小分子免疫调节剂 L—2—(3—羟基—4—羟甲基苯基)—甘氨酸前体物 DL—2—(3—羟基—4—羟甲基苯基)—N—乙酰基甘氨酸的合成

本文通过对小分子免疲调节剂L—2—(3—羟基—4—羟甲基苯基)—甘氨酸(英文名:Forphenicinol)前体物DL—2—(3—羟基—4—羟甲基苯基)—N—乙酰基甘氨酸的合成方法研究,探索了终产物Forphenicinol 的合成路线及实验方法、并通过对各步产物之红外光谱分析,确证各步产物之主要特征吸收峰,为今后的合成工作打下良好之基础、     

来自地衣的2个新化合物及其抗氧化活性

药用地衣长松萝(Usnea longissimaAch.)是藏、蒙、维、傣等多个民族以各自的传统医药理论为依据,应用于医疗实践中的传统药材之一,其次生代谢产物有二苯骈呋喃类、缩酚酸类、单环苯酚类、甾醇类、三萜类、脂肪酸类及多糖等,具有抗菌、抗肿瘤、抗氧化、抗病毒、抗血栓活性、抗血小板、免疫调节、抗辐射生物活性等。该研究采用ABTS和DPPH 2种方法对长松萝干燥枝状地衣体中提取分离纯化得到的2个新化合物(4aR,9bS)-2,6-二乙酰基-3,4a,7,9-四羟基-8,9b-二甲基-1-氧代-1,4,4a,9b-四氢二苯骈呋喃酮(1)、4-[3-(7-乙酰基-4,6-二羟基-3,5-二甲基-2-氧代-2,3-二氢苯骈呋喃基)]-4-[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-3-氧代丁酸乙酯(2)进行抗氧化活性测定。结果2个化合物均表现不同程度的抗氧化能力,并显示浓度依赖性。2种方法测定结果:化合物(1)的ABTS法测试总抗氧化能力为3.53 mmol/LTrolox;DPPH自由基半数清除浓度(IC50)为0.31 mmol/L,化合物(2)的ABTS法测试总抗氧化能力为12.39 mmol/LTrolox;DPPH自由基半数清除浓度(IC50)为0.01 mmol/L,标准品2,6-二叔丁基-4-甲基苯酚(BHT)半数清除浓度(IC50)为0.16 mmol/L。     

海南粗榧内生真菌S15的细胞毒活性产物

对海南粗榧(Cephalotaxus hainanensis Li)内生真菌S15次生代谢产物进行了研究。用多种柱色谱技术从S15的发酵液中分离得到2个化合物,根据波谱数据和理化性质将其鉴定为乙酰基细胞松弛素D(1)和细胞松弛素D(2),对其进行生物活性测试,发现2种细胞松弛素对肿瘤细胞SGC-7901有强细胞毒活性。     

高选择性不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺的重组氧化还原酶催化性质及其酶促转化

【目的】通过表达多种重组立体选择性氧化还原酶,分析其催化不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的性质,从而构建酶促合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)的反应体系。【方法】基于已有立体选择性氧化还原酶重组大肠杆菌,通过Ni离子亲和层析法纯化得到重组氧化还原酶,以DKTP为底物,考察不同重组氧化还原酶对DKTP的催化活性和选择性,进一步对高选择性酶促合成(S)-DHTP的重组酶CR2进行性质分析,并考察其在最适条件下不对称还原DKTP的过程。【结果】筛选获得产物构型为(S)-型的催化活性最高的酶为CR2,该酶米氏常数Km为0.135 mmol/L,kcat/Km为3.689 L/(mmol·s),最适p H 8.4(0.1 mol/L三乙醇胺缓冲液),最适反应温度为35°C,在10-45°C条件下和p H 7.5-8.5较为稳定,Zn2+离子对酶活有促进作用。CR2催化DKTP不对称还原反应6 h后,DHTP的产率达92.1%、光学纯度达99.9%。【结论】基于活性和选择性分析,获得不对称还原DKTP的目标酶CR2,其催化特性有利于高立体选择性还原DKTP生成度洛西汀中间体(S)-DHTP,从而为进一步提高酶促不对称还原DKTP的转化效率提供研究基础。     

