指数线性聚合酶链式反应法制备焦磷酸测序反应的单链模板

建立了一种简单的焦磷酸测序用单链模板制备方法,以包含SNP6位点一段78bp序列为对象,采用非热启动Taq酶进行指数线性PCR扩增,通过加入甘油、BSA等PCR增强剂增加反应的效率和特异性,设计反应液A和B处理PCR产物中干扰焦测序的限制性引物、未完全反应的产物、焦磷酸和dNTPs等杂质, 处理后1～2 μL PCR产物就可直接用于焦测序检测.测定了BRCA1基因中5个乳腺癌相关的SNP位点,获得的图谱无非特异性信号,测得序列与参考序列一致,能够进行SNP分析,表明本方法可以制备高质量焦测序单链模板,且使焦测序的成本显著降低,操作更为简便,减少了操作过程中样本间的交叉污染,有利于焦测序样品预处理的自动化.     

Reliable Synthesis of Various Nucleoside Diphosphate Glycopyranoses

A reliable and high yielding synthetic pathway for the synthesis of the biologically highly important class of nucleoside diphosphate sugars (NDP‐sugars) was developed by using various cycloSal‐nucleotides 1 and 9 as active ester building blocks. The reaction with anomerically pure pyranosyl‐1‐phosphates 2 led to the target NDP‐sugars 20 – 45 in a nucleophilic displacement reaction, which cleaves the cycloSal moiety in anomerically pure forms. As nucleosides cytidine, uridine, thymidine, adenosine, 2′‐deoxy‐guanosine and 2′,3′‐dideoxy‐2′,3′‐didehydrothymidine were used while the phosphates of D ‐glucose, D ‐galactose, D ‐mannose, D ‐NAc‐glucosamine, D ‐NAc‐galactosamine, D ‐fucose, L ‐fucose as well as 6‐deoxy‐D ‐gulose were introduced.     

A combined experimental and theoretical study of the pH‐dependent binding mode of NAD+ by water‐soluble molecular clips

The highly selective recognition process of NAD⁺ and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum‐chemical approach. The strongly pH‐dependent complexation‐induced ¹H NMR shifts of the guest molecule indicate that in buffered aqueous solution at pH = 7.2 the nicotinamide ring, the active site of NAD⁺, is preferentially bound inside the cavity of the molecular clip, whereas in pure water under slightly acidic conditions both units (the nicotinamide ring as well as the adenine moiety) are located outside the cavity. The latter finding is explained by a competing self‐aggregation of NAD⁺ which prohibits the recognition process. In addition, the investigation of the NAD⁺ fragments NMNA, NMN, and AMP as well as the comparison of measured and computed chemical shieldings provides information on possible binding modes. Under equal conditions the binding of NADH to the molecular clip is significantly weaker than that of NAD⁺, so that the oxidation states (NAD⁺/NADH) can be distinguished by the molecular clips. The nucleotides NMN and AMP are bound less strongly by the molecular clips than NAD⁺. The weaker binding indicates that multiple aromatic π?π and cation?π host–guest interactions only possible in NAD⁺ have a synergetic effect on the complex stability. In addition to the inhibition of the cofactor NAD⁺, a further implication is the development of sensors since a quenching of fluorescence is observed for specific molecular clips by the addition of NAD⁺. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of Sugar and Nucleoside Analogs and Evaluation of Their Anticancer and Analgesic Potentials

Chemical modification of sugars and nucleosides has a long history of producing compounds with improved selectivity and efficacy. In this study, several modified sugars (2–3) and ribonucleoside analogs (4–8) have been synthesized from α-d-glucose in a total of 21 steps. The compounds were tested for peripheral anti-nociceptive characteristics in the acetic acid-induced writhing assay in mice, where compounds 2, 7, and 8 showed a significant reduction in the number of writhes by 56%, 62%, and 63%, respectively. The compounds were also tested for their cytotoxic potential against human HeLa cell line via trypan blue dye exclusion test followed by cell counting kit-8 (CCK-8) assay. Compound 6 demonstrated significant cytotoxic activity with an IC₅₀ value of 54 µg/mL. Molecular docking simulations revealed that compounds 2, 7, and 8 had a comparable binding affinity to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. Additionally, the bridged nucleoside analogs 7 and 8 potently inhibited adenosine kinase enzyme as well, which indicates an alternate mechanistic pathway behind their anti-nociceptive action. Cytotoxic compound 6 demonstrated strong docking with cancer drug targets human cytidine deaminase, proto-oncogene tyrosine-protein kinase Src, human thymidine kinase 1, human thymidylate synthase, and human adenosine deaminase 2. This is the first ever reporting of the synthesis and analgesic property of compound 8 and the cytotoxic potential of compound 6.     

