【作者】 李俊芬；

【导师】 董川；

【作者基本信息】 山西大学 ， 分析化学， 2008， 博士

【摘要】 第一章:简述了DNA的组成及结构特征;小分子与DNA相互作用的主要模式及影响因素;生物碱的结构与性质;以光谱法为主,对应用于生物碱等小分子与DNA相互作用领域的各种分析技术的研究理论及现状进行了综述。第二章:在不同极性和不同酸度介质中考察了巴马汀的荧光光谱和紫外光谱特性。随着溶剂极性的增加,巴马汀的溶解性增大,吸收峰强度明显增大且红移。巴马汀的荧光发射光谱受溶剂极性的影响也很显著。随着溶剂极性的降低,荧光发射光谱的第二个发射带紫移并且荧光强度显著提高,而第一个发射带几乎不受影响。由于本身荧光较弱,pH对巴马汀溶液的吸收和荧光光谱影响都较小。量子产率在二氯甲烷和乙腈溶剂中达到最大。结果表明,巴马汀对周围介质环境的敏感程度较高。第三章:以盐酸巴马汀作为探针,采用紫外吸收光谱、荧光光谱方法为主要手段,结合电化学方法系统地研究了溶液中巴马汀与小牛胸腺DNA的相互作用,探讨了作用方式和作用机理。化合物与目标靶分子结合后,荧光发射强度、吸光度和荧光偏振等光谱参数产生了变化。巴马汀本身荧光很弱,但有DNA存在时,荧光被显著增强。虽然吸收光谱表明巴马汀与ctDNA之间符合嵌插模式,但荧光偏振、阴离子猝灭实验却更符合沟槽键合。研究表明,巴马汀与ctDNA相互作用的模式为混合模式,即嵌插、沟槽和静电三种作用方式都不同程度存在,其中沟槽键合是主要作用方式。巴马汀的异喹啉端可能插入到DNA双螺旋的碱基对中,分子的另一部分伸出沟槽外。利用荧光滴定数据,计算巴马汀与ctDNA的结合常数为2.52（±0.15）×10⁴L/mol,结合位点数为0.16。第四章:以固体基质发光光谱法,研究了盐酸巴马汀的光谱性质,详细考察了实验条件对盐酸巴马汀固体基质室温荧光（PS-RTF）和磷光光谱（PS-RTP）的影响。实验表明,以未经任何预处理的慢速定量滤纸做基质,巴马汀能直接产生强的RTF而不产生磷光;以TIAc做重原子微扰剂,巴马汀则可发射出强的室温磷光,最大激发波长为350nm,最大发射波长为522nm、615nm。当重原子浓度高于2mol/L、pH值在6-7时,巴马汀的PS-RTP信号最强。在最佳实验条件下提出了巴马汀的PS-RTP测定法。在此基础上,利用PS-RTP和PS-RTF光谱法,详细研究了巴马汀与ctDNA的非共价复合作用,探讨了它们之间的作用机理和作用模式,进一步确定巴马汀与DNA之间的作用方式为混合模式。第五章:以固体基质发光光谱法,研究了盐酸小檗碱和盐酸药根碱的光谱性质,详细考察了实验条件对它们PS-RTF、PS-RTP的影响。小檗碱和药根碱能直接在慢速定量滤纸上产生强的室温荧光。TINO₃做重原子微扰剂,小檗碱可发射强的磷光,其最大激发波长λ_ex为353nm,最大发射波长λem为520nm、619nm。而药根碱未观察到磷光发射。在此基础上,利用固体基质室温发光光谱,结合流体中吸收光谱和荧光光谱法,详细研究了小檗碱和药根碱与ctDNA的非共价复合作用,求出了结合常数、结合位点,并考察了DNA对小檗碱和药根碱的荧光及磷光偏振的影响,探讨了它们之间的作用方式和作用机理。结果表明,两种生物碱与DNA的作用方式均为嵌插模式;结合常数分别为1.84（±0.12）×10⁴L/mol和0.95（±0.08）×10⁴L/mol,与DNA的作用大小为小檗碱＞药根碱。第六章:采用紫外光谱、荧光光谱、荧光偏振,热变性等手段,对白屈菜红碱和血根碱与ctDNA的键合作用、作用方式进行了研究。研究发现,在pH=5.40时,白屈菜红碱和血根碱以离子形式存在,DNA的加入可使白屈菜红碱和血根碱的荧光强度显著发生变化。紫外光谱出现明显的减色和红移现象,证明两者与DNA的相互作用方式是嵌插方式。荧光偏振和DNA变性实验同样证实白屈菜红碱和血根碱是通过嵌插方式与ctDNA相互作用的,即两种生物碱分子可以插入到DNA双螺旋中。在一定离子强度下,该嵌插作用会减弱。荧光滴定实验测得白屈菜红碱和血根碱与DNA的本征键合常数分别为2.98（±0.10）×10⁵L/mol和8.86（±0.18）×10⁵L/mol,结合位点数分别约为0.210和0.18。此外,循环伏安曲线表明,两种化合物与DNA之间还存在着非特异性的静电作用。第七章:采用荧光光谱法和吸收光谱法,在接近人体生理条件的Tris-HCl缓冲溶液中研究了盐酸吗啡与ctDNA的相互作用。考察了浓度、酸度和温度等因素对盐酸吗啡与ctDNA的相互作用的影响,探讨了相互作用方式和作用机理。离子强度影响、阴离子猝灭剂（亚铁氰化钾）、荧光偏振实验及DNA变性实验均表明,盐酸吗啡与DNA的相互作用属嵌插模式,DNA对盐酸吗啡的猝灭机理属静态猝灭。相互作用的结合常数和结合位点数分别为5.65×10³L/mol和1.08,表明盐酸吗啡与DNA的结合能力不大,可以和DNA形成1∶1的络合物。第八章:考察了QN的荧光光谱特性。选用QN为发光探针,采用紫外光谱、荧光光谱、荧光偏振等光谱方法研究了QN与ctDNA的相互作用,考察了影响其相互作用的各种因素,探讨了作用机理和作用方式。研究发现,该探针在与目标靶分子结合后其光谱参数产生了变化,如荧光发射强度、荧光量子产率和偏振等。DNA存在时,QN的荧光被显著猝灭,吸收光谱出现减色现象。QN与DNA的本征键合常数为1.25×10⁵L/mol,结合位点数为0.15,一分子的QN可以和大约3个碱基对作用。初步证实QN是通过嵌插方式与ctDNA作用的。QN和QD与ctDNA的作用模式和作用强度类似。第九章:总结全文,提出下一步工作设想。更多还原

【Abstract】 Chapter 1 In this chapter,following questions were elaborated:the composition of DNA and characteristics of its structure;binding modes of small molecules with DNA and fators affect their interactions.Furthermore,the theoretical research and present situations of some common techniques used in this field were reviewed.Chapter 2 The fluorescence and absorption spectra behaviors of palmatine in different solvents with different polarities and under different pH were studied.The results show that,the absorption intensity of palmatine increases and red shifts with the increase