甘肃地区回族人群18个STR基因座的遗传多态性

研究了D5S818、D8S1179、D7S820、CSF1PO、D2S1338、D3S1358、v WA、D21S11、D16S539、Penta E、TPOX、TH01、D19S433、D18S51、FGA、D6S1043、D13S317、D12S391等18个短串联重复序列(short tandem repeats,STR)基因座在甘肃地区回族人群的遗传多态性。采用荧光标记复合扩增及毛细管电泳技术对1 038名甘肃地区回族无关个体18个STR基因座进行分析。研究结果显示,1 038名甘肃地区回族个体在18个STR基因座上,共检出223种等位基因,982种基因型,其分布均符合Hardy-Weinberg平衡(P0.05),18个基因座的杂合度(H)介于0.601~0.929之间,匹配概率(Pm)介于0.012~0.213之间,个体识别概率(DP)介于0.787~0.988之间,多态信息含量(PIC)介于0.550~0.920之间,非父排除概率(PE)值介于0.292~0.854之间。本文研究结果对甘肃地区回族人群群体遗传学及法医学后续研究应用具有参考价值。     

锡尾矿真菌的次生代谢产物研究

采用正相硅胶柱色谱、凝胶柱色谱、高效液相色谱等方法对一株来自锡尾矿的真菌的发酵产物进行分离,共获得14个化合物,依据理化性质及波谱数据分析,分别鉴定为司他弗林(1)、布雷非德菌素E(2)、布雷非德菌素H(3)、布雷非德菌素D(4)、(2S,3S)-3,5-二羟基-2-[(Z)-4-(羟甲基)-8-甲基-3,7-二烯-1-基]-2-甲基-3,4,8,9-四氢吡喃并[2,3-e]异吲哚-7(2H)-酮(5)、5-((1R,2R,5R,6R)-2,5-二羟基-7-氧杂二环[4.1.0]庚烷-2-基)-3-((1S,2S,4aR,6S,8a S)-2,6-二甲基-1,2,4a,5,6,7,8,8a-八氢萘-1-羰基)-4-羟基吡啶-2(1H)-酮(6)、(3s)-6,8-二羟基-3-甲基-3,4-二氢-1H-苯并吡喃-1-酮(7)、腺苷(8)、4-羟基苯甲醛(9)、5-氨基-2-羟基苯乙酸甲酯(10)、尿嘧啶核苷(11)、乙酰司他弗林(12)、尼克酰胺(13)、(S)-7-苯甲基-6-甲基-6,7-二氢苯并[6,7][1,4]二氮杂卓[2,1-b]喹唑啉-5,13-二酮(14)。采用MTT法检测,结果表明化合物5具有一定的抗肿瘤活性,其对HT-29、A-549和MCF-7三种细胞的ED50分别为21.59、23.45μg/m L和37.36μg/m L。     

ERα特异性抑制剂MPP降低小鼠2-细胞胚G1/S期过渡相关因子Nanog、cyclin D1和cyclin E水平

目的观察雌激素受体(ERa)特异性抑制剂甲基哌啶吡唑(methyl-piperidino-pyrazole,MPP)对小鼠2-细胞胚G1/S期过渡相关因子Nanog、cyclin D1和cyclin E表达水平的影响,阐明ERa对小鼠2-细胞胚G1/S期过渡的作用,为进一步探讨ERa在小鼠植入前胚合子基因组激活中的作用机制提供理论依据。方法收集昆明雌性小鼠与雄性小鼠交配所得的受精卵,分别置于无MPP的培养液和含20mmol/L MPP的培养液中培养,获取体外发育的2-细胞胚,采用免疫荧光染色技术检测细胞内Nanog、cyclin D1和cyclin E水平。结果免疫荧光染色结果显示,含20mmol/L MPP培养液培养的受精卵其体外发育2-细胞胚内Nanog、cyclin D1和cyclin E免疫反应性明显降低。结论 ERa可通过调控小鼠2-细胞胚内Nanog、cyclin D1和cyclin E水平,影响细胞从G1期到S期的过渡。     

