胍变及脲变α-淀粉酶的研究 Ⅰ.用高效疏水色谱法研究变性机理和复性效率

用疏水性强弱不同的两种色谱柱对７．０ｍｏｌ／Ｌ盐酸胍及８．０ｍｏｌ／Ｌ脲变性的α－淀粉酶变体和在疏水色谱介质表面上折叠的中间体进行了分离和复性。通过研究和比较发现,两者的变性机理和形成折叠中间体的个数以及复性效率均不相同。在用疏水性较弱的疏水色谱柱对脲变α－淀粉酶的折叠中间体进行分离时,得到了疏水性接近连续的、数目很多的中间体。用疏水性较强的疏水色谱柱对胍变α－淀粉酶进行复性的效果较好。还研究了柱温变化对其折叠、分离效果和复性效率的影响。     

胍变及脲变α-淀粉酶的研究 Ⅰ.用高效疏水色谱法研究变性机理和复性效率

用疏水性强弱不同的两种色谱柱对７．０ｍｏｌ／Ｌ盐酸胍及８．０ｍｏｌ／Ｌ脲变性的α－淀粉酶变体和在疏水色谱介质表面上折叠的中间体进行了分离和复性。通过研究和比较发现，两者的变性机理和形成折叠中间体的个数以及复性效率均不相同。在用疏水性较弱的疏水色谱柱对脲变α－淀粉酶的折叠中间体进行分离时，得到了疏水性接近连续的、数目很多的中间体。用疏水性较强的疏水色谱柱对胍变α－淀粉酶进行复性的效果较好。还研究了柱温变化对其折叠、分离效果和复性效率的影响。     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

用疏水色谱对还原型胍变性牛胰岛素的折叠特性研究

用疏水相互色谱(HPHIC)对还原胍变性牛胰岛素在疏水界面上的折叠与复性进行了研究.结果表明,采用普通流动相时,对还原胍变胰岛素的复性效果较差,而采用氧化型流动相可使其复性效率提高到66%,并用反相色谱(RPLC)、紫外吸收光谱、荧光光谱及MALDI-TOF对其复性效果进行了验证.同时与体积排阻色谱(SEC)和稀释法对还原胍变胰岛素的复性结果进行了比较.结果表明,SEC根本无法使还原胍变胰岛素复性,而稀释法的复性效率仅有2%.这进一步表明HPHIC是变性蛋白复性的有效工具,变性蛋白在疏水界面折叠过程中,蛋白质与固定相之间的疏水相互作用对蛋白折叠起着关键性的作用,是蛋白折叠的主要驱动力.     

高效弱阳离子交换色谱法对脲还原变性溶菌酶的折叠研究

用高效弱阳离子交换色谱(HPWCX)对脲还原变性溶菌酶(Lys)进行了复性研究. 在流动相中脲浓度固定为4.0 mol•L^－1和选用对天然态蛋白有稳定作用的硫酸铵为盐或置换剂时, 在蛋白浓度为15.0～50.0 mg•mL^－1时, HPWCX法比稀释法活性回收率高. 为了提高Lys的质量及活性回收率对所用色谱条件进行了优化研究, 当蛋白起始浓度为20.0 mg•mL^－1时, Lys的质量回收率和活性收率分别为97.8%和95.4%. 表明此种方法简便且有可能对其他还原变性蛋白的复性具有通用性.     

液相色谱Z值对溶菌酶分子构象变化的定量表征

目前, 研究蛋白分子的构象变化用得最多的是光谱法, 如荧光光谱、圆二色谱和核磁共振谱等[1]. 由于蛋白质分子构象变化与其在色谱中的保留行为直接相关[2], 所以色谱法也成为研究蛋白质分子构象变化的一种新方法[3]. 此外, 计量置换理论中的Z值也已被成功地用于表征生物大分子的构象变化[4]. 用Z值研究蛋白质分子构象变化的优点是可以使用不纯的样品, 这是因为在测定Z值的过程中会与其它组分相分离. 使用Z值还会对蛋白分子构象变化进行定量表征[5,6]. 本文以溶菌酶(Lys)为目标蛋白, 用色谱法[反相液相色谱(RPLC)和弱阳离子交换色谱(WCX)]系统地研究了Lys在不同变性(胍变非还原、脲变非还原、胍变还原和脲变还原)环境中因疏水性及电荷分布的不同对Lys分子构象变化的影响, 并用Z值进行了定量表征.     

Z及logI对离子交换色谱中脲变溶菌酶分子构象变化的表征

以溶菌酶(Lys)为目标蛋白,用计量置换保留理论(SDT-R)中的参数Z和logI对弱阳离子交换色谱(WCX)中“准天然态”和脲变还原与非还原的两种变性状态的Lys的分子构象变化进行了表征。发现在流动相中含有脲时,蛋白的保留仍服从SDT理论,可以准确测定在特定脲浓度条件下Lys的Z及logI值。结果表明,3种分子构象状态的Lys的Z值均随脲浓度改变呈现不连续变化;“准天然态”Lys在同一脲浓度条件下的Z值比变性状态的大,logI比变性状态的小,而非还原变性态和“准天然态”Lys的Z和logI值比较接近。还     

