【作者】 张来斌；

【导师】 步宇翔；

【作者基本信息】 山东大学 ， 理论与计算化学， 2010， 博士

【摘要】 荧光光谱法具有快速、简便、灵敏度高、选择性好等优点,在生物化学和分子生物学中具有重要的应用。天然DNA碱基的荧光量子产率非常低,不能直接利用。因此,为了利用荧光光谱技术来研究DNA的构象动力学以及其他分子与DNA之间的相互作用,设计和合成具有荧光特性的碱基类似物具有重要意义。本文主要以几类新设计的DNA碱基类似物为研究对象,在对这些体系几何结构和电子结构研究的基础上,运用含时密度泛函理论(TD-DFT)探讨了它们的紫外吸收光谱和荧光光谱,并考察了生物微环境(包括水合效应、与脱氧糖环相连、碱基配对和碱基堆积等)对其电子光谱性质的影响。通过对以上性质的研究,我们可以对实验结果进行解释,进一步探索它们在生物技术方面的实际应用,并为以后的相关实验提供一定的指导信息。通过研究,取得了一些有意义的研究成果,主要成果和创新简述如下：1.研究了“苯”扩环y-碱基的电子光谱性质并考察了与脱氧核糖相连及与天然碱基形成WC碱基对产生的影响。首先,对单体的紫外吸收光谱进行了定量解析归属。计算得到的吸收波长和荧光波长与实验值符合较好。四个碱基的最低激发态均具ππ*特征,主要对应于H→L跃迁。yA、yT和yT-m的S1态具有平面结构,而yG和yC具有非平面的S1结构。脱氧核苷的最低激发态对应于单体的第一激发态。总的来说,与脱氧核糖相连将会使y-碱基的最大激发波长和荧光波长发生红移。而且,与脱氧核糖相连将会使yA的荧光强度有所增大(荧光量子产率增大),而使yT和yC荧光强度有所减小(荧光量子产率减小)。四个WC碱基对的基态均具有平面结构,其最低激发态实质上与y-碱基单体的第一激发态一样,因此是局域激发。yAT和yTA的S1态具有平面结构,而yCG和yGC的S1态具有螺旋扭曲的非平面结构,主要是由y-碱基组分氨基的角锥化造成的。研究表明形成WC氢键对yA、yT和yC的最大激发波长和荧光波长没有太大影响,但是将会使yG单体的最大激发波长和荧光产生较大蓝移。2.在对y-碱基的研究基础之上,将研究延伸到“萘”扩环yy-碱基之上,以探讨yy-碱基的电子光谱性质,并考察其与y-碱基性质的区别。首先,对单体气相和甲醇溶液中的最低十个单重激发态进行了解析归属。所有四个碱基的最低激发态均具ππ*特征,主要对应于H→L跃迁。yyC、yyG和yyA的S1态具有非平面结构,而yyT的S1态具有平面结构。yyC和yyT的最低nπ*具有高度非平面结构,与基态相比,激发对应的羰基键长分别增长了0.07和0.075 (?)对yyC和yyT来说,计算得到的激发能和荧光波长与已报道的实验值符合较好。此外,我们发现可以用PCM模型和气相基态结构来很好地模拟四个yy-碱基甲醇溶液中的紫外吸收光谱。计算表明,在甲醇溶液中,yyA和yyG的荧光波长分别为439和562 nm,表明yyA是一个绿色荧光团,yyG是一个黄色荧光团。甲醇溶液使yyC、yyT和yyA的低激发ππ*态产生红移,却使yyG的低激发ππ*态发生蓝移。然而甲醇溶液对四个yy-碱基一致的效应是使其最大吸收波长的吸光度大大增强。四个WC碱基对的基态均具有平面结构,其最低激发态实质上与yy-碱基单体的第一激发态一样,因此是局域激发。yyAT和yyTA的S1态具有平面结构,而yyCG和yyGC的S1态具有螺旋扭曲的非平面结构,主要是由yy-碱基组分氨基的角锥化造成的。研究表明形成WC氢键对yyA、yyT和yyC的最大激发波长和荧光波长没有太大影响,但是将会使yyG单体的最大激发波长和荧光产生较大蓝移。此外,我们发现甲醇溶液对y-碱基电子吸收光谱的影响与其对yy-碱基的影响类似,即其使yC、yT和yA的电子吸收光谱发生红移,而使yG的吸收光谱发生蓝移。与y.碱基相比,yy-碱基的最大激发波长和荧光波长均发生较大的红移,而且其最大激发波长的吸光度较y-碱基发生了降低,但是其最大吸收波长的吸光度却比y-碱基大得多。3.对我们小组设计的若干鸟嘌呤类似物(A1-A4,具体结构参见正文信息)以及天然鸟嘌呤和xG的电子光谱性质进行了研究,并考察了与胞嘧啶配对产生的影响。研究表明：一、扩环碱基的最低单重激发态为ππ*态,均对应于H→L跃迁,与天然G相比,它们的最大激发波长均发生红移,而荧光峰均发生蓝移；二、其中三个类似物(A1-A3)的S1态具有与G相类似的高度非平面结构,而另外两个(A4和xG)的S1态具有近平面结构,H和L轨道的不同特点,导致了它们激发态结构的差异；三、所有新设计的G-类似物的Stokes Shift均比天然G小,说明它们具有较强的分子刚性,而且它们的荧光量子产率预期比天然鸟嘌呤的大；四、当考虑C配对产生的效应时,这些碱基可以分为两组。第一组由G、A1和A2组成,与C配对使它们的最低单重激发态和荧光发射过程的振子强度分别下降了27％-60％和19-23％。而且,对A1C和A2C碱基对来说,吸收过程中由嘌呤的H到胞嘧啶的L的电荷转移态与第一激发态(局域激发ππ*)的能量差与天然GC相应的值相差无几。另外的三个碱基(A3,A4和xG)组成第二组,与C配对使它们的最低单重激发态和荧光发射过程的振子强度分别提高了11％-15％和3-20％。此外,由嘌呤的H到胞嘧啶的L的电荷转移态与第一激发态(局域激发ππ*)的能量差比第一组相应的值大得多。总的来说,如果与C配对不改变这些单体的无辐射过程,那么它将降低G、A1和A2的荧光量子产率,而增大A3、A4和xG的荧光量子产率。4.系统研究了具有直接应用可能性的胞嘧啶类似物x-胞嘧啶的激发态性质,并检验了生物微环境,包括水合作用、与脱氧核糖相连、与G形成WC碱基对和碱基堆积对其激发态性质的影响。计算表明,最低激发态为ππ*态,主要对应于H→L跃迁。其第一激发态(S1态)具有非平面的结构。计算得到激发能和荧光波长与实验值符合较好。实验中观察到的250 nm以下的强烈吸收带对应于计算得到的xC的最大吸收波长,即220 nm的吸收带；水合作用对xC的吸收光谱具有增色效应,而且其将导致最大激发波长和荧光波长分别蓝移12和8 nm；脱氧核糖对xC的吸收光谱的低能区域有增色效应,而对其高能区域有减色效应。而且,与脱氧核糖相连将会使xC的荧光红移7 nm与鸟嘌呤形成WC碱基对将使xC的最大激发波长和荧光波长发生蓝移,并使相应的振子强度增大。而且,对xCG水合,将使最大激发波长和荧光波长发生进一步的蓝移。总的来说,水合作用、与脱氧核糖相连和与鸟嘌呤形成WC碱基对均可能增大xC的荧光量子产率,而碱基堆积将导致碱基之间的轨道混合,从而使最低激发态和荧光过程的振子强度均发生很大的降低,相应的能量也会发生一定的红移。在堆积结构中,xC的荧光量子产率预期将通过静态猝灭机制而降低。更多还原