1株杀鲑气单胞菌杀鲑亚种对甲酸乙酯的降解研究

甲酸乙酯广泛存在于化工行业的废水中,对生物体及环境都具有很大的危害。微生物降解是处理甲酸乙酯的重要方法之一。采用富集培养法和平板划线法从活性污泥中筛选出一株能降解甲酸乙酯的菌株ETH-3。经形态观察、生理生化试验和16S rDNA鉴定,确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。通过摇瓶实验测定菌株ETH-3的最佳生长环境条件以及其对甲酸乙酯的降解能力,结果表明,该菌株最佳生长温度为25-30℃、最佳生长pH为8左右。该菌株具有较强降解甲酸乙酯能力,在30℃时,36 h内对0-10 500 mg/L的甲酸乙酯可达完全降解。经GC-MS对代谢产物的分析发现,甲酸乙酯会先被微生物代谢为甲酸和乙醇,后甲酸和乙醇会被微生物进一步降解为CO_2和H_2O。     

N,N,N-三甲基壳聚糖甲基硫酸盐的合成及理化性质的测定

壳聚糖与甲醛、甲酸反应得到N,N-二甲基壳聚糖,然后以硫酸二甲酯为季铵化试剂反应得到N,N,N-三甲基壳聚糖甲基硫酸盐(TMCMS),用IR1、H NMR和元素分析对其结构进行了表征。元素分析结果表明其季铵化度为74.6%,差示扫描量热法和热重分析法结果表明其热稳定性比壳聚糖差,但其水溶性明显优于壳聚糖,25℃时在水中的溶解度可达20 mg/mL,浓度为2 mg/mL时在pH 3～12范围内无沉淀产生。     

固定化细胞拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的研究

【目的】筛选鉴定一株产酯酶用于选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,利用该菌株固定化细胞催化拆分外消旋底物。【方法】通过富集培养、罗丹明B平板初筛及复筛培养获得一株选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,通过对其形态、生理生化特征及16S r DNA序列分析,确立该菌株系统发育地位。优化了利用硅藻土-戊二醛吸附交联法对该菌体细胞固定化的条件,研究固定化细胞催化性质及操作稳定性。【结果】该菌为革兰氏阴性菌,鉴定其为甲基球状菌属(Methylopila)。固定化体系最优条件:聚乙烯亚胺0.15%(V/V),戊二醛0.2%(V/V),硅藻土6 g/L,菌体质量浓度100 g/L。与游离细胞相比,固定化细胞最适p H由8.0变为8.5,最适温度由35°C变为40°C,p H稳定性和温度稳定性都有所提高。Cu~(2+)、Mn~(2+)、Ca~(2+)能促进酶活,Zn~(2+)、Fe~(2+)抑制酶活。固定化细胞的有机溶剂耐受性较游离细胞有所提高。动力学分析细胞固定化后Km值变大,底物亲和力降低。利用固定化细胞水解(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯,底物浓度200 g/L,反应20 h,保留构型为S型,得率47.8%,对映体过量值ees为99.4%,重复使用12次后仍保留初始酶活的80%以上。【结论】开发了利用Methylopila sp.cxzy-L013固定化细胞择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的工艺,该工艺具有良好的工业应用前景。     

Strategy and Design of Novel Reagents for the Fluorometric Analysis of Biomolecules

This article covers molecular designs to develop several new fluorometric reagents and their applications to increase the sensitivities up to the picomole level using HPLC for the measurement of biomolecules. The methods were designed to demonstrate the physiological activities, for example (1) N-(9-acridinyl)maleimide (NAM) for the measurement of SH, –S–S–, and sulfite such as cysteine, (2) diphenyl-1-pyrenylphosphine (DPPP) for the hydroperoxides in lipids, serum, tissues, and foodstuffs, (3) 9-bromomethylacridine (9-BrMA), (4) 2-(anthracene-2,3-dicarboxylimide)ethyltrifluoromethane sulfonate (AE-OTf) for carboxylic acids, and (5) The chiral fluorometric labelling reagent (S)-( + )-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid (TBMB) to identify the chiralities of amino acids, sugars, and mono- and diacylglycerols.     

