运用含有偶氮苯的自组装单层膜在光化学条件下可逆地控制细胞黏附

细胞生物学研究的一个重要方向是动态地控制细胞在基底上的黏附。最近，随着表面化学的研究深入，尤其是对烷基硫醇在金基底上形成自组装单层膜（self-assembled monolayers, SAMs）这一体系的研究，使得人们能在分子水平的表面上控制细胞黏附。精氨酸-甘氨酸-天冬氨酸（arginine-glycine-aspartate, RGD）序列首先是从细胞外基质蛋白中分离出来的，能够识别并非共价结合细胞膜表面的整合素受体，从而促进细胞黏附。以前的一些工作已经证实，将含有RGD的肽链连接到SAMs表面之后，能够生物特异性地黏附动物细胞。已有的手段比如光照、电压、加热、微电极、微流控以及表面纳米形貌的梯度变化，都不能真正实现可逆地控制细胞黏附，原因是这些方法所用的化学有限；这些方法也不能得到完全抗拒细胞黏附的表面，原因是这些方法产生的表面缺陷等不完整。用两种不同波长的光（紫外光和可见光）照射偶氮苯，偶氮苯会发生可逆的光致异构变化，因此，偶氮苯的光致异构性质可以用来可逆地控制细胞在表面黏附。运用含有偶氮苯的混合SAMs，偶氮苯的末端连接GRGDS肽，混合SAMs中是以末端为六聚乙二醇的硫醇为背景，该SAMs修饰而成的表面能够黏附或者抗拒细胞黏附，其表面黏附性质取决于SAMs中偶氮苯的构象。该方法提供了一种在分子水平的表面上我们所了解到的唯一能可逆控制细胞黏附的方法，该方法需要用到的光源来自于标准荧光显微镜所配置的汞灯。 为了实现在金基底表面可逆的控制细胞黏附，我们合成了如下三个化合物： 由于化合物1的溶解性很差，几乎在所有溶剂里都不溶，所以不能直接用化合物1制备SAMs；化合物2能高效地抗拒细胞的黏附；化合物3的偶氮苯末端是活化酯，能够连接GRGDS肽，从而控制细胞黏附。 将化合物2和化合物3以一定的比例均匀混合在金基底表面形成SAMs，然后将GRGDS肽连接到偶氮苯(反式)的末端（通过GRGDS肽的甘氨酸上的伯胺基与偶氮苯末端的活化酯反应），从而得到细胞黏附的表面。用紫外光照射该细胞黏附表面5-10小时，随着偶氮苯的构象由反式变为顺式，偶氮苯末端的GRGDS肽淹没在化合物2的六聚乙二醇中，得到抗拒细胞黏附的惰性表面。再用可见光照射该惰性表面1个小时，随着偶氮苯的构象由顺式变为反式，原先埋没在六聚乙二醇中的GRGDS肽伸展至单层膜的末端，又得到了细胞黏附的表面。因此，该表面能完全可逆地控制细胞在金表面黏附。 An important area in cell biology is the dynamic control of cell adhesion on substrates. Recent advancements in surface chemistry, in particular, self-assembled monolayers (SAMs) of alkanethiols on gold substrates, have permitted unprecedented control of cell adhesion via molecularly defined surfaces. The tri-peptide sequence arginine-glycine-aspartate (RGD), initially isolated from the extracellular matrix (ECM) proteins, can recognize and non-covalently bind with integrin receptors on cell membranes to promote cell adhesion. Some previous work has demonstrated that RGD peptide grafted on SAMs can allow bio-specific adhesion of mammalian cells that mimic natural adhesion. Existing technologies such as light, voltage, heat, microelectrodes, microfluidic systems and surface gradient of nanotopography, either cannot realize fully reversible control of cell adhesion, due to the limitation in the chemistry used, or cannot yield a surface completely resistant against cell adhesion, due to the imperfection of surfaces. Azobenzenes undergo reversible photo-induced isomerization rapidly at two different wavelengths of light (UV and visible light), it therefore potentially allows the reversible control of cell adhesion on a surface. By using a mixed SAMs presenting azobenzene groups terminated in GRGDS peptides in a background of hexa(ethylene glycol) groups, the surface can either accommodate or resist cell adhesion depending on the conformation of the azobenzene embedded in SAMs. This method provides the only means we know to control cell adhesion reversibly on a molecularly well-defined surface by using light generated by a mercury lamp equipped on standard fluorescence microscopes. To realize the reversible control of cell adhesion on gold surface, we synthesized three kinds of compounds as following, We found that it was difficult to obtain SAMs directly from compound 1 because of its poor solubility in almost all kinds of solvents; compound 2 can resist cell adhesion efficiently; compound 3 presents an azobenzene terminated with NHS-activated ester, which can couple with a GRGDS peptide to control cell adhesion. After coating a gold surface with compound 2 and 3 in appropriate ratios to form a SAM followed by coupling the GRGDS peptides with NHS-activated esters at the end of azobenzene (E configuration) resulted in a cell-adhesive SAM. Irradiating this cell-adhesive SAM with UV light for 5-10 h converted the E configuration of azobenzene into the Z form, the GRGDS peptides becoming masked in the PEG, resulting in a cell-resistant surface. These SAM could again support cell adhesion as a result of the conformational switch of azobenzene from Z to E with the irradiation of visible light for 1 h. This surface, therefore, allows completely reversible control of cell adhesion on a gold surface.

