天冬酰胺合成酶B抑制剂高效液相色谱筛选方法的建立与应用

建立了一种快速、高效测定天冬酰胺合成酶B(AS-B)酶活性的反相高效液相色谱法(RP-HPLC)。酶反应体系中的氨基酸经2,4-二硝基氟苯(DNFB)柱前衍生,通过RP-HPLC测定酶反应体系前后底物及产物的变化来分析酶的活性。采用的色谱柱为Agilent C18柱(250 mm×4.6 mm,5 μm),以50 mmol/L醋酸钠缓冲液(pH 6.2)-乙腈(体积比为15:85)为流动相,流速为1.0 mL/min,柱温为30 ℃,检测波长365 nm,于6 min内实现了各组分的基线分离。通过该方法测定反应动力学参数进行AS-B的抑制定量分析。将已知AS-B抑制剂L-谷氨酸-γ-甲酯作用于酶反应体系,测得的抑制剂的抑制常数与文献值相接近,证明该方法可用于AS-B抑制剂的筛选。     

气相色谱-质谱法同时测定乳酸和7种糖的研究

建立了气相色谱-质谱(GC-MS)法同时测定乳酸和葡萄糖、葡萄糖-6-磷酸、果糖、半乳糖、乳糖、1,5-脱水山梨醇、山梨醇7种糖的方法.选择核糖醇为内标,进样前先进行肟化反应再进行硅烷化反应.采用DB-5熔融石英毛细管柱,升温程序为初始温度70℃,保持4 min,以8℃/min的速率升至300℃,保持3 min.在优化...     

果糖-6-磷酸-2-激酶的色氨酸残基的研究

NBS滴定PFK-2的紫外光谱的结果表明,该酶可滴定Trp残基数为4个其中2个是活性必需的。 用295nm的紫外光激发果糖-6-磷酸-2-激酶(PFK-2),在335nm附近有较强的发射荧光。该荧光能较专一反映PFK-2的Trp基团的状态。底物果糖-6-磷酸对该荧光不影响,ATP引起荧光吸灭。最大吸灭幅度为30％左右。 采用荧光滴定方法测定ATP与酶的结合,得出解离常数(K_i)在低浓度ATP时为0.033mmol/L,高度时为0.23mmol/L。表明该结合具有负协同性。Mg~(2+)、无机磷均降低ATP与酶的K_i值而表现为激活因子;果糖-6-磷酸和果糖-2,6-二磷酸酸对K_i值不影响。ATP结合后引起了该酶构象上的较大的变化。 在ATP保护下,PFK-2对NBS的失活速度稍有增加。丙烯酰胺荧光滴定的结果是,在有无ATP时的Stern-Volmer常数(K_(SV))皆为4.0。     

小鼠肝果糖6-磷酸,2-激酶的纯化及动力学研究

经过超离心,聚乙二醇分级沉淀及DEAE-Sephadex,Blue-Sepbarose。磷酸纤维素三种柱层析等一系列过程,纯化了小白鼠肝脏的果糖6-磷酸,2-激酶。经凝胶过滤和SDS聚丙烯酰胺电泳方法测出该酶是由两个相同亚基构成的分子量为110000的蛋白。Mg~(2+)是其活性所必需的,活化方式呈正协同性。酶与底物的结合方式,对果糖6-磷酸表现正协同性,对ATP无协同性,其Km值随果糖6-磷酸的浓度减小而增大,表明酶的作用为顺序机制。活性中心有一个必需氨基酸残基与结合ATP有关,结合后,其侧链pKa由9.5移至9.8。     

鸡肝果糖-6-磷酸-2-激酶的性质研究

本文经过匀浆、聚乙二醇分级沉淀和DEAE-Sephadex A-50柱层析分离步骤,进一步用蓝色三嗪染料琼脂糖柱层析分离,得到了电泳均一的鸡肝果糖-6-磷酸-2-激酶(EC2.7.2.105)。该酶的性质为:(1)对果糖-6-磷酸的底物饱和曲线为双曲线型,无机磷降低果糖-6-磷酸的Km,而不影响V_(max);(2)对底物ATP的结合具有负协同性,Hill系数为0.56;(3)受低浓度Mg~(2+)的激活,但受高浓度Mg~(2+)的抑制,在EDTA存在下不表现活力;(4)在30℃内较稳定,高于40℃下易失活;(5)在pH7—9范围内活性比较平稳,pH高于9.0时,活性上升,而在大于9.5后,活性很快下降;(6)对胰蛋白酶较敏感,但ATP对其有显著的保护作用。     

