谷胱甘肽过氧化酶模拟物2—位碲桥联环糊精的合成及催化作用

以β-环糊精（CD）为酶模型，将Te引入β-环糊精中，成功地合成出一种新的水溶性好，活力高的谷胱甘肽过氧化物酶（GPX）小分子模拟物2－TeCD，并对其结构进行了表征，采用Wilson辅酶偶联法，间接测定了2－TeCD催化还原型谷胱甘肽（GSH）还原H2O2的GPX活力为46.7U/μmol，与文献报道的数据相比，2－TeCD的GPX活力最高，通过考察2－TeCD催化GSH还原H2O2反应的动力学，发现反应初速度对底物浓度的双倒数曲线为一组平行线表明2－TeCD所遵循的催化剂可能为三转移乒乓机制，通过考察自由基捕获剂2，4－二叔丁基甲基苯酚对酶促和自发反应速率的影响，发现2－TeCD催化的酶促反应为非自由基机理。通过考察酶不可逆抑制剂碘乙酸对酶促反应速率的影响，发现2－TeCD催化反应过程中不生成碲醇中间体，由此推测出2－TeCD的催化循环经历碲硫化合物，次碲酸硫酯和次碲酸中间体，该催化循环与含硒GPX小分子模拟物所经历的催化循环不同，以及环糊精对底物具有识别与结合的能力，可能是2－TeCD具有高GPX活力的主要原因。     

谷胱甘肽过氧化物酶模拟物碲化透明质酸的合成及催化动力学研究

 以透明质酸(HA)作为谷胱甘肽过氧化物酶(GPX)的酶模型,将碲(Te)引入HA中,合成了一种新型的高活力的GPX模拟物碲化透明质酸(TeHA). 用红外光谱和核磁共振技术对TeHA的结构进行了研究,证明Te的修饰位点位于HA的N-乙酰氨基葡萄糖的羟甲基(-CH2OH)上. 采用Wilson辅酶偶联法测定得到TeHA催化还原型谷胱甘肽(GSH)还原H2O2的GPX活力为163.6 U/μmol, 高于文献报道的其它模拟酶. TeHA还能够催化GSH还原异丙苯基过氧化物(CuOOH)和叔丁基过氧化物(t-BuOOH)的反应,并且CuOOH为该模拟酶的最适底物. 通过研究TeHA催化GSH还原三种不同过氧化物的反应动力学发现, TeHA的催化遵循乒乓机制.     

具有GPX活力的抗体片段Fv的制备和性质

具有谷胱甘肽(GSH)结合部位的鼠抗体3H₄(IgM)经胃蛋白酶水解,产生分子量为25000的抗体Fv片段,用荧光滴定法测定了它与GSH的亲和常数K_a=1.17×10₇L/mol.该片段经苯甲基磺酰氟活化,再经NaHSe作用,其结合部位的丝氨酸被突变为谷胱甘肽过氧化物酶(GPX)的催化基团硒代半胱氨酸.突变后的Fv片段表现出很高的GPX活性,其活力高达2500U/μmol,称为Fv抗体酶.动力学分析表明,Fv酶的最适温度为55℃,最适pH为7.0,催化机制为乒乓机制,米氏常数分别为:K_m(GSH)=4.16×10^-3mol/L,K_m(H₂O₂)=2.8×10^-4mol/L.     

活力高于天然谷胱甘肽过氧化物酶的含硒抗体酶

还原型谷胱甘肽(GSH)的巯基和两个羧基分别与2,4-二硝基氯苯(DNCB)和甲醇反应,合成出半抗原Hp2;通过戊二醛将Hp2连到牛血清白蛋白(BSA)上,合成出全抗原Ag2;吸收光谱法测出Hp2在BSA上的平均连接量为30.2mol/mol.用标准的单克隆抗体(McAb)制备技术,制备出阳性McAb(IgG)-4G3;4G3对GSH和半抗原Hp2的解离常数(K_D)表明,单克隆抗体4G3对GSH具有较强的亲和力.对4G3进行两步化学诱变(Chemical Mutation),诱变后即为具有谷胱甘肽过氧化物酶(GPX)活性的含硒抗体酶(m4G3),m4G3的GPX活力为9337U/μmol,是兔肝GPX(RL-GPX,5780U/μmol)的 1.6倍,曾报道的含硒抗体酶m4A4(1239 U/μmol)的7.5倍,制备出活性高于天然酶的抗体酶,证实了半抗原设计思想.将m4G3拆分成Fab和Fc片段,发现m4G3的活性中心位于Fab片段上,m4G3的硒代半胱氨酸(Se-Cys)含量为1.9 mol/mol.     

