectroscopic features of the [(MIII)(μ‐O)2(MIII)′]2+ core. In particular, the unprecedented nucleophilic reactivity of the symmetric [NiIII(μ‐O)2NiIII]2+ complex and the decay of the asymmetric [NiIII(μ‐O)2CoIII]2+ core through aromatic hydroxylation reactions represent a new domain for high‐valent bis(μ‐oxido)dimetal reactivity.

Hydroxyl radical induced transformation of phenylurea herbicides: A theoretical study

Aromatic ring hydroxylation reactions occurring during radiolysis of aqueous solutions are studied on the example of phenylurea herbicides by Density Functional Theory calculations. The effect of the aqueous media is taken into account by using the Solvation Model Based on Density model. Hydroxyl radical adds to the ring because the activation free energies (0.4–47.2kJmol−1) are low and also the Gibbs free energies have high negative values ((−27.4) to (−5.9)kJmol−1). According to the calculations in most of cases the ortho- and para-addition is preferred in agreement with the experimental results. In these reactions hydroxycyclohexadienyl type radicals form. In a second type reaction, when loss of chlorine atom takes place, OH/Cl substitution occurs without cyclohexadienyl type intermediate.

A Systematic Account on Aromatic Hydroxylation by a Cytochrome P450 Model Compound I: A Low-Pressure Mass Spectrometry and Computational Study.

Cytochrome P450 enzymes are heme-containing mono-oxygenases that mainly react through oxygen-atom transfer. Specific features of substrate and oxidant that determine the reaction rate constant for oxygen atom transfer are still poorly understood and therefore, we did a systematic gas-phase study on reactions by iron(IV)-oxo porphyrin cation radical structures with arenes. We present herein the first results obtained by using Fourier transform-ion cyclotron resonance mass spectrometry and provide rate constants and product distributions for the assayed reactions. Product distributions and kinetic isotope effect studies implicate a rate-determining aromatic hydroxylation reaction that correlates with the ionization energy of the substrate and no evidence of aliphatic hydroxylation products is observed. To further understand the details of the reaction mechanism, a computational study on a model complex was performed. These studies confirm the experimental hypothesis of dominant aromatic over aliphatic hydroxylation and show that the lack of an axial ligand affects the aliphatic pathways. Moreover, a two-parabola valence bond model is used to rationalize the rate constant and identify key properties of the oxidant and substrate that drive the reaction. In particular, the work shows that aromatic hydroxylation rates correlate with the ionization energy of the substrate as well as with the electron affinity of the oxidant.

Development of Heteroatom-introducing Methodologies Based on Ate Complex Formation: Perfluoroalkylation, Borylation, Aromatic Hydroxylation, and Amination Reactions.

We have developed regio- and chemoselective heteroelement-introducing reactions based on ate complex formation. This review is focused on novelly designed reactive species for efficient access to perfluoroalkylated aliphatic and aromatic compounds as well as Csp2-borylated compounds.

Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions.

