亲和毛细管电泳法对单胺氧化酶与犬尿胺特异性相互作用的研究

采用亲和毛细管电泳技术对粗提单胺氧化酶(MAO)与其专一性底物犬尿胺(Kyn)的相互作用进行研究,并引入MAO抑制剂氯吉兰对该特异性相互作用进行验证.结果表明,随着缓冲液中MAO浓度的增大,Kyn相对两个内标的迁移时间比率(RMTR)逐渐减小.而加入氯吉兰后,Kyn的RMTR值比未加入抑制剂时有所增大.说明MAO活性位...     

Cu催化吲哚啉的炔丙基烷基化/脱氢对映选择性合成手性N-炔丙基吲哚化合物(英文)

基于吲哚啉的Cu催化不对称炔丙基烷基化及DDQ脱氢策略,成功合成了手性N-炔丙基吲哚化合物.通过使用一个结构刚性的酮亚胺三齿P,N,N-配体,反应获得了很好的对映选择性.该方法反应条件温和、底物适用范围广、产物收率高、立体选择性好,为手性N-炔丙基吲哚化合物的合成提供了一条简捷、高效的新途径.     

毛细管电泳法测定单胺氧化酶活性

应用毛细管电泳技术,建立了快速测定单胺氧化酶（MAO）活性的方法。研究对分离缓冲液pH值、浓度、毛细管表面改性剂十四烷基三甲基溴化铵（TTAB）浓度等影响因素进行优化,探讨了方法的可行性,确立了最佳分离条件。以70cm×50μm（i．d．）未涂敷熔融石英毛细管为色谱分离柱,运行电压15kv,运行缓冲液：0．5mmol／LTTAB,磷酸盐缓冲液（50mmol／L,pH 10．5）;紫外检测波长：214nm。MAO催化犬尿胺（Kyn）反应的产物4-羟基喹啉（4-HQ）的浓度和峰面积的线性范围为5～1000μmol／L,相关系数为0．9997,相对标准偏差（RSD）小于5．6％（n=5）,检出限为2μmol／L（S／N=3）。实验证明,此方法可以用于生物样品中MAO催化活性的检测。     

环胺类骨架的联二萘酚配体催化炔烃对N-二苯基膦酰亚胺不对称加成反应研究

本文发展了一种新的简便方法,用于制备系列含环胺类骨架的联二萘酚(BINOL-Cycloamine)配体。此类配体联合Et_2Zn,可用于催化端基炔对N-二苯基膦酰亚胺的不对称加成反应,合成炔丙基胺类产物。其中筛选出的配体能有效催化合成炔丙基胺类产物,产率高达85%,对映选择性ee值57%。     

无溶剂下胺、炔、炔反应选择性构筑季碳炔丙基胺（英文）

在无溶剂下以胺、炔、炔为原料通过"一锅法"串联的反应方便、高效地合成了季碳炔丙基胺.脂肪炔和芳香炔都可以适用于该反应体系.在脂肪炔反应体系中, AgOTf催化促进胺-炔-炔"马氏"反应进程;当芳香炔作为反应底物时,CuBr_2/Zn(OTf)_2用于共催化剂.该串联反应展示出优异的原子利用效率,为季碳炔丙基胺的制备提供了一种极具有吸引力的绿色合成方法.     

手性磷酰胺类化合物不对称催化合成α-芳基丙醇类化合物

对一系列磷酰胺类化合物进行了考察,发现以芳香醛类化合物与二乙基锌作为底物,手性磷酰胺类化合物4a(N-(1R,2R)-2-(异丙基氨基)-1,2-二苯基乙基)-P,P-二苯基次膦酰胺)作为催化剂,通过发生不对称还原反应合成具有生理活性的α-芳基丙醇类化合物.手性磷酰胺类化合物合成所使用的起始物料不仅廉价易得,而且催化效...     