of the polarity of the solvents.The fluorescence intensity and spectra of palmatine are affected by the polarity of the solvents obviously as well.Only the second fluorescence emission peak blue shifts and increases with the decrease of the polarity of solvents.Effects of pH on the spectra of absorption and fluorescence are not great.The fluorescence quantum yields of palmatine in different solvents were measured.It can be drawn from this chapter that palmatine is sensitive to the surrounding environment.Chapter 3 The interaction of palmatine with DNA was studied mainly by absorption and fluorescence spectrometry.Many spectral parameters change after the addition of DNA, such as the intensity of fluorescence,absorbance,fluorescence quantum yield and polarization,etc.It is found that palmatine acts as a light switch,its fluorescence is very weak in the absence of DNA,but in the presence of DNA,its fluorescence is obviously enhanced.After the addition of DNA,the absorption spectra of palmatine red shift and its absorbance decreases,but its polarization and fluorescence quenching extent by KI do not increase.The binding constant was measured and the binding mode and mechanism were discussed.The results show that the binding mode is mix-mode,that is to say,intercalating, groove,and electrostatic binding are all exist.Among them,groove binding is a major factor affecting the binding ability.Binding constant measured is 2.52（±0.15）×10⁴L/mol, and binding site number is 0.16.Chapter 4 The PS-RTP spectra properties of palmatine were investigated.Experiments conditions were studied in detail.Palmatine can emit strong RTF on the slow speed filter paper directly.Using TIAc as perturber,it can emit strong room temperature phosphorescence.Its maximum excitation is 350nm,emission are 522nm and 615nm. When the concentration of heavy atom higher than 2mol/L and in pH 6-7 medium,its RTP intensity reaches to the maximum.PS-RTP analytical method was established under the optimum experimental conditions.At the same time,the interaction of palmatine with DNA was investigated by PS-RTP and PS-RTF.The effect of quencher to the luminescence intensity was studied,and the binding mechanism and binding mode were discussed.This chapter further demonstrates that the binding mode of palmatine with DNA is mixed mode.This is in accordance with the conclusion in chapter 3.Chapter 5 The PS-RTP and PS-RTF spectra properties of berberine and jiatrorrhizine were investigated.Experiment conditions were studied in detail.Berberine and jiatrorrhizine can emit strong RTF on the slow speed filter paper directly.Using TINO₃ as heavy atom perturbation,berberine can emit strong RTP.Its maximum excitation is 353nm, maximum emission are 520nm and 619nm.The RTP of jiatrorrhizine is not observed under the same condition.At the same time,the interactions of berberine and jiatrorrhizine with DNA was investigated by PS-RTP and PS-RTF techniques,absorption and