四川蜡瓣花、密花樫木、四川溲疏及云南豆腐柴的化学成分研究

本论文对四川蜡瓣花 (Corylopsis willmottiae Rehd. et Wils.)、密花樫木[Dysoxylum densiflorum (Blume) Miq.]、四川溲疏 (Deutzia setchuenensis Franch)及云南豆腐柴 (Premna yunnanensis W. W. Smith)的化学成分进行了研究。通过色谱分离得到44个化合物。主要基于波谱数据鉴定了它们的结构，其中1个为新化合物。 1．从四川蜡瓣花全株的95%乙醇提取物中共分离鉴定了13个化合物，它们是：1-O-(3-O-甲基没食子酸)-岩白菜素(1)、11-O-没食子酰基岩白菜素(2)、 11-O-紫丁香基岩白菜素(3) 、岩白菜素(4)、4-O-没食子酰基岩白菜素(5) 、4,11-O-二没食子酰基岩白菜素 (6)[14]、β-谷甾醇 (7)、acetyl aleuritolic acid (8)、(-)-表没食子儿茶素没食子酸酯(9)、对羟基苯甲酮 (10)、 11-香豆酸酰岩白菜素 (11)[19]、丁香酸 (12)和没食子酸 (13)。其中1为新化合物。 2．从密花樫木根的95%乙醇提取物中共分离纯化了13个化合物，它们是：β-白檀酮(14)、richenone (15)、β-谷甾醇 (7)、cabraleadiol (16)、β-香树脂醇 (17)、龙脑香醇酮 (18)、cabraleadiol monoacetate (19)、cabraleone (20)、3β-hydroxy-5 -pregnen-20-one (21)、3β-hydroxy-5α-pregnan-20-one (22)、cabraleahydroxylactone (23)、川楝子甾醇B (24)、表儿茶素 (25)。 3．从四川溲疏全株95%乙醇提取物中共分离11个化合物，鉴定了其中的9个化合物。它们是：β-谷甾醇 (7)、白桦酯醇(26)、齐墩果酸(27)、hydrangetin (28)、肉桂酸 (29)，齐墩果酸-3-O-β-D-吡喃葡萄糖醛酸苷(30)、β-胡萝卜苷 (31)、齐墩果酸-3-O-（β-D-吡喃葡萄糖醛酸-6-正丁酯）(32)、齐墩果酸-3-O-β-D-吡喃葡萄糖醛酸-28-O-β-D-吡喃葡萄糖苷 (33)。 4．从云南豆腐柴95%乙醇提取物中分离得到12个化合物，分别为白桦脂醇 (25)、7-羟基黄烷酮 (34)、松属素 (35)、2’,4’-羟基查儿酮 (36)、高良姜素-3-甲醚 (37) 、高良姜素-3,7-二甲醚 (38)、异甘草素-4-甲醚 (39)、豆蔻明 (40)、乔松酮 (41)、异甘草素 (42)、arjunolic acid (43)、槲皮素3-O-β-D-木糖苷(44)。 5．综述了1976年以来樫木属植物化学成分和活性研究的概况。 Phytochemical investigation on Corylopsis willmottiae, Dysoxylum densiflorum, Deutzia setchuenensis, and Premna yunnanensis, led to the isolation of 44 compounds, 1 of which was new one. 1. One new compound was isolated from 95% ehanolic extrat of the whole plants of C. willmottiae, identified as 11-O-(3-O-methylgalloyl)-bergenin (1). The twelve known compounds isolated were 11-O-galloylbergenin (2), 11-O-syringylbergenin (3), bergenin (4), 4-O-galloylbergenin (5), 4,11-di-O-galloylbergenin (6), β-sitosterol (7), acetyl aleuritolic acid (8), (-)-epigallocatechin 3-O-gallate (9), 1-(4-hydroxyphenyl) ethanone (10), 11-O-coumaroylbergenin (11), syringic acid (12), gallic acid (13). 2. Thirteen compounds were isolated from 95% ethanol extract from the roots of D. densiflorum and identified as β-amyrenone (14), richenone (15), β-sitosterol (7), cabraleadiol (16), β-amyrin (17), hydroxydammarenone-Ⅱ (18), cabraleadiol monoacetate (19), cabraleone (20), 3β-hydroxy-5-pregnen-20-one (21), 3β-hydroxy-5α-pregnan-20-one (22), cabraleahydroxylactone (23), toosendansterol B (24) and (-)-epicatechin (25). 3. Eleven compounds were isolated from ethanol extract of D. Setchuenensis. Nine were identified as β-sitosterol (7), betulin (26), oleanolic acid (27), hydrangetin (28), cinnamic acid (29), oleanolic acid 3-O-β-D-glucuronopyranoside (30), β-daucosterol (31), oleanolic acid 3-O-β-D-glucuronopyranoside-6-O-butyl ester）(32), oleanolic acid 3-O-β-D-glucuronopyranosyl-28-3-O-β-D-glucopyranoside (33). 4. Twelve compounds were isolated from ethanol extract of P. yunnanensis and identified as betulin (26), 7-hydroxyflavanone (34), pinocembrin (35), 2’,4’-dihydroxychalcone (36), galangin 3-methyl ether (37), galangin 3,7-dimethyl ether (38), isoliquiritigenin 4-methyl ether (39), cardamonin (40), pinostrobin (41), isoliquiritigenin (42), arjunolic acid (43), quercetin 3-O-β-D-lyxosopyranoside (44). 5. Chemical constituents and biological activities of the genus Dysoxylum (Meliaceae) were reviewed during 1976-2009.

野桂花的化学成分研究

首次从野桂花（Osmanthus yunnanensis Fr. P. S. Green）地上部分95%乙醇提取物中通过色谱分离得到20个化合物, 其中化合物20为新化合物。基于波谱数据它们被鉴定为(E)-阿魏酸二十烷基酯(1)、β-谷甾醇(2)、羽扇豆醇(3)、齐墩果酸(4)、7-oxo-β-sitosterol(5)、乙酰齐墩果酸(6)、(6′-O-palmitoyl)-sitosterol 3-O-β-D-glucoside(7)、rotundioic acid(8)、地榆糖甙Ⅱ(9)、27-O-(E)-对羟基肉桂酰-28-齐墩果酸(10)、27-O-(Z)-对羟基肉桂酰-28-齐墩果酸(11)、hycandinic acid ester(12)、绿原酸丁酯(13)、4,5-二咖啡酰奎尼酸丁酯(14)、4,5-dihydroxyprenyl caffeate(15)、28-O-β-D-glucopyranosyl rotundioic acid (16)、4-(6-O-caffeoyl-β-D-glucopyranosyloxy)-5-hydroxyprenyl caffeate (aohada-glycoside C, 17)、 4-β-D-glucopyranosyloxy-5-hydroxy-prenyl caffeate (aohada-glycoside A, 18)、β-胡萝卜甙(19)以及3-[O-β-D-(6-O-咖啡酰吡喃葡萄糖)]-甲基-2-烯-γ-内酯 (20)。化合物13、14、15和17有较强的α-葡萄糖甙酶抑制活性。当浓度为1 mg/ml时，它们对α-葡萄糖甙酶的抑制分别为61.5%、95.5%、72.1%、62.6%，活性高于阿卡波糖。 综述了木犀属植物化学成分及1993年以来苯丙素甙类化合物活性研究进展。 Twenty compounds were isolated from the 95% ethanol extract of the aerial parts of Osmanthus yunnanensis Fr. P. S. Green by chromatography for the first time. On the basis of spectral data, they were identified as (E)-ferulic acid eicosyl ester (1), β-sitosterol (2), lupenol (3), oleanolic acid (4), 7-oxo-β-sitosterol (5), acetyloleanolic acid (6), (6′-O-palmitoyl)-sitosterol 3-O-β-D-glucoside (7), rotundioic acid (8), ziyu glycosideⅡ (9), 3β-hydroxy-27-p-(E)-coumaroyloxy-olean-12-en-28-oic acid (10), 3β-hydroxy-27-p-(Z)-coumaroyloxyolean-12-en-28-oic acid (11), hycandinic acid ester (12), chlorogenic acid butyl ester (13), 4,5-di-O-caffeoylquinic acid butyl ester (14), 4,5-dihydroxyprenyl caffeate (15), 28-O-β-D-glucopyranosyl rotundioic acid (16), 4-(6-O-caffeoyl-β-D-glucopyranosyloxy)-5-hydroxyprenyl caffeate (aohada- glycoside C, 17), 4-β-D-glucopyranosyloxy-5-hydroxyprenyl caffeate (aohada- glycoside A, 18), β-daucosterol(19) and 3-[O-β-D-(6-O-caffeoylglucopyranosyl)]- methyl-2-en-γ-lactone (20). Compound 20 is a new one. Compounds 13, 14, 15 and 17 inhibit α-glucosidase with corresponding inhibitory rate of 61.5%, 95.5%, 72.1% and 62.6% at a concentration of 1 mg/ml, higher than acarbose. The chemical studies on Osmanthus genus and bioactivities of phenylpropanoid glycosides were summarized.

