【作者】 张淑琴；

【导师】 傅家谟； 程平；

【作者基本信息】 上海大学 ， 环境工程， 2013， 博士

【摘要】 金属离子在生物体内起着重要的作用，例如，金属离子对DNA双螺旋结构起着非常重要的稳定作用；核苷是DNA和RNA的基本结构单位，核苷类化合物具有重要的生物学功能，它们参与了生物体内几乎所有的生化反应过程，而这些反应过程的发挥往往离不开金属离子，金属离子在核苷类化合物的生物合成、构象维持、功能发挥与调控等方面起着重要的作用。因此，研究金属离子与核苷分子的相互作用一直受到人们的关注，但是关于金属离子和核苷在气相条件下的相互作用研究的报道却很少。质谱技术可用于气相中研究金属离子与生物相关的分子配合物，特别是电喷雾质谱技术（ESI-MS）已成为一系列关于氨基酸等小生物分子甚至是复杂生物分子如多肽和蛋白质分析研究的重要实验手段；而量子化学方法可以从理论上解释某些实验现象，预测一些反应的过程和结果，并且能从理论上给出实验中难以观测到的如寿命很短的反应中间体或过渡态的结构，从而为实验研究提供帮助和理论支持。本文采用ESI-MS结合密度泛函理论计算（DFT）进行本课题的分析研究。将通过气相条件下环境中常见的重金属铜离子与五种核苷分子相互作用的ESI-MS实验分析，考察一系列的铜-核苷相互作用产物的构型，能量等各种特性，研究铜离子与核苷分子的相互作用机制，同时还对相互作用产物的碰撞诱导解离（CID）过程进行了系统研究，为讨论在相关生命现象中这种相互作用的机理提供一定的科学依据。主要研究内容和结论如下：1)通过ESI-MS实验分析了五种核苷与Cu（NO₃）₂的水/甲醇（30/70）溶液，各种离子[CuLn]²⁺,[Cu（L-H）Ln]⁺,[LH]⁺,[L2H]⁺,[BH]⁺,[CuL（MeOH）_n]²⁺和[CuL（NO₃）_n]⁺基本都可以在质谱图中观察到；在铜离子与核苷分子的相互作用中，共价配位键作用在铜离子与胞苷和鸟苷分子的相互作用中表现突出，且铜离子与胞苷的ESI-MS图谱清晰，谱图识别产物匹配度高。2)对铜离子与胞苷相互作用的ESI-MS谱图中离子峰强度产物的结构及特征进行了CID研究发现：[L+H]⁺的CID分析可以观察到脱去NH₃, H₂O和CONH等小分子的反应，而[Cu（L-H）Ln]⁺（n=0,1）仅观察到糖甙键断开脱去糖环基团或糖环被打破后脱去某个小分子基团的反应；配体间质子转移在所有的[CuLn]²⁺（n=2-6） CID过程中均可发生,而其他解离反应则包括电荷减少的裂解和中性小分子基团的脱去等；[CuL（MeOH）]²⁺的CID可以看到中性甲醇分子的脱去和三种电荷减少的解离过程；[CuLn]²⁺（n=4-6）的CID过程中有质子化胞苷二聚体[L2H]⁺的产生，其结构是由两个胞苷通过氢键作用形成的。3)对铜离子与胞苷相互作用的ESI-MS谱图产物中[CuLn]²⁺与[LnH]⁺的配合物构型的稳定性进行DFT理论分析发现：Cu²⁺易于与胞苷分子以共价配位键结合形成较为稳定的配合物[CuLn]²⁺（n=1-4），[CuLn]²⁺由Cu²⁺以4配位形式在一个平面形成配合物形式较为稳定，其中N（3）和O（7）同时为胞苷分子的螯合位点，此时[CuLn]²⁺稳定性顺序为[CuL2]²⁺>[CuL]²⁺>[CuL4]²⁺>[CuL3]²⁺；而质子H+也非常易于与胞苷分子以氢键形式结合形成质子化多聚体[LnH]⁺（n=1-4），[LnH]⁺的稳定性顺序为[L2H]⁺>[LH]⁺>[L3H]⁺>[L4H]⁺；ESI-MS谱图中离子峰强度与稳定性顺序基本一致，但是没有观察到[CuL]²⁺可能是由于铜更倾向于形成4配位的形式，因此[CuL]²⁺与1-2个甲醇分子极易进一步配位，形成更为稳定的复合物分子[CuL（MeOH）1-2]²⁺；而离子峰[LH]⁺强度较大可能是由于Cu²⁺-胞苷配合物的CID过程产生的。4) DFT计算和空间结构分析比较铜离子与胞苷和鸟苷分子的配合物[CuLn]²⁺与[CuGn]²⁺的同分异构体，研究发现：鸟苷分子倾向于以N（7）与铜离子形成Cu-N配位，然后与其它鸟苷分子以氢键结合形成较为稳定的铜-鸟苷配合物；胞苷则是以N（3）和O（7）同时与铜离子配位，并在ESI-MS图中表现出较高的离子峰强度。通过对[CuLn]²⁺与[CuGn]²⁺（n=2-4）进行CID反应途径计算分析，在较低碰撞能量下显示[CuLn]²⁺的CID过程以质子转移为主，而[CuGn]²⁺则是电子重排后发生电子转移。理论计算结果还表明胞苷三配体易于发生质子转移失去一个质子配体[CuL（L-H）]⁺，而鸟苷三配体则是生成脱去一个分子自由基阳离子的二配产物[CuG2]⁺。5)采用多级CID和DFT计算分析质子化胞嘧啶阳离子的同分异构体，结果表明质子化胞嘧啶阳离子和中性胞嘧啶分子在整体结构上很接近，但由于质子化氢的加入，整个胞嘧啶阳离子中的电荷进行了重新分配，质子化加氢后嘧啶环上与加氢的N（3）相连的两个C-N键长略有增加，而嘧啶环上其他的键长都有所紧缩。在气相中不同质子化胞嘧啶阳离子构型的转化过程有O-H键的σ旋转、嘧啶环上杂原子之间[1,3]-质子转移和[1,4]-质子转移等。采用类似方法对其他质子化核苷和质子化核苷碱基的多级CID过程分析和计算，结果均发现质子化核苷打破糖甙键，CID过程脱去糖环；解离后，质子化核苷碱基的一次CID过程基本为脱去HNCO、H₂O、CN2H₂、NH₃和HCN等小分子的反应，此外质子化核苷碱基（胞嘧啶，鸟嘌呤，胸腺嘧啶和尿嘧啶）的多级CID过程均有脱去CO的反应，而质子化腺嘌呤的多级解离产物均为HCN。本论文的创新点：1)采用ESI-MS质谱技术分析了核苷分子与铜离子在气相条件下相互作用的产物，同时结合高精度DFT计算在理论上准确分析了产物的构型及特征；2)率先对金属离子与生物配体复合体产物的构型及稳定性进行了分析研究，确定了复合物种金属离子与配体的化学键特征；3)采用多级CID对质子化胞嘧啶等核苷碱基进行了综合分析，基本掌握核苷碱基在生命体内的迁移降解转化、新陈代谢机制。更多还原

【Abstract】 It’s well known that metal ions play an important role in vivo, for example,inDNA, the metal ionscan affect the stability of the DNA double helix structure; Asthe basic structural unit of DNA and RNA, the nucleoside compounds are involved inalmost all of the biochemical reaction process in vivo, and the reactions often dependson the metal ions, whcihplay an important role in biological synthesis, conformationmaintain nucleoside, function and regulation. Thus, the studies of the