【Abstract】 Fluorescence spectroscopic techniques are extremely powerful tools for wide applications in biochemistry and biology due to their fascinating properties, including high speed, simplicity and convenience, exquisite sensitivity and selectivity. As we all know, natural nucleobases display extremely low fluorescent quantum yields and ultra-short excited-state lifetimes in both solution and gas phase. Thus, in order to more easily probe DNA/RNA strand conformational dynamics and the interactions between DNA/RNA and other molecules with spectroscopic techniques, the creation of fluorescent DNA base analogues is very important. In this work, the electronic spectroscopic properties of several kinds of newly designed fluorescent base analogues were investigated theoretically. Also considered were the effects of the Micro-Biologic environments, including the water solvent, linking to deoxyribose, base pairing, and base stacking on their photophysical characters. Understanding of the excited-state properties of these analogues and effects of perturbations induced by the biologic environment is helpful in finding ways for their direct usefulness of their fluorescence. In addition, the theoretical predictions are helpful in understanding the photophyscical properties of the newly designed DNA oligonucleotides and explaining experimental data in the future. Furthermore, it may help in the design and synthesize of new fluorescent base analogues. Some interesting phenomena and characters have been observed through the investigations, which can be described as follows:1. Firstly, the electronic spectroscopic properties of y-bases were investigated. Also examined were the effects of linking to deoxyribose and hydrogen bonding to their natural complementary bases. For the isolated bases, the calculated excitation and emission energies are in good agreement with the measured data where experimental results are available. The lowest singlet excited state of these bases is ofππ* character, which is mainly dominated by the configuration HOMO (H)→LUMO (L). The S1 geometries of yA, yT, and yT-m are found to be planar, while those for yG, yG-t2, and yC are nonplanar. In general, binding with deoxyriboses red-shifts the absorbance maxima and the fluorescence emission of the y-bases. Furthermore, the linking with deoxyriboses increases the fluorescence intensity of yA, but decreases those of yT and yC. The ground-state geometries of the WC base pairs (yAT, yTA, yGC, and yCG) are found to be planar, and the calculated interaction energies are very close to those of natural base pairs, indicating that the y-bases can pair with their natural complementary partners to generate stable base pairs. The S1 geometries of yAT and yTA base pairs are found to be quasi-planar, while those for yCG and yGC show propeller-twisted nonplanar structures resulting from the pyramidalization of the amino groups that belong to the y-base moieties. In general, base pairing does not have a significant effect on the fluorescence emissions of yA, yC, and yT, but blue shifts the fluorescence emission of yG by 22 nm.2. Based on the investigations of the y-bases, we extend our work to the yy-bases. Also examined were the effects of methanol solution and hydrogen bonding to their natural complementary bases, and the results were compared with those of y-bases. The nature of the lowest ten singlet transitions of the yy-bases were revealed in both gas phase and methanol solution. The lowest singlet excited state of these bases is ofππ* character, which is mainly dominated by the configuration H→L. The S1 geometries of yyC, yyG, and yyA are found to be nonplanar, while that for yyT is planar. The geometries of the lowest nn* states of yyC and yyT are highly nonplanar, involving the elongation