GSH特异的人单链抗体计算机辅助结构研究

以还原型谷胱甘肽 (GSH)的两种衍物作为半抗原 ,从半人工合成的人单链抗体库中筛选得到了多株特异结合的单链抗体 .从中选择亲合力最强的单链抗体进行了基因的重组和测序 .根据由核苷酸测定结果而推导的氨基酸残基序列 ,用计算机模拟分析了两株单链抗体的空间结构 .结果显示单链抗体以二聚体形式存在 ,与单链抗体蛋白SDS_PAGE电泳结果相一致 .抗原结合部位———重链CDR3区位抗体的表面 ,且形成一个凹腔 ,因此可推测位于CDR3区的丝氨酸最可能参与硒化反应     

具有谷胱甘肽过氧化物酶活性的含硒人源单链抗体的制备

以谷胱甘肽(GSH)为靶抗原, 从噬菌体展示人源单链抗体库中筛选人源单链抗体(scFv). 经3轮筛选后, 用ELISA方法检测出5个(2, 11, 16, 24, 32 )可以和GSH结合的克隆. PCR产物的电泳和测序结果表明, 只有3个克隆(11, 16, 24)具有完整的scFv编码基因. 选取和GSH结合力高的克隆11的scFv 编码基因组装到表达载体pPELB上, 在大肠杆菌Rosetta中进行可溶性表达, 用Ni2+螯合亲和层析纯化scFv-11, 免疫点印迹结果证实该抗体能与GSH特异结合. 通过化学突变将scFv-11的丝氨酸转变成硒代半胱氨酸(Sec)后, 获得了具有谷胱甘肽过氧化物酶(GPX)活力的含硒(Se)人源单链抗体(Se-scFv-11), 其活力为351 U/μmol.     

通过快速定点突变表征人源含硒单链抗体酶的底物结合部位

为了对人源含硒单链抗体酶Se-scFv-B3的底物结合部位和催化基团进行研究, 在理论预测的基础上, 通过快速定点突变法分别在2个理论预测的底物结合部位(位点1和位点2)内选定Ala180和Ala44定点突变为丝氨酸(Ser). 2个突变体蛋白经化学修饰将Ser转变成谷胱甘肽过氧化物酶(GPX)的催化基团硒代半胱氨酸(Sec)后, 前者的GPX活力达到了Se-scFv-B3的2倍多, 而后者的GPX活力没有明显提高, 这表明位点1可能是主要的底物结合部位, 与理论预测的结果一致.     

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.     

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.     

疏水腔修饰法及高活力GPX抗体酶的研究：Ⅱ.半抗原及全抗原的制备和表征

自Jencks抗体酶的概念出现以来，已有80多种化学反应被证实可以用抗体来催化［‘”j．一般合成抗体酶的方法是通过选择合适的过渡态类似物来诱导抗体酶，这种方法产生的抗体酶大部分活力很低，我们曾对3个具有谷脱甘肽（GSH）过氧化物酶（GPX）特性的含硒抗体酶进行了报道「”‘a，半抗原是，2，3是根据天然抗体酶活性中心的结构设计合成的，它们对GPX的底物GSH具有不同程度的疏水修饰．随着半抗原疏水程度的增加，通过单抗技术和化学修饰而得的3个抗体酶的相应活力分别为兔肝GPX的0．2，1·6和8．5倍．在上述结果基础上，我们提出疏…     

具有cGPX活力的含硒抗体酶的酶学及动力学性质研究

通过单克隆抗体制备技术得到三株特异结合半抗原4(GSH-S-DNP二苄酯)的单克隆抗体HB4,HB5和HB7.抗体经两步化学诱变得到具有细胞谷胱甘肽过氧化物酶(cGPX)活性的含硒抗体酶mHB4,mHB5和mHB7,活力分别为170,1867,32U/μmol.其中mHB5的活力是天然兔肝cGPX的0.32倍,m4A4的1.51倍.等离子体-质谱(ICP/MS)测得每分子含硒抗体酶分子中大约存在2个硒原子.mHB5的最适pH为8.6～8.8.在pH值范围为7.0和37℃条件下,mHB5催化GSH和H₂O₂或t-ROOH反应的二级速率常数为:_k+1(H₂O₂)9.71×10⁶L/(mol·min),_k+1(t-ROOH)5.99×10⁵L/(mol·min).mHB5使非酶催化反应速率提高了9.8×10⁶和3.7×10⁵倍.     

人源化甲状腺素脱碘酶单链抗体的结构模拟

以四碘甲状腺素原氨酸为半抗原,从人噬菌体展示半合成单链抗体库中筛选与半抗原有特异结合性的单链抗体E3,并测定了E3的一级结构,同时用计算机模拟了其空间结构.     