N,N-二(8-黄酮甲基)香叶胺类化合物的合成及生理活性

设计合成了4个N,N-二（8-黄酮甲基）香叶胺类化合物,所有目标化合物的结构均经1H NMR、MS和元素分析测试技术确证。采用MTT法考察了目标化合物对K562（白血病细胞）和SMMC7721（肝癌细胞）2种肿瘤细胞的体外抑制活性,结果表明,所测化合物对2种肿瘤细胞都有体外抑制活性,其中N,N-二(3′,4′-二甲氧基-8-黄酮甲基）香叶胺(1c)的活性最好,IC50值分别为5.78和3.85 μmol/L,N,N-二（4′-氟-8-黄酮甲基）香叶胺(1a)和化合物1c对K562（白血病细胞）的体外抑制活性明显优于商品药物美法仑(Melphalan)。以溴化乙锭(EB)为荧光探针测定了化合物1c与鲱魚精DNA有较强的相互作用。     

N,N-二(8-黄酮甲基)香叶胺类化合物的合成及其细胞毒性

设计合成了4种N,N-二(8-黄酮甲基)香叶胺类化合物,所有目标化合物的结构均经1HNMR、MS和元素分析测试技术确证。采用MTT法考察了目标化合物对K562(白血病细胞)和SMMC7721(肝癌细胞)2种肿瘤细胞的体外抑制活性。结果表明,所测化合物对2种肿瘤细胞均有体外抑制活性,其中N,N-二(3′,4′-二甲氧基-8-黄酮甲基)香叶胺(1c)的活性最好,IC50值分别为5.78和3.85μmol/L,N,N-二(4′-氟-8-黄酮甲基)香叶胺(1a)和化合物1c对K562(白血病细胞)的体外抑制活性明显优于商品药物美法仑(Melphalan)。以溴化乙锭(EB)为荧光探针观测到化合物1c与鲱魚精DNA有较强的相互作用。     

氟代索拉非尼的合成及其抗肿瘤活性研究

以N-甲基-4-氯-2-吡啶甲酰胺为原料,经过4步共合成4个化合物(S-1,S-2,R-1和R-2),其中2个为新的化合物(S-1和R-2)。经过1H NMR,13C NMR,HR-MS等方法对其结构表征。最后通过CTG法,测试4种化合物对四种人肝癌细胞(PLC/PRF/5,Hep3B,HepG2,BEL-7402)的抑制活性。结果表明:S-1,S-2,R-1和R-2均表现较明显的对4种细胞的抑制活性,且呈现出浓度依赖关系。IC50值从1304nM到11228nM。其中化合物R-1(瑞格非尼)对PLC/PRF/5和HepG2细胞,S-1对Hep3B细胞的抑制活性,R-2对HepG2的细胞活性均较高于原药索拉非尼。     

2-炔基苯胺与二芳基碘鎓盐的选择性芳基化/环化串联反应（英文）

有机高价碘试剂是一类环境友好、制备简单且性质温和的有机合成新试剂.近年来,有机高价碘试剂因表现出新颖、独特的反应性能而受到化学工作者广泛关注,成为有机合成重要研究领域之一.二芳基碘鎓盐是有机高价碘试剂的一个重要组成部分,是一类具有较高普适性的芳基化试剂,可用于羰基化合物、烯烃、炔烃和杂原子亲核化合物等的芳基化反应.目前,二芳基碘鎓盐作为芳基化试剂对具有单一芳基化位点化合物的芳基化已经有了非常广泛而深入的研究.对于具有两个甚至多个芳基化位点的化合物(如同时具有胺基和炔基),其芳基化选择性问题仍缺乏系统研究.特别是在多个芳基化位点共存时如何能够使芳基化发生在某一特定位点仍然是一大难题.这限制了二芳基碘鎓盐作为芳基化试剂更广泛的应用.因此,我们选用2-炔基苯胺(含有胺基和炔基两个芳基化位点)作为原料,通过溶剂的选择以及溶液酸碱性的调控来改变不同芳基化位点的反应活性,通过催化剂的调变来改变二芳基碘鎓盐芳基化反应的能力,从而找出最优条件实现底物分子的选择性芳基化反应,并利用剩余活性位点实现分子内的环化反应,从而实现芳基化-环化串联反应合成一系列N-芳基吲哚类化合物.在对模型底物进行条件筛选实验时发现,以2-乙基辛酸铜(Cu(OCOC8H17)2)作催化剂,二异丙基乙基胺(DIPEA)作碱,1,2-二氯乙烷(DCE)为溶剂,反应以最高93%的收率得到1,2-二苯基吲哚.使用该最优反应条件,一系列2-炔基苯胺都能与二芳基碘鎓盐很好地发生反应并且以良好到优秀的产率(71%–98%)得到目标产物N-芳基吲哚.此外,2-炔基苯胺与非对称的二芳基碘鎓盐也能发生反应,实验结果证明为位阻较小的芳基对胺基进行了N-芳基化反应.通过空白实验和对比实验,我们提出了可能的反应机理:二芳基碘鎓盐在铜催化剂作用下转化为亲电性的芳基活性中间体,该中间体与底物的胺基发生芳基化反应,然后芳基化产物在铜催化剂作用下环化生成N-芳基吲哚.该反应很好地解决了同时具有胺基和炔基两个芳基化位点的底物与二芳基碘鎓盐反应时C-芳基化和N-芳基化的竞争问题,选择合适反应条件使N-芳基化反应优先进行,为二芳基碘鎓盐的选择性芳基化反应提供了很好的实例,并为其它具有多个芳基化位点化合物的选择性芳基化反应提供了途径.     