谷胱甘肽过氧化物酶模拟物2-位碲桥联环糊精的合成及催化作用

 以β－环糊精（CD）为酶模型，将Te引入β－环糊精中，成功地合成出一种新的水溶性好、活力高的谷胱甘肽过氧化物酶（GPX）小分子模拟物2－TeCD，并对其结构进行了表征．采用Wilson辅酶偶联法，间接测定了2－TeCD催化还原型谷胱甘肽（GSH）还原H2O2的GPX活力为46．7U／μmol，与文献报道的数据相比，2－TeCD的GPX活力最高．通过考察2－TeCD催化GSH还原H2O2反应的动力学，发现反应初速度对底物浓度的双倒数曲线为一组平行线，表明2－TeCD所遵循的催化机制可能为三转移乒乓机制．通过考察自由基捕获剂2，4－二叔丁基甲基苯酚对酶促和自发反应速率的影响，发现2－TeCD催化的酶促反应为非自由基机理．通过考察酶不可逆抑制剂碘乙酸对酶促反应速率的影响，发现2－TeCD催化反应过程中不生成碲醇中间体．由此推测出2－TeCD的催化循环经历碲硫化合物、次碲酸硫酯和次碲酸中间体．该催化循环与含硒GPX小分子模拟物所经历的催化循环不同，以及环糊精对底物具有识别与结合的能力，可能是2－TeCD具有高GPX活力的主要原因．     

GSH对两种谷胱甘肽过氧化物酶模拟物活性影响的研究

设计并合成了谷胱甘肽过氧化物酶(GPX)模拟物6A,6A’-二苯胺-6B,6B’-二硒桥联-β-环糊精(6-AnSeCD). 采用双酶偶联法测定GPX的活力结果显示, 6A,6A’-二环己胺-6B,6B’-二硒桥联-β-环糊精(6-CySeCD)催化谷胱甘肽还原H2O2和枯烯H2O2的活力均比6-AnSeCD的高. 为了进一步考察6-CySeCD和6-AnSeCD与GSH之间的相互作用, 进行了分子动力学(MD)模拟和分子对接研究. 结果表明, 与GSH的结合使GPX模拟物的构象发生变化, 这种改变可能是影响桥连GPX模拟物催化活性的关键因素.     

2-位碲桥联环糊精的制备、表征及其谷胱甘肽过氧化物酶活性的研究

该文以三种母体环糊精(CD),即α-、β-和γ-CD为修饰模板,将功能性基团有机碲引入到环糊精次面的2位羟基上,制备得到了三种具有谷胱甘肽过氧化物酶(GPX)活性的GPX模拟物。采用元素分析、红外光谱、核磁共振等手段对三种环糊精衍生物的结构进行了表征。运用GPX经典双酶体系法测定了三种环糊精衍生物的GPX活性,实验结果表明三者均具有很高的催化活性,其中2-位碲桥联γ-环糊精(2-Te-γ-CD)具有最高的GPX活性,其催化谷胱甘肽(GSH)还原过氧化氢(H2O2),叔丁基过氧化氢(t-BuOOH)和枯烯过氧化氢(CuOOH)的活力分别是传统"小分子硒酶"Ebselen的80.5,333.3和118.3倍。     

基于γ-环糊精的谷胱甘肽过氧化物酶模拟物的构建及催化作用

基于对天然谷胱甘肽过氧化物酶(GPX)结构与功能的理解,我们利用超分子化学的方法和原理,选择γ-环糊精为骨架,通过引入催化基团硒或碲,设计并合成了7种基于γ-环糊精的新型谷胱甘肽过氧化物酶(GPX)模拟物,并采用元素分析、红外光谱、核磁共振等手段对其结构进行了详细的表征和确认。运用GPX经典双酶体系法测定了它们的GPX活性,实验结果表明:6,6’双碲桥联γ-环糊精(6-diTe-γ-CD)表现出了最高的GPX活性,其催化GSH还原过氧化氢(H2O2)、叔丁基过氧化氢(t-BuOOH)和枯烯过氧化氢(CuOOH)的活力分别是传统小分子硒酶Ebselen的147.3、1897.9和663.9倍,该结果是目前报道的环糊精GPX模拟物中酶活力最高的。     

以透明质酸为骨架的新型谷胱甘肽过氧化物酶(GPX)模拟酶的制备及性质研究

用修饰法合成以透明质酸为骨架的两种新型GPX模拟酶: 硒化透明质酸SeHA及碲化透明质酸TeHA. 用红外光谱和核磁共振波谱对模拟酶的结构进行研究, 证明其修饰位点位于透明质酸的N-乙酰氨基葡萄糖的—CH2OH. 用二硫代双硝基苯甲酸(DTNB)法测定模拟酶的硒含量为1.2%. 通过模拟酶对3种不同底物过氧化氢(H2O2)、过氧化氢正丁烷(t-BuOOH)和过氧化氢异丙苯(CuOOH)的催化活性的研究结果表明CuOOH为该反应的最佳底物. 研究模拟酶催化谷胱甘肽(GSH)还原3种过氧化物的动力学发现, 反应速率与底物浓度的双倒数曲线均为平行的直线, 说明模拟酶反应的动力学机制与天然GPX相同, 为乒乓机制. 用2,4-二叔丁基甲基苯酚(BHT)法证明了该催化反应为非自由基机理, 且模拟酶不易被碘乙酸抑制.     