5-氟尿嘧啶-β-环糊精结肠靶向前体药物的研究

5-氟尿嘧啶(5-Fluorouracil， 5-FU)是一种抗代谢药物，广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症，但其首过代谢显著、亲脂性较低，选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作，包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin，简称CD)，可被结肠中的糖苷酶特异性地降解成小分子糖，而胃和小肠中由于缺乏相应的酶而使环糊精不被降解，这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基，易进行化学修饰，将药物与环糊精通过共价键结合制成前药，使其在胃和小肠中不降解，而在盲结肠中被特异性的酶降解释出药物，达到结肠靶向释药的目的。研究表明，环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体，通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联，合成了系列5-FU-β-CD前体药物，并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时，还研究了前体药物的体外释药性质。具体内容包括： 1. 含有羧基的5-FU衍生物中间体的合成：(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联：分别通过以6-OTs-β-CD为中间体的取代法和活化酯法，合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中，通过改变原料的比例，合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究：分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为，并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

绵参和地榆的化学成分研究

本论文由三章组成。第一章为综述，概述了植物中环烯醚萜类化合物的研究进展；第二和第三章为实验论文，分别报道了唇形科药用植物绵参和蔷薇科药用植物地榆的化学成分研究。 第一章概述了植物中环烯醚萜类化合物的研究成果，主要包括结构类型及药理活性等方面。 第二章包括两个部分。第一部分报道了藏药绵参（Eriophyton wallichii Benth）地上部分甲醇提取物的化学成分。采用正、反相硅胶柱层析等各种分离方法，从中共分离出7个化合物，有6个化合物为首次从该植物中分离得到，分别为β-谷甾醇（1），夏至草苦素（marrubiin，2），乌苏酸（3），cimigoside（4），5-deoxyantirrhinoside（5），8-表马钱子酸葡萄糖苷（8-epiloganic acid，6）和apigenin 7-(6''-p-coumaroyl)glucoside（7）。第二部分，采用高效液相色谱－质谱联用技术对绵参地上部分的甲醇提取物进行了分析，通过标准品对照紫、外光谱分析以及多级质谱分析与文献对照鉴定了8个成分，分别是：8-epiloganic acid（Ⅰ），quercitrin 3-glucoside-7-(6''-p-coumaroyl)glucoside（Ⅱ），ajugoside(I) （Ⅲ），chrysoeriol 7-O-E-p-coumaroyl-3-O-b-D-glucoside（Ⅳ），helichrysoside（Ⅴ），生物碱（Ⅵ），apigenin 2,3-dihydrogen-7-(6''-p-coumaroyl) glucoside（Ⅶ），apigenin 7-(6''-p-coumaroyl) glucoside（Ⅷ）。 第三章报道了中药地榆根部乙醇提取物正丁醇相的化学成分，通过正、反相硅胶柱层析等各种分离方法，从中分离得到8个化合物，分别为3,4¢- O-二甲基逆没食子酸（8），3,3¢,4¢-O-三甲基逆没食子酸（9）和3,4¢-O-二甲基逆没食子酸-4-O-b-D-木糖苷（10），19a-羟基-3-O-(a-L-阿拉伯糖)乌苏酸-28-O-b-D-葡萄糖苷（11）， 3b-[(a-L-arabinopyranosyl)oxy]-urs-11,13(18)-dien-28-oic acid b-D- glucopyranosyl ester（13），3-O-a-L-arabinopyranosyl-urs-12,18(19)-dien-28-oic acid b-D-glucopyranosyl ester（14），儿茶素（15），还有一种可能是皂苷11的工作产物（12）。 This dissertation consisted of three chapters. The first chapter elaborated the progress of iridoids occurring in plants. The later two chapters respectively elaborated the chemical constituents of Eriophyton wallichii Benth. and Sanguisorba officinalis L. The first chapter is a review of the research progress of iridoids occurring in plants, which includes their structure and pharmacology. The second chapter consisted of two parts. The first part is about the chemical constituents of methanol extraction from the aerial parts of Eriophyton wallichii Benth. Seven compounds were isolated and identified. Among them, the compounds of marrubiin, ursolic acid, cimigoside, 5-deoxyantirrhinoside, 8-epiloganic acid，apigenin 7-(6''-p-coumaroyl)glucoside were firstly reported in this plant. A HPLC-MSn method was developed for rapid identification of major compounds of Eriophyton wallichii. A total of 8 peaks in the chromatograms were unequivocally determined (peaks 1, 8) or tentatively identified (peaks 2-7) based on the detailed UV and tandem mass spectra analysis. Seven components were identified as 8-epiloganic acid（Ⅰ），Quercitrin 3-glucoside-7-(6''-p-coumaroyl)glucoside（Ⅱ），ajugoside(I)（Ⅲ），Chrysoeriol 7-O-E-p-coumaroyl-3-O-b-D-glucoside（Ⅳ），helichrysoside（Ⅴ），apigenin 2,3-dihydrogen-7-(6''-p-coumaroyl) glucoside（Ⅵ），apigenin 7-(6''-p-coumaroyl) glucoside（Ⅶ）。 The third chapter elaborated the chemical constituents of methanol extraction from Sanguisorba officinalis L, eight compounds were isolated from this plant by repeat column chromatography over silica gel. These compounds were identified as 3,4′-O-dimethylellagic acid, 3,3′,4′-O-trimethylellagic acid, 3,4′-O-dimethylellagic acid-4-O-b-D-xyloside, 3b-O-a-L-arabinopyranosyl-19a- hydroxyl-urs-12-en-28-oic acid 28-b-D-glucopyranoside, 3b-[(a-L-arabinopyranosyl)oxy]-urs-11,13(18)-dien- 28-oic acid b-D-glucopyranosyl ester，3-O-a-L–arabinopyranosyl-urs-12,18(19) -dien-28-oic acid b-D-glucopyranosyl ester, catechin.

