Reaction mechanism of the vitamin K-dependent glutamate carboxylase: a computational study

In the reaction cycle of glutamate carboxylase, vitamin K epoxidation by O2 has been proposed to generate a very strong base able to remove a proton from the gamma carbon of a Glu residue, thus yielding a Glu-based carbanion that readily reacts with CO2. We have used hybrid density functional theory to study this appealing mechanism. Our calculations show a very exergonic four-step mechanism with the reaction of (triplet) O2 with the singlet vitamin K anion as the rate-limiting step, with a rate similar to the experimental value. Our study also establishes the need to apply continuum models when performing the optimization of minimum-energy crossing points between potential energy surfaces of different multiplicities for enzyme model systems.     

Reactivity of the >P‐O− nucleophiles toward arylmethyl chloride systems*†

The reactions of sodium dimethyl and diisopropyl phosphite, as well as dibenzylphosphinite with 4‐nitrobenzyl chloride, 9‐chlorofluorene, and diphenylchloromethane were studied in detail by the isolation and identification of all the products, and the examination of the effects of the solvents on the product distribution. The results of the performed experiments are compatible with the proposed mechanism: a >P‐O⁻ anion acts toward an arylmethyl chloride as a base and abstracts a proton to form a carbanion, which can then participate in the SET processes to produce carbon‐centered radicals. Additionally, the >P‐O⁻ reagent can act as a carbon‐centered radical trap if it is present in a high enough concentration. © 1999 John Wiley & Sons, Inc. Heteroatom Chem 10: 431–439, 1999     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

1,1‐Disilyl Alcohols as d1 Synthons: Harnessing the 1,2‐Brook Rearrangement

1,1‐Disilyl alcohols like 6 give the silyl ethers like 9 on treatment with base and alkyl halides, in a reaction which may be formulated as the alkylation of the Brook‐rearranged carbanion 8 . The products can be oxidised to give ketones like 10 , showing that this Brook‐rearranging system supplies a controlled d¹ synthon of the acyl anion class. The alcohols can be prepared from the acid chloride 12 and dimethyl(phenyl)silyllithium, but the intermediate anion 21 need not be worked up; it can be used directly in the alkylation step.     

Rearrangements of the carbanions derived from allyl benzyl thioether

The metalation of allyl benzyl thioether involves the benzylic or the allylic hydrogens. The benzylic carbanion undergoes a rapid[2,3] sigmatropic shift whereas the allylic carbanion gives rise to various rearrangements, among them migration of the allylic unit to the para position with allylic inversion. The temperature dependence of the ratio of products arising from the benzylic carbanion vs those from the allylic carbanion shows that the allylic-to-benzylic carbanion transformation occurs only under special conditions: (a) with slow addition of the base; (b) with thioether in excess relative to the base, and (c) on raising the temperature of the reaction medium from ?78° to ?15°. In the last instance, the proton transfer is intramolecular as shown with labeled thioethers. The extent of the different rearrangements depends on the temperature and solvent. A choice of mechanism cannot be made at this time for the para migration 5→9a. A leaving group effect on the reaction regioselectivity of the carbanion from allyl methyl thioether with benzyl halides has been noticed. The presence of dibenzyl indicates that, in addition to S_N2 reactions, some electron transfer process is occurring.     

Base-Catalyzed Dehydration of 3-Substituted Benzene cis-1,2-Dihydrodiols: Stabilization of a Cyclohexadienide Anion Intermediate by Negative Aromatic Hyperconjugation