反相高效液相色谱法测定Arthrobacter sp.黄嘌呤氧化酶活性

报道了采用反相高效液相色谱法(RP-HPLC)测定节杆菌(Arthrobacter sp.)黄嘌呤氧化酶活性的有效方法. 将酶初提液与含有黄嘌呤的反应体系在37 ℃下反应20 min, 反应终止后通过HPLC测定产物尿酸生成量的变化来分析酶的活性. 通过流动相组成、pH和柱温等分离条件的优化, 确定了最佳的色谱检测条件以NH4H2PO4 (50 mmol/L, pH 7.5)溶液为流动相, 流速1 mL/min, 柱温25 ℃, 检测波长290 nm. 为深入研究微生物细胞内黄嘌呤氧化酶提供了高效检测手段.     

部分还原和分步序列测定法定位虎纹捕鸟蛛毒素-Ⅳ二硫键

结合部分还原和分步序列测定法确定了虎纹捕鸟蛛毒素-的二硫键配对方式.在pH=3和40℃的条件下与还原剂三羧甲基磷酸(TCEP)反应10min,利用RP-HPLC分离并分别收集含有一对和两对二硫键被还原的中间体,分别与0.5mol/L碘乙酰胺溶液(pH=8.3)反应1min,使游离巯基烷基化后,测定各中间体的氨基酸序列,从而确定虎纹捕鸟蛛毒素-的3对二硫键分别为Cys2-Cys17,Cys9-Cys24和Cys-Cys31(1-4,2-5和3-6).     

2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

利用含羰基化合物与2,4-二硝基苯肼在酸性条件下反应得到的产物腙对紫外-可见光有吸收的特性,采用柱前衍生高效液相色谱法测定了1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数。酶反应产物1-脱氧-D-木酮糖-5-磷酸在碱性磷酸酶的作用下生成去磷酸化产物1-脱氧-D-木酮糖,然后与2,4-二硝基苯肼衍生成腙。衍生化反应的适宜条件为:HClO4浓度为1.5%,反应温度37℃,反应时间60 min,2,4-二硝基苯肼与1-脱氧-D-木酮糖-5-磷酸的摩尔比为6∶1。色谱条件:0～17 min,40%～80%甲醇;18～20 min,40%甲醇。结果表明,本方法具有较高的灵敏度和良好的线性关系,1-脱氧-D-木酮糖-5-磷酸的检出限为1.0 mg/L,在0.005～1.0 g/L范围内线性相关系数为0.999,相对标准偏差小于5.0%(n=3),标准回收率为102.22%。用本方法测得的1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数Km、Vmax和Kcat与文献报道一致。     

以变色酸2R为底物测定辣根过氧化物酶的研究

变色酸 2R(CT2R)是一种具有电化学活性的物质 ,在碱性条件下 ,该物质在滴汞电极上 - 6 6 0mV(vs.SCE)左右产生还原波 ,加入H2 O2 和辣根过氧化物酶 (HRP)后 ,该物质被氧化生成具有非电化学活性的物质 ,导致溶液中游离的CT2R浓度降低 ,相应还原波波高降低 ,HRP在一定含量范围内与伏安峰的降低值具有良好的线性关系。通过测定波高的降低值可测定HRP ,测定游离HRP的线性范围是 4 .0× 10 -5～ 4 .0× 10 -3 g/L。对酶促反应的动力学进行了研究 ,反应的米氏常数Km =1.38mmol/L ,最大反应速率Vmax=5 3.9nA/min。     

三磷酸腺苷生物发光法快速测定食品中细菌的含量

建立了测定食品中细菌三磷酸腺苷(ATP)的生物发光分析方法.生物发光原理基于萤光素酶、虫萤光素、氧气、ATP和Mg2+的催化氧化反应.产生的光信号强度与ATP含量成一定关系.考察了各种物理化学参数对反应的影响.反应体系最优化条件:pH 7.4、牛血清白蛋白(BSA)浓度为1.0 g/L和室温反应.方法检出限为1.0×10-12mol/L,线性范围1.0×10-9～1.0×10-11mol/L,分析时间30 min,批内变异和批间变异分别小于4%和5%.研究了部分食品中细菌检测的样品前处理方法,应用于糕点和饮料样品中细菌的测定,加标回收率范围为82.1%～115%.检测结果与传统培养方法检测结果相关性良好.     

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.     

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.     

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.     

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.     

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.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.