Preparation of Non-natural L Amino Acids via Deracemization of DL-Amino Acids Catalyzed by Immobilized D-amino Acid Oxidase

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶（D-AAO）对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明：D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

Catalysis of Extracellular Deamination by a FAD-Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.     

细胞色素P450蛋白酶催化下环己胺脱氨基反应机理的理论研究

在细胞色素P450蛋白酶催化的一系列二级胺和三级胺的脱烷基、脱氨基反应中,环己胺的脱氨基反应有着重要的生理意义,该反应中涉及Cα-H键氧化过程的机理细节在过去几十年中一直存在较大争议,且迄今尚未见相关理论计算报道. 针对P450催化的环己胺脱氨基反应,一系列的密度泛函理论（DFT）计算结果及以相关计算数据为基础进行了动力学同位素效应计算结果显示：环己胺底物首先经过一个酶促的Cα-H键氧化反应生成一种甲醇胺,之后又通过一个水分子的辅助在非酶环境中进行了脱氨基反应,生成环己酮和氨气. 其中第一步Cα-H键氧化反应涉及了氢原子传递（HAT）过程以及后续无垒的氧反弹过程,且四重态和二重态的能量十分接近,从而证明了Cα-H键氧化反应涉及了“双态反应”（TSR）机理,同时相关的动力学同位素效应计算结果也显示了较大的KIE（动力学同位素效应）值,符合HAT的机理特征.     

A computational mechanistic study of the deamination reaction of melamine

A detailed computational study of the deamination reaction of melamine by OH^–, n H₂O/OH^–, n H₂O (where n = 1, 2, 3), and protonated melamine with H₂O, has been carried out using density functional theory and ab initio calculations. All structures were optimized at M06/6‐31G(d) level of theory, as well as with the B3LYP functional with each of the basis sets: 6‐31G(d), 6‐31 + G(d), 6‐31G(2df,p), and 6‐311++G(3df,3pd). B3LYP, M06, and ω B97XD calculations with 6‐31 + G(d,p) have also been performed. All structures were optimized at B3LYP/6‐31 + G(d,p) level of theory for deamination simulations in an aqueous medium, using both the polarizable continuum solvation model and the solvation model based on solute electron density. Composite method calculations have been conducted at G4MP2 and CBS‐QB3. Fifteen different mechanistic pathways were explored. Most pathways consisted of two key steps: formation of a tetrahedral intermediate and in the final step, an intermediate that dissociates to products via a 1,3‐proton shift. The lowest overall activation energy, 111 kJ mol^?1 at G4MP2, was obtained for the deamination of melamine with 3H₂O/OH^?.     

Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account

Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.     

Radical‐mediated cytosine and 5‐methylcytosine hydrolytic deamination reactions

The mechanisms of cytosine and 5‐methylcytosine hydrolytic deamination reactions in the gas phase have been investigated. The rate‐determining steps of the reactions are found by density functional theory (DFT). The lower barriers of hydrogen and hydroxyl radical‐mediated 5‐methylcytosine deamination make the C‐5 site of 5‐methylcytosine the hot spot for spontaneous mutations. The hydrogen radical inhibits cytosine and 5‐methylcytosine hydrolytic deamination reactions, while the hydroxyl radical clearly leads to an increased risk of genetic mutagenesis. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

用循环伏安法对彩色显影剂氧化产物脱氨反应的研究

本文讨论了用循环伏安法测定电化学反应之后的化学反应(EC反应)速率的方法。测定了pH1.85～12.5范围内彩色显影剂CD-2的脱氨速率。结果表明,在酸性溶液中(pH<5),随着pH的增加脱氨速率减小;在碱性溶液中(pH>7),随着pH的增加脱氨速率增大;在中性或弱酸性溶液中pH5—7的范围内几乎与pH无关。同时讨论了脱氨反应机理。     

Thermal deamination kinetics of tris(ethylenediamine)nickel(II) sulphate in the solid-state