interactionsbetween metal cations and nucleosides have attained widespread attention, but fewwere studied in the gas phase.Electrospray Ionization mass spectrometry （ESI-MS） has become the importantmeans to anlyze the small biological molecules such as amino acids, and evencomplex biological molecules such as peptides and proteins; and the quantumchemistry calculationcan be used to study the chemical related issues which couldexplain the experimental phenomena, forecast the reaction process and result, andgive some reaction intermediates or reaction transition state structures which was veryhard to be obseved in experiment.In this disseration, ESI-MS and density functional methods （DFT） was used toinvestigate ithe interactions between environmental heavy metal copper ions and fivekinds of nucleoside molecules in the gas phase. The studies include the interactionproduct configurations, the interaction mechanism, and the products of collisioninduced dissociation （CID） which would provides some scientific guidance for therelevant life phenomena.The main research contents and conclusions as follows:1. Five Nucleosides and Cu（NO₃）₂water/methanol （30/70） solutions were seperatelyanalyzed by ESI-MS. The meatal cations of [CuLn]²⁺,[Cu（L-H）Ln]⁺,[LH]⁺,[L2H]⁺,[BH]⁺,[CuL（MeOH）_n]²⁺ and [CuL（NO₃）_n]⁺could be clearly showed in the massspectra; the covalent coordination bond had showed evidently effect on theinteraction of the copper ions with cytidine and guanosine; and the spectra strengthof the complex of copper ions with cytidine was high.2. Further analysis for the product of the divalent copper ions and cytidine of itscomplex structure and characteristics was described on a lower collision energy: NH₃, H₂O and CONH stripped off could be observed in the [L+H]⁺CID progress. agroup of small molecules broken off after the sugar ring off could be observed inthe [Cu（L-H）Ln]⁺（n=0,1） CID progress. Ligand proton transfer could be occured inall the[CuLn]²⁺（n=2-6） CID process, and other channels including the charge toreduce cracking and neutral molecules group off. Neutral methanol moleculesstripped off and three charge reduced processes could be observed in the[CuL（MeOH）]²⁺ CID progress. Proton cytidine dimmers [L2H]⁺could be observedin the [CuLn]²⁺（n=4-6） CID process, which might be bonded by the hydrogenbonding interactions of two cytidine molecules.3. DFT study for complex configuration of [CuLn]²⁺ and [LnH]⁺was described. It wasfound that the Cu²⁺ was easily liganded with cytidine molecules by covalentcoordination bond to form stable complexes on chelation sites N（3）and O（7） ofcytosine at the same time on a plane. And the proton H+was easily bonded cytidinemolecular by hydrogen bonds to form a protonated poly [LnH]⁺（n=1-4）. Thecalculated stability orders are [CuL2]²⁺>[CuL]²⁺>[CuL4]²⁺>[CuL3]²⁺ and[L2H]⁺>[LH]⁺>[L3H]⁺>[L4H]⁺which are consistent quite well with the peakintensity distribution in the mass spectrum. The [CuL]²⁺ was not observed mightbecause that the high strength of [CuL（MeOH）1-2]²⁺ was easily observed