离子液体的制备及其在酶催化反应中的应用

离子液体,尤其是非水溶性离子液体可以作为一种溶剂或酶的载体用于非水相酶促反应中,也可以用于双相体系中的酶促反应。本文概括性介绍了常见离子液体的制备,总结和讨论了离子液体中酶的活性、稳定性、反应选择性以及各类酶在离子液体中的催化反应行为。离子液体的物性及其与酶的相容性对酶本身及酶促反应都有很大的影响。在非水相酶促反应中,离子液体的极性作用不遵从通常用来判别大多数有机物溶剂行为的规则,比如lgP规则。     

离子液体中的生物催化反应

本文综述了近年来离子液体中生物催化反应的研究进展。离子液体作为新的绿色溶剂,用于生物催化反应具有以下特点:在离子液体中酶有良好的稳定性、选择性和反应活性。离子液体可溶解极性大的反应物, 产物易分离,酶和离子液体可重复使用。对离子液体中的生物催化进行了展望。     

离子液体中脂肪酶催化酯类合成的新进展

作为21世纪最具发展潜力的绿色溶剂,离子液体用于酶促合成的反应介质具有得天独厚的优势.与传统的有机溶剂相比,离子液体能够提高脂肪酶的稳定性和选择性,减少有机合成中的副反应和有毒气体的产生,后处理简单,可重复利用.参考10年来的文献,从脂肪酶催化酯类合成的常用离子液体种类、过程因素及场/反应器强化等方面进行了综述,同时展望了离子液体在未来酯类酶促合成领域的发展趋势.     

离子液体中糖酯类化合物的酶法合成

糖酯作为一种非离子型表面活性剂, 由于特殊的两亲结构, 已广泛应用于医药、食品及化妆品工业. 除作为表面活性剂外, 一些糖酯及其衍生物还具有抗菌及抗肿瘤等生物活性. 酶法合成糖酯反应条件温和, 选择性高. 有机介质中酶促糖酯合成的困难在于糖类化合物的低溶解度. 离子液体作为新型绿色反应介质, 用于糖酯类化合物的酶法合成具有许多优点. 综述了近年来该领域的研究进展.     

在非水介质中进行酶促反应的几个重要问题

介绍了在非水介质中酶促反应的最新进展。较深入地讨论了介质性质对酶的催化活性及立体选择性的影响，有机溶剂中必需水含量对酶催化反应的重要性，描述了非缓冲体系中的盐及其它极性添加剂对酶活性的影响，介绍了在非均相的低共熔体系中进行酶促合成肽的研究。     

离子液体微乳液体系的应用研究

室温离子液体具有诸多优异的物理化学性质及功能,是一类备受关注的新型介质和材料,应用于诸多领域。特别是近年来,由离子液体参与形成的微乳液因其在生物、医药、催化以及材料制备等领域具有潜在的应用前景而备受关注。本文综述了近年来咪唑类离子液体作为极性、非极性和表面活性剂组分,分别取代微乳液体系中的水相、油相和表面活性剂相,形成的一系列新型的微乳液体系的研究进展,归纳了水、有机溶剂、高聚物、助表面活性剂、温度等因素对离子液体微乳液性质的影响。重点介绍了离子液体微乳液的热点应用,包括以离子液体微乳液液滴为模板合成纳米材料,离子液体微乳液作为酶反应的介质及其在有机反应等方面的研究进展。     

含离子液体［bmim］Cl的反应介质中马肝醇脱氢酶的催化特性

 研究了马肝醇脱氢酶(HLADH)在含离子液体1-丁基-3-甲基咪唑氯酸盐(［bmim］Cl)的反应介质中的催化特性. 以乙醇为底物时,该酶在［bmim］Cl含量≤0.15 g/ml的体系中的活力高于在不含离子液体的体系中的活力; 离子液体浓度过高(＞0.15 g/ml)对酶活性有明显的抑制作用. 反应温度和pH对含离子液体的反应介质中酶活力的影响规律与不含离子液体时的规律相似. 与不含离子液体的反应介质相比, HLADH在含0.05 g/ml ［bmim］Cl的体系中催化乙醇氧化的活化能下降,酶反应的Vmax和Km均升高. 反应体系中低浓度(≤0.1 g/ml)的离子液体能提高酶的热稳定性,但高浓度(＞0.1 g/ml)的离子液体可降低酶的热稳定性. 紫外二阶导数光谱显示,在含不同浓度离子液体的反应介质中酶分子构象的变化有较大的差异.     

离子液体中固定化光合细菌催化不对称还原反应的研究

采用苯乙酮为模式底物,选用了3种典型的离子液体作为反应介质系统研究了离子液体与缓冲液构成的均相及两相体系中固定化光合细菌催化不对称还原反应的特性．通过对构建的离子液体反应体系进行条件优化,发现与水相及有机相相比,离子液体作为生物催化反应介质更有利于还原反应的进行,并且离子液体及固定化细胞易回收重复利用．研究结果表明,在...     

