【作者】 刘绪峰；

【导师】 官文超；

【作者基本信息】 华中科技大学 ， 材料物理与化学， 2007， 博士

【摘要】 利用[C60]富勒烯的各种衍生化方法,将水溶性基团或某些生物活性的组份引入到C₆₀上,合成生物相容的C₆₀衍生物,研究它们的生物活性,已经成为[C60]富勒烯化学及富勒烯生物学应用的一个重要课题。本文利用C₆₀的胺化反应,C₆₀与亚氨基二乙酸酯类的光化学反应,C60 Bingel环加成反应以及C₆₀与叠氮化物的环加成反应,合成和制备了几种未见文献报道的生物相容性的C₆₀衍生物。通过对它们抗氧化、自由基清除及细胞保护等活性的研究,为寻找具有优良抗氧化活性的C₆₀衍生物提供基础性的数据。同时,通过对这些生物学性质的影响因素及构效规律的探讨和分析,为生物相容性C₆₀衍生物分子的设计和合成提供基本的信息。主要内容包括: 1.C₆₀与氨基酸类物质在甲苯/乙醇混和溶剂中的亲核加成反应制得水溶性的C₆₀氨基酸衍生物1_a, 1_b, 1_c和1_d,它们的加成度分别为:n=5（1_a）, 5（1_b）, 4（1_c）和3（1_d）。C₆₀氨化反应的加成度与氨基酸碳链的空间阻碍及加成产物在反应介质中的溶解性等因素有关。氧自由基的清除实验、体外培养小鼠胸腺细胞实验、及红细胞氧化性溶血实验表明:1_a, 1_b, 1_c和1_d都具有抗氧化及清除自由基的活性,而且剂量效应明显。上述实验也表明:C60氨基酸衍生物（1_a和1_b; 1_a, 1_c和1_d）抗氧化活性的大小都有如下规律: 1_a > 1_b ;1_a > 1_c > 1_d表明,C₆₀氨基酸加成物加成基团的空间位阻及加成度增加所导致的烯键电荷密度增大是影响其抗氧化及自由基清除活性的内在因素。2.C₆₀吡咯烷羧酸衍生物2_b和3_b经由C₆₀与亚氨基二乙酸甲酯(NH（CH₂COOMe）₂的光化学反应,N-烃基化反应及水解和酸化等步聚制得,产率分别为65%和53% （基于C60吡咯烷衍生物2_a和3_a）。2_b和3_b在0³×10^-4 mol/L浓度范围内,能明显清除化学发光体系中产生的超氧阴离子O₂^-·和羟基·OH等自由基,它们的清除能力并无明显差异。相同的C₆₀母体结构—单加成[6,6]-闭环结构决定了它们相似的自由基清除能力。C₆₀吡咯烷二羧酸2_b和三羧酸3_b在极性介质中容易聚集。用电导法测定了2b和3b钠盐的临界聚集浓度（CAC）,分别为3.58×10^-4 mol/L （2_b）和3.33×10^-4 mol/L （3_b）。透射电子显微镜（TEM）、静态光散射（SLS）、UV-vis等方法进一步证实了2_b和3_b的这一聚集行为。2_b和3_b CAC的差异、聚集体粒径大小的不同,表明C60单加成衍生物加成基团中羧基（COOH）数目的多少对其聚集行为的影响。氧自由基的清除实验及红细胞氧化性溶血实验表明:2_b和3_b聚集行为的发生导致了它们抗氧化及自由基清除能力下降。3.合成了C₆₀基Ebselen衍生物7 （产率53%）和10（产率42%,以消耗的[60]富勒烯计）。MTT法及LDH法测试表明,7和10及相应的C₆₀、和Ebselen衍生物均能抑制H₂O₂诱导的大鼠大脑皮层神经细胞的损伤,而且7和10和保护作用要强于相应的C₆₀、及Ebselen衍生物。NADPH-RD酶偶联分析法的测试表明,7和10的GPx活力也均高于相应的C₆₀、及Ebselen衍生物。C₆₀基Ebselen衍生物比相应的C₆₀、及Ebselen衍生物表现出更强的抗氧化与神经保护活性,这一活性的增强应归因于它的自由基清除能力的提高及GPX活力的增强。因此,通过选择合适的抗氧化组份并将它们引入到C₆₀是获得具有更强的抗氧化性的C₆₀基衍生物的有效方法。更多还原