革兰氏阴性细菌群体感应信号分子AHL结构类似物的合成及活性研究

基于组氨酸的结构基础,以82.7～85.2％的产率合成了4种N-酰基高丝氨酸内酯(AHL)的结构类似物(His-7,His-8,His-10和His-12),经IR、1 H NMR、13 C NMR和MS确认了产物的结构.将四种化合物与紫色色杆菌共培养考察其对革兰氏阴性细菌群体感应系统的影响.结果表明:His-7对紫色...     

固体饮料中一种新型卡巴地那非类似物的定性鉴别和定量分析

采用超高效液相色谱-四极杆-飞行时间高分辨质谱（UPLC-Q-TOF MS）、超高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱（UPLC-LTQ/Orbitrap MS）筛查出固体饮料中一种新型的、结构未知的非法添加卡巴地那非类似物。经核磁共振（Bruker）一维、二维谱和文献分析,确定该物质为N-苯基丙氧苯基卡巴地那非。通过高效液相色谱-串联质谱（HPLC-MS/MS）外标法定量分析表明,N-苯基丙氧苯基卡巴地那非在2～50 ng/mL质量浓度范围内线性关系良好,相关系数（r2）为0.999 1,方法检出限为0.05 mg/kg,定量下限为0.1 mg/kg,在低、中、高3个加标水平下的平均回收率为89.2%～93.1%,相对标准偏差（RSD）为1.8%～3.1%。采用该方法测定10批次固体饮料,发现5批次阳性样品,N-苯基丙氧苯基卡巴地那非的检出含量为214～880 mg/kg。该方法的准确度高、灵敏度好,可作为筛查非法添加卡巴地那非类似物的有效方法。     

Anticancer Cytotoxic Activity of Bispidine Derivatives Associated with the Increasing Catabolism of Polyamines

Polyamine (PA) catabolism is often reduced in cancer cells. The activation of this metabolic pathway produces cytotoxic substances that might cause apoptosis in cancer cells. Chemical compounds able to restore the level of PA catabolism in tumors could become potential antineoplastic agents. The search for activators of PA catabolism among bicyclononan-9-ones is a promising strategy for drug development. The aim of the study was to evaluate the biological activity of new 3,7-diazabicyclo[3.3.1]nonan-9-one derivatives that have antiproliferative properties by accelerating PA catabolism. Eight bispidine derivatives were synthetized and demonstrated the ability to activate PA catabolism in regenerating rat liver homogenates. However, only three of them demonstrated a potent ability to decrease the viability of cancer cells in the MTT assay. Compounds 4c and 4e could induce apoptosis more effectively in cancer HepG2 cells rather than in normal WI-38 fibroblasts. The lead compound 4e could significantly enhance cancer cell death, but not the death of normal cells if PAs were added to the cell culture media. Thus, the bispidine derivative 4e 3-(3-methoxypropyl)-7-[3-(1H-piperazin-1-yl)ethyl]-3,7-diazabicyclo[3.3.1]nonane could become a potential anticancer drug substance whose mechanism relies on the induction of PA catabolism in cancer cells.     

硅基催化结构合成的策略

Two approaches to synthesize silicon-based catalytic structures that aim at capturing the properties and functionalities of natural enzymes are described in this brief review:unit-by-unit synthesis of macromolecular units and templating/imprinting synthesis of nanocages. The unit-by-unit approach mimics the peptide synthesis method,offers atomic control of the structure,but is inefficient in synthesizing large structures such as nanocages. The templating/imprinting method is more suitable for nanocages at the sacrifice of atomic control,and the nanocages obtained are shown to possess properties exhibited by enzyme cavities.     

基于引物延伸反应进行SNP基因分型的电化学方法

引物延伸反应的高特异性使其成为单核苷酸多态性(SNP)基因分型的最常用方法. 本文利用引物延伸反应, 通过二茂铁标记的dUTP将二茂铁引入到延伸的产物中, 用一条捕获探针将延伸产物捕获到电极表面, 用差分脉冲伏安法对电极表面的二茂铁进行检测, 从而实现了SNP基因分型. 考察了延伸反应的退火温度、聚合酶用量以及DNA杂交温度等因素的影响. 应用该方法对β-地中海贫血基因密码子28位单碱基突变进行检测, 获得了满意的基因分型结果. 该方法检测限可达到0.86 fmol/L, 是一种简便、快速且灵敏的SNP分型方法.