无溶剂体系中的脂肪酶催化反应研究进展

酶作为一种生物催化剂，具有专一性强、催化效率高、在常温常压等温和条件下能进行操作等优点，从而引起众多学者的兴趣．传统酶学研究的是酶在水溶液中的催化行为，这使得人们产生了误解，认为只有在水溶液中酶才能保持活性，有机溶剂会使酶变性失活．1984年，美国麻省理工工学院的科学家Klibanov首次发现有许多酶可用于有机溶剂，从而解决了有机化合物在水介质中的低溶解性限制酶催化剂应用的问题，酶不仅能够在有机溶剂中保持稳定，而且还显示出很高的催化转酯活力．这一发现为酶学研究和应用带来了又一次革命性飞跃，从而促进了非水酶学的兴起，使之成为目前重要的酶技术之一．其中研究较多的是脂肪酶，脂肪酶不仅能催化油脂水解，而且在非水介质中也能催化油脂的酯交换反应，用来提高油脂的品位．随着研究的深入和学科的交叉，各种新的酶促反应体系也相继被发现，大大拓宽了酶催化反应的应用范围，使酶法合成逐步发展成为与化学法合成相互补充的合成方法．近年来非水介质中的不同反应体系的酶催化反应，主要有：有机溶剂体系、反相胶束体系、超临界体系、气相体系等，其中无溶剂体系中的酶促反应是非水体系中酶促反应的特例．     

基于离子液体共溶剂效应的氯过氧化物酶催化氧化邻苯二胺的研究

通过缓冲液-离子液体混合溶剂中氯过氧化物酶(CPO)催化氧化邻苯二胺(OPD)的产物结构和性能的表征,证实此酶促反应的产物为2,3-二氨基吩嗪(DAP);OPD-H2O2-CPO反应体系有望用于荧光酶联免疫分析;酶动力学分析表明以咪唑类离子液体(ILS)为共溶剂时,CPO对底物的亲和力及对底物识别的专一性都得以改善,从而有效提高了产物收率;酶促反应主要受CPO的稳定性及酶的用量等因素控制.在最佳条件下,产率可达81.16%.     

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters,determined by ultra-high-performance liquid chromatography–tandem mass spectrometry

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC–MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters.     

Studies on the enzymatic hydrolysis of polyesters I. Low molecular mass model esters and aliphatic polyesters

The bio-catalysed cleavage of ester bonds in low molecular mass model esters and aliphatic polyesters was studied in detail with the aim to gain improved information about the underlying mechanism and the parameters controlling polyester degradation. Among various hydrolytic enzymes the lipase of Pseudomonas species (PsL) was chosen for the investigations. In the heterogeneous phase system the specific hydrolysis rate of the esters was constant as long as free substrate surface was available. In addition to aliphatic low molecular mass model esters, also cycloaliphatic and aromatic esters were cleaved by PsL, indicating that a steric hindrance of the enzymatic ester cleavage is not the predominant controlling factor in polyester degradation. However, the cleavage rates of the aliphatic model esters are larger by more than an order of magnitude. For aliphatic polyesters the temperature difference between the melting point of the polymer and the temperature where degradation takes place (ΔT_mt), turned out to be the primary controlling parameter for polyester degradation with the lipase. Only if ΔT_mt<30 °C, a measurable enzymatic degradation rate is found. ΔT_mt can be regarded as a measure of the mobility of the polyesters chains in the crystalline domains, necessary for the access of the esters to the active site of the lipase. Though aliphatic homopolyesters are seemingly very similar with regard to their chemical structure and reactivity of the ester bonds, their enzymatic degradation rates still differ significantly even at the same ΔT_mt. These differences have obviously to be attributed to small changes in the chemical structure, as, for instance, the C number of the aliphatic diacid.     

Qualitative analysis of phenols and alcohols in complex samples after derivatization to esters of ferrocene carboxylic acid by gas chromatography with mass spectrometric detection

Phenols and alcohols in complex petrochemical samples are derivatized to esters of ferrocene carboxylic acid in a rapid and simple reaction. These esters show several characteristic ions of high intensity in gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI-MS) which can be used for the identification of the analyte. A differentiation between alcohols and phenols is possible due to the McLafferty rearrangement shown only by the alcohol esters. Selective ion monitoring of m/z 213 yields a phenol-selective chromatogram and m/z 230 an alcohol-selective chromatogram. For all iron containing fragments, the isotopic pattern of iron can be observed which enhances the reliability of the peak identification. The ferrocene esters of 11 alcohols, 20 alkylphenols (including octyl- and nonylphenol), phenylphenol, naphthol and hydroxyphenanthrene, several chloro- and all mononitrophenols were synthesized as well as the esters of pentadeutero- and two fluorinated phenols. Their fragmentation pattern under EI ionization is studied and a GC-ion trap-MS system was optimized for simultaneous use of the full scan mode and an MS/MS experiment in the same run. This provides for a very high selectivity in the detection of the esters and makes available the complete mass spectra without any additional measurements. The benefits of the simple and rapid derivatization procedure in combination with this powerful detection method are demonstrated for selected petrochemical samples. Several alkylphenols could be successfully identified in such samples and molecular information about unknown phenolic components could be easily obtained.     