西藏胡黄连和鸡矢藤的化学成分及黄芪多糖的提取工艺研究

本论文由四部分组成，前三部分为实验论文，第四部分为文献综述。第一、二部分分别报道了中药西藏胡黄连和鸡矢藤的化学成分研究结果。从两种药用植物中共分离和鉴定了32个化学成分，其中3个为新化合物。第三部分为黄芪多糖的提取工艺研究。第四部分概述了近年来植物多糖的研究进展。 第一章为西藏胡黄连化学成分研究。通过正、反相硅胶柱层析等分离方法从药用植物西藏胡黄连（Picrorhiza scrophulariiflora Pennell）的根茎中共分离纯化出7个化合物。运用MS、1H-NMR、13C-NMR、DEPT、HSQC和HMBC等现代谱学方法，结合理化分析对这些化合物的结构进行了分析鉴定。7个化合物中有两个是酚性的葡萄糖苷类成分：西藏胡黄连酚苷D (1)、4-O-β-D-(6-O-vanilloyl glucopyranosyl) vanillic acid (6)；四个苯乙基苷类化合物：plantamajoside (2)、plantainoside D (3)、西藏胡黄连苷A (4) 和西藏胡黄连苷F (5)；一个苯基小分子化合物：香豆酸甲酯 (7)。其中化合物1和5未见文献报道，确定为新化合物；化合物3为首次从该种植物中分到。 第二章为鸡矢藤化学成分研究。从鸡矢藤（Paederia scandense (Lour) Merrill）全草中分离出25个化合物，通过理化常数和波谱数据鉴定了它们的结构。25个化合物中包括一个蒽醌类成分：茜根定-1-甲醚 (1)；两个香豆素：异东莨菪香豆素 (2)和5-羟基-8-甲氧基吡喃香豆素 (3)；两个香豆素-木脂素化合物：臭矢菜素 B (4)和臭矢菜素 D (5)；一个木脂素：异落叶松树脂醇 (6)；两个黄酮：diadzein (7)和蒙花苷 (8)；三个三萜类化合物：齐墩果酸 (9)、乌苏酸 (10)和 3-O-β-D-吡喃葡萄糖基乌苏烷 (11)；三个甾体及其糖苷：b-谷甾醇 (12)、胡萝卜苷 (13)和(24R)-豆甾-4-烯-3-酮 (14)；六个小分子化合物：对羟基苯甲酸 (15)，咖啡酸 (16)，香豆酸 (17)，丁烯二酸 (18)，3,5-二甲氧基-4-羟基苯甲酸(19)，咖啡酸-4-O-β-D-吡喃葡萄糖苷(20)；五个环烯醚萜类化合物：鸡矢藤苷 (21)，鸡矢藤酸 (22)，鸡矢藤酸甲酯 (23)，saprosmoside E (24)和paederoside B (25)。其中化合物25未见文献报道，为新化合物。化合物1～8、11、14、15～20为首次从该化合物中分离得到。同时对鸡矢藤中环烯醚萜类化合物做了高效液相-串联质谱(HPLC-MSn)分析，探讨了这类化合物的质谱裂解规律。 第三章为黄芪多糖的提取工艺研究。首先确定了黄芪多糖含量的测定方法，并进行了方法学验证；其次探讨了黄芪中黄芪多糖的提取工艺，确定以酶法-Sevag法联用来去除黄芪多糖中的蛋白质，可使其提取物中黄芪多糖总含量达到70％以上。 第四章为近年来植物多糖的研究进展。主要包括植物多糖的提取纯化、多糖的定性定量检测方法、多糖的结构分析和多糖的药理活性。 This dissertation consists of four parts. The first and second parts reports the studies on the chemical constituents of medicinal plants of Picrorhiza Scrophulariiflora and Paederia scandens. The third part is about the extract technique of Astragalan Polysaccharide (APS). The last part reviews the progress of the studies on plant polysaccharides. 　 The first chapter is about the chemical constituents of P. Scrophulariiflora which is widely used as an important medicine to treat various immune-related diseases. A new phenyl glycoside, scrophenoside D (1) and a new phenylethyl glycoside, scroside F (5), together with five known compounds, plantamajoside (2), plantainoside D (3), scroside A (4), 4-O-β-D-(6-O-vanilloylglucopyranosyl) vanillic acid (6); and methyl-p-coumarate (7) were isolated from the stems of P. scrophulariiflora. Their structures were elucidated by spectroscopic and chemical methods. The second chapter is about the chemical constituents of medicinal herb of P. scandens. Twenty-five compounds were isolated and purified by normal and reversed phase silica gel column chromatography. By physicochemical properties and spectral analysis, their structures were identified as rubiadin-1-methylether (1), isoscopoletin (2), 5-hydroxyl-8-methoxyl-coumarin (3), cleomiscosin B (4), cleomiscosin D (5), isolariciresinol (6), diadzein (7), linarin (8), oleanolic acid (9), ursolic acid (10), 3-O-β-D-glucopyranosyloxyl-ursane (11), b-sitosterol (12), b-daucosterol (13), (24R)-stigmast-4-ene-3-one (14), p-hydroxyl-benzoic acid (15), caffic acid (16), coumaric acid (17), trans-butenedioic acid (18), 3,5-dimethoxyl-4-hydroxylbenzoic acid (19), caffeic acid 4-O-β-D-glucopyranoside (20), paederoside (21), paederosidic acid (22), paederosidic acid methyl ester (23), saprosmoside E (24), paederoside B (25). Among them, compound 25 is a new compound. Compounds 1～8、11、14、15～20 were isolated from this plant for the first time. Futhermore, we studied the HPLC-MSn analysis and investigation of fragmentation behavior of the sulfur-containing iridoid glucosides. The third chapter is about the extracting process of Astragalan Polysaccharide (APS). The method of the content determination is built. The optimum condition of extraction of polysaccharides from Radix Astragali is defined and the more effective way to remove protein is combined enzyme method with Sevag method, by which the content of polysaccharides extract can be up to 70%. The last part is a review of the research progress of the plant polysaccharides, which includes its extraction, isolation, purification, determination, structure analysis, and pharmacology.

尼泊尔水东哥、水麻、木姜冬青和青荚叶的化学成分研究

活性筛选中发现尼泊尔水东哥 (Saurauia napaulensis DC.) 树皮95%乙醇提取物具有α-淀粉酶抑制活性、水麻(Debregeasia orientalis) 枝叶95%乙醇提取物显示血管紧张素转化酶(ACE)抑制活性、青荚叶(Helwingia japonica (Thunb.) Dieter.) 95%乙醇提取物的中小极性部分显示蛋白酪氨酸磷酸酯酶(PTP)1B抑制活性。为全面了解它们的成分及相关活性成份，主要运用硅胶柱层析方法从这三个植物分离得到39个化合物，通过波谱分析或与已知品对照的方法对其进行了鉴定。对木姜冬青(Ilex litseaefolia Hu et Tang)的成分做了进一步的研究，取得了如下结果。 1. 从尼泊尔水东哥树皮的95%乙醇提取物分离并鉴定12个化合物： auranamide、aurantiamide benzoate、齐墩果酸、β-谷甾醇、β-胡萝卜甙、乌苏酸、2α,3α-二羟基-12-烯-28-乌苏酸、2α,3β,24-三羟基-12-烯-28-乌苏酸、(2S,3S,4R,10E)-2-[(2'R)-2' -hydroxytetracosanoylamino] -10-octadecene -1,3,4-triol、 2α,3α,24-三羟基-12-烯-28-齐墩果酸、2α,3β-二羟基-12-烯-28-乌苏酸和2α,3α,24-三羟基-12-烯-28-乌苏酸。 2. 从水麻枝叶的95%乙醇提取物分离并鉴定了18个化合物：棕榈酸、二十烷酸、二十烷酸甲酯、β-谷甾醇、Monogynol A、桦木酸、Hederagenin、β-胡萝卜甙、18αH-19(29)-烯-3-酮-乌苏烷、3，4-开环-20(30)-烯-乌苏烷-3-酸、Pomolic acid，表儿茶素、儿茶素、槲皮素、槲皮素-3-O-β-D-吡喃葡萄糖苷、紫丁香苷、紫丁香酚苷和山萘酚-3-O-芸香糖。儿茶素、槲皮素和槲皮素-3-O-β-D-吡喃葡萄糖苷为具有ACE抑制活性的成分。 3. 从木姜冬青95%乙醇提取物的乙酸乙酯部分分离并鉴定了5个化合物： 2-O-β-D-吡喃葡萄糖-6,2´-二羟基-4,4´-二香草酰氧甲基-1,1´-二苯醚（冬青苷）和四个已知化合物：七叶内酯、香草酸、3,4-二甲氧基苯乙酸和vanilloylcalleryanin。冬青苷为新化合物。 4. 从青荚叶95%乙醇提取物的中小极性部分分离并鉴定了9个化合物：β-谷甾醇、β-胡萝卜苷、羽扇豆醇、桦木醇、桦木酸、棕榈酸甘油酯、桂皮酸、6αH-4-烯-3-酮-豆甾醇和6βH-4-烯-3-酮-豆甾醇。 5. 对1985-2006年间天然二苯醚类化合物及活性研究进展进行综述. The in vitro test indicated that the 95% ethanolic extract of the barks of Saurauia napaulensis DC showed α-amylase inhibitory activity, the 95% ethanolic extract of the whole plants of Debregeasia. orientalis showed angiotensin converting enzyme (ACE) inhibitory activity and some fractions of the 95% ethanolic extract of the aerial parts of Helwingia japonica showed protein tyrosine phosphatase (PTP)1B inhibitory activity. In order to investigate components and active compounds of the three plants, they were chemically studied mainly using. Thirty-nine compounds were isolated predominantly by column chromatography identified by spectral methods or comparing them with authentic samples. Further investigation of Ilex litseaefolia Hu et Tang was carried out. Major results are as follows: 1. Twelve compounds were isolation from the 95% ethanolic extract of the barks of S. napaulensis DC. They were identified as auranamide, aurantiamide benzoate, oleanolic acid, β-sitosterol, β-daucosterol, ursolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyurs-12-en-28-oic acid, (2S,3S,4R,10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1,3,4-triol, 2α,3α,24 -trihydroxyolean-12-en-28-oic acid, 2α,3β-dihydroxyurs-12-en-28-oic acid, and 2α,3α,24-trihydroxyurs-12-ene-28-oic acid, respectively, by spectral methods or comparing them with authentic samples. 2. Eighteen compounds were isolation from the 95% ethanolic extract of the whole plants of D. orientalis. They were identified as palmitic acid, henicosanoic acid, henicosanoic acid methyl ester, β-sitosterol, monogynol, betulinic acid, hederagenin, β-daucosterol, 18αH-urs-20(30)-en-3-one, 3,4-seco-urs-20(30)-en-3-oic acid, pomolic acid, (-)-epicatechin, (+)-catechin, quercetin, quercetin 3-O-β-D-glucopyranoside, syringin, syringiaresinol digloside and kaempferol-3-O-rutinose. (+)-Catechin, quercetin and quercetin 3-O-β-D-glucopyranoside were the ACE inhibitory active components. 3. Further phytochemical investigation of the ethyl acetate parts of 95% ethanolic extract of the whole plant of I. litseaefolia afforded 2-O-β-D-glucopyranose-4,4´-di-vanilloyloxymethyl-2,6´-dihydroxy-1,1´-diphenyl ether (ilexiside), esculetin, vanillic acid, 3,4-dimethoxybenzylacetic acid and vanilloylcalleryanin. Ilexiside was new compound. 4. Nine compounds were isolation from the 95% ethanolic extract of the whole plant of H. japonica: β-sitosterol, β-daucosterol, lupeol, betulin, betulinic acid, glycerol monopalmitate, cinnamic acid, stignast-4-en-6β-3-one and stignast-4-en-6α-3-one 5.Diphenyl ether compounds from nature between 1985-2006 were summarized.