of the associated carbonyl group by 0.07 and 0.075 A, respectively. The calculated excitation and emission maxima of yyC and yyT agree well with the measured data. In methanol solution, the fluorescence from yyA and yyG would be expected to occur around 539 and 562 nm, respectively, suggesting that yyA is a green-colored fluorophore, while yyG is a yellow-colored fluorophore. It was found that the methanol solution can red-shift both the absorption and emission maxima of yyA, yyT, and yyC, but blue-shift those for yyG. There is consensus that the intensity of the strongest absorption peak is greatly increased after solvation. Also it was found that the strategy of using the PCM model combined with the gas phase ground-state structures can well reproduce the absorption spectra of yy-bases in methanol solution. The ground-state geometries of the WC base pairs (yAT, yTA, yGC, and yCG) are found to be planar, and their lowest transitions are essentially identical to the first transition of free yy-base, and therefore can be classified as local excitations of the yy-bases. The S1 geometries of yyAT and yyTA base pairs are found to be quasi-planar, while those for yCG and yGC show propeller-twisted nonplanar structures resulting from the pyramidalization of the amino groups that belong to the yy-base moieties. Generally, though the base pairing has no significant effects on the absorption and fluorescence maxima of yyA, yyC, and yyT, it blue-shifts those for yyG. In addition, it was found that the methanol solution has a similar effect on the absorption spectra of the y-bases compared with those for yy-bases, that is to say, the methanol solution can red-shift the absorption of yA, yT, and yC, but blue-shift that for yG. Compared with y-bases, both the absorption and emission maxima of yy-bases are red-shifted. Furthermore, the intensity of the excitation maxima corresponding to yy-bases are lower compared with those for y-bases, while the intensity of the strongest absorption bands are much higher than those of y-bases.3. Thirdly, in order to understand the electronic spectroscopic properties of the guanine (G) analogues designed by our group,4 analogues named A1-A4 are explored using ab initio calculations, and the results were compared with those of natural G and xG. The pairing effect with their complementary base, cytosine, on both the absorption and emission processes were examined as well. For the isolated bases, the onset absorption peaks of these newly designed analogues are red-shifted compared with that of natural guanine, while the fluorescence wavelengths are blue-shifted. The calculated excitation energies are in good qualitative agreement with measured data where experimental results are available. In general, the S1 singlet excited states are nonplanar for these newly designed base analogues, and the Stokes-Shifts are much smaller than that for guanine, suggesting they have stronger rigidity of molecule than guanine. Therefore, the fluorescence quantum yields of these analogues are expected to be higher than that of natural guanine. When the effect of the pairing with C is taken into account, these bases can be divided into two groups. The first group includes G, Al, and A2 (one-bond-intercalated at the C5 site), and their parings with C could reduce the