Über halogen- und stickstoffhaltige Derivate aliphatischer Carbonsäuren, 7. Mitt.: Über Gewinnung und Reaktionen von α-Nitrocarbonsäureestern

Esters of α-bromo-α-methyl carboxylic acids are reacted with NaNO₂ in water/alcohol to give esters of the corresponding α-nitro carboxylic acid. Some of these nitro esters can be characterized as amides. Activating groups (benzyl or acetyl group) decrease the stability of such tertiary α-nitro esters and give consecutive reactions.     

Determination of zinc O,O′-dialkyl dithiophosphate lubricant additives via the corresponding methyl and p-nitrobenzylic ester derivatives using GC-MS, GC-NPD and HPLC-MS

 A quantitative fingerprinting of automotive lubricants with respect to zinc dialkyl dithiophosphates (ZDP), major anti-wear/antioxidant additives, is presented. ZDPs in lubricant solutions are converted into the corresponding methyl and p-nitrobenzylic esters, respectively. After removal of the lubricant matrix the methyl esters are submitted to gas chromatography (GC). Mass spectrometric detection (MS) and comparison with reference methyl esters enable the characterisation of practical ZDP mixtures with respect to alkyl chain length and isomery of the single components. Overall recovery rates are higher than 90% and phosphorus-selective detection (NPD) allows a quantitative determination down to 0.1 pg/μl. The p-nitrobenzylic esters may be analysed by HPLC. Identification and quantification is performed by on-line HPLC-UV and HPLC-MS (APCI) with a determination limit of 20 pg/μl. The ZDP quantification via the methyl esters is applied to seven lubricants from the German market. The method is applicable to used oils allowing the monitoring of ZDP consumption during engine operation. Received: 22 March 1996/Revised: 19 June 1996/Accepted: 21 June 1996     

Analysis of alcohols, as dimethylglycine esters, by electrospray ionization tandem mass spectrometry

Dimethylglycine (DMG) esters are new derivatives for the rapid, sensitive and selective analysis of primary and secondary alcohols, in complex mixtures, by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their development was inspired by the use of the complementary dimethylaminoethyl esters for the trace, rapid analysis of fatty acids. DMG esters are simply prepared by heating a dichloromethane solution of the imidazolide of dimethylglycine, containing triethylamine, and an alcohol. DMG esters of long-chain fatty alcohols, isoprenoidal alcohols and hydroxy-acids are analysed by electrospray ionization tandem mass spectrometry with a precursor ion of m/z 104 scan. Diols, glyceryl esters, glyceryl ethers and some sterols are analysed by a neutral loss of 103 Da scan. Trimethylglycine (TMG) ester iodides, prepared by alkylation of DMG esters with methyl iodide, are more sensitive derivatives for molecules containing secondary alcohol groups, such as cholesterol and gibberellic acid. They are analysed by a precursor ion of m/z 118 scan. DMG or TMG derivatives were shown to be at least comparable and sometimes an order of magnitude more sensitive than N-methylpyridyl ether derivatives for ESI-MS/MS analysis of the different classes of alcohols. Applications of these derivatives for the diagnosis of inherited disorders and the analysis of natural products are presented.     

HPLC of fatty acid esters of mono- and polyhydric alcohols. Part 2: Preparative separation

This paper demonstrates how simply and rapidly fatty acid esters of mono- and polyhydric alcohols can be separated quantitatively in preparative quantities on Lobar® RP-8 packed columns. After the separation of unknown mixtures, the isolated esters are identified from spectroscopic data (IR/NMR) and, after saponification of the ester components (fatty acids and alcohols), from the retention times of gas and high-pressure liquid chromatographic separations. Thus, in particular, sparingly volatile or nonvolatile partial esters of polyhydric alcohols, as well as the long-chained full esters, can be determined qualitatively and quantitatively. The following fatty acid esters of mono- and polyhydric alcohols have been separated: the i-propylesters of the laurinic and myristinic acids, the i-butyl-, i-octyl- and i-octadecyl-esters of the palmitinic and stearic acids, the mono- and di-fatty acid esters of the 1,3-bis-(2-hydroxyethyl)-5,5-dimethylhydantoins, the mono-, di- and tri-esters of the trimethylalpropane and the full esters of the pentaerythrite.     

Glycidyl fatty acid esters in food by LC-MS/MS: method development

An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70-150 μg/kg for each analyte using 10 mg sample and 1-3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5-10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to the survey of glycidyl fatty acid esters in food products on the Canadian market.     

Preparation of azidoaryl- and azidoalkyloxazoles for click chemistry

A series of azidoaryl- and azidoalkyl(diphenyl)oxazole scaffolds were warranted for biofilm inhibition studies. Cyclization of azidoaryl- or azidoalkyl esters of benzoin with ammonium acetate in acetic acid gives 2-azidoaryl- or 2-azidoalkyl-4,5-diphenyloxazoles. The azidoaryl esters are prepared from the corresponding azidocarboxylic acids/acid chlorides while the azidoalkyl esters are prepared from the corresponding haloalkyl esters.