【作者】 王雪丽；

【导师】 李正强；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 以meso-位具有不同侧链的卟啉化合物四-[3-甲氧基-4-(N-咔唑)正丁氧苯基]卟啉(4C4-TPP)和四-[3-甲氧基-4-(N-咔唑)正己氧苯基]卟啉(4C6-TPP)为研究对象,利用吸收光谱、荧光光谱、圆二色谱和拉曼光谱探讨卟啉侧链基团在聚集体形成、聚集体结构及其与DNA结合中所起的作用。在一定浓度范围内,4C4-TPP和4C6-TPP在有机溶剂四氢呋喃中以单体分子存在,溶于水-四氢呋喃混合溶剂中卟啉分子趋于聚集。特定水-四氢呋喃(tetrahydrofuran,THF)体积比中4C6-TPP以典型的自组装动力学而聚集。4C6-TPP,THF和水三者相互作用形成笼状结构,4C6-TPP分子处于笼中,侧链苯基团与中心环的共轭程度增加,聚集体整体结构对称,圆二色信号消失。增加水-四氢呋喃混合溶剂中的离子强度诱导4C6-TPP聚集。降低pH值,导致4C6-TPP质子化进而聚集。以四苯基卟啉和咔唑基团为模型化合物深入研究质子化诱导4C6-TPP聚集的机理,并提出质子化4C6-TPP聚集体的结构模型:借助于外加酸根阴离子,卟啉中心环与侧链之间以非共价键作用,分子间规则排列形成J-聚集体。4C4-TPP和4C6-TPP侧链插入小牛胸腺DNA(calf thymus DNA,ctDNA),4C4-TPP与ctDNA的作用不涉及复杂的过程,但是4C6-TPP与ctDNA以负协同模式分两步相互作用,其结合常数远大于4C4-TPP与ctDNA的结合常数。4C4-TPP和4C6-TPP在聚集及其与ctDNA作用中的差异有力的证明了卟啉侧链不仅是形成聚集体结构的关键因素,还影响其与DNA的作用。更多还原

【Abstract】 Porphyrin aggregate through non-covalent self-assembly is a common phenomenon and has a crucial role in many systems, especially in the fields of preparation of materials mimic biological systems and photodynamic therapy. Control the structure of porphyrins aggregate and study on the interaction of porphyrin and DNA play an important role in expanding application of porphyrin in the field of biology and medicine.Research on porphyrin aggregation and interaction of porphyrin with DNA has drawn greater attention with the expansion of its application. A large number of different structures of porphyrins are synthesized as research objects including natural porphyrins and others obtained by changing the substituent of porphyrin. The most common object is porphyrin with the meso-substituents. The porphyrins aggregation type, aggregation model, factors affecting aggregation, the interaction mode of porphyrin based on the spectroscopy with DNA has basically clear through the joint efforts of researchers. But many of the problems still exist, what merits in-depth study. Especially, the solvent induction to porphyrin aggregation and the influence of substituent on the interaction of porphyrin with DNA have been hot, retroactive issues. Recent studies show that solvent induce porphyrin aggregation througth polar interaction of substituents with solvent moleculars resulting in different morphology of aggregation. The chemical structure of peripheral substituents appears to play a more important role than merely their size in determining the binding preferences of porphyrins. An effective way to resolve these problems is that selecting appropriate solvent and modulating the size of peripheral substituents to explore the origin of the role of substituents on the aggregation and interaction of porphyrin with DNA.The structure of 4C4-TPP (meso- tetrakis [3-methoxy-4-(N-carbazyl) n-buxyloxyphenyl] porphyrin, 4C4-TPP) and 4C6-TPP (meso-tetrakis [3-methoxy-4-(N-carbazyl) n-hexyloxyphenyl] porphyrin, 4C6-TPP) are similar, the only difference is that the size of substituent of the later is longer than that of former. Hence, they are chose to be the model to study on the solvent induction of porphyrin aggregation and the effect of substituent on the interaction of porphyrin with DNA.Thus, this paper uses 4C4-TPP and 4C6-TPP as cases study to elucidate the solvent effects on the aggregation and the interaction of porphyrin with DNA through measured by absorption spectrum, fluorescent spectrum, Circular Dichorism (CD) and Raman spectrum. In special solvent conditions, 4C6-TPP tend to aggregate through autocatalytic-like kinetics and the location of the phenyl groups plays a key role in the determination of the structure of the aggregate. Based on the understanding of porphyrin aggregation, 4C4-TPP and 4C6-TPP are interacted with calf thymus-DNA (ctDNA) to validate the conclusion that the chemical structure of peripheral substituents appears to play a more important role than merely their size in determining the binding preferences of porphyrins.(a) Study on the porphyrin aggregationThe term molecular self-assembly can be defined as the spontaneous association of two or more molecules under thermodynamic equilibrium resulting in the generation of well-defined aggregates (strict self-assembly) or of extended polymolecular assemblies (self-organization) by means of noncovalent interactions such as hydrogen bonds, metal-coordination orπ-πinteractions. Porphyrin tends to aggregate through self-assembly under special solution conditions.4C4-TPP and 4C6-TPP tend to be monomer in organic (tetrahydrofuran, THF) in certain range of concentration. 