Evidence that a 1,2-dihydroxycyclohexadienide anion is stabilized by aromatic "negative hyperconjugation" is described. It complements an earlier inference of "positive" hyperconjugative aromaticity for the cyclohexadienyl cation. The anion is a reactive intermediate in the dehydration of benzene cis-1,2-dihydrodiol to phenol. Rate constants for 3-substituted benzene cis-dihydrodiols are correlated by σ(-) values with ρ = 3.2. Solvent isotope effects for the reactions are k(H(2)O)/k(D(2)O) = 1.2-1.8. These measurements are consistent with reaction via a carbanion intermediate or a concerted reaction with a "carbanion-like" transition state. These and other experimental results confirm that the reaction proceeds by a stepwise mechanism, with a change in rate-determining step from proton transfer to the loss of hydroxide ion from the intermediate. Hydrogen isotope exchange accompanying dehydration of the parent benzene cis-1,2-dihydrodiol was not found, and thus, the proton transfer step is subject to internal return. A rate constant of ～10(11) s(-1), corresponding to rotational relaxation of the aqueous solvent, is assigned to loss of hydroxide ion from the intermediate. The rate constant for internal return therefore falls in the range 10(11)-10(12) s(-1). From these limiting values and the measured rate constant for hydroxide-catalyzed dehydration, a pK(a) of 30.8 ± 0.5 was determined for formation of the anion. Although loss of hydroxide ion is hugely exothermic, a concerted reaction is not enforced by the instability of the intermediate. Stabilization by negative hyperconjugation is proposed for 1,2-dihydroxycyclohexadienide and similar anions, and this proposal is supported by additional experimental evidence and by computational results, including evidence for a diatropic ("aromatic") ring current in 3,3-difluorocyclohexadienyl anion.     

Making thiamin work faster: acid-promoted separation of carbon dioxide

The conjugate of thiamin and benzoylformate, mandelylthiamin (MTh), undergoes decarboxylation about 106 times slower than the analogous enzymic intermediate. It has now been discovered that the decarboxylation of MTh is accelerated by the acid component of pyridine and 4-picoline buffers. There is no role for a proton donor to stabilize the transition state for decarboxylation: catalysis must be achieved by the acid's trapping the product carbanion, preventing recarboxylation. This requires that diffusion of CO2 is rate-determining, and that protonation of the carbanion allows this to occur. This interpretation correctly predicts that the same acid components will prevent a fragmentation reaction by protonating the intermediate, which fragments only as the conjugate base.     

Establishing the kinetic competency of the cationic imine intermediate in nitroalkane oxidase

The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2-(2H2)-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.     

The Mechanism of the Wolff-Kishner Reduction,Elimination, and Isomerization Reactions

The mechanism of the Wolff-Kishner reaction is discussed in the light of accumulated information about the kinetics, thermodynamics, and solvent effects of the process. Wolff-Kishner reduction under normal conditions is thought to involve the trans hydrazone anion, which, in the rate-determining step, exhibits a more or less concerted proton capture at the carbanion terminal and undergoes a solvent-induced proton loss at the nitrogen terminal. The stereochemistry of the Wolff-Kishner reduction is believed to be affected by the presence of “ortho” substituents and by a change to a nonpolar, aprotic reaction medium. The mechanism elaborated for the Wolff-Kishner reduction is extended to the Wolff-Kishner elimination and isomerization reactions. Conformational analysis of the intermediates permits correlation of the competitive reduction and elimination processes with the structures of acyclic systems.     

On the role of the conserved aspartate in the hydrolysis of the phosphocysteine intermediate of the low molecular weight tyrosine phosphatase

The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine phosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, general base-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked. However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do not reveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of water is difficult to reconcile with the known gas-phase proton affinities and solution phase pK(a)'s of aspartic acid and water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. To understand the hydrolysis mechanism, we have used high-level density functional methods of quantum chemistry combined with continuum electrostatics models of the protein and the solvent. Our calculations do not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygen directly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transition state, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to proton transfer to the base is greatly diminished; the aspartate can abstract a proton only after the transition state, a result consistent with experimental solvent isotope effects for this enzyme and with established precedents for phosphomonoester hydrolysis.     

Transition‐metal‐free Chemoselective Oxidative C−C Coupling of the sp3 C−H Bond of Oxindoles with Arenes and Addition to Alkene: Synthesis of 3‐Aryl Oxindoles,and Benzofuro‐ and Indoloindoles