oscillator strengths of both the firstππ* transitions by 27%-60% and the fluorescence emissions by 19%-23%. In addition, the energy gaps between the first local excitedππ* state and the charge transfer state (from the H of purine to the L of cytosine) are close to each other for their corresponding base pairs. The other three bases (A3, A4, and xG) considered here make up the second group, which are two-bonds-intercalated at the C5 sites. The pairing with C increases the oscillator strengths of both the firstππ* transitions by 11%-15% and the fluorescence emissions by 3%-20%, and the corresponding energy gaps are much larger than that of the GC pair. In general, if the pairing with C does not affect the nonradiative process, it can reduce the fluorescence quantum yield for G, A1, and A2, but enhance it for A3, A4, and xG.4. Finally, the excited-state properties of the size-expanded cytosine analogue xC were investigated. The cytosine analogue xC by benzo-homologation is fluorescent with a fluorescent quantum yield of 0.52. Most importantly, it was demonstrated that a DNA polymerase is able to read the chemical information stored in xC and that the full replication machinery of E. coli is able to recognize the sequence encoded by xC correctly and efficiently, suggesting that it is a promising candidate for direct usefulness. Clearly, an in-depth understanding of the excited-state properties and the effects of perturbations induced by the biologic environments, the water solvent, linking to deoxyribose, base pairing, and base stacking is very important and helpful in finding ways for its direct usefulness of its fluorescence. Therefore, a detailed and systemic computational study on the properties of xC with the aim of gaining more insight into the properties of the excited states by investigating the dependence of absorption and emission spectra on the biologic environments mentioned above. For isolated xC, the nature of the low-lying singlet transitions are discussed and the calculated electronic absorption peaks and emission maximum are in good agreement with reported experimental values. The experimental observed absorption band for dxC below 250 nm in methanol solution must be related to the calculated 223 nm in the gas phase. Water hydration and hydrogen bonding to G are demonstrated to have hyperchromic effect on the excitation maximum of xC. It was found that hydration blue-shifts the excitation maximum and fluorescence by 12 and 8 nm, respectively. Overall, linking to deoxyribose has a hyperchromic effect in the low energy region and a hypochromic effect in the high energy region on the absorption of xC. Similarly, base paring with G blue shifts the fluorescence of xC by 0.09 eV (9 nm), and hydration of xCG blue shift the fluorescence of xC further (by 0.19 eV,17 nm). Furthermore, the fluorescent quantum yield would be increased after hydration, linking to deoxyribose, and base paring with G. In the case of the stacked configurations, a significant decrease of the oscillator strength as well as a red shift of the dipole-allowed transition with respec(?)to free xC is observed in all cases. Because of the strong mixing of the molecular orbitals, the fluorescence quantum yield of xC are expected to be lowered in the stacked complexes due to a static quenching mechanism.更多还原