量子点与人源抗谷胱甘肽单链抗体的连接与表征

用已构建的表达载体pPELB-B3, 在大肠杆菌Rosetta中可溶性表达人源抗谷胱甘肽(GSH)单链抗体B3(scFv-B3), 经Ni2+螯合亲和层析纯化后, 用点印迹法验证了其与GSH结合的特异性. 将水相合成的半导体纳米粒子(半导体量子点, QDs)在N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)的作用下, 与scFvs连接. 光谱分析和膜印迹结果表明, scFvs成功地共价连接到QDs表面, 所得的QD-scFvs复合物能够较好地识别GSH. 荧光显微镜观察QD-scFvs与人乳腺癌细胞MCF-7的作用结果, 初步判断QD-scFvs能够跨膜进入细胞.     

单链抗体PS-9的基因克隆、表达和免疫特性鉴定

成功地构建了鼠抗人胰腺癌单克隆抗体PS-9的单链抗体可变区基因(V_H-linker-V_L),并将该基因克隆到噬菌体表达载体pCANTAB5的外壳蛋白g3p基因中,使单链抗体以融合蛋白的形式表达在噬菌体的表面。免疫学鉴定结果表明,这种噬菌体抗体仍保留着亲代抗体的免疫特性,能与人粘液细胞癌LS-174-T细胞表面抗原特异结合。这项研究结果有助于鼠源性单克隆抗体的临床应用以及肿瘤导向诊断和治疗。     

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%.     

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.     

A general and convenient synthesis of 4-(tosylmethyl)semicarbazones and their use in amidoalkylation of hydrogen,heteroatom, and carbon nucleophiles

A general synthesis of previously unknown semicarbazone-based α-amidoalkylating reagents, 4-(tosylmethyl)semicarbazones, has been developed. The synthesis involved three-component condensation of semicarbazones of aliphatic or aromatic aldehydes with the same or other aldehydes and p-toluenesulfinic acid. The scope and limitations of this reaction were investigated. The compounds obtained were demonstrated to be an efficient α-(4-semicarbazono)alkylating agents. They were reacted with H- (sodium borohydride), O- (sodium methylate), S- (sodium phenylthiolate), N- (pyrrolidine, sodium succinimide), P- (trialkyl phosphites), and C-nucleophiles (sodium diethyl malonate) to give the corresponding products of the tosyl group substitution, 4-substituted semicarbazones, including analogues of nitrofurazone. Among the prepared compounds tested in vitro for antibacterial and antifungal activity, three nitrofuryl-containing semicarbazones exhibited high biological activities with minimum inhibitory concentration (MIC) values of 8–32 μg/mL.     

Synthesis of novel chiral bidentate hydroxyalkyl-N-heterocyclic carbene ligands and their application in palladium-catalyzed Mizoroki–Heck couplings and asymmetric addition of diethylzinc to benzaldehyde

A small library of new chiral bidentate hydroxyalkyl-imidazolium salts 1 is conveniently synthesized on multi-gram scale from inexpensive and commercially available chiral pool amino acids. The corresponding carbenes, generated by deprotonation of imidazolium salts 1, in combination with palladium(II) chloride were tested in the Mizoroki–Heck coupling reaction. The most significant results in terms of yields and reactivities were achieved with low catalyst loading. The catalytic activities of these imidazolium salts were also investigated in the asymmetric addition of diethylzinc to benzaldehyde. The use of MgO nanoparticles as an additive in conjunction with these ligands played a crucial role in increasing the efficiency of these reactions.     

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.     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

Microwave-assisted copper-catalyzed stereoselective ring expansion of three-membered heterocycles with α-diazo-β-dicarbonyl compounds

Microwave-assisted copper-catalyzed ring expansions of three-membered heterocycles with α-diazo-β-dicarbonyl compounds were investigated. Thiiranes generated 3-acyl-5,6-dihydro-1,4-oxathiines in the presence of copper sulfate and trans-3-acyl-5,6-dihydro-1,4-oxathiines as stereospecific products for 1,2-disubstituted cis-thiiranes through an intramolecular S_N2 process. Oxiranes gave rise to 2-acyl-5,6-dihydro-1,4-dioxines under the catalysis of copper hexafluoroacetylacetonate and cis-3-acyl-5,6-dihydro-1,4-dioxines as stereospecific products for 1,2-disubstituted cis-oxiranes via an intimate ion-pair mechanism. The current method provides a direct and simple strategy in efficient preparation of 3-acyl-5,6-dihydro-1,4-oxathiines and 2-acyl-5,6-dihydro-1,4-dioxines, important agents in medicinal and agricultural chemistry, from readily available thiiranes and oxiranes, respectively.     

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.     

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.