Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.     

Halogenation of aromatic compounds: thermodynamic, mechanistic and ecological aspects

Biological halogenation of aromatic compounds implies the generation of reducing equivalents in the form of e.g. NADH. Thermodynamic calculations show that coupling the halogenation step to a step in which the reducing equivalents are oxidized with a potent oxidant such as O₂ or N₂O makes the halogenation reaction thermodynamically feasible without the input of additional energy in the form of e.g. NADH. In a current model on the halogenation of tryptophan to 7-chloro-l-tryptophan NADH and O₂ are proposed as co-substrates in a reaction in which the aromatic compound is oxidized via an epoxide as intermediate. The thermodynamic calculations thus indicate that such a route hinges on mechanistic insights but has no thermodynamic necessity. Furthermore the calculations suggest that halogenation of tryptophan and other aromatic compounds should be possible with N₂O, and possibly even with nitrate replacing O₂ as the oxidant.     

Catalysis by modified polyethylenimine of nucleophilic substitution reactions of azide ions

The reactions of azide ion with 4-chloro-3,5-dinitrobenzoate and with 4-acetoxy-3-nitrobenzoate catalyzed by laurylethyl polyethylenimine were examined at pH 5.72 and 25°C. In the presence of the polymer, marked accelerations were observed for nucleophilic attack of azide ion in the aromatic substitution reaction, but only small enhancements appeared in the acylation reaction. A detailed rate analysis indicates that the intrinsic reactivity of the reactants in the aromatic substitution reaction is increased in the environment of the polymer about 100 times more than is the reactivity of reactants in the acylation reaction. This difference is ascribed to the influence of the apolar environment of the polymer matrix on the more delocalized charge distribution in the transition state of the aromatic substitution reaction.     

芳香族香料化合物生物合成研究进展

芳香族化合物在香料中占很大的比重,传统生产方式有化学合成和植物提取。化学合成依赖于石油资源,并具有环境不友好、反应条件恶劣等缺点。植物提取方法受限于植物资源,且占用耕地。近年来,随着代谢工程和合成生物学技术的发展,利用可再生原料,微生物合成芳香族香料化合物成为一种新的生产方式。文中介绍了大肠杆菌和酵母菌等模式微生物合成芳香族香料的研究进展,包括利用莽草酸途径合成香兰素等,聚酮途径合成覆盆子酮等。综述重点介绍了生物合成途径解析、人工合成途径创建及代谢调控等,为微生物发酵法生产芳香族香料化合物提供参考。     

The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.     

Synthesis of stable cysteine-heterodisulphides

The reaction of Fmoc-Cys(Npys)-OH under acidic conditions with several thiols has been studied. Results show that aromatic thiols give stable heterodisulphides.     

Synthesis of stable cysteine-heterodisulphides

Summary The reaction of Fmoc-Cys(Npys)-OH under acidic conditions with several thiols has been studied. Results show that aromatic thiols give stable heterodisulphides.     

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.     

Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.     

木质素的微生物降解机制

研究微生物降解木质素的反应机理,可以从根本上解释微生物或酶对木质素的作用过程,对提高木质素降解效率,治理环境污染等具有非常重要的意义。从木质素结构的差异出发,总结了近年来研究木质素微生物降解机制所采用的主要模型化合物、研究方法,概述了微生物对木质素的三大作用机理:侧链氧化、去甲基化和芳香环断裂,以及参与这三个反应的主要微生物。     

Studies on Browning Reactions between Sugars and Amino Compounds

N-d-Glucosyl-p-aminobenzoic acid has been found to form melanoidins in methanol solution acidified with hydrogen chloride at 25°. From the reaction mixture 5-hydroxymethyl-furfural (HMF) has been isolated and identified as its 2,4-dinitrophenylhydrazone. So, comparisons between the browning reaction of HMF or furfural with aromatic amine and that of the corresponding n-glycosides have been made under the same condition. From the results obtained, it has been shown that, under the described condition, furfural is almost inactive for browning, while on the contrary, HMF is active and plays an important role in the browning reaction.     

The amidase activity of Candida antarctica lipase B is dependent on specific structural features of the substrates

The hydrolytic activity of Candida antarctica lipase B (CAL-B) was studied using 15 amides with different linear saturated acyl residues and substituents in the aromatic amine. A strong dependence of the hydrolysis rate on the length of the acyl residue and the substituents groups in the aromatic ring of the amides was demonstrated, with the highest hydrolytic initial reaction rates found for the C₁₀ acyl derivates and benzylamides. The C₁₀ benzylamide, an amide without substituents in the aromatic ring was hydrolyzed almost as fast as capsaicin and five times faster that the corresponding C₁₀ vanillyl derivative. Therefore, a benzylamide bearing the non-linear unsaturated acyl residue of capsaicin (8-methyl-6-nonanenoic acid) was synthesized. This substrate was hydrolyzed four times faster than capsaicin. Although it has been widely claimed that lipases rarely display amidase activity, with this contribution we demonstrate that the amidase activity of CAL-B is dependent on the structural features of the substrate.     

The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution.

