How Does the Axial Ligand of Cytochrome P450 Biomimetics Influence the Regioselectivity of Aliphatic versus Aromatic Hydroxylation?

How deep is your orbital? Density functional theory studies on the axial ligand effect of aliphatic versus aromatic hydroxylation of ethylbenzene by iron–oxo complexes with a variable axial ligand show that strong (anionic) ligands pull the metal inside the plane of the haeme and destabilise cationic intermediates through orbital interactions (see picture).

     

How Lanthanide Ions Affect the Addition–Elimination Step of Methanol Dehydrogenases

The recently discovered methanol dehydrogenase, XoxF, is a widespread enzyme used by methylotrophic bacteria to oxidize methanol for carbon and energy, and requires lanthanide ions for its activity. This enzyme represents an essential component of methanol utilization by both methanol- and methane-utilizing bacteria. The present investigation looks on the electronic, energetic and geometrical behavior of the methanol dehydrogenase from Methylacidiphilum fumariolicum SolV, which is strictly dependent on early lanthanide metals with +3 oxidation states, by examining enzyme-substrate complexes of all the lanthanides. We focus on the catalytic reaction mechanism of two methanol dehydrogenases having as cofactor europium and ytterbium belonging to the mid- and later- series of lanthanides, in comparison with the methanol dehydrogenase containing the cerium, one early lanthanide. Our results provide evidence for the influence of the lanthanide contraction effect in all the elementary steps of the catalytic reaction mechanism. This indication may prove useful for developing new catalytic machineries of enzymes that adopt new-to-nature transformations.     

Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.     

The Role of Cytochrome P450 AbyV in the Final Stages of Abyssomicin C Biosynthesis

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.     

Early Main Group Metal Catalysis: How Important is the Metal?

Organocalcium compounds have been reported as efficient catalysts for various alkene transformations. In contrast to transition metal catalysis, the alkenes are not activated by metal–alkene orbital interactions. Instead it is proposed that alkene activation proceeds through an electrostatic interaction with a Lewis acidic Ca²⁺. The role of the metal was evaluated by a study using the metal‐free catalysts: [Ph₂N^?][Me₄N⁺] and [Ph₃C^?][Me₄N⁺]. These “naked” amides and carbanions can act as catalysts in the conversion of activated double bonds (C?O and C?N) in the hydroamination of Ar? N?C?O and R? N?C?N? R (R=alkyl) by Ph₂NH. For the intramolecular hydroamination of unactivated C?C bonds in H₂C?CHCH₂CPh₂CH₂NH₂ the presence of a metal cation is crucial. A new type of hybrid catalyst consisting of a strong organic Schwesinger base and a simple metal salt can act as catalyst for the intramolecular alkene hydroamination. The influence of the cation in catalysis is further evaluated by a DFT study.     

Early Main Group Metal Catalysis: How Important is the Metal?

Organocalcium compounds have been reported as efficient catalysts for various alkene transformations. In contrast to transition metal catalysis, the alkenes are not activated by metal–alkene orbital interactions. Instead it is proposed that alkene activation proceeds through an electrostatic interaction with a Lewis acidic Ca²⁺. The role of the metal was evaluated by a study using the metal‐free catalysts: [Ph₂N⁻][Me₄N⁺] and [Ph₃C⁻][Me₄N⁺]. These “naked” amides and carbanions can act as catalysts in the conversion of activated double bonds (CO and CN) in the hydroamination of Ar NCO and R NCN R (R=alkyl) by Ph₂NH. For the intramolecular hydroamination of unactivated CC bonds in H₂CCHCH₂CPh₂CH₂NH₂ the presence of a metal cation is crucial. A new type of hybrid catalyst consisting of a strong organic Schwesinger base and a simple metal salt can act as catalyst for the intramolecular alkene hydroamination. The influence of the cation in catalysis is further evaluated by a DFT study.     

Theoretical Study on the Mass Transfer of Metal Ions in Ion Exchange Process

The kinetics of ion exchange between Ca2 , Mg2 , Co(Ⅱ), Cu(Ⅱ),Ni(Ⅱ), Fe(Ⅲ), y3 or Sm3 , respectively, in 0.50 mol/L HC1 and H on macrorecticular sulfonic ion exchange resin and the kinetics of the same reactions (M -H exchange) when Mn(Ⅱ) coexisted in resin phase as accelerating ion were studied. The accelerating effect manifested and its rule are consistent with the accelerating effect theory based on the concept concerned with adsorption electrical double layer which has been suggested in a previous paper published.     

Cytochrome P450 Catalyzed Oxidative Hydroxylation of Achiral Organic Compounds with Simultaneous Creation of Two Chirality Centers in a Single C?H Activation Step

Regio‐ and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio‐, diastereo‐, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.     

How Hot are Your Ions in TWAVE Ion Mobility Spectrometry?

Effective temperatures of ions during traveling wave ion mobility spectrometry (TWIMS) analysis were measured using singly protonated leucine enkephalin dimer as a chemical thermometer by monitoring dissociation of the dimer into monomer, as well as the subsequent dissociation of monomer into a-, b-, and y-ions, as a function of instrumental parameters. At fixed helium cell and TWIMS cell gas flow rates, the extent of dissociation does not vary significantly with either the wave velocity or wave height, except at low (<500 m/s) wave velocities that are not commonly used. Increasing the flow rate of nitrogen gas into the TWIMS cell and decreasing the flow rate of helium gas into the helium cell resulted in greater dissociation. However, the mobility distributions of the fragment ions formed by dissociation of the dimer upon injection into the TWIMS cell are nearly indistinguishable from those of fragment ions formed in the collision cell prior to TWIMS analysis for all TWIMS experiments. These results indicate that heating and dissociation occur when ions are injected into the TWIMS cell, and that the effective temperature subsequently decreases to a point at which no further dissociation is observed during the TWIMS analysis. An upper limit to the effective ion temperature of 449 K during TWIMS analysis is obtained at a helium flow rate of 180 mL/min, TWIMS flow rate of 80 mL/min, and traveling wave height of 40 V, which is well below previously reported values. Effects of ion heating in TWIMS on gas-phase protein conformation are presented.     