非天然β-氨基酸L-7-(N-苄氧羰基)氨基-3-(N-叔丁氧羰基)氨基-正庚酸的合成

以 Nε-苄氧羰基保护的 L -赖氨酸 ( L - Lys( Z) - O H)为原料 ,经过混合酸酐活化 ,与重氮甲烷反应合成重氮酮 ,再经 W olff重排 ,合成了具有光学活性的 L- 7- ( N -苄氧羰基 )氨基 - 3- ( N-叔丁氧羰基 )氨基 -正庚酸     

Study on the polarographic catalytic wave of vitamin P in the presence of persulfate and its application

The polarographic catalytic wave of vitamin P in the presence of persulfate was studied by linear potential scan polarography and cyclic voltammetry. Vitamin P yielded a single reduction wave in acidic aqueous solution, which was ascribed to a 2e(-), 2H(+) reduction of the carbonyl group in the C-4 position. Actually, the carbonyl group C=O first underwent a 1e(-), 1H(+) reduction to form a neutral free radical, and the further 1e(-), 1H(+) reduction of the free radical was simultaneous with its following chemical reactions. When S(2)O(2-)(8) was present, the free radical of vitamin P was oxidized by both S(2)O(2-)(8) and its reduction intermediate, the sulfate radical anion SO(*-)(4), to regenerate the original, which resulted in the production of a polarographic catalytic wave of vitamin P. Based on this catalytic wave, a novel method for the determination of vitamin P was proposed. In 0.02 M tartaric acid-sodium tartrate (pH 3.3) buffer containing 5.0 x 10(-3) M K(2)S(2)O(8), the peak potential of the catalytic wave was -1.42 V (vs SCE) and the peak current was rectilinear to the vitamin P concentration in the range of 8.0 x 10(-9)-1.0 x 10(-6) M (r = 0.9994, n = 13). The catalytic wave of 2.0 x 10(-7) M vitamin P enhanced the polarographic current 70 times compared with the corresponding reduction wave. The detection limit was 2.0 x 10(-9) M, and the relative standard deviation at the 2.0 x 10(-7) M level was 0.7% (n = 15). The proposed method was used for the determination of vitamin P content in the pharmaceutical preparation of tablets and the medicinal plant Sophora japonica L. without previous separation.     

Analysis of 6-hydroxy-2-aminocaproic acid (HACA) as a specific marker of protein oxidation: The use of <Emphasis Type="Italic">N(O,S)</Emphasis>-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry

Summary. An alteration of low density lipoprotein (LDL) apolipoprotein (apo) B-100 structure by direct oxidative modification is an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification because a lack of specific assays. The use of N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters for a rapid and sensitive determination of 6-hydroxy-2-aminocaproic acid (HACA), a highly specific marker of metal catalyzed protein oxidation, by using standard gas chromatography/electron impact mass spectrometry, is discussed. The derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. Femtomole levels of HACA can be reproducible measured in different LDL preparations subjected to oxidative damage in the presence of iron or copper. HACA determination compares well with the measurement of carbonyl groups that are generally accepted as a nonspecific index of protein oxidation. Thus, the method could prove to be a sensitive assay for studying specific apoB-100 modification.     

Quantitative gas—liquid chromatography of neutral sugars in human serum glycoproteins : Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma

A precise and accurate gas—liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars -fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis times (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m × 2 mm I.D. column with 2.0% Silar-7 CP on Chromosorb W AW 80–100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50W-X8 (H⁺) ion-exchange resin in 0.5 N HCI at 100° for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of <86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC—mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.     