4C4-TPP is monomer when the concentration is less than 3.34×10-6M; 4C6-TPP is monomer as the concentration is less than 4.49×10-6M. Exceeding this range, porphyrins tend to generate aggregate resulting from the intermolecular or intramolecular force.In special aqueous-organic solution, 4C6-TPP tends to aggregate with autocatalytic-like kinetic process. The increased conjugation of the meso-phenyl rings with the porphyrin core brings about the red shift of the electronic absorption and the increase of phenyl mode in Raman spectra. Solvent effects on the 4C6-TPP aggregate result equally in the non-planarity of the porphyrin and its substituents and the close stacking of the porphyrin monomers, then CD signal disappear. The complex of 4C6-TPP and water had the possibility to form a structure like a cage deduced by the classical clathrate structures, in which 4C6-TPP molecules existed inside the cavity composed of water. The hydroxyl group of the 4C6-TPP can be expected to form a strongly directional hydrogen bond with the water framework driving the rotation of the phenyl groups to be coplanar with the porphyrin core. In order to form hydrogen bonds, the alkyl and carbazole groups rotated to a position resulting in the equal compromise of the non-planarity of the porphyrin and substituents and the close stacking of porphyrin monomers.Study on the effect of pH and ionic strength to the aggregation of 4C6-TPP in aqueous-organic solution. Increasing ionic strength, the structure of aggregate tends to change. Upon addition of HCl, the pronated 4C6-TPP was found to form J-aggregate and exhibit“hyper”spectra as witnessed by a red-shifted Soret band and one broad band in 694 nm. Fluorescence spectra were deeply affected by protonation and the one-band emission converted to two-band emission. Comparison of the Raman spectra of the protonated 4C6-TPP and non-protonated indicated the former exhibited some new bands in the region of 200 cm-1 and 900 cm-1 involved in the Cmeso-Cphenyl stretching vibration and deformation of porphyrin ring. These new bands tended to be more intense with HCl concentration increased. The experimental evidence showed the dependence of the protonation and aggregation behavior on the concentration of HCl and there were obviously differences between the aggregation of partly protonated and fully protonated species. The nonplanar deformations of porphyrin core were responsible for the red-shifted electronic spectra. Based on the constructed model of TPP and carbazole, the fully protonated J-aggregate of 4C6-TPP involved the porphyrin core and substituents of carbazoles.(b) Study on the interaction of porphyrin and DNAThe interaction of 4C4-TPP and 4C6-TPP with calf thymus-DNA (ctDNA) is explored by UV-vis, fluorescence and CD spectra. The results suggest that 4C4-TPP and 4C6-TPP that porphyrin outside binds ctDNA but peripheral substituents intercalated. The interaction of 4C4-TPP and ctDNA generate the red shift of Soret band (△λ=3nm) and isobestic point at 445nm. For 4C6-TPP, there is no isobestic point resulted from its interaction with ctDNA, but show the great red shift of Soret band (△λ=14nm) and absorption of 4C4-TPP interacted with ctDNA. The isobestic point in the absorption imply that the interaction of 4C4-TPP with ctDNA does not involve complex binding process and the binding constants by calculation is 2.44×103 L?mol-1。However, the Scatchard plots reveal that binding of 4C6-TPP to ctDNAshows negative cooperativity. Thus this binding involves two stages: (a) initial binding to ionic sites on the ctDNA driven by ionic interaction of4C6-TPP with ionic sites on the ctDNA; (b) binding of peripheral alkyl substituents of the porphyrins to hydrophobic patches close to the ionic sites in the ctDNA. The difference of binding showed by 4C4-TPP and 4C6-TPP suggest the chemical structure of peripheral substituents plays a more important role than merely their size in determining the binding preferences of porphyrins.In summary, this paper uses 4C6-TPP and 4C6-TPP with similar structure but different substituents as cases study to elucidate the solvent effects on the porphyrin aggregation and interaction of porphyrin with ctDNA. All the results demonstrate the peripheral substituents play a key role in the determination of the structure of the aggregate and the interaction of porphyrin with DNA. Thus, it has great significance to realize the possibility of controlling the size, the structural, and spectroscopic features of aggregation of porphyrins and expanding application of porphyrin in the fields of preparation of materials mimic biological systems and photodynamic therapy. Meanwhile, it provides a theoretical basis for further research the interaction mechanism of porphyrin with interaction.更多还原