具有环氧化酶-2选择性抑制活性的多取代蒽醌的合成

1-甲基-2-甲氧羰基-3, 6, 8-三羟基-7-甲氧基蒽醌是从唐菖蒲干球茎中分离到的具有环氧化酶-2选择性抑制活性的多取代蒽醌类化合物。本文试图合成该化合物，实现了其类似物的合成，同时发现了几个未见报道的反应。 1．通过Diels-Alder 反应合成了关键中间体——3-甲基-5-羟基-1, 2, 4-苯三甲酸三甲酯，1-COOMe选择性水解产物与1, 2, 3-三甲氧基苯进行分子间Friedel-Crafts反应的产物再进行分子内Friedel-Crafts反应得到了目标产物的类似物1-甲基-2-甲氧羰基-3-羟基-6，7，8-三甲氧基蒽醌（路线1）。目标产物及其它类似物的合成正在进行中。 2．以乙酰乙酸甲酯和巴豆醛为原料，经过Michael加成、分子内的Aldol反应、芳香化、选择性甲酰化和还原反应，得到关键中间体2-甲基-3-羟甲基-6-甲氧基苯甲酸甲酯及其衍生物。通过该化合物与3，4，5-三甲氧基苯甲酸甲酯进行Friedel-Crafts烷基化反应得到了多取代的二苯基甲烷衍生物，拟进一步关环合成目标化合物（路线2）。 3．发现邻甲氧基苯甲酸甲酯中酯甲基可以被正丁基锂和仲丁基锂中烷基交换生成相应的酯，反应的机理不明确。当使用叔丁基锂时，得到的是邻甲氧基苯基叔丁酮，这个方法可以用来合成芳基叔丁酮类化合物。 4．以2-苄氧基-6-甲基苯甲酸甲酯为原料进行氯甲基化反应时，以苯和二氯乙烷作溶剂，发生了苄基的迁移和芳环的偶联，分别得到2，2'-二甲基-3，3'-二甲氧羰基-4，4'-二羟基联苯和2，2'-二甲基-3，3'-二甲氧羰基-4，4'-二羟基-5，5'-二苄基联苯。这是对称联苯合成的新方法。 5．水杨酸羟基邻对位的选择性甲酰化可以分别通过水杨酸和水杨酸甲酯用HMTA/CF3COOH来实现。 6．Lewis酸催化3，4，5-三甲氧基苄醇环化成1, 2, 3, 6, 7, 8, 11, 12, 13-nonamethoxyl-10,15-dihydro-5H-trbibenzo [a, d, g] cyclononene (NDTC)，产率(54%)高于已有方法（12%）。 Methyl 3,6,8-trihydroxy-7-methoxy-1-methylanthraquinone-2-carboxylate is a new COX-2 selective inhibitor isolated from Gladiolus gandavensis. Two strategies were investigated to synthesis this compound, in which some important reactions were discovered. 1. The key intermediate 5-hydroxy-3-methylbenzene-1,2,4-tricarboxylic acid 2,4-dimethyl ester was prepared via Diels-Alder reaction followed by selective hydrolysis of 1-COOMe. This compound was coupled with 1,2,3-trimethoxybenzene and the product undergo intramolecular Friedel-Crafts reaction to give methyl 3-hydroxy-5,6,7-trimethoxy-1-methylanthraquinone-2-carboxylate (1st route). The target compound and other analogues are being prepared with the same procedure. 2. The key intermediates methyl 3-hydroxymethyl-6-methoxy-2-methylbenzoate and its derivatives were prepared starting from crotonaldehyde and methyl acetoacetate via Michael addition, intramolecular aldol reaction, aromatization, formylation and reduction. The intermediates were coupled respectively with derivatives of gallic acid to give polysubstituted diphenylmethane. However, attempts to cyclize these compounds to the target compounds and analogues were not successful (2nd route). 3. In the process for ortho-lithiation of methyl 2-methoxybenzoate, the substrate converted respectively to n-butyl 2-methoxybenzoate and sec-butyl 2-methoxybenzoate when n-BuLi and sec-BuLi were used. However, tert-BuLi reacted with methyl 2-methoxybenzoate afford 2-methoxyphenyl tert-butyl ketone, which could be used to synthesize aryl tert-butyl ketones. 4. The transformtion of methyl 2-benzoxy-6-methylbenzoate to dimethyl 4,4'-dihydroxy-2,2'-dimethylbiphenyl-3,3'-dicarboxylate in benzene, and dimethyl 5,5'-dibenzyl-4,4'-dihydroxy-2,2'-dimethylbiphenyl-3,3'-dicarboxylate in 1,2-dichloroethane in the presence of ZnCl2 provides a new method for the synthesis of symmetric biphenyl. 5. The formylation of salicylic acid at C-5 and methyl 2-hydroxybenzoate at C-3 could be regioselectively realized by using HMTA/CF3COOH. 6. Racemic 1, 2, 3, 6, 7, 8, 11, 12, 13-nonamethoxyl-10, 15-dihydro-5H-trbibenzo [a, d, g] cyclononene was prepared via Lewis acids catalyzed trimerization of 3, 4, 5-trimethoxylbenzyl alcohol with yield (54%) higher than the reported procesure (12%).