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the biodegradation of the xenobiotic compound pentachlorophenol by Sphingobium chlorophenolicum. The mechanism of this transformation is of interest because it is unusual and difficult, and because aerobic microorganisms rarely catalyze reductive dehalogenation reactions. Tetrachlorohydroquinone dehalogenase is a member of the glutathione S-transferase superfamily. Many enzymes in this superfamily are capable of catalyzing nucleophilic aromatic substitution reactions. On the basis of this precedent, we have considered a mechanism for tetrachlorohydroquinone dehalogenase that involves a nucleophilic aromatic substitution reaction, either via an S(N)Ar mechanism or an S(RN)1-like mechanism, in the initial part of the reaction. Mechanistic studies were carried out with the wild type enzyme and with the C13S mutant enzyme, which catalyzes only the initial steps in the reaction. Three findings eliminate the possibility of a nucleophilic aromatic substitution reaction. First, the product of such a reaction, 2,3,5-trichloro-6-S-glutathionylhydroquinone, is not a kinetically competent intermediate. Second, the enzyme can carry out the reaction when the substrate is deprotonated at the active site. Nucleophilic aromatic substitution should not be possible when the substrate is negatively charged. Third, substantial normal solvent kinetic isotope effects on k(cat) and k(cat)/K(M,TriCHQ) are observed. Nonenzymatic and enzymatic nucleophilic S(N)Ar reactions typically show inverse solvent kinetic isotope effects.     

Constitutive and repressivle enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates.

Synthesis of five of the enzymes of the common pathway of aromatic biosynthesis has been shown to be unaffected by either the aromatic amino acids--the product of the first reaction (3-deoxy-D-arabinoheptulosonic acid-7-phosphate) or the product of the last reaction (chorismate)--or by the state of regulator gene loci tyrR. On the other hand, the rate of synthesis of these enzymes, and of several other enzymes for which repression control was inactive because of mutations in relevant regulator genes, was found to change with growth rate. These changes were found to correlate at faster growth rates than those observed in glucose minimal medium with the alterations in the relative frequencies of the corresponding structural genes which occur at these growth rates. It was found that when wild-type cells were grown at these faster growth rates in medium lacking the aromatic amino acids, complete derepression of the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase occurred, in strong contrast to the situation when wild-type cells are grown in glucose minimal medium.     

Synthesis of cyclohexylpuromycin and its reaction with N-acetylphenylalanyl-transfer ribonucleic acid on rat liver ribosomes.

1. Cyclohexylpuromycin, an anlogue of puromycin in which a cyclohexane ring replaces the aromatic benzene ring of the L-phenylalanyl moeity of the nucleoside., has been synthesized and examined for its ability to release N-acetylphenylalanine from tRNA attached to rat liver ribosomes. 2.dl-Cyclohexylpuromycin was active in reacting with N-[3H]acetylphenylalanyl-tRNA on rat liver ribosomes to form N-E13H]lacetylphenylalanycyclohexypuromycin. 3. The reaction product N-acetylphenylalanylcyclohexylpuromycin and the corresponding analogue N-acetylphenylalanylpuromycin were chemically synthesized for evaluation of the structure of the released N-acetylphenylalanyl-containing material. 4. The results obtained suggest that the model of Raacke (1971) for purmycin reactivity needs further examination with regard to the role played by the aromatic ring system of the Lphenylalanyl moiety of the nucleoside     

Steroid modifications. II. The effect of ruthenium tetroxide on several aromatic steroids

The ability of ruthenium tetroxide to form a double dibenzylic oxidation product with ring A aromatic steroids has been tested further. The reaction was found to be limited to estratrienes possessing only a 3-acyloxy moiety on ring A. Other substituents resulted in the oxidative degradation of ring A.     

Reaction and substrate specificity of recombinant pig kidney Dopa decarboxylase under aerobic and anaerobic conditions

Dopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO(2) abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O(2) on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a k(cat) approximately half of that measured in the presence of O(2), and is accompanied by a decarboxylation-dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O(2). Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O(2), the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached.     

Studies on a new metabolite and its oxidation in the presence of ascorbic acid

1. A number of tissues, in particular, brain, liver, and kidney, incubated aerobically in vitro as slices or ground suspensions produce a compound which combines with p-aminobenzoic acid in acid solution to form a yellow color. 2. A study of this reaction in rat brain has shown that this compound can be produced when washed boiled brain protein is incubated aerobically with ascorbic acid. The latter acts as a catalyst to break the linkage between the protein and the compound. Oxygen is taken up in the process. 3. A number of aromatic hydroxy compounds such as epinephrine and catechol inhibit the reaction. Cyanide has little or no effect. No reaction occurs anaerobically. 4. The occurrence of the reaction in some animals has been described.     

Photochemistry and photophysics of the endoperoxide of mesodiphenylhelianthrene: a contribution to the localization of the S1(π*σ*) state of aromatic endoperoxides

The endoperoxide of mesodiphenylhelianthrene MDHPO has been studied in detail with respect to fluorescence and photo-induced rearrangement. MDHPO proved to be non-fluorescent, although its absorption spectrum is dominated at the low energy side by a strong ππ* band with a maximum at 429.5 nm. Irradiation of that band effects rearrangement to the corresponding diepoxide MDHDO, a reaction typical for S(1)(π*σ*) excited endoperoxides (EPOs). The absorption spectrum of the product MDHDO is blue shifted by only 3.5 nm. MDHDO has the same extended planar aromatic system like its precursor MDHPO, but MDHDO fluoresces strongly. These results set the excitation energy of the S(1)(π*σ*) state of MDHPO to ≤23?000 cm(-1), which is considered to be a generally realistic value of the S(1)(π*σ*) state energy of aromatic EPOs. The main reaction of S(1)(π*σ*) excited MDHPO is, however, chemical deactivation to ground state MDHPO via an oxygen biradical. The sequence of O-O bond opening and closing is the general way of repopulation of the S(0) state of aromatic EPOs from S(1)(π*σ*) excited states.