【作者】 秦艺旻；

【导师】 邹国林；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 卟啉不仅以其独特的生物活性存在于自然界,而且因其结构具有奇异的性能与癌细胞有特殊的亲合力,在医学研究中可作为检测癌症的光敏剂和抗癌药物。其中阳离子卟啉由于具有核酸的结合和切割特性,吸引了人们极大的兴趣。本文从合成和应用研究中经常使用的阳离子卟啉出发,包括5,10,15,20—四（1—甲基—4—吡啶基）卟啉甲苯磺酰盐（TMPyP）和5,10,15,20—四（1—乙氧羰基甲基—4—吡啶基）卟啉四氯盐（H₂TEOCMPyP）及其金属配合物,研究了亲水性阳离子卟啉与蛋白质和核酸的相互作用,探讨了它们在转运、肿瘤选择性及DNA损伤方面的相关特性。在此基础上,为提高其性能,改变了1—2个侧链基团,合成了3种新型阳离子卟啉化合物,进一步研究了它们的抗微生物和抗肿瘤活性。测试了几种新型卟啉对DNA的结合和损伤作用,并对比了它们与临床常用的癌症化疗药物阿霉素（ADM）对脂质体的过氧化损伤作用,从分子水平对卟啉的抗微生物和抗肿瘤机制进行了初步探讨。 本文的工作内容和结果总结如下: 结合荧光光谱和吸收光谱,研究了卟啉与人血清白蛋白（HSA）的相互作用,确定了反应的结合常数和授体-受体间的结合距离;采用光学生物传感器法获得卟啉与固定化HSA相互作用的动力学与热力学常数,比较了25℃时该方法与荧光光谱法得到的结合常数,发现两种方法具有可比性。卟啉主要通过静电相互作用与HSA结合,结合常数在10⁵L/mol左右,金属和侧链的疏水性对卟啉与HSA的亲合力有影响,疏水作用力仍在一定程度上发挥作用。卟啉结合在HSA疏水腔外部,与疏水腔保持一定的距离。 应用IAsys光学生物传感器研究了两种水溶性阳离子卟啉与固定化的麦胚凝集素（WGA）的相互作用。20℃时自由卟啉和相应的锌卟啉的结合常数分别为更多还原

【Abstract】 Porphyrin derivatives have been used as photosensitizers in photodynamic therapy, a new approach developed for the treatment of cancer and bacterial infections. Cationic porphyrins are of considerable interest owing to their possible biomedical applications and the capability of nucleic acid binding and cleavage. In this paper, we studied the interaction between biomacromolecules with some water-soluble porphyrins and their metal complex. These porphyrin derivatives have been widely used in synthesis and application. We concluded their properties of transport, tumor selectivity and DNA damage. Depend on thesis, hydrophobic and bioactive substituents were introduced to enhance the antimicrocial activity and antitumor abilities. The novel cationic porphyrins were tested for their capability of DNA binding and cleavage. The lipid peroxidation induced by these porphyrins was compared with that induced by adriamycin. The mechanism of porphyrin bioactivities was further discussed.The studies in this paper are concluded as below:The binding of three water-soluble cationic porphyrins to human serum albumin (HSA) was investigated by absorption spectrum and fluorescence spectroscopy. Meanwhile, an optical biosensor with a stirred cuvette has been used to monitor theinteraction between immobilized HSA and these porphyrins. The binding constants at 25 ℃ obtained from biosensor analysis were compared with those from fluorescence spectroscopy. The interactions were further investigated at temperatures between 15 and 30 ℃. The thermodynamics parameters, changes of free energy (AG), enthalpy (AH) and entropy (AS), were evaluated from equilibrium data. Although the interaction was governed primarily by electrostatic forces, the differences in porphyrin structure changed their binding modes to some extent. The metal core and the hydrophobicity of side chains affected the affinity to HSA. Porphyrins studied here could bind outside hydrophobic loop with definite distance. In addition, the optical biosensor was proved well suited for investigating the interaction between such porphyrins and HSA.An optical biosensor with a stirred cuvette has also been used to monitor the interaction of immobilized wheat germ agglutinin (WGA) with two water-soluble cationic porphyrins. The association constants (Ka) of the free base porphyrin and its Zn( II) form were 2.66 and 27.31 X 105 M~-1 at 20 ℃ respectively. The interactions of the free base porphyrin were further investigated at temperatures between 15 and 37 ℃. The thermodynamics parameters, changes in free energy, enthalpy, entropy and heat capacity, were evaluated from equilibrium data. The binding was driven by entropic contribution, and showed strong enthalpy-entropy compensation. It was governed primarily by hydrophobic forces. Besides, the free base porphyrin could bind to the same or vicinal sites for iV-acetyl-D-glucosamine on WGA, but the metal porphyrin might not.The binding modes of several cationic porphyrin derivatives to calf thymus DNA and polynucleotide were analysed by absorption spectrum. Free porphyrins intercalated into base pairs of DNA and metal porphyrins tended to external bind to DNA groove. An optical biosensor was used to study the interaction of immobilized synthesized DNA segment and Zn(II) porphyrin. The kinetic and thermodynamic data showed that the binding process was mainly drived by coulombic force anddesolvation. Agarose gel electrophoresis was used to check the cleavage of plasmid DNA mediated by porphyrin derivatives. The results indicated that the cleavage ability to DNA was irrelative to the binding mode, and mostly attributed to the hydroxyl free radical induced by porphyrins.The novel /weso-substituted cationic porphyrins were covalent bonded with methoxycarbonylphenyl and 1-phenylpiperazine. The antimicrobial and antitumor activities of these porphyrins were largely enhanced. The bindings of these porphyrins to calf thymus DNA were studied by combining absorption spectrum, fluorescence and circular dichroism methods. Introduction of phenylpiperazines at /weso-position has apparently affected the mode of the binding. The capabilities of lipid peroxidation and DNA cleavage showed the same tendence as their antimicrobial and antitumor activities, which suggested that porphyrin derivatives could cause damage to the membrane structures and DNA and interfere with the cell growth.The lipid peroxidation induced by ADM, Fe3+ and ferritin was investigated by absorption spectrum. The results indicated that the lipid peroxidation mediated by ADM’Fe(III) was more effective than ADM. Iron played a key role in the process. Meanwhile, ADM could stimulate Fe-dependent lipid peroxidation. The inhibition of superoxide dismutase and some -OH scavenges indicated the participation of -OH. Deferoxamine could chelate iron and largely inhibit the lipid peroxidation, which further proved the importance of iron. Moreover, ADM could promote horse spleen ferritin to release iron and lead to a stronger peroxidation effect, which implied a possible way for ADM cardiotoxicity in vivo.更多还原