Monoamine Oxidase Inhibitor Screening by Proteoliposome Capillary Electrophoresis

In this study, a new strategy for screening of enzyme inhibitors based on the interaction between enzyme and substrate was established. The bioactive proteoliposome reconstituted by monoamine oxidase (MAO) and liposome was added in the running buffers of capillary electrophoresis (CE) as pseudostationary phase to simulate the interaction between MAO and its substrate kynuramine (Kyn). The results showed that the relative migration time ratio (RMTR) of Kyn decreased obviously with increase of the proteoliposome, indicating strong interaction between proteoliposome and Kyn. After adding MAO inhibitors into the running buffers containing proteoliposome, the decline of the RMTR value of Kyn became slower. This occurred because MAO was inhibited to some degree by the inhibitors, weakening the interaction between MAO and Kyn. However, adding compounds which do not inhibit MAO activity into the running buffers did not affect the RMTR of Kyn obviously. The results obtained demonstrate that the method developed could characterize the inhibition of MAO inhibitors. Compared with traditional inhibitor screening methods, it does not need incubation outside the column to complete the enzyme catalytic reaction, so analysis is faster and less sample is consumed. With further development, the method might be chosen as a useful tool for screening of membrane protein inhibitors and identification of proteins associated with various diseases.     

Proteoliposome-based capillary electrophoresis for screening membrane protein inhibitors

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.     

New bioluminogenic substrates for monoamine oxidase assays

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.     

On-column synthesis coupled to affinity capillary electrophoresis for the determination of binding constants of peptides to glycopeptide antibiotics

Binding constants of the glycopeptide antibiotics teicoplanin (Teic), ristocetin (Rist), and vancomycin (Van), and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column ligand and receptor synthesis coupled to affinity capillary electrophoresis (ACE) or partial filling ACE (PFACE). In the first technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-D-Ala-D-Ala species are first synthesized using on-column techniques. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and buffer, respectively. Upon electrophoresis, the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the glycopeptide in the running buffer and the plug of Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Subsequent analysis of the change in the electrophoretic mobility (mu) or relative migration time ratio (RMTR) of the peptide relative to the non-interacting standards, as a function of the concentration of the antibiotic, yields a value for the binding constant. In the second technique, derivatives of the glycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the column has been partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides, a plug of buffer followed by two separate plugs of reagents are injected. The order of the reagent plugs containing the antibiotic and two non-interacting standards and the anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in RMTR of the new glycopeptide relative to the non-interacting standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant.     

Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.     