【Abstract】 Making use of various functionalizations of [60]fullerene, introducing solubilising groups or potentially biologically effective components to [60]fullerene to synthesize biologically compatible C₆₀ derivatives, and investigating their biological activites have recently become a topic of importance in the fields of [60]fullerene chemistry and its biological applications. In this paper, amination reaction, photochemical reaction, and （Bingel）cycloaddition of C₆₀, respectively, with amino-acids, iminodiacetic alkyl esters, azides, and malonates were carried out to prepare a few biologically compatible C₆₀ derivatives. Their antioxidative and radical scavenging properties, and their protective effects on culturing cells were studied. The studies may provide basic data for finding better antioxidative C₆₀ derivatives. Meanwhile, we also discussed and analyzed influencing factors of these biological properties and structure-activity relationships in order to obtain basic information for designing and synthesizing biologically compatible C60 derivatives.This paper has the following main aspects:1. Nucleophilic addition of C₆₀ with amino-acids in toluene/ethanol gave the corresponding C₆₀ amino-acid adducts （1_a, 1_b, 1_c and 1_d）, their addition number （n） determined by elemental analysis: n=5 （1_a）, 5 （1_b）, 4 （1_c） and 3 （1_d）. Addition number （n） of amination reaction of C₆₀ was influenced by steric hindering of hydrocarbon chains of amino-acids and solubility of C₆₀ adducts in reaction media.The experimentations of eliminating active oxygen radicals in chemical systems, mouse thymus cell cultures, and H2O2-induced erythrocytes oxidative hemolysis demonstrated that C₆₀ amino-acid adducts （1_a, 1_b, 1_c and 1_d ） possess antioxidative and radical scavenging properties. With the increase of concentrations, their antioxidative and radical scavenging abilities increased.These experimentations also showed that antioxidative potencies of the C₆₀ amino-acid adducts were with the same sequence: 1_a > 1_b ; 1_a > 1_c > 1_dThe consistencies in the sequence reveal that steric hindering of addition groups of C60 amino-acid adducts and enhanced electron densities of the conjugated bonds of C₆₀ core are intrinsic factors of influencing their antioxidative and radical scavenging abilities.2. The synthesis of fulleropyrrolidine carboxylic acid 2_b and 3_b were conveniently achieved in a 65% and 53%yield （relative to 2_a and 3_a）, by a multiple-step process: photochemical reaction of C₆₀ with iminodiacetic methyl ester (NH（CH₂COOMe）₂, further N-Alkylation, hydrolysis and acidification.2_b and 3_b, in the concentration range of 0 ³×10^-4 mol/L, have obvious scavenging activities against superoxide(O₂^-·)and hydroxyl （·OH） radicals generated in chemiluminescence systems. However, 2_b and 3_b were found to have no obvious differences in radical scavenging efficiency. The similar radical scavenging potencies can reasonably be ascribed to their identical [6,6]-closed structure of parent C₆₀.Fulleropyrrolidine carboxylic acid 2_b and 3_b easily form aggregate in polar media. Critical aggregation concentrations （CAC） of their sodium salts were determined by conductivity measurements, which were 3.58×10^-4 mol/L （2_b） and 3.33×10^-4 mol/L （3_b）. Their aggregation behaviors were further proved using UV-Vis absorption, static light scattering （SLS） and transmission electron microscopic （TEM） techniques. CAC and aggregate sizes of 2_b and 3_b were different from each other, indicating that the aggregate is effected by the number of carboxyl groups connected to fulleropyrrolidine mono-adduct derivatives.The experimentations of eliminating active oxygen radicals in chemical systems, and H₂O₂-induced erythrocytes oxidative hemolysis demonstrated that aggregation behavior of 2_b or 3_b resulted in the decline of their antioxidative and radical scavenging potencies. 3. C₆₀-based ebselen derivatives 7 （53%） and 10 (42%, based on consumed C₆₀ ) were synthesized.MTT assay and LDH leakage assay showed that 7 and 10 and their corresponding C₆₀, ebselen derivatives can inhibit H₂O₂-induced neuronal injury, and C₆₀-based ebselen derivatives （7, 10） possess stronger nuroprotective potencies than parent C₆₀, or ebselen derivatives alone. NADPH-RD coupled enzymatic assay also showed that the GPX activities of 7, or 10 were higher than those of parent C₆₀, or ebselen derivatives.The C₆₀-based ebselen derivatives showed stronger nuroprotective potency than parent C₆₀, or ebselen derivatives alone. This enhanced nuroprotective potencies can reasonably be attributed to both improvement of its free radical scavenging capabilities and improvement of GPX-like activities. Therefore, selecting appropriate antioxidant components and introducing them to [60]fullerenes, should be an effective methods of obtaining more biologically effective C₆₀-based antioxidants.更多还原