Synthesis and characterization of acetacetic derivatives of flullerene

Acetacetic derivatives of fullerene were obtained by the reaction of C-60 with acetacetic ester, after separation with silica gel column chromatography, in good yields. Fullerenols with 13-15 hydroxyl groups were-isolated as the by-products of this reaction. Detailed experimental conditions and the structural characterizations of these new compounds were discussed.

A New Cadinane Sesquiterpene from the Marine Brown Alga Dictyopteris divaricata

A sample of the marine brown alga D. divaricata collected off the coast of Yantai (P. R. China) was dried, powdered, and extracted with the mixture of CHCl3 and MeOH (1: 1, v/v). By a combination of silica gel and Sephadex LH-20 column chromatography and preparative TLC, a new cadinane sesquiterpene 1,4-epoxymuurolan-5 beta-ol (1) was isolated from this species. Its structure was established by detailed MS and NMR spectroscopic analysis, as well as comparison with literature data.

海带根中α-glucosidase 和PTP-1B 靶向生物活性物质的分离纯化及其抗糖尿病作用研究

海带根是一种治疗糖尿病的民间中药，在沿海地区有很长的民间用药历史。食用海带根能够有效降低糖尿病患者的血糖，起到治疗作用。本文目的在于发现海带根中抗糖尿病的天然活性物质并分析它们在糖尿病治疗中的靶点；进一步开发一种低价且无毒副作用的化学类新药或中药新药。 α-glucosidase和 PTP-1B是II型糖尿病的两个重要靶点，海带根提取物能同时作用于这两个靶点。通过抑制这两种酶，降低血糖水平，85%乙醇粗提物对两种酶的IC50分别为1589ug/ml、IC50 1271ug/ml。乙酸乙酯相和石油醚相分别抑制α-glucosidase和 PTP-1B，IC50分别为380ug/ml和220ug/ml。因此以α-glucosidase和 PTP-1B的抑制活性为导向，用天然产物化学的方法对活性成分进行追踪分离，寻找单体活性物质进而鉴定其结构。由于乙酸乙酯相具有α-glucosidase抑制活性，用硅胶柱层析（石油醚：丙酮5：1、1：1），（二氯甲烷：甲醇60：1、20：1、5：1），凝胶柱层析Sephadex LH20（二氯甲烷：甲醇1：1），HPLC （80% 甲醇-水），对α-glucosidase抑制剂进行分离，得到组分IC50 为3.6ug/ml。用质谱仪和核磁共振确定结构。 生物活性测定结果表明α-glucosidase和 PTP-1B是两种不同的物质，分别位于乙酸乙酯相和石油醚相。光照实验和高温实验表明抑制α-glucosidase的活性成分对光照和温度敏感。光照48h或者50℃ 12h而且对α-glucosidase的抑制活性显著降低，TLC检测并用FeCl3显色初步表明抑制α-glucosidase的活性成分可能是多数酚类物质。动物实验显示在1450ug/kg剂量下，乙酸乙酯相能够显著降低糖尿病小鼠血糖，与阴性对照组差异极显著（P<0.01）。表明，海带根提取物在体内和体外均呈现出抗糖尿病活性，是一种潜在的抗糖尿病药物。