fluorescence spectrometry were used as well.Binding constants were determined by absorption and fluorescence spectrometry,and the binding mechanism and mode were discussed. Intercalation was proved as the binding mode.The evaluation of binding constants shows that,the binding affinities of the two protoberberines with DNA are in the order of berberine＞jiatrorrhizine.Chapter 6:The interactions of chelerythrine and sangunarine with DNA were mainly studied by absorption,fluorescence spectrometry,fluorescence polarization,and denatured DNA methods.Their binding modes and binding affinities with ctDNA were researched. The results show that at pH5.40,the addition of DNA leads to the increase of the fluorescence intensity of chelerythfine,while the intensity of sangunarine is obviously quenched.The phenomena of hypochromicity and bathochromic shifts of their absorption spectra prove that their binding modes with DNA are both intercalation.Same result can be drawn from the fluorescence polarization experiments.The two alkaloids can intercalate into the double helix of DNA.Under some degree of ion strength,this affinity will become weak.Binding constants for chelerythrine and sangunarine with DNA are 2.98×10⁵L/mol and 8.86×10⁵ L/mol respectively,binding site are 0.210 and 0.18 respectively.In addition,electroanalytical method were studied,and it shows that there exists nonspecific electrostatic binding.Sangunarine shows stronger binding ability with DNA than chelerythrine.Chapter 7 The interaction of morphine with DNA was studied by absorption, fluorescence spectrometry,fluorescence polarization,and denatured DNA methods in neutral Tris-HCl buffer.The results show that morphine forms stable complex with DNA. The effects of concentration,pH and temperature on the interaction of morphine with DNA were investigated.The binding mode and binding mechanism were discussed. Experiments of ion strength,anion quencher,fluorescence polarization and denatured DNA all indicate that the binding mode is intercalation.With the increase of temperature, the quenching extent of DNA to morphine becomes weak.This proves that the fluorescence quenching of morphine is in accordance with static mechanism.In addition, the binding constant and binding site were measured.The results show that the binding affinity is not very big and morphine can form 1:1 complex with DNA.Chapter 8 The fluorescence spectra behaviors of QN were researched.Selecting QN as luminescence probe,the interaction of QN with DNA was studied by means of absorption, fluorescence spectroscopy and fluorescence polarization.Factors effected the interaction was studied,and the mechanism and binding mode were discussed.It is found that the fluorescence intensity is quenched greatly,and the fluorescence polarization increases.QN can binds to DNA by intercalation mode.Chapter 9 This chapter summarized all the experimental results in this thesis.Further works were proposed as well.更多还原