Synthesis of 17O isotope labeled Leu-enkephalin and 17O n.m.r

Oxygen-17 isotope was introduced into the alpha-carboxyl group of glycine, 1-phenylalanine, 1-leucine and 1-tyrosine by acid catalyzed exchange of 17O from H2O(17) or by acid hydrolysis of respective amino acid methyl esters in H2O(17). Quantitative enrichment of glycine was achieved by acid hydrolysis of amino acetonitrile in H2O(17). For alpha-amino protection in amino acids t-butoxycarbonyl (Boc) group was employed for 17O labeled enkephalin synthesis. Five analogues of Leu-enkephalins (I-V) labeled with 17O at different amino acid residues were synthesized by solid phase method. 17O n.m.r. spectra were measured at 24.4 and 67.8 MHz for Leu-enkephalins 17O labeled at Gly2 and Phe4 positions. A downfield shift was observed for 17O labeled Gly2 Leu-enkephalin upon heating. This shift is indicative of the rupture of intramolecular hydrogen bonds. The preliminary results confirm the hypothesis that an intramolecular hydrogen bond exists between the carbonyl group of Gly2 and NH group of Leu5.     

Combining mutations in HIV-1 protease to understand mechanisms of resistance

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.     

Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

Characterization of 2-amino-2,6-dideoxy-D-glucose as a constituent of the lipopolysaccharide antigen of Pseudomonas aeruginosa immunotype 4.

The title lipopolysaccharide was freed from its lipid A component by mild, acid hydrolysis, to give a polysaccharide fraction that was subsequently hydrolyzed completely to afford a mixture of neutral sugars and amino sugars. The amino sugars were separated, and identified as 2-amino-2-deoxy-D-galactose, 2-amino-2,6-dideoxy-galactose as a 2:1 mixture of the D and L enantiomers, and 2-amino-2,6-dideoxy-D-glucose. A reference sample of 2-amino-2,6-dideoxy-D-glucose was synthesized by an improved preparative route. Among the lipopolysaccharide antigens of the seven recognized immunotypes of Pseudomonas aeruginosa, 2-amino-2,6-dideoxyglucose is also characterized as a constituent of two others, types 3 and 5.     

Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by carbonyl carbon nuclear magnetic resonances three phenylalanyl residues

The carbonyl carbon NMR signals of the Phe residues in Streptomyces subtilisin inhibitor (SSI) were selectively observed for [F]SSI, in which all phenylalanines were uniformly labeled with [1-13C]Phe. The three enhanced resonances in the spectrum of [F]SSI were unambiguously assigned to the specific sites in the amino acid sequence by means of 15N,13C double-labeling techniques. Namely, the resonances at 174.9 and 172.6 ppm (in D2O, pH 7.3, 50 degrees C) showed the satellite peaks due to 13C-15N spin coupling in the spectra of [F,GS]SSI and [F,A]SSI, in which Ser/Gly and Ala residues were labeled with [15N]Gly/Ser and [15N]Ala, respectively, together with [1-13C]Phe. The carbonyl groups of Phe-97 and Phe-111 are involved in peptide bonds with the amino nitrogens of Ser-98 and Ala-112, respectively. These results clearly indicate that the signals at 174.5 and 172.6 ppm are due to Phe-97 and Phe-111, respectively. The signal at the lowest field (177.1 ppm) was thus assigned to the carboxyl carbon of the C-terminal Phe-113. The lifetimes of the amide hydrogens of the three Phe residues and their C-terminal-side neighbors (Ser-98 and Ala-112) were investigated by using the effect of deuterium-hydrogen exchange of amide on the line shapes (DEALS) for the Phe carbonyl carbon resonances. In this method, the NMR spectra of [F]SSI dissolved in 50% D2O (pH 7.3) were measured at various temperatures, and the line shape changes caused by deuteriation isotope shifts were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)