The ubiquitous ATP synthase uses an electrochemical gradient to synthesize cellular energy in the form of ATP. The production of this electrochemical gradient relies on liposoluble proton carriers like ubiquinone (UQ), which is used in the respiratory chains of eukaryotes and proteobacteria. The biosynthesis of UQ requires three hydroxylation reactions on contiguous positions of an aromatic ring. In Escherichia coli, each of three UQ flavin monooxygenases (FMOs), called UbiF, UbiH, and UbiI, modifies a single position of the aromatic ring. This pattern of three hydroxylation reactions/three proteins has been accepted as a paradigm in UQ biology. Using a phylogenetic analysis, we found that UbiF, UbiH, and UbiI are detected only in a small fraction of proteobacteria, and we identified two new types of UQ FMOs: UbiM, which is distributed in members of the alpha, beta, and gamma classes of proteobacteria, and UbiL, which is restricted to members of the alphaproteobacteria. Remarkably, the ubiL and ubiM genes were found in genomes with fewer than three UQ hydroxylase-encoding genes. We demonstrated, using biochemical approaches, that UbiL from Rhodospirillum rubrum and UbiM from Neisseria meningitidis hydroxylate, respectively, two and three positions of the aromatic ring during UQ biosynthesis. We conclude that bacteria have evolved a large repertoire of hydroxylase combinations for UQ biosynthesis, including pathways with either three specialist enzymes or pathways with one or two generalist enzymes of broader regioselectivity. The emergence of the latter is potentially related to genome reduction events. IMPORTANCE UQ, a key molecule for cellular bioenergetics that is conserved from proteobacteria to humans, appeared in an ancestral proteobacterium more than 2 billionyears ago. UQ biosynthesis has been studied only in a few model organisms, and thus, the diversity of UQ biosynthesis pathways is largely unknown. In the work reported here, we conducted a phylogenomic analysis of hydroxylases involved in UQ biosynthesis. Our results support the existence of at least two UQ hydroxylases in the proteobacterial ancestor, and yet, we show that their number varies from one to four in extant proteobacterial species. Our biochemical experiments demonstrated that bacteria containing only one or two UQ hydroxylases have developed generalist enzymes that are able to catalyze several steps of UQ biosynthesis. Our study documents a rare case where evolution favored the broadening of an enzyme's regioselectivity, which resulted in gene loss in several proteobacterial species with small genomes.

Participation of Electron Transfer Process in Rate-Limiting Step of Aromatic Hydroxylation Reactions by Compound I Models of Heme Enzymes.

Hydroxylation reactions of aromatic rings are key reactions in various biological and chemical processes. In spite of their significance, no consensus mechanism has been established. Here we performed Marcus plot analysis for aromatic hydroxylation reactions with oxoiron(IV) porphyrin π-cation radical complexes (compound I). Although many recent studies support the mechanism involving direct electrophilic attack of compound I, the slopes of the Marcus plots indicate a significant contribution of an electron transfer process in the rate-limiting step, leading us to propose a new reaction mechanism in which the electron transfer process between an aromatic compound and compound I is in equilibrium in a solvent cage and coupled with the subsequent bond formation process.

Comprehensive evaluation of the photo-transformation routes of trans-resveratrol

Liquid chromatography (LC) combined with accurate mass spectrometry (MS), based on the use of a hybrid quadrupole time-of-flight (TOF) MS system, is employed to systematically investigate the photo-transformation routes of trans-resveratrol. Experiments were performed in quartz tubes, containing ethanolic solutions (12% v/v) of the precursor compound, exposed to different ultraviolet (UV) sources and to solar light. Time-courses of trans-resveratrol and transformation products (TPs) were investigated by direct injection of different reaction times aliquots in the LC–QTOF–MS system. Structural elucidation of detected TPs was derived from interpretation of their accurate product ion scan spectra. Trans-resveratrol labelled with 13C6 in the mono-hydroxylated ring was also employed to further confirm the exact positions of some substituents in the generated TPs. In addition to the well-known trans-/cis-isomerization process, three different main reactions pathways were noticed under all the investigated conditions: (1) water addition to the exocycle double bond followed by oxidation to a ketone and cleavage of the molecule, (2) intramolecular cyclization to render a trihydroxylated phenanthrene, and (3) oxidation of the phenanthrene-like derivative to generate an orto-diquinone. Both, the trihydroxylated phenanthrene and the orto-diquinone underwent further aromatic hydroxylation reactions. The above transformation routes were also noticed for cis-resveratrol and the two analogue phytoalexins piceid and piceatannol. In addition to above transformation pathways, under solar light exposure, resveratrol underwent a molecular re-arrangement rendering the so-called resveratrone, whose structure consists of two fused aromatic rings bonded to a linear chain containing a carbonyl group conjugated with a double bond.