Novel Thiosemicarbazone Derivatives: In Vitro and In Silico Evaluation as Potential MAO-B Inhibitors

MAO-B inhibitors are frequently used in the treatment of neurodegenerative diseases such as Parkinson’s and Alzheimer’s. Due to the limited number of compounds available in this field, there is a need to develop new compounds. In the recent works, it was shown that various thiosemicarbazone derivatives show hMAO inhibitory activity in the range of micromolar concentration. It is thought that benzofuran and benzothiophene structures may mimic structures such as indane and indanone, which are frequently found in the structures of such inhibitors. Based on this view, new benzofuran/benzothiophene and thiosemicarbazone hybrid compounds were synthesized, characterized and screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro fluorometric method. The compounds including methoxyethyl substituent (2b and 2h) were found to be the most effective agents in the series against MAO-B enzyme with the IC₅₀ value of 0.042 ± 0.002 µM and 0.056 ± 0.002 µM, respectively. The mechanism of hMAO-B inhibition of compounds 2b and 2h was investigated by Lineweaver–Burk graphics. Compounds 2b and 2h were reversible and non-competitive inhibitors with similar inhibition features as the substrates. The K_i values of compounds 2b and 2h were calculated as 0.035 µM and 0.046 µM, respectively, with the help of secondary plots. The docking study of compound 2b and 2h revealed that there is a strong interaction between the active sites of hMAO-B and analyzed compound.     

Simultaneous determination of hydrochlorothiazide and several inhibitors of angiotensin-converting enzyme by capillary electrophoresis

A capillary electrophoresis method was developed for the simultaneous determination of hydrochlorothiazide and several angiotensin-converting enzyme (ACE) inhibitors: enalapril, lisinopril, quinapril, fosinopril, ramipril, and cilazapril. The most critical parameter is the pH of the running buffer. Separation was performed on a fused-silica capillary (52 cm total length x 75 microm I.D.) using a sodium phosphate buffer (pH 7.25; 100 mM). The method was successfully applied to the quantitative determination of these compounds in their corresponding pharmaceutical formulation. The method was validated in terms of linearity of response, reproducibility and accuracy.     

High concentrations of sodium chloride facilitate the use of immobilized chymotrypsin for separating virgin forms of specific trypsin inhibitors.

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.     

毛细管区带电泳测定D1蛋白酶的活性及其抑制剂先导化合物的筛选

建立了毛细管区带电泳技术快速测定D1蛋白酶活性及其抑制剂先导化合物的筛选方法。实验选用未涂层熔融石英毛细管(43 cm 5mm)和磷酸盐缓冲溶液(50 mmol/L, pH 3.0)作为分离介质,运行电压18 kV,测定了D1蛋白酶的活性并对部分抑制剂先导化合物进行了筛选。结果表明, ITP26和ITP21两种异噁唑噻唑哌啶类先导化合物对蛋白酶活性具有抑制作用,抑制率分别为26%和13%。     

Analysis of monoamine oxidase enzymatic activity by reversed-phase high performance liquid chromatography and inhibition by beta-carboline alkaloids occurring in foods and plants

Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)-containing enzyme located at the outer membranes of mitochondria that catalyzes the oxidative deamination of biogenic and xenobiotic amines. We have used a chromatographic method to measure MAO-enzymatic activity by using kynuramine as a non-selective substrate with its MAO-oxidation product subsequently analyzed by RP-HPLC-DAD and HPLC-mass spectrometry (MS). This method was applied to study the kinetic parameters, inhibition and reaction products of MAO recombinant enzymes in presence of tetrahydro-beta-carboline and beta-carboline alkaloids occurring in foods, plants and mammals. Analysis by HPLC showed that tetrahydro-beta-carbolines or beta-carbolines were not modified by MAO. Several beta-carbolines such as tryptoline (1,2,3,4-tetrahydro-beta-carboline) and 1-methyltryptoline (1-methyl-1,2,3,4-tetrahydro-beta-carboline) were inhibitors of MAO-A; instead their corresponding 6-hydroxy-derivatives (6-hydroxytryptoline and 6-hydroxy-1-methyltryptoline) lacked this activity. Tetrahydro-beta-carboline-3-carboxylic acids were unable to inhibit MAO enzymes. In contrast, their oxidation products, i.e. the fully aromatic beta-carbolines (norharman and harman), acted as good inhibitors of MAO. Two tetrahydro-beta-carbolines (i.e. tryptoline and 1-methyltryptoline) occurring in foods were isolated by solid-phase extraction (SPE) and RP-HPLC from selected samples of sausages and the corresponding extracts exhibited good inhibition properties over MAO-A. These results suggest that beta-carbolines from foods, plants, and mammals may exert inhibitory actions on MAO enzymes.