Lipase-Catalyzed Synthesis of Carboxylic Amides: Nitrogen Nucleophiles as Acyl Acceptor

Summary.  The lipase-catalyzed aminolysis of carboxylic esters is a fairly general reaction that has been performed with a wide range of esters and amines, generally in anhydrous organic media to avoid undesirable hydrolysis of the ester. Alternatively, carboxylic amides can be synthesized by lipase mediated condensation of carboxylic acids and amines if an excess of either reactant is avoided. Chiral carboxylic esters have been resolved by lipase-catalyzed aminolysis. In the majority of these resolutions, Candida antarctica lipase B has been employed as the catalyst. A range of chiral amines has been resolved by lipase mediated acylation, using mainly the lipases from C. antarctica (B type) and Pseudomonas species. The enantiorecognition was frequently found to depend critically on the acylating agent and the reaction medium. Received December 20, 1999. Accepted January 1, 2000     

酶促水解法拆分布洛芬乙酯的工艺研究

以布洛芬乙酯为反应底物,探索了酶促水解法拆分布洛芬的工艺。以曲拉通X-100为表面活性剂,利用南极假丝酵母脂肪酶B（CAL-B）催化拆分外消旋布洛芬乙酯,并对表面活性剂含量、有机溶剂种类和添加量、酶量、缓冲溶液pH、温度及时间等反应条件进行了优化;得到的最佳反应条件为：在pH为9.0的缓冲溶液中,添加80 mg曲拉通X-100、50 μL二氯甲烷和15 mg CAL-B,于30℃下反应48 h;在该条件下,S-布洛芬乙酯的剩余率（C）为65%,布洛芬乙酯的对映体过量值（ees）为94%。     

Resolution of Ibuprofen Catalyzed with Free and Immobilized Lipases

Enzymatic hydrolyses of ibuprofen esters produced 99% optically pure S-(+)-ibuprofen. The ee values of two enantiomers of ibuprofen were determined on a HPLC equipped with a chiral column.     

Lipase-Catalyzed Solvent-Free Amidation of Phenolic Acids

A series of N-alkyl-substituted amides, based on various phenolic acids, have been synthesized by the condensation of equimolar amounts of phenolic acids with different alkyl amines in the presence of Candida antarctica lipase at 60–90 °C in 16–20 h. The reactions were carried out in a solvent-free system without the use of any activating agents. All the products were obtained in appreciable amounts and the yields for different compounds varied between 75.6% and 83.5%. The synthesized compounds were characterized using spectroscopy techniques, namely infrared and NMR (¹H and ¹³C).     

Lipase-Catalyzed Regio- and Stereoselective Acylation of Hydroxy Groups in Steroid Side Chains

Summary.  Lipase from Pseudomonas cepacia (PCL) catalyzes the regio- and stereoselective acylation of primary and secondary hydroxy groups in steroid side chains under irreversible transesterification conditions with vinyl acetate in organic solvents. Received December 20, 1999. Accepted December 31, 1999     

有机相脂肪酶催化的有机物合成反应

综述了近２０年为有机介质中脂肪酶催化反应在酯合成，酯交换、内酯合成、多肽合成、高聚物合成及立体异构体拆分等有机合成领域的应用。参考文献７１篇。     

有机溶剂中脂肪酶催化的2—辛醇动力学拆分

利用有机溶剂中假单胞菌脂肪酶催化的对映选择性酯化反应，对外消旋（Ｒ，Ｓ）－２－辛醇进行了光学拆分。分别考察了冻干时酶缓冲液的ｐＨ值和浓度以及２－辛醇酯化的酰基给体（脂肪酸），有机溶剂，反应温度等因素对脂及酶催化性能的影响。结果表明，冻干时酶缓冲液的ｐＨ值和浓度对酶的活力影响很大，而对选择性影响较小。以ｐＨ值为８．３，浓度为２０ｍｍｏｌ／Ｌ的磷酸盐缓冲液最为适合。     

Rapid Estimation of Enantioselectivity in Lipase-catalyzed Resolution of Glycidyl Butyrate Using pH Indicator

A simple method for rapid estimation of the enantioselectivity of lipase in resolution of chiral esters is described. The enantioselectivity of lipase can be estimated rapidly through comparing the dif-ference of hydrolysis rates for the racemic ester and its slow reacting enantiomer under the same condition because the difference mainly depends on the enantioselective ratio(E values). The higher the enantiose-lectivity of enzyme, the larger the difference of hydrolysis rate. The bromothymol blue(BTB) can be used as pH indicator for microplate reader to monitor the formation of acid in lipase-catalyzed hydrolysis ofesters. This method has been successfully used to rapidly estimate the enantioselectivity of several lipases in the resolution of glycidyl butyrate.     