海洋红藻多管藻和松节藻的化学成分及抗氧化活性研究

海洋生物具有产生丰富多样的次生代谢产物的能力，其中红藻门松节藻科海藻卤代次生代谢产物以其结构新颖、生物活性独特引起了天然产物化学家的重视。 本论文对海洋红藻多管藻和松节藻进行了化学成分研究，综合利用各种色谱学方法 (硅胶柱层析、反相硅胶柱层析、凝胶Sephadex LH-20柱层析、半制备高效液相色谱以及重结晶等) 和现代波谱学技术 (IR、UV、EI-MS、FAB-MS、HR-ESI-MS、CD、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)，共分离鉴定了100个化合物，发现25个新化合物。 从多管藻中分离鉴定38个化合物 (24个溴酚化合物)，其中7个新化合物 (均为溴酚化合物)，包括1个菲并呋喃结构溴酚 (P1), 2个二氢菲结构溴酚 (P2, P3)，1个含 5,7-dihydrodibenzo[c,e]oxepine 结构溴酚 (P4)和3个简单溴酚 (P5, P6, P7)。P1 (urceolatin) 属首例报道的具有菲并呋喃结构的天然产物，从该种中分离的化合物P12 和 P13 可能是其生源合成的前体。P2和P3为第二例报道的具有二氢菲结构的溴酚化合物。 从松节藻中分离并鉴定了62 个化合物，其中18 个为溴酚类新化合物，44 个为已知化合物。化合物具有多变的取代基团，包括2 个脲基吡咯烷酮溴酚化合物 (R1, R2), 4 个γ-脲基丁酸溴酚化合物 (R3-R6)，5 个酰胺溴酚化合物 (R7, R8, R9, R13, R14)，1 个溴酚砜化合物 (R12), 1 个Xanthene 溴酚化合物 (R10)和5 个简单溴酚化合物 (R11, R15, R16, R17, R18)。R1、R2 是首例报道的含有脲基吡咯烷酮片段的天然产物，R10 为首次报道的溴代Xanthene 类天然产物。 对分离到的化合物进行了清除DPPH 和ABTS两种自由基活性的筛选。结果发现溴酚类天然产物具有显著的DPPH自由基清除活性，其中R3 的IC50 仅为3.3 μM, 其活性强度约为阳性对照BHT (IC50 为82.1 μM) 的24倍。另外，溴酚类天然产物对ABTS自由基有较强的清除活性，R2 的TEAC（Trolox efficency activity capacity）值为5.2 mM，约为阳性对照 (ascorbic acid, 1.02 mM) 的 5 倍。初步的构效关系研究发现，稠环分子、多羟基和邻位甲氧基等结构特点能有效增强DPPH 自由基清除活性；特殊取代基如脲基、吡咯烷酮等含有氮原子的基团，能有效增强ABTS 自由基清除活性，多羟基、溴代等结构特点也使其活性有所增强。 本研究结果丰富了海藻卤代化合物的结构类型，为多管藻和松节藻的合理利用提供了一定的科学依据。

两株海藻内生真菌次生代谢产物及其生物活性研究

海洋微生物拥有丰富多样的次生代谢途径，其中海洋生物内生真菌次生代谢产物研究日益受到天然产物化学界的重视。本论文以菌丝体生物量、发酵产物重量、抗菌与细胞毒活性、薄层色谱分析结果以及高效液相色谱分析结果等为评价依据对采自青岛沿海的13株海藻内生真菌在四种液体培养基上的静置发酵产物进行了综合评价，并从中选择了黑曲霉Aspergillus niger EN-13（分离自褐藻囊藻Colpomenia sinuosa）和杂色曲霉A. versicolor EN-7（分离自褐藻鼠尾藻Sargassum thunbergii）两株真菌进行了30升规模发酵（分别采用GPYM培养基和PDB培养）和化学成分的研究，对分离得到的大部分化合物进行了初步的生物活性筛选。 发酵提取物采用常规的硅胶柱层析、反相硅胶柱层析，凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱以及重结晶等分离手段，得到单体化合物。利用各种现代波谱技术(IR、UV、EI-MS、FAB-MS、HR-ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)并结合化学方法从两种菌株发酵提取物中鉴定了55个化合物的结构。其中从菌株A. niger EN-13分离鉴定了31个化合物，发现9个新化合物，包括2个鞘酯类化合物（AN-1~2）、3个萘并-γ-吡喃酮类化合物（AN-3~5）、3个苯乙基取代的α-吡喃酮类化合物（AN-17, AN-19~20）和1个甾体Diels-Alder加成产物（AN-21），另有1个新的天然环二肽（AN-27）被分离鉴定；从菌株A. versicolor EN-7分离鉴定了24个化合物，发现2个新化合物，为蒽醌AV-12与AV-17，另外，从前一菌株（A. niger EN-13）中鉴定的2个新鞘酯类化合物（AN-1~2）在A. versicolor EN-7中也被再次分离到。 对大部分单体化合物进行了抗菌活性、DPPH自由基清除活性和细胞毒活性测试。结果显示新化合物AN-1、AN-5和AN-20具有弱或中等强度的抑制白色念珠菌生长的活性，AN-4、AN-5、AN-21显示了弱或中等强度的抑制黑曲霉生长的活性，AV-12、AV-17显示了弱的抑制大肠杆菌生长的活性。在DPPH自由基清除活性筛选中，AN-5显示了中等强度的活性，其EC50为109.3 mM，与阳性对照BHT相近（EC50为81.8 mM）。其它部分已知化合物在抗菌和DPPH自由基清除活性的筛选中也显示了弱或中等强度的活性。在针对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549的体外细胞毒活性筛选中，所测样品均未显示显著活性。

Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis

Three sulfated polysaccharide fractions (F1, F2, and F3) were isolated from Porphyra haitanesis, an important economic alga in China, through anion-exchange column chromatography and their in vitro antioxidant activities were investigated in this study. Galactose was the main sugar unit of the three fractions. The analytical results indicated that polysaccharide fractions from P. haitanesis had similar chemical components to porphyran from other species, but differed in their high sulfate content. The sulfate content of F1, F2 and F3 was 17.4%, 20.5% and 33.5% respectively. All three polysaccharide fractions showed antioxidant activities. They had strong scavenging effect on superoxide radical, and much weaker effect on hydroxyl free radical. Lipid peroxide in rat liver microsome was significantly inhibited, and H2O2 induced hemolysis of rat erythrocyte was partly inhibited by F1, F2 and F3. Among them, F3 showed strongest scavenging effect on superoxide radical; F2 had strongest effect on hydroxyl radical and lipid peroxide.

Steroids from green alga Chaetomorpha basiretorsa Setchell

Six steroids have been isolated from ethanolic extract of green alga Chaetomorpha basiretorsa Setchell by a combination of repeated normal phase silica gel and Sephadex LH-20 gel column chromatography as well as recrystallization. Using spectroscopic methods including MS and NMR, their structures were determined as beta-lawsaritol (1), saringosterol (2), 24-hydroperoxy-24-vinyl-cholesterol (3), beta-stigmasterol (4), stigmast-4-en-3 alpha, 6 beta-diol (5), 29-hydroxystigmasta-5, 24 (28)-dien-3 beta-ol (6). All these compounds were obtained from this genus for the first time and they were inactive (IC50 > 10 mu g /ml) against KB, Bel-7402, PC-3M, Ketr 3 and MCF-7 cell lines.

Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica

Fucoidan, a group of sulfated heteropolysaccharide, was extracted from Laminaria japonica, an important economic alga species in China. Three sulfated polysaccharide fractions (F1, F2, and F3) were successfully isolated through anion-exchange column chromatography and had their antioxidant activities investigated employing various established in vitro systems, including superoxide and hydroxyl radical scavenging activity, chelating ability, and reducing power. Chemical analysis suggested that F1 and F3 were heteropolysaccharide in which galactose was the major component, while F2 was a typical fucoidan. All fractions possessed considerable antioxidant activity, and F1, F2 and F3 had stronger antioxidant ability than fucoidan in certain tests. The correlation between the sulfate content and scavenging superoxide radical ability was positive. Available data obtained with in vitro models suggested that the ratio of sulfate content/fucose was an effective indicator to antioxidant activity of the samples. (C) 2007 Elsevier B.V. All rights reserved.

The chemical constituents from red alga Gymnogongrus flabelliformis Harv.

Eight compounds were isolated from red alga Gymnogongrus flabelliformis Harv. In normal phase silica gel, Sephadex LH-20 gel column chromatography, reverse phase HPLC, and recrystallization. Based on MS and 1D NMR spectroscopic data, their structures were determined as: stigmast-4-en-3-one (I), cholest-4-en-3-one (II), cholesterol (III), uracil (IV), uridine (V), adenosine (VI), succinic acid (VII), and 5-hydroxy-4-methyl-5-pentyl-2,5-dihydro-furan-2-on (VIII). All of them were obtained from this species for the first time. Cytotoxicity of these compounds was screened using standard MTT method, but all the compounds were inactive (IC50 > 10 mu g/ml).

Structural studies on a novel fucogalactan sulfate extracted from the brown seaweed Laminaria japonica

A low molecular weight fucogalactan, obtained from the brown seaweed Laminaria japonica, was separated into three fractions (LF1, LF2 and LF3) by DEAE-Sepharose FF column chromatography. All three fractions contained predominantly fucose, sulfate group and galactose. The results showed that the main fraction LF2 consisted of L-fucose, D-galactose and sulfate at a molar ratio 6:1:9. Structural study on the LF2 was carried out by NMR spectroscopy. The backbone of LF2 was primarily (1 -> 3)-linked alpha-L-fucopyranose residues (75%) and a few (1 -> 4)-alpha-L-fucopyranose linkages (25%). The branch points were at C-4 of 3-linked alpha-L-fucopyranose residues by beta-D-galactopyranose unites (35%, molar ratio) or at C-2 of 3-linked alpha-L-fucopyranose residues by non-reducing terminal fucose unites (65%, molar ratio). Sulfate groups occupied at position C-4 or C-2, sometimes C-2, 4 to fucose residues, and C-3 and/or C-4 to galactose residues. The structure of LF2 was supposed as following: [GRAPHICS] (C) 2010 Elsevier B.V. All rights reserved.