A transition‐metal (TM)‐free and halogen‐free NaOt Bu‐mediated oxidative cross‐coupling between the sp³ C−H bond of oxindoles and sp² C−H bond of nitroarenes has been developed to access 3‐aryl substituted and 3,3‐aryldisubstituted oxindoles in DMSO at room temperature in a short time. Interestingly, the sp³ C−H bond of oxindoles could also react with styrene under TM‐free conditions for the practical synthesis of quaternary 3,3‐disubstituted oxindoles. The synthesized 3‐oxindoles have also been further transformed into advanced heterocycles, that is, benzofuroindoles, indoloindoles, and substituted indoles. Mechanistic experiments of the reaction suggests the formation of an anion intermediate from the sp³ C−H bond of oxindole by tert ‐butoxide base in DMSO. The addition of nitrobenzene to the in‐situ generated carbanion leads to the 3‐(nitrophenyl)oxindolyl carbanion in DMSO which is subsequently oxidized to 3‐(nitro‐aryl) oxindole by DMSO.     

A theoretical study of the mechanism of phosphine-catalyzed hydroalkoxylation of methyl vinyl ketone

The mechanism of phosphine-catalyzed hydroalkoxylation of the methyl vinyl ketone has been investigated by the second-order M?ller-Plesset perturbation theory and the conductor-like polarized continuum model. The free energy reaction profiles of the reaction in both gas phase and solution phase are explored and compared. Our results suggest that the first stage of the studied reaction is the generation of the base (the methoxide anion) with the help of trialkylphosphine, and the second stage is the hydroalkoxylation of the methyl vinyl ketone catalyzed by this base. In the first stage, trialkylphosphine first adds to the methyl vinyl ketone to form a phosphonium enolate intermediate and then this species deprotonates a methanol molecule to generate a methoxide anion. Both steps involve free energy barriers of about 20 kcal/mol. In the second stage, both the addition of the methoxide anion to the methyl vinyl ketone and the proton transfer process from methanol to the methoxyl enolate anion intermediate have activation free energies of about 16 kcal/mol. The reaction in the second stage is exothermic by 10.2 kcal/mol at room temperature. A comparison of the free energy reaction profiles in the gas phase and the solution phase demonstrated that the generation of the methoxide anion could only occur in the presence of the polar solvents. The mechanism proposed in the present work is in reasonable agreement with the known experimental facts.     

Nucleophilic Addition to CC Bonds,Part X

A mechanistic model is presented for the base‐catalyzed intramolecular cyclization of polycyclic unsaturated alcohols of type A to ethers D (Scheme 1). The alkoxide anion B is formed first in a fast acid‐base equilibrium. For the subsequent reaction to D , a carbanion‐like transition state C is proposed. This mechanism is in full agreement with our results regarding the influence of substituents on the regioselectivity and the rate of cyclization. We studied the effect of alkyl substituents in allylic position (alkylated endocylic olefinic alcohols 1 – 3 ) and, especially, at the exocyclic double bond ( 12 – 15 ). The fastest cyclization (k_rel=1) is 12 → 16 , which proceeds via a primary carbanion‐like transition state ( E : R¹=R²=H). The corresponding processes 13 → 17 and 14 → 17 are characterized by a less‐stable secondary carbanion‐like transtition state ( E : R¹=Me, R²=H, or vice versa) and are slower by a factor of 10⁴. The slowest reaction (k_rel ca. 10⁻⁶) is the cyclization 15 → 18 via a tertiary carbanion‐like transition state ( E : R¹=R²=Me).     

弱碱柔能克刚:动力学去质子官能化反应

碳氢键的去质子官能化反应是碳碳键构建最常用的方法,是一种重要的碳氢键活化方式.近年来,碱催化碳碳键形成反应在含弱酸性碳氢键化合物作为亲核试剂的底物拓展方面取得了重要进展.强碱性试剂或催化剂是实现这些弱酸性碳氢键官能化反应的关键.根据酸碱平衡理论,相对较强的碱才能够对弱酸性碳氢键发生去质子化反应,形成较大浓度的碳负离子中间体,进而发生亲核反应.相对较弱的碱不足以对弱酸性碳氢键进行去质子化反应,然而尽管碳负离子中间体可能浓度很低,但应该仍然存在于反应体系中.如果可以选择性地进行热力学有利的化学转化,碳负离子中间体的浓度将会下降并引起去质子化平衡的重新构建.结合碳负离子中间体不可逆的转化和去质子平衡的重新构建,弱酸性碳氢键就可以在弱碱条件下实现缓慢却持续不断的去质子官能化反应.为区别于强碱条件下、通过热力学稳定碳负离子中间体的传统碳氢键去质子官能化反应,我们将这种在弱碱条件下、通过热力学不利的碳负离子中间体转化和酸碱平衡重新构建实现的弱酸性碳氢键的官能化反应称为动力学去质子官能化反应.本文总结了碳氢键去质子官能化反应研究现状和本研究团队近年来在弱碱条件下的动力学去质子官能化反应研究进展.     