五枫藤、小驳骨和川西茶藨的化学成分研究

八月瓜属植物五枫藤(Holboellia latifolia Wall.)和驳骨草属植物小驳骨(Gendarussa vulgaris Nees)均为药用植物, 前者化学成分研究不深入, 后者的化学成分未见报道。川西茶藨(Ribes takare D. Don)为茶藨子属植物, 没有化学成分的报道。本论文对三个植物的化学成分和活性成分进行了研究, 主要通过色谱方法分离得到了48 个化合物, 采用波谱分析或与已知标准品对照等手段鉴定了它们的结构, 其中有1 个新的原小檗碱类化合物和3 个新的联苯类化合物,发现了具有细胞毒活性和α-葡萄糖苷酶抑制活性的化合物。1、从五枫藤地上部分的95%乙醇提取物中分离得到了12 个化合物: 五加苷K (1)、hederagenin 3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2)、β-萘乙酸(3) 、3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4) 、3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5) 、3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid 28-O- β-D-glucopyranoside (6)、lup-20(29)-en-3-one (7)、lupeol (8)、β-谷甾醇(9)、齐墩果酸(10)、乌苏酸(11)、β-胡萝卜苷(12)。化合物1 对Lu-06、N-04 和Bre-04 癌细胞株的GI50 分别是0.77µg/mL、1.26 µg/mL 和1.55 µg/mL, 化合物2 对N-04 癌细胞株的GI50 为2.44 µg/mL。2、从小驳骨地上部分的95%乙醇提取物中分离得到了1 个原小檗碱类新化合物13-hydroxyl gusanlung A (25), β-谷甾醇(9)、齐敦果酸(10)、β-胡萝卜苷(12)、棕榈酸(1-)甘油酯(13)、棕榈酸(14)、阿苯哒唑(15)、阿苯哒唑砜(16)、阿苯哒唑亚砜(17)、aurantiamide acetate (18)、华良姜素(19)、芫花素(20)、(-)-丁香树酯醇(21)、gusanlung B (22) 、eupteleasaponinsⅤ acetate (23)、gusanlungA (24)、刺五加苷E (26)、岩白菜素(27)、咖啡酸(28)。化合物25 对肝癌细胞株(HepG2) 的GI50 为2.08 µg/mL。3、从川西茶藨地上部分的95%乙醇提取物中分离鉴定了22 个化合物: β-谷甾醇(9) 、β- 胡萝卜苷(12) 、O-acetyloleanolic aldehyde (29),4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30) 、3', 5-dimethoxy-3, 4-methylenedioxybiphenyl (31) 、桦木醇(32) 、6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33)、3'-hydroxy-5-methoxy-3,4-methylenedioxybiphenyl (34) 、7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35)、桦木醛(36)、没食子酸(37) 、6β- 羟基-4- 烯-3- 酮- 豆甾醇(38) 、5α, 8α-epidioxy-(22E,24R)-ergosta-6, 22-dien-3β-ol (39)、verrucofortine (40)、6-methoxycalpogoniumisoflavone A (41)、2-羟基二苯甲酮(42)、桦木酸(43), 3, 5-二甲氧基苯甲酸-4-O-β-D-吡喃葡萄糖苷(44)、洋芹素(45)、刺槐素(46)、水杨酸(47)、洋芹素-5-O- β-D-葡萄糖苷(48), 化合物30、31 和35 为新的联苯化合物。化合物30的α-葡萄糖苷酶抑制率为10.2% (1.00 mg/mL); 化合物35 的抑制率为17.2% (1.00mg/mL)。4、综述了1960 年以来原小檗碱类化合物药理活性研究进展。 Plants Holboellia latifolia Wall and Gendarussa vulgaris Nees, are used as folkmedicine. Ribes takare D. Don belongs to the genus Ribes. The three plants have notbeen chemically studied in detail. Chemical and bioactive study of three plants led tothe isolation of 48 compounds by chromatography. Their structures were elucidatedon the basis of spectroscopic evidence or comparison with authentic samples. Amongthe 48 componds isolated one protoberberine alkaloid and three biphenyls are newones. Cytotoxic and α-glucosidase inhibitory compounds had been found.1. Twelve compounds were isolated from the 95% ethanol extract of the aerial partof H. latifolia Wall. They were characterized as fellow: eleutheroside K (1),hederagenin-3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2),2-naphthyl acetic acid (3),3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4), 3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5),3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid-28-O- β-D-glucopyranoside (6),lup-20(29)-en-3-one (7), lupeol (8), β-sitosterol (9), oleanolic acid (10), ursolicacid (11), and β-daucosterol (12). Compound 1 showed moderate cytotoxicity againstLu-06 (GI50, 0.77 µg/mL), N-04 (GI50, 1.26 µg/mL) and Bre0-4 (GI50=1.55 µg/mL)and compound 2 showed moderate cytotoxicity against N-04 (GI50=2.44 µg/mL).2. A new protoberberine alkaloid, 13-hydroxyl gusanlung A (25), was isolated fromthe aerial part of Gendarussa vulgaris Nees, together with β-sitosterol (9), oleanolicacid (10), β-daucosterol (12), glycerol monopalmitate (13), palmific acid (14),albendazole (15), albendazole sulphone (16), albendazole sufloxide (17), aurantiamideacetate (18), kumatakenin (19), genkwanin (20), (-)-syringaresinol (21), gusanlung B(22), eupteleasaponinsⅤ acetate (23), gusanlung A (24), eleutheroside E (26),bergenin (27) and caffeic acid (28). Compound 25 showed cytotoxicity against HepG2 cells (GI50, 2.08 µg/mL).3. Phytochemical study of the Ribes takare D. Don led to the isolation of three newbiphenyls, 4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30), 3', 5-dimethoxy-3,4-methylenedioxybiphenyl (31) and 7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35), along with nineteenknown compounds, β-sitosterol (9), β-daucosterol (12), O-acetyloleanolic aldehyde(29), betulin (32), 6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33),3'-hydroxy-5-methoxy-3, 4-methylenedioxybiphenyl (34), betulinic aldehyde (36),gallic acid (37), stigmast-4-en-6β-ol-3-one (38), 5α, 8α-epidioxy-(22E, 24R)-ergosta-6,22-dien-3β-ol (39), verrucofortine (40), 6-methoxycalpogonium isoflavone A (41),2-hydroxybenzophenone (42), betulinic acid (43), 3,5-dimethoxygallic acid-4-O- β-D-glucopryranoside (44), apigenin (45), acacetin (46), salicylic acid (47) andapigenin-5-O- β-D-glucopryranoside (48). α-Glucosidase inhibitory rates ofcompound 30 and 35 were respectively 10.2% and 17.2% at a concentration of 1.00 mg/mL).4. Pharmacological activities of protoberberines were summarized.