标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用

设计合成融合表达标签谷胱甘肽S-转移酶(GST)的二价亲和标记试剂,用于功能化磁珠后位点选择性固定化标签GST,为磁分离筛选配体混合物库提供固定化融合靶蛋白的候选方案。 为减少疏水配体在标签GST活性位点的结合,需同时占据标签GST双活性中心内疏水结合位点并发生共价修饰的二价亲和标记试剂。以双苯环为疏水定位基、溴乙酰基为巯基修饰基团、羧基为连接官能团得单价标记试剂,以二乙基三胺为连接臂将单价标记试剂与连接臂两端伯胺连接得标签GST的对称二价亲和标记试剂,再以线性三胺连接臂中间的氨基与羧基磁珠偶联得功能化磁珠。 表征目标化合物对标签GST的标记动力学、结合比;功能化磁珠对标签GST的不可逆固定化动力学和固载容量,及将磁珠表面二价亲和标记试剂转变成还原型谷胱甘肽(GSH)加合物后对标签GST可逆固定化的效果;以碱性磷酸酶及疏水荧光配体为模型考察磁珠固定化标签GST后的非特异结合。 目标化合物对标签GST半抑制浓度为(22±0.2) μmol/L,其与GSH的饱和加合物半抑制浓度为(0.41±0.06) μmol/L,二者与标签GST二聚体结合比接近1：1。 功能化磁珠对标签GST不可逆及可逆固定化的容量均接近25 mg/g磁珠。 偶联GST的磁珠对蛋白非特异吸附很弱,再进一步用单价亲和标记试剂和GSH加合物封闭固定化标签GST剩余的活性位点后对疏水小分子也无显著结合。 结果表明,所设计二价亲和标记试剂功能化磁珠适合用于标签GST及其融合表达蛋白的位点选择性固定化。     

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%.     

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.     

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.     

Copper catalyzed/mediated synthetic methodology for ebselen and related isoselenazolones

Scope of the copper catalyzed/mediated selenium-nitrogen coupling reaction has been studied for the synthesis of isoselenazolones. It is noticed that the 2-chloro, 2-bromo-, and 2-iodo-aryl amides substrates can be exploited in the selenium-nitrogen coupling reaction by employing 25-100 mol % of CuI/1,10-phenanthroline (L) and potassium carbonate as a base in DMF. Furthermore, electron rich 2-chloro-arylamides also underwent selenium-nitrogen coupling reaction to give biologically important selenium-nitrogen heterocycles. Also, copper-catalyzed selenium-nitrogen coupling reaction has been meticulously applied for the synthesis of diaryl diselenides having methoxy, amine, and amide functionality from respective aryl iodides in the presence of stoichiometric amount of succinimide as an external Se-N coupling partner.     

Knoevenagel condensation catalyzed by novel Nmm-based ionic liquids in water

A series of novel N-methyl morpholine (Nmm) based ionic liquids with 1,2-propanediol group were synthesized and used as catalysts for Knoevenagel condensation at room temperature in water. Under the effect of the catalyst, various aldehydes or aliphatic ketones could react with a wide range of activated methylene compounds well, including malononitrile, alkyl cyanoacetate, cyanoacetamide, β-diketone, barbituric acid, 2-arylacetonitrile and thiazolidinedione. Furthermore, most of the products could be separated just by filtrating and washing with water. Additionally, the catalyst is recyclable and applicable for the large-scale synthesis.     

Construction of polyheterocyclic spirotetrahydrothiophene derivatives via sulfa-Michael/aldol cascade reaction

A series of polyheterocyclic spirotetrahydrothiophene derivatives were obtained in moderate to excellent yields via a catalyst-free sulfa-Michael/aldol cascade reaction of chalcones 1 and commercially available 1,4-dithiane-2,5-diol 2 under mild conditions. We also present the first asymmetric sulfa-Michael/aldol cascade reaction of chalcones 1 and commercially available 1,4-dithiane-2,5-diol 2 with moderate to good enantioselectivities catalyzed by readily available chiral phase-transfer catalysts (PTCs).     

Suzuki-Miyaura reactions of the soluble guanylate cyclase inhibitor NS2028: a non-product specific route to C-8 substituted analogues

Both soluble guanylate cyclase (sGC) inhibitors ODQ 1 and NS2028 2 are synthesized via improved protocols. In the former case treating 3,4-dihydroquinoxalin-2(1H)-one oxime 8, which can be prepared in two steps from 1,2-benzenediamine, with 1,1′-carbonyldiimidazole (CDI) gives the dihydro-ODQ 10 that in the presence of KMnO₄ oxidises to give ODQ 1 in an overall yield of 46% starting from 1,2-benzenediamine. In the latter case, the synthesis affords NS2028 2 from 2-amino-4-bromophenol 3 in three steps with an overall yield of 85% and avoids the need for chromatography. Furthermore, Suzuki-Miyaura reaction conditions are described that enable the preparation of 8-aryl and 8-heteroaryl derivatives of NS2028 directly from NS2028 2. Finally, demethylation of the 8-(methoxyphenyl) substituted analogues afforded the 8-(hydroxyphenyl) derivatives 40-42. All new products are fully characterised.