用疏水色谱复性并同时纯化蛋白质的机理及其应用

变性蛋白表面的疏水氨基酸残基有与疏水色谱固定相(STHIC)颗粒相互作用的倾向, 两者之间的疏水相互作用能够抑制变性蛋白分子间的相互聚集. 同时疏水色谱固定相还能在分子水平上给变性蛋白分子提供足够高的能量, 使其瞬时脱水并折叠成其天然构象或不同的折叠中间体. 变性蛋白在疏水界面上的折叠不仅取决于其氨基酸之间的特异性相互作用及疏水色谱固定相的结构, 而且还取决于固定相和流动相之间的协同作用. 同时, 还提出了高效疏水相互色谱(HPHIC)进行蛋白折叠的机理及其进行蛋白折叠时能实现质量控制的原理. 在适当的色谱条件下, HPHIC 可使几种变性蛋白一步实现复性及同时纯化. 此外, 还设计制造出了直径比柱长大得多的实验室型和制备型“变性蛋白复性及同时纯化装置, USRPP”, 该“装置”具有完全除去变性剂、使蛋白质复性, 与杂蛋白分离及易于回收变性剂的“一石四鸟”功能. 该“装置”对变性蛋白的复性和纯化效率与通常使用的长柱相当. 在制备规模情况下, 该“装置”可以在低压梯度条件下简便、快速、而经济地应用于重组蛋白药物的制备. 文中以重组人干扰素-γ为例, 说明了制备型“装置”在其复性及同时纯化生产工艺中的应用.     

体积排阻色谱法研究变性溶菌酶分子复性过程中集聚体的形成

在体积排阻色谱柱上研究了还原剂存在时脲和盐酸胍变性的3种溶菌酶溶液的复性和分离过程。当变性液中原始溶菌酶浓度大于10 g/L时,变性溶菌酶在体积排阻色谱柱上除了复性为与未变性溶菌酶出峰时间相同的复性态溶菌酶分子外,还形成了溶菌酶折叠中间体的二分子集聚体。这个结果得到了用稀释法复性时溶菌酶的蛋白电泳检测结果的支持。与稀释法复性相比较,用体积排阻色谱法复性时所形成的折叠中间体二分子集聚体的量要远远低于用稀释法所形成的集聚体的量。     

用于变性蛋白质复性及同时纯化的简易型色谱柱的研制及其应用

研制出一种简易型色谱柱并通过装填大颗粒的疏水色谱填料对色谱柱的性能进行了考察,该柱的外形和操作如同传统的液相色谱柱,但它却在生物大分子分离方面有着和高效液相色谱相似的分辨率。另外,只要给该柱装填合适的固定相,比如疏水相互作用色谱固定相,便可用于蛋白质的复性及同时纯化。实验考察了该色谱柱的结构、操作和性能,包括柱压、柱寿命及对蛋白质的分辨率等。以溶菌酶为模型蛋白质,实验测得其在初始浓度为50.0 g/L时的质量回收率和活性回收率分别为(96.6±1.3)%和(101.1±6.0)%。这种简易型色谱柱价格低廉     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

Toward new camptothecins. Part 6: Synthesis of crucial ketones and their use in Friedländer reaction

In the context of the preparation of camptothecin and luotonin A analogs, the synthesis of some key keto-precursors and their use in Friedländer condensation are described. This paper also focuses on the stability of these keto intermediates and emphasizes the major differences between indolizinones and pyrroloquinazolinones series. Noteworthy is also the report of some original structures isolated as by-products of some experiments.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

KMnO4-mediated oxidative CN bond cleavage of tertiary amines: Synthesis of amides and sulfonamides

KMnO₄-mediated oxidative CN bond cleavage of tertiary amines producing secondary amine was introduced, which was trapped by electrophiles (acyl chloride and sulfonyl chloride) to form amides and sulfonamides. The reaction could take place at mild condition, tolerating a wide range of function groups and affording products in moderate to excellent yields.     

Chemistry of heterocyclic compounds at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, over 50 years (Review)

The review contains a concise historical account and information on the most significant researches undertaken by the staff at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences on the Chemistry of Heterocyclic Compounds. Dedicated to Academician of the Russian Academy of Sciences B. A. Trofimov on his 70th jubilee. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1443–1502, October, 2008.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.     

Selective syntheses of 2H-1,3-oxazines and 1H-pyrrol-3(2H)-ones via temperature-dependent Rh(II)-carbenoid-mediated 2H-azirine-ring expansion

The Rh(II)-catalyzed reaction of 2-carbonyl-substituted 2H-azirines with ethyl 2-cyano-2-diazoacetate or 2-diazo-3,3,3-trifluoropropionate provides an easy access to 2H-1,3-oxazines and 1H-pyrrol-3(2H)-ones. These compounds can be selectively prepared from the same starting material using temperature as the only varied parameter. The 2-azabuta-1,3-diene intermediate, a common precursor for both heterocyclic products, isomerizes into 2H-1,3-oxazine under kinetic control, while 1H-pyrrol-3(2H)-one is the sole product of the reaction at elevated temperatures. According to DFT-calculations a one-atom oxazine ring contraction involving ring-opening to a 2-azabuta-1,3-diene intermediate, followed by a 1,5- and 1,2-prototropic shift leads to the consecutive formation of imidoylketene and azomethine ylide, which then further undergo cyclization to the pyrrole derivative.     

Synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives

Different approaches for the synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives from simple amino acids have been investigated. A library of 33 precursors for the preparation of N-hydroxy pyrazinones was obtained in moderate to good yields.