手性有机小分子路易斯碱催化剂的设计、合成及其在亚胺不对称还原中的应用

手性胺是合成天然产物和手性药物的重要中间体，亚胺的不对称催化还原是制备光学活性手性胺的最直接有效的方法之一。但是，由于C＝N双键的反应活性较弱以及容易发生E/Z异构等问题，亚胺的不对称催化还原具有很大的挑战性，既具有高对映选择性又具有宽广底物普适性的催化剂很少。 本文分别由手性脯氨酸、哌啶酸、哌嗪酸以及氨基醇出发，设计和合成了一系列结构新颖、合成简便、性能优良的酰胺类有机小分子路易斯碱催化剂，以廉价的三氯氢硅为氢源，用这些催化剂催化亚胺不对称还原，得到了非常优良的收率、对映选择性和前所未有的底物普适性。 文献研究认为，除N-甲酰基外，分子内含有芳香酰胺是能催化亚胺还原的有机小分子路易斯碱催化剂具有较高对映选择性的必要条件，我们研究发现N-甲酰脯氨酸非芳香酰胺类催化剂（包括结构简单的C2-对称型脯氨酰胺类催化剂），对N-芳基酮亚胺的还原可获得达86%的对映选择性，远高于同类芳香酰胺催化剂，证明N-甲酰非芳香酰胺类路易斯碱催化剂在亚胺还原中也能得到高的对映选择性。 在进一步研究中，我们以手性六元哌啶酸为模板，分别设计合成了N-甲酰哌啶酸芳香酰胺和N-甲酰哌啶酸非芳香酰胺两类催化剂，其中芳香酰胺催化剂（S）-N-（甲酰基）哌啶-2-酸-1-萘基酰胺（28）和非芳香酰胺催化剂（2S,1'S,2'S）-N-（甲酰基）-哌啶-2-酸（1',2'-二苯基-2'-乙酰氧基-乙基）酰胺（30）显示出非常优良的催化活性和对映选择性，对于N-芳基芳香酮亚胺的还原，无论是缺电子体系还是富电子体系，绝大部分都能得到很高的收率（达98%）和对映选择性（达96% ee)。特别值得一提的是30对一些脂肪族亚胺和α,β-不饱和亚胺的还原，虽然底物为E/Z混合物，也能得到很高的收率（达93%）和对映选择性（达95% ee)，这样的底物普适性在过渡金属催化体系中也是前所未有的。 现有的催化亚胺还原的高对映选择性催化体系大多仅适用于甲基酮亚胺底物，对位阻较大的非甲基酮亚胺很难获得好的结果。我们以L-哌嗪酸为模板设计和合成出的（S）-N-（甲酰基）-哌嗪-2-酸-4-对叔丁基苯磺酰基-苯基酰胺不但对N-芳基甲基酮亚胺有很好的对映选择性（达90% ee)，而且对于大位阻的N-芳基非甲基酮亚胺有更好的对映选择性（达97% ee)。该催化剂与30在底物普适性方面具有很好的互补性。 我们还设计了基于1,2-二苯基氨基醇为模板的新型N-甲酰路易斯碱有机小分子催化剂，首次发现结构简单的N-甲酰（1S,2R）二苯基氨基醇能较好的催化N-芳基酮亚胺，最高可以得到82%的对映选择性。 针对我们设计合成的结构新颖、性能优良的催化剂，我们对催化机理进行了探讨和解释，提出了几个假想的机理模型。 Catalytic enantioselective reduction of imines represents one of the most straightforward and efficient methods for the preparation of chiral amines, an important intermediate for the synthesis of natural products and chiral drugs. However, asymmetric reduction of imines remains a big challenge and highly enantioselective catalysts with a satisfactorily broad substrate scope remain elusive. Factors contributing to the difficulty of this transformation include the weak reactivity of the C=N bond and the existence of inseparable mixtures of E/Z isomers. Starting from chiral proline, pipecolinic acid, piperazine-2-carboxylic acid and 1,2-diphenyl amino alcohol, a series of structurally simple and easily prepared amides were developed as highly effective Lewis basic organocatalysts for the asymmetric reduction of imines with trichlorosilane as the reducing agent, which promoted the reduction of N-aryl imines with high yields and excellent enantioselectivities with an unprecedented substrate spectrum. In the literature, it has been believed that besides the N-formyl group, the existence of an arylamido group in the structure of Lewis basic organocatalysts is a prerequisite for obtaining high enantioselectivity in the catalytic reduction of imines. However, we found that the N-formyl-L-prolinamides bearing non-arylamido groups, including structurally simple C2-symmetric tetraamides, could also work as effective Lewis basic catalysts to promote the asymmetric reduction of ketimines with high enantioselectivities (up to 86% ee), which are even more enantioselective than the analogues with arylamido groups. In further studies, we developed novel N-formamides with arylamido groups and non-arylmido groups as Lewis basic catalysts using the commercially available L-pipecolinic acid as the template. The catalysts (S)-1-formyl-piperidine-2-carboxylic acid naphthylamide 28 and (2S,1'S,2'S)-acetic acid 2-[(1-formyl-piperidine-2-carbonyl) -amino]-1,2-diphenyl-ethyl ester 30 were found to promote the reduction of a broad range of N-aryl imines in high yields (up to 98%) and excellent ee values (up to 96%) under mild conditions. Furthermore, catalyst 30 also exhibited high enantioselectivities (up to 95% ee) for the challenging aliphatic ketimines and α,β-unsaturated imines despite that these imines exist as E/Z isomeric mixtures. The broad substrate spectrum of this catalyst is unprecedented in catalytic asymmetric imine reduction, including transition-metal-catalyzed hydrogenation processes. Many of the currently available highly enantioselective catalytic systems only tolerate methyl ketimines, which gave poor results for bulkier non-methyl ketimines. Starting from L-piperazine-2-carboxylic acid, we developed (S)-4-(4-tert- butylbenzenesulfonyl)-1-formyl-N-phenyl-piperazine-2-carboxamide as highly enantioselective Lewis basic catalysts for the hydrosilylation of both methyl ketimines and steric bulky non-methyl ketimines. Moreover, higher enantioselectivities were obtained for non-methyl ketimines than methyl ketimines under the catalysis of this catalyst. Thus, this catalyst system complements with 30 in terms of the substrate scope. We also found that easily accessible (1R,2S)-N-formyl-1,2-diphenyl- 2-aminoethanol worked as an effective Lewis basic catalyst in the enantioselective hydrosilylation of ketimines, affording high enantioselectivities (up to 82% ee) for a broad range of ketimines. To rationalize the high efficiencies of the structurally novel catalysts we developed, several catalytic models have been proposed.

N-烷基氮杂糖及其衍生物的合成

多羟基哌啶类化合物通常称为氮杂糖，由于与糖结构的相似性，亚胺基环醇表现出强的糖苷酶和糖基转移酶抑制活性，可调控在生物识别及酶结构控制中起到重要作用的糖蛋白的生物合成与水解。因此这类抑制剂有望成为与糖代谢紊乱有关的疾病的治疗药物，如：抗糖尿病、抗肿瘤、抗溶酶体贮积症及抗病毒感染（包括艾滋病）等药物。正是由于氮杂糖的重要生物活性及诱人的药用开发前景，近年来，有关氮杂糖及其衍生物的合成、生物活性及应用研究备受关注。 本论文探索了一系列的作为潜在的迈克加成中间体1-C-乙酰甲基/甲氧羰基甲基-5-N-取代呋喃核糖碳苷衍生物在碱的作用下先发生β-消除反应，接着发生分子内的迈克加成反应生成1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物及1-C-甲氧羰基甲基-N-取代氮杂吡喃糖碳苷衍生物的方法，该转变过程为先通过β-消除得到非环状的α/β不饱和共轭酮或酯的中间体，接着5-N-取代氨基与分子内的α/β不饱和共轭酮或酯发生分子内的1,4-亲核加成，其中，2'-酯的环加成立体选择性的得到β型1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物，而2'-酮的环加成得到立体异构体1-C-乙酰甲基-N-取代氮杂吡喃糖碳苷衍生物。此外，该类N-取代氮杂吡喃糖碳苷衍生物进一步脱除保护基，得到了一系列新的N-取代氮杂吡喃糖衍生物，拓展了氮杂吡喃糖碳苷分子库。 中间体1-C-(2'-oxoalkyl)-5-N-alkylated glycoribofuranoside的合成是由核糖为原料，通过对其结构修饰，在C-5氮原子上先引入不同的取代基，在C-1上引入乙酰甲基或甲氧羰基甲基。C-5取代氨基的引入通过两种方法：(a) 5-取代链状脂肪氨基可由链状的伯胺直接与5-甲磺酰基发生SN2亲核取代得到；(b) 5-取代芳香氨基可通过芳香醛与C-5氨基缩合再由硼氢化钠还原得到。2'-酰基的引入通过烯丙基氧化得到：2'-酮羰基由醋酸汞和琼斯试剂氧化得到；2'-酯基由高锰酸钾氧化再碘甲烷的作用下得到。 The polyhydroxylated piperidines, commonly be called azasugars. Iminocyclitols and their derivatives have exhibited remarkable biological activity to inhibit glycosidase-processing enzymes, with resulting potential chemotherapeutic applications against diabetes, cancer, lysosomal storage disorders and viral infections including AIDS. Recently, because of the important biological activity and excellent foreground on pharmaceutical application, great attention has been attracted to the synthesis of the new derivatives and analogues. In this dissertation, 1-C-(2'-oxoalkyl)-5-N-substituted-glycoribofuranosides, which used as latent substrates for intramolecular hetero-Michael addition, were converted to 2-ester and 2-ketone aza-C-glycopyranosides by base treatment. The transformation was achieved through β-elimination to an acyclic α/β-conjugated ketone or ester, followed by an intramolecular hetero-Michael addition by the 5-N-alkylated amino group. The 2-ester cycloaddition was highly stereoselective in favor of an equatorial 1-C-substitution while the 2-ketone cycloaddition was produced a pair of stereoisomers of 2′-ketonyl aza-C-glycoside. Additionally, the resultant different N-alkylated aza-C-glycopyranosides could be further prepared for various azasugar library constructions by removal of protecting groups. Synthesis of the key intermediate 1-C-(2'-oxoalkyl)-5-N-alkylated glycoribo- furanoside involved the introduction of 5-substituted amino and 1-C-2′-oxoalkyl groups from D-ribose. The 5-alkylated amino was introduced through two methods: (a) the 5-aliphatic series amino synthesized by the nucleophilic substitution of 5-mesylate using neat ethylamine, propylamine, butylamine, and hexylamine, (b) the 5-aromatic series amino synthesized by various aromatic aldehydes with C-5 amino under NaBH4 reduction. The 1-C-2′-oxoalkyl groups were introduced through oxidation of the ally group: the 1-C-allyl group was oxidized with Hg(OAc)2 and Jones reagent to the 2-ketonyl C-glycoside; the 1-C-allyl group was oxidized with KMnO4 and CH3I/NaHCO3 to 1-C-methyl acetate glycoside.