Resolution of 2-Octanol via Lipase-catalyzed Enantioselective Acetylation in Organic Solvents

The lipases from different sources were screened for their ability to catalyze the resolution of 2-octanol in organic solvents with vinyl acetate as the acylating reagent. The medium effect has been studied on the irreversible transesterification with varying water activity(aw). The influence of vinyl acetate concentration on it has also been investigated. Under the optimal conditions, the enantiomeric ratio(E value) of pseudomonas fluorescence lipase (PFL) exceeded 200 with an enantiomeric excess (e. e. ) of S-2-octanol above 99% at a 51% degree of conversion.     

有机相中脂肪酶催化有机硅醇转酯反应机理

首次探讨了皱摺假丝酵母脂肪酶CRL催化有机硅醇及其碳结构类似物与脂肪酸酯的转酯反应机理。结果表明,反应符合乒乓机制,底物中硅原子的存在并未改变脂肪酶催化转酯反应的机制。给出了CRL催化1-三甲基硅烷基乙醇及其碳结构类似物3,3-二甲基-2-丁醇与丁酸正戊酯间的转酯反应动力学方程。     

Improvement of Properties of Pseudomonas sp. Lipase in Resolution of 2-Octanol

IntroductionThe demand for the high purity of chiral com-pounds has led to the increasing use of biocatalystsand the increasing research in order to obtain en-zymes with a higher activity and selectivity.In thisfield the utilization of lipases has been proved to beone of the simplest but most promising techniquesfor the enantioselective conversion of various hy-drophobic acids and alcohols in the last decade.There has been a considerable interestin improvingthe enantioselectivity of lipases an…     

Novel Carboxyl Esterase Preparations for the Resolution of Linalyl Acetate

Summary.  Biocatalytic resolution of the tertiary terpene alcohol (±)-linalool was accomplished via hydrolysis of its corresponding acetate ester using two highly enantiospecific enzymes (E > 100). The latter were identified in a crude cell-free extract of Rhodococcus ruber DSM 43338 and could be separated by (partial) protein purification. Since they showed opposite enantiopreference, they were termed (R)- and (S)-linalyl acetate hydrolase (LAH). The activity and selectivity of the enzyme preparations was markedly dependent on the fermentation conditions. Received November 18, 1999. Accepted January 21, 2000     

脂肪酶催化作用下酰胺化反应的立体规则性研究

提出了在LipasefromCandidaAntrarctica(CAL)脂肪酶的催化作用下,含有手性中心C原子的胺类化合物酰胺化的异构选择性规则.与氨基相连的碳原子的立体结构对酰胺化反应有很大的影响.型胺类化合物呈R-型优先选择,型胺类化合物呈S-型优先选择.     

The Resolution of Enantiomeric Ibuprofen on Chiral Stationary Phases Containing N-Arylcarbamoyl Derivatives of Phenylglycine

A series of fourteen anilide derivatives of ibuprofen were resolved on six chiral stationary phases (CSPs) derived from N-arylcarbamoyl derivatives of (S)-phenylglycine. Excellent chiral resolutions were achieved on these CSPs. The ionic-type CSPs showed better chiral recognition abilities than the corresponding covalent-type CSPs, and the CSP bearing two chiral centers has better performance than the CSPs bearing only one chiral center. The highest separation factor was achieved using the ionic-type CSP bearing two chiral centers for the resolution of the 3,5-dinitroanilide derivative of ibuprofen. This result is better than those reported in literature for the resolution of ibuprofen on the CSPs derived from amino acids, According to the chromatographic behaviors, the hydrogen bonding interaction, the π-π interactions provided by the phenyl groups in CSPs bearing one chiral center, and the phenylethylcarbamoyl moiety in CSPs bearing two chiral centers dominate the chiral recognition.     

Quantitative High Resolution Electron Microscopy

 With the resolution becoming sufficient to reveal individual atoms, HREM is now entering the stage where it can compete with X-ray methods to quantitatively determine atomic structures of materials without much prior knowledge, but with the advantage of being applicable to aperiodic objects such as crystal defects. In our view the future electron microscope will be characterised by a large versatility in experimental settings under computer control such as the illumination conditions (TEM-STEM), CBED, detecting conditions (diffraction, image, ptychography) and many other tunable parameters such as focus (g), voltage, spherical aberration (C _s ), beam tilt, etc. Since modern detectors can detect single electrons, also the counting statistics is known. The only limiting factor in the experiment will be the total number of electrons that interact with the object during the experiment due to the limitations in the exposure time or in the object damage. However, instrumental potentialities will never be exploited fully if not guided by an experimental strategy. Here intuitive guidelines can be very deceptive. For instance an image made with the best electron microscope (C _s  = 0) at the best focus (g = 0) from the best object (phase object) would show no contrast at all. Hence, questions such as what is the best C _s , focus, object thickness, etc. can only be answered properly if done using a method of experiment design.     