Nouveaux complexes de structure η4-trimethylenemethane obtenus a partir du tetracarbonylferrate de sodium par insertion d'allene dans une liaison metalcarbone

The mechanism of formation of α,β-unsaturated ketones in the reaction of disodium tetracarbonylferrate, with an alkyl bromide and allene, is investigated; new intermediate complexes are isolated. In the anion resulting from insertion of allene into an ironacyl bond the negative charge is delocalised over the metallic center and the organic ligand and confirms an η⁴-trimethylenemethane type behaviour. Reactions with (CH₃)₃SiCl and proton yield neutral η⁴-trimethylenemethane complex. In the case of proton a subsequent sigmatropic shift yields a η⁴-heterodiene complexe. The anion is isolated as its ionic cristalline PPN⁺ salt.     

Mechanistic investigation and DFT calculation of the new reaction between S-methylisothiosemicarbazide and methyl acetoacetate

A study on the synthesis and mechanistical aspects of formation of 3-methyl-5-oxo-3-pyrazolin-1-carboxamide (MOPC) starting from S-methylisothiosemicarbazide hydrogen iodide and methyl acetoacetate was performed. In the alkaline aqueous solution, the intermediate methyl acetoacetate S-methylisothiosemicarbazone undergoes substitution of CH₃S^? anion by hydroxide anion, cyclization, carbanion formation, and elimination of methanol, thus yielding corresponding Na-enolate salt of pyrazol-5-one derivative. The structure of the compound obtained after protonation of the formed enolate salt was determined by means of spectroscopic techniques and single-crystal X-ray diffraction analysis. The mechanism of conversion of methyl acetoacetate S-methylisothiosemicarbazone into MOPC was investigated by means of the B3LYP functional, and it was found that the reaction is thermodynamically controlled.     

Unraveling the Mechanism and Regioselectivity of the B12‐Dependent Reductive Dehalogenase PceA

PceA is a cobalamin‐dependent reductive dehalogenase that catalyzes the dechlorination of perchloroethylene to trichloroethylene and then to cis‐dichloroethylene as the sole final product. The reaction mechanism and the regioselectivity of this enzyme are investigated by using density functional calculations. Four different substrates, namely, perchloroethylene, trichloroethylene, cis‐dichloroethylene, and chlorotheylene, have been considered and were found to follow the same reaction mechanism pattern. The reaction starts with the reduction of Co^II to Co^I through a proton‐coupled electron transfer process, with the proton delivered to a Tyr246 anion. This is followed by concerted C?Cl bond heterolytic cleavage and proton transfer from Tyr246 to the substrate carbon atom, generating a Co^III?Cl intermediate. Subsequently, a one‐electron transfer leads to the formation of the Co^II?Cl product, from which the chloride and the dehalogenated product can be released from the active site. The substrate reactivity follows the trend perchloroethylene>trichloroethylene?cis‐dichloroethylene?chlorotheylene. The barriers for the latter two substrates are significantly higher compared with those for perchloroethylene and trichloroethylene, implying that PceA does not catalyze their degradation. In addition, the formation of cis‐dichloroethylene has a lower barrier by 3.8 kcal mol^?1 than the formation of trans‐dichloroethylene and 1,1‐dichloroethylene, reproducing the regioselectivity. These results agree quite well with the experimental findings, which show cis‐dichloroethylene as the sole product in the PceA‐catalyzed dechlorination of perchloethylene and trichloroethylene.     