猪场废水短程硝化反硝化-厌氧氨氧化脱氮研究

畜禽废水是农村水环境污染的主要来源之一，其处理的难点在于脱氮。传统生物脱氮法具有能耗高、需大量外加碳源等缺点，开发低成本、高效率的新型生物脱氮技术具有重要意义。 本研究将短程硝化反硝化和厌氧氨氧化两种脱氮新技术结合，让前者为后者创造去除可降解COD、降低总氮负荷、调整pH、调整氨氮和亚硝酸盐氮浓度比例等进水条件，而后者可在无需外加碳源的条件下进一步脱氮，二者结合可成为高氨氮、低C/N废水脱氮的新途径。 试验以低碳氮比猪场废水为研究对象，首先进行了短程硝化反硝化预处理研究，同时启动并运行调控厌氧氨氧化反应器，最后以经过短程硝化反硝化预处理的猪场废水为进水，进行厌氧氨氧化脱氮考察。实验表明：（1）短程硝化反硝化作为厌氧氨氧化的预处理工序是可行的。猪场废水通过短程硝化反硝化，可以达到基本去除可生化COD、部分脱氮、控制出水氨氮和亚硝酸盐氮浓度之比在1︰1左右、pH在7.5～8.0的目的， COD和总氮平均去除率分别为64.3%、49.1%,出水可达到厌氧氨氧化反应的进水要求。（2）采用模拟废水启动厌氧氨氧化反应器，经过5个月左右的运行调控，反应器启动成功并稳定运行，最高总氮去除率为87.1%，总氮容积去除率最高达到0.14kg/m3.d；整个稳定阶段，氨氮、亚硝酸盐氮、硝酸盐氮的变化量之比为1︰1.21︰0.33。（3）经过短程硝化反硝化预处理的猪场废水厌氧氨氧化脱氮效果稳定，氨氮、亚硝酸盐氮、总氮、COD的平均去除率分别为93.0%、99.4%、84.6%、18.1%，处理效果与模拟废水处理系统相比无明显变化。（4）经过短程硝化反硝化预处理后，猪场废水中残留有机物成分在厌氧氨氧化反应过程中无显著变化，主要为酯类和烷烃类物质；残留有机物对厌氧氨氧化效果无明显影响。（5）采用PCR技术进行特殊功能菌种检测，结果表明模拟废水处理系统和猪场废水处理系统的菌群中均含有厌氧氨氧化菌和好氧硝化菌；通过blast比对，厌氧氨氧化菌扩增序列与未培养的Planctomycetales菌和Candidatus Brocadia fulgida菌16S rRNA部分序列相似性分别为95％、90%。（6）MPN法菌种计数结果显示，模拟废水处理系统和猪场废水处理系统的菌群中均含有硝化细菌、亚硝化细菌和少量反硝化菌，实验条件下的微生物系统是一个厌氧氨氧化菌与好氧硝化菌、反硝化菌共存的系统。 Poultry wastewater is one of the main source of water pollution in rural areas，and nitrogen removal is the most difficult part in treating poultry wastewater. There are some disadvantages in traditional nitrogen removal, such as high energy consumption and more additional organic carbon. It is important to develop ecolomical and efficient technologyies. Shortcut nitricfication/denitrification, as a pretreatment process, was combined with Anammox in this research, so that part of total nitrogen and most degradable COD could be removed by the former, and further nitrogen removal could be implemented by the latter. The combination of the two technologies was a new approach to treat wastewater with high ammonium and low C/N. Piggery wastewater with low C/N was treated in lab-scale experiment. Firstly, shortcut nitrification/denitrification was investigated, and Anammox reactor was started up successfully at the same time. Then piggery wastewater after pretreatment was treated by Anammox. The results showed :(1) It was feasible to take nitrification/denitrification as the pretreatment process of Anammox. By using this process, part of total nitrogen and COD were removed, the ratio of ammonium and nitrite reached around 1︰1 and the pH was about 7.8, which were favorable for Anammox. The average removal percentage of COD and total nitrogen were about 64.3% and 49.1%, respectively. (2) Simulated wastewater was used to start up Anammox reactor. The reactor was started up successfully within 5 months and stable performance was achieved. The highest nitrogen removal reached 87.1% and the biggest volumetric total nitrogen removal rate reached 0.14kg/m3.d. The average ratio of ammonium, nitrite and nitrate was 1:1.21:0.33. (3)Taking the effluent of shortcut nitrification/denitrification as the influent, the nitrogen removal efficiency of Anammox was stable, and the the average removal percentage of ammonium, nitrite, total nitrogen and COD were 93.0%, 99.4% , 84.6% and 18.1%, respectively, which had little difference with that by using simulated wastewater..(4) After pretreatment, the residual organic carbon in piggery wastewater showed no obvious change during the Anammox process, and the main organic compounds were saturated hydrocarbon and ester, which had no obvious negative effect on Anammox process.(5) By PCR technology, the existence of Anammox bacteria was confirmed and the aerobic nitrifying bacteria was found to coexist as well. The result of blast showed that the identities of Anammox bacterium to part of 16S rRNA sequence of uncultured Planctomycetales bacterium and Candidatus Brocadia fulgida bacterium were 95% and 90%, respectively.（6）By MPN method, nitrite oxidizer, ammonium oxidizer and denitrification bacteria were detected in both simulated and piggery wastewater treatment system of Anammox, and the microorganism system was composed of Anammox bacteria，aerobic bacteria and denitrification bacteria together.

红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.