来源于类芽孢杆菌属新型耐有机溶剂脂肪酶的克隆、表达与性质研究（英文）

脂肪酶(EC 3.1.1.3)全称为三酰基甘油水解酶,是一类能够将长链脂肪酸甘油酯水解成脂肪酸和二甘酯、单甘酯或甘油的酯键水解酶.它除了能够水解脂肪外,还具有催化酯化反应、酯交换反应、酸解反应、醇解反应以及氨解等反应的性质.在脂肪酶催化的反应中,通常用有机溶剂代替水.有机溶剂可以转移合成反应的平衡方向,通过溶剂工程修饰酶的选择性能够提高底物的溶解度、有机相产物的回收率、酶的热稳定性.但有机溶剂对酶活性和稳定性有不同程度的影响.因此,寻找在有机溶剂中表现出高活性和稳定性的脂肪酶是一个亟待解决的重要课题.由于微生物种类多、作用底物专一性强,且微生物来源的脂肪酶一般分泌到胞外,因此微生物脂肪酶是工业用脂肪酶的重要来源.目前,微生物脂肪酶的研究主要集中于根霉属(Rhizopus)、曲霉属(Aspergillus)、青霉属(Penicillium)、毛霉属(Mucor)、地霉属(Geotrichum)、假丝酵母属(Candida)、假单胞菌属(Pseudomonas)、伯克霍尔德菌属(Burkholderia)等具有工业应用价值的菌株.很少有类芽孢杆菌属所产脂肪酶进行相关酶学性质的研究.我们以Paenibacillus pasadenensis CS0611为出发菌株,在全基因序列草图中得到了一个新型脂肪酶基因lp2252.以Paenibacillus pasadenensis CS0611基因组为模板,设计特异性引物对目标序列进行扩增,并成功将其插入到表达载体p ET-28a中得到含有目的基因的重组质粒.在E.coli BL21(DE3)中,脂肪酶lp2252经0.1mmol/L的IPTG诱导后在20°C实现了高水平表达.重组脂肪酶的活性约为野生型的1631倍.用镍离子亲和层析柱快速、高效地纯化了两端带有组氨酸标签的重组脂肪酶,回收率为63.5%,纯化因子为10.78.纯化后的脂肪酶最适温度为50°C,在20-40°C范围内具有良好的稳定性.最适pH值为7,属于中性脂肪酶,同时在pH 3.0-8.0间具有较高稳定性.在金属离子如钙、镁离子和一些非离子表面活性剂的作用下,其活性有所提高.此外,纯化后的脂肪酶可被一系列水溶性有机溶剂激活,例如一些短链醇.而对某些水不溶性有机溶剂,其也具有高度的耐受性.综上所述,本文所涉新型脂肪酶在非水相催化领域具有广泛的应用和前景.     

Studies on Hybrid Organic/Inorganic Nanocomposite Gels Using Photoluminescence Techniques

Summary.  Transparent nanocomposite gels made of hybrid organic/inorganic polymers, synthesized through the sol-gel method, composed of poly-(ethylene oxide) or poly-(propylene oxide) chains, and grafted on silica through urea bridges, have been studied by steady-state and time-resolved photoluminescence techniques. These nanocomposite materials consist of two distinguished subphases, an organic and an inorganic one. The volume fraction of the organic (polyether) subphase is larger than that of the inorganic (silica) subphase, and it increases with increasing polyether chain size. The condensation of the silica subphase provides luminescent entities emitting light by electron-hole recombination on delocalized states associated with the active chemical species of the urea bridges. Materials with smaller polyether chains are more luminescent than such with longer polyether chains. Divalent or trivalent cations introduced into these materials enhance the luminescence intensity by solubilization close to the silica cluster surface and thus by decreasing surface defects and the ensuing quenching mechanism. Received June 23, 2000. Accepted (revised) July 18, 2000     

Immobilization of Candida antarctica lipase B on Polystyrene Nanoparticles

Polystyrene (PS) nanoparticles were prepared via a nanoprecipitation process. The influence of the pH of the buffer solution used during the immobilization process on the loading of Candida antarctica lipase B (Cal‐B) and on the hydrolytic activity (hydrolysis of p‐nitrophenyl acetate) of the immobilized Cal‐B was studied. The pH of the buffer solution has no influence on enzyme loading, while immobilized enzyme activity is very dependent on the pH of adsorption. Cal‐B immobilized on PS nanoparticles in buffer solution pH 6.8 performed higher hydrolytic activity than crude enzyme powder and Novozyme 435.

     

Synthesis of Amphiphilic Guanylated Polymers as Potential Gene Delivery Carriers

Abstract

We have developed novel polymeric systems that aggregate in aqueous media to form nanospheric particles. Our polymeric system constitutes guanidine functional groups and poly(ethylene glycol) (PEG) units. The guanidine group is an important structural component in many biologically active compounds. Because of their strongly basic character, guanidines are fully protonated under physiological conditions. The positive charge thus imposed on the molecule forms the basis for specific interactions between the ligand and receptor or the enzyme and substrate. The PEG units may form a characteristic micelle structure with a hydrophilic shell layer surrounding the core of the polyion complex (PIC) formed between DNA and the cation segments (PIC micelles).