Microcoulometric Determination Of Total Organo-Chlorinepesticide And Polychlorinated Biphenyl Residues In Grey Seal (Halichoerus-Grypus) Blubber

A procedure for estimating total organochlorine pesticide and PCB residue in seal blubber at concentrations of greater than 1μg g-1 of lipid is described. Lipid is cleaned up by alumina column chromatography, and the halogen concentration of the resulting hexane eluace is determined by combustion and microcoulometry. Results are similar to those obtained by gas chromatographic analysis and can be used to interpolate between results so obtained when data on specific organochlorine compounds is not required for each sample. The organochlorine residues recovered in this manner did not constitute all the halogen determined by combustion and microcoulometry of seal lipid. Analysis by the total halogen procedure was 2.5 tunes faster than the rate achieved with a combination of liquid and gas chromatography operated manually; the requirements for laboratory equipment and space for sample preparation are reduced.

The isolation and purification of tris-2,2'-bipyridine complexes of ruthenium(II) containing unsymmetrical ligands

Monomeric ruthenium(II) complexes [Ru(L)3]2+ containing unsymmetric bipyridine ligands [Where L = 5-methyl-2,2'-bipyridine (L1), 5-ethyl-2,2'-bipyridine (L2), 5-propyl-2,2'-bipyridine (L3), 5-(2-methylpropyl)-2,2'-bipyridine (L4), 5-(2,2-dimethylpropyl)-2,2'-bipyridine (L5) and 5-(carbomethoxy)-2,2'-bipyridine (L6)] have been studied and the meridional and facial isomers isolated by the use of cation-exchange column chromatography (SP Sephadex C-25) eluting with either sodium toluene-4-sulfonate or sodium hexanoate. The relative yield of the facial isomer was found to decrease with increasing steric bulk, preventing the isolation of fac-[Ru(L5)3]2+. The two isomeric forms were characterized by 1H NMR, with the complexes [Ru(L1-3)3]2+ demonstrating an unusually large coupling between the H6 and H4 protons. Crystals suitable for X-ray structural analysis of [Ru(L1)3]2+ were obtained as a mixture of the meridional and facial isomers, indicating that separation of this isomeric mixture could not be achieved by fractional crystallisation. The optical isomers of the complex [Ru(L3)3]2+ were chromatographically separated on SP Sephadex C-25 relying upon the inherent chirality of the support. It is apparent that chiral interactions can inhibit geometric isomer separation using this technique.

Rational design of a dual-mode optical and chemical prodrug

Purpose. The purpose of this study is to demonstrate the rational design and behaviour of the first dual mode optical and chemical prodrug, exemplified by an acetyl salicylic acid-based system. Methods. A cyclic 1,4-benzodioxinone prodrug was synthesised by reaction of 3,5-dimethoxybenzoin and acetyl salicoyl chloride with pyridine. After purification by column chromatography and recrystallization, characterization was achieved using infrared and NMR spectroscopies, mass spectrometry, elemental analysis and single crystal X-ray diffraction. Light-triggered drug liberation was characterised via UV-visible spectroscopy following low-power 365 nm irradiation for controlled times. Chemical drug liberation was characterised via UV-visible spectroscopy in pH 5.5 solution. Results. The synthetic method yielded pure prodrug, with full supporting characterisation. Light-triggered drug liberation proceeded at a rate of 8.30 10j2 sj1, while chemical, hydrolytic liberation proceeded independently at 1.89 10j3 sj1. The photochemical and hydrolytic reactions were both quantitative. Conclusions. This study demonstrates the first rational dual-mode optical and chemical prodrug, using acetyl salicylic acid as a model, acting as a paradigm for future dual-mode systems. Photochemical drug liberation proceeds 44 times faster than chemical liberation, suggesting potential use in drug-eluting medical devices where an additional burst of drug is required at the onset of infection.

Solventless synthesis of acyl phosphonamidates, precursors to masked bisphosphonates

A series of acyl phosphonamidates, the synthetic precursors to bisphosphonates, have been readily prepared from phosphoramidite type reagents and a range of acid chlorides. These reactions were performed using solventless conditions, where purification was easily achieved using column chromatography with yields ranging from 71-90%. Furthermore, we have demonstrated that these acyl phosphonamidates could be used for the preparation of unsymmetrical bisphosphonates, which do date are scarcely reported in the literature.

Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.