Drug Metabolism by Cytochrome P450 Enzymes: What Distinguishes the Pathways Leading to Substrate Hydroxylation Over Desaturation?

Cytochrome P450 enzymes are highly versatile biological catalysts in our body that react with a broad range of substrates. Key functions in the liver include the metabolism of drugs and xenobiotics. One particular metabolic pathway that is poorly understood relates to the P450 activation of aliphatic groups leading to either hydroxylation or desaturation pathways. A DFT and QM/MM study has been carried out on the factors that determine the regioselectivity of aliphatic hydroxylation over desaturation of compounds by P450 isozymes. The calculations establish multistate reactivity patterns, whereby the product distributions differ on each of the spin‐state surfaces; hence spin‐selective product formation was found. The electronic and thermochemical factors that determine the bifurcation pathways were analysed and a model that predicts the regioselectivity of aliphatic hydroxylation over desaturation pathways was established from valence bond and molecular orbital theories. Thus, the difference in energy of the O?H versus the O?C bond formed and the π‐conjugation energy determines the degree of desaturation products. In addition, environmental effects of the substrate binding pocket that affect the regioselectivities were identified. These studies imply that bioengineering P450 isozymes for desaturation reactions will have to include modifications in the substrate binding pocket to restrict the hydroxylation rebound reaction.     

How Is a Metabolic Intermediate Formed in the Mechanism‐Based Inactivation of Cytochrome P450 by Using 1,1‐Dimethylhydrazine: Hydrogen Abstraction or Nitrogen Oxidation?

A precise understanding of the mechanism‐based inactivation of cytochrome P450 enzymes (P450s) at the quantum mechanical level should allow more reliable predictions of drug–drug interactions than those currently available. Hydrazines are among the molecules that act as mechanism‐based inactivators to terminate the function of P450s, which are essential heme enzymes responsible for drug metabolism in the human body. Despite its importance, the mechanism explaining how a metabolic intermediate (MI) is formed from hydrazine is not fully understood. We used density functional theory (DFT) calculations to compare four possible mechanisms underlying the reaction between 1,1‐dimethylhydrazine (or unsymmetrical dimethylhydrazine, UDMH) and the reactive compound I (Cpd I) intermediate of P450. Our DFT calculations provided a clear view on how an aminonitrene‐type MI is formed from UDMH. In the most favorable pathway, hydrogen is spontaneously abstracted from the N2 atom of UDMH by Cpd I, followed by a second hydrogen abstraction from the N2 atom by Cpd II. Nitrogen oxidation of nitrogen atoms and hydrogen abstraction from the C? H bond of the methyl group were found to be less favorable than the hydrogen abstraction from the N? H bond. We also found that the reaction of protonated UDMH with Cpd I is rather sluggish. The aminonitrene‐type MI binds to the ferric heme more strongly than a water molecule. This is consistent with the notion that the catalytic cycle of P450 is impeded when such an MI is produced through the P450‐catalyzed reaction.     

Cytochrome P450 119 Compounds I Formed by Chemical Oxidation and Photooxidation Are the Same Species

Compound I from cytochrome P450 119 prepared by the photooxidation method involving peroxynitrite oxidation of the resting enzyme to Compound II followed by photooxidation to Compound I was compared to Compound I generated by m-chloroperoxybenzoic acid (MCPBA) oxidation of the resting enzyme. The two methods gave the same UV/Visible spectra, the same products from oxidations of lauric acid and palmitic acid and their (ω-2,ω-2,ω-3,ω-3)-tetradeuterated analogues, and the same kinetics for oxidations of lauric acid and caprylic acid. The experimental identities between the transients produced by the two methods leave no doubt that the same Compound I species is formed by the two methods.     

Discovery of a Dual Function Cytochrome P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis

The 1,3-enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn ( 1 ) is a 1,3-enyne-containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3-enyne biosynthesis in 1 , and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1 . A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis-diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3-enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five-carbon prenyl chain, rather than a polyprenyl chain, to a p-hydroxybenzoic acid acceptor.     

Suzuki Coupling Reactions in Neat Water as the Solvent: Where in the Biphasic Reaction Mixture Do the Catalytic Reaction Steps Occur?

Many reports on water‐compatible palladium catalysts have appeared in the recent literature. For hydrophobic substrates, mixtures with pure water are biphasic, and it is widely not regarded that the elusive locality of the catalytic process (in water, the organic layer, or at the phase boundary) has an important impact on the mechanism and efficiency of the reaction. In the present work, for the first time systematic variation of reaction parameters has been performed for Suzuki coupling experiments with chloro‐ and bromoarenes in pure water. The investigations are not only aimed at the factors influencing the catalytic activity, but also at the effects that may occur particularly in water/organic biphasic media, and on the question as to in which of the two liquid phases the reaction takes place. These investigations have revealed that dilution of the base (in the aqueous layer) and the Pd species (in the organic layer) are detrimental to the reaction, and that phase‐transfer processes play a major role in the overall mechanism. A series of experiments with variation of parameters like precatalyst hydrophilicity, organic and water phase volume, additives, stirring rate, base concentration, and so forth, indicate that for the systems under study the reaction occurs in the organic layer. The water phase needs to be present to dissolve and provide polar reactants, and re‐absorb side products. The results encourage to pay more regard to the question of phase locality of coupling reactions in water in general.