as [CuL]²⁺ liganded with methanol molecules; While high strength of [LH]⁺might be thebivalent Cu²⁺-cytidine complexes CID products.4. Combining with the stability analysis of the spatial structure of the isomers[CuLn]²⁺（n=2-4） of cytidine and guanosine complexes, CID fragmentationpathway analysis and calculation results showed that the guanosine ligand site wasN（7） with copper ions which preferred Cu-N ligand bond, and then the guanosinecomplex molecules would ligand the third or more guanosine by hydrogen bonds toform a more stable copper-guanosine complexes; while cytidine ligand complexespreferred ligand number4or6at the ligand site both of the N（3） and carbonyl O（7）,which showed a higher peak intensity of the ESI-MS. Protonated cytidine andradical guanosine cationic products were respectively removed on the Low energyCID process. CID of Cytidine ligand dissociation channel was mainly showedProton Transfer process, while the guanosine ligand dissociation was the electrontransfer process after the electronic rearrangement for [CuGn-1]⁺.。5. Combined with the stability analysis of the spatial structure of the isomeric by DFT method, the protonation of cytosine isomers of cation configurations was analyzed.the overall structure of the protonation cytosine cations and the neutral cytosinemolecule were very close, but the hydrogen proton changed and the cytosine in thecationic charge redistributed. bond length of the two C-N connected with N（3）would be increased slightly as hydrogenation happened on the protonation of thepyrimidine ring, while the pyrimidine ring on other bond length was tightened. Inthe gas phase, sigma transformation process of the O-H bond,[1,3]-proton transferand [1,4]-proton transfer of the pyrimidine ring would observed on differentprotonation of cytosine cationic configurational. CID fragmentation pathwayanalysis of [LH]⁺and [BH]⁺and calculation results showed that the N-glycosidicbond broken in LH, the sugar ring was also broken off in CID dissociate reaction；After dissociation, some little molecules of HNCO、H₂O、CN2H₂、NH₃and COwould be stripped off on the following channels of the CID process of nucleobase[BH]⁺; In addition, CO stripped process would happened on the multi-stage CIDchannels of the nulceoside base（cytosine, guanine, thymine and uracil）, and theHCN stripped process would happened on the multi-stage CID channels of adenine.The innovation points of this paper:1．ESI-MS was used for the analysis of the products of different nucleotidemolecules with copper ions in the gas phase, and also combined with highprecision accurate DFT calculation, the product configurations were sucessfulprovided.2．For the first time, the statbility of the complexes of copper ion with the nucleotidemolecules were investigated in the gas phase, and the characteristics of theinteractions were confirmed.3．The multiple mass spectrometry method and DFT calculation was used to analyzethe CID of the protonated cytosine, adenine, etc., and the their dissociationmechanism was investigated.更多还原