A FMO-controlled reaction path in the benzil-benzilic acid rearrangement

Reaction paths for the title rearrangement along with its methyl analogue were investigated by density functional theory calculations. The reaction model is R-CO-CO-R + OH(-)(H2O)4 --> R2C(OH)-COO- + (H2O)4 (R = Me and Ph), where the water tetramer is employed both for solvation to OH- and for the proton relay along hydrogen bonds. The reaction is composed of OH- addition, C-C rotation, carbanion [1,2] migration, and proton relay toward the product anions. The rate-determining step was calculated to be the carbanion migration. Apparently, carbanion [1,2] migration is unlikely relative to the carbonium ion one. However, LUMOs of the 1,2-diketones have large and nodeless lobes at the reaction center, the C1-C2 bond. The specific LUMO character is reflected both in the [2+1]-like one-center nucleophilic addition and in the carbanion [1,2] shift. The proton relay involved in the isomerization from the oxo intermediate to the carboxylate was calculated to take place via the water tetramer.     

Phototriggered Release of a Leaving Group in Ketoprofen Derivatives via a Benzylic Carbanion Pathway,But not via a Biradical Pathway

2‐Acetoxymethyl‐2‐(3‐benzoylphenyl)propionic acid (KP‐OAc) was used as a model to elucidate the solvent‐mediated photochemistry mechanism of Ketoprofen (KP). In solutions with a low concentration of water, KP‐OAc exhibits a benzophenone‐like photochemistry, reacting with water molecules through some reaction to form a ketyl radical intermediate. In neutral solutions with a high concentration of water or acidic solutions, KP‐OAc undergoes a photodecarboxylation reaction with the assistance of water molecules or with the catalysis of perchloric acid to directly generate a biradical intermediate that cannot induce the phototrigger reaction to release the AcO^? group. Therefore, the lifetime of the biradical intermediate of KP‐OAc is almost same as that of the biradical intermediate formed from KP in the same kinds of solutions. However, the photodecarboxylation of KP‐OAc in phosphate buffer solution directly produces the benzylic carbanion intermediate, which can induce the phototrigger reaction to release the AcO^? group. Therefore, the lifetime of the biradical intermediate of KP‐OAc is significantly shorter than the lifetime of the biradical intermediate of KP in phosphate buffer solution. Interestingly, the investigation of the photochemistry of KP‐OAc not only verifies the solvent‐mediated photochemistry mechanism of KP but also provides some new insight into the potential of using this kind of platform for phototrigger applications. The biradical intermediate is not the key species leading to the phototrigger reaction but the benzylic carbanion species is the key reactive intermediate that can mediate the phototrigger reaction of KP‐OAc. Therefore, a change in the pH of the solutions can be utilized to switch on and switch off the photorelease reactions of KP derivative phototrigger compounds.     

Evidence of a borderline region between E1cb and E2 elimination reaction mechanisms: a combined experimental and theoretical study of systems activated by the pyridine ring

We report a combined experimental and theoretical study to characterize the mechanism of base-induced beta-elimination reactions in systems activated by the pyridyl ring, with halogen leaving groups. The systems investigated represent borderline cases, where it is uncertain whether the reaction proceeds via a carbanion intermediate (E1cb, A(xh)D(H) + D(N)) or via the concerted loss of a proton and the halide (E2, A(N)D(E)D(N)) upon base attack. Experimentally, the Taft correlation for H/D exchange, in OD(-)/D(2)O with noneliminating substrates (1-methyl-2-(2-Xethyl)pyridinium iodide), is used to predict the expected values of the rate constants for the elimination reactions with N-methylated substrates and F, Cl, Br as the leaving group. The comparison indicates an E1cb irreversible mechanism with F, but the deviation observed with Cl and Br does not allow a conclusive assignment. The theoretical calculations show that for the N-methylated substrate with a fluoride leaving group the elimination proceeds via formation of a moderately stable carbanion. No stable anionic intermediate is instead found when the leaving group is Cl or Br, as well as for any of the nonmethylated species, indicating a concerted elimination. The methylated substrate with Cl shows however only a moderate increase in reactivity compared to the fluorinated substrate, despite the change in mechanism. Very interestingly, our analysis of the computed two-dimensional potential energy surface for the reaction with a F leaving group indeed evidences the lack of a net distinction between the E1cb and E2 reaction paths, which appear to merge smoothly into each other in these borderline cases.