Thermogravimetric techniques have been used to study the kinetics of thermal deamination of tris(ethylenediamine)nickel(II) sulphate. The complex was synthesized and characterized by various chemical and spectral techniques. Thermal decomposition studies were carried at different heating rates (5, 10, 15 and 20°C min⁻¹) in dynamic air. The complex undergoes a four-stage decomposition pattern. The stages are not well resolved. Decomposition path can be interpreted as a two-stage deamination, and a two-stage decomposition. Reaction products at each stage were separated and identified by means of IR and XRD. The morphology of the complex and the residue were studied by means of SEM. Final residue of the decomposition was found to be crystalline NiO. The deamination kinetics was studied using model-free isoconversional methods viz., Friedman, Flynn-Wall-Ozawa (FWO) and Kissinger-Akahira-Sunose (KAS) methods. It is observed that the activation energy varies with the extent of conversion; indicating the complex nature of the deamination reaction.     

New insight into the substituent effects on the hydrolytic deamination of saturated and unsaturated cytosine

Ab initio calculations were carried out to understand the effect of electron donating groups (EDG) and electron withdrawing groups (EWG) at the C5 position of cytosine (Cyt) and saturated cytosine (H₂Cyt) of the deamination reaction. Geometries of the reactants, transition states, intermediates, and products were fully optimized at the B3LYP/6-31G(d,p) level in the gas phase as this level of theory has been found to agree very well with G3 theories. Activation energies, enthalpies, and Gibbs energies of activation along with the thermodynamic properties (ΔE, ΔH, and ΔG) of each reaction were calculated. A plot of the Gibbs energies of activation (ΔG^‡) for C5 substituted Cyt and H₂Cyt against the Hammett σ-constants reveal a good linear relationship. In general, both EDG and EWG substituents at the C5 position in Cyt results in higher ΔG^‡ and lower σ values compared to those of H₂Cyt deamination reactions. C5 alkyl substituents ( H,  CH₃,  CH₂CH₃,  CH₂CH₂CH₃) increase ΔG^‡ values for Cyt, while the same substituents decrease ΔG^‡ values for H₂Cyt which is likely due to steric effects. However, the Hammett σ-constants were found to decrease at the C5 position of cytosine (Cyt) and saturated cytosine (H2Cyt) on the deamination reaction. Both ΔG^‡ and σ values decrease for the substituents Cl and Br in the Cyt reaction, while ΔG^‡ values increase and σ decrease in the H₂Cyt reaction. This may be due to high polarizability of bromine which results in a greater stabilization of the transition state in the case of bromine compared to chlorine. Regardless of the substituent at C5, the positive charge on C4 is greater in the TS compared to the reactant complex for both the Cyt and H₂Cyt. Moreover, as the charges on C4 in the TS increase compared to reactant, ΔG^‡ also increase for the C5 alkyl substituents ( H,  CH₃,  CH₂CH₃,  CH₂CH₂CH₃) in Cyt, while ΔG^‡ decrease in H₂Cyt. In addition, analysis of the frontier MO energies for the transition state structures shows that there is a correlation between the energy of the HOMO–LUMO gap and activation energies.     

Electroreductive Cross-Coupling of Trifluoromethyl Alkenes and Redox Active Esters for the Synthesis of Gem-Difluoroalkenes

An electroreductive access to gem-difluoroalkenes has been developed through the decarboxylative/defluorinative coupling of N-hydroxyphtalimides esters and α-trifluoromethyl alkenes. The electrolysis is performed under very simple reaction conditions in an undivided cell using cheap carbon graphite electrodes. This metal-free transformation features broad scope with good to excellent yields. Tertiary, secondary as well as primary alkyl radicals could be easily introduced. α-aminoacids L-aspartic and L-glutamic acid-derived redox active esters were good reactive partners furnishing potentially relevant gem-difluoroalkenes. In addition, it has been demonstrated that our electrosynthetic approach toward the synthesis of gem-difluoroalkenes could use an easily prepared Kratitsky salt as alkyl radical precursor via a deaminative/defluorinative carbofunctionalization of trifluoromethylstyrene.