【作者】 李慧芳；

【导师】 步宇翔；

【作者基本信息】 山东大学 ， 理论与计算化学， 2009， 博士

【摘要】 DNA是储存、复制和传递遗传信息的主要物质基础,具有自组装、严格自我复制等优良性质。这些性质保证了其作为遗传物质的稳定性,也为其作为构建纳米级分子器件的基本结构单元提供了结构基础。但是辐射以及氧化等外界环境因素会破坏DNA的稳定性,改变DNA的碱基结构,甚至会导致DNA链状结构的断裂。如果这些破坏的碱基没有修复或者被不正确的修复,那么会直接导致细胞死亡甚至癌症的发生。作为磷酸根骨架的抗衡离子,金属离子会渗透到DNA的沟状结构中影响到DNA碱基对的性质,从而发挥其生物功能。另外随着遗传的分子机理以及DNA可以作为纳米分子器件的功能材料逐渐被认识,为了获得在某特定方面优于天然DNA性质的分子,DNA分子设计和修饰合成工作也受到了广泛的关注。本文重点讨论了金属抗衡离子渗透、氧化、辐射破坏以及扩环修饰等造成的一系列非常态DNA碱基的核磁、稳定性以及电荷迁移等性质。从而进一步的探索外界环境以及人工修饰等因素对天然DNA碱基性质的影响,并取得了一些有意义的研究成果,具体如下:1.我们首先利用密度泛函量子化学计算和分子动力学模拟相结合的方法研究了金属抗衡离子对核苷碱基对间N-H...N氢键单元中^2hJ_NN自旋偶合以及δ（¹H）/Δδ(¹⁵N)化学位移的影响。结果表明结合到天然G-C或者A-T碱基对上的金属阳离子使碱基间氢键的NMR参数发生了很大的变化,从而可以利用来识别W-C碱基对是否受到金属抗衡离子的干扰。金属离子渗透所引起的最基本的变化是使^2hJ_NN自旋偶合增大,Δδ(¹⁵N)化学位移差减小,δ（¹H）化学位移向高场方向移动。而金属离子的作用却导致A-T碱基对间N-H...N氢键的NMR参数发生了相反的变化。并且变化量的大小与所引入金属离子的路易斯碱性密切相关。对于两类碱基系列(M^z+GC和M^z+AT),计算所得到的NMR参数之间存在着很好的线性相关性,并且NMR参数值的大小取决于定位在N原子上的电子供体轨道（LPN）和电子受体σ^*（N-H）轨道之间的能隙以及二者之间的二阶微扰能。另外,^2hJ_NN自旋偶合的变化还会取决于从LP（N）自然键轨道迁移到σ^*（N-H）反键轨道上电荷量,即从嘌呤到嘧啶碱基之间的电荷迁移量。因此可以看出A-T和G-C碱基间NMR参数不同的变化趋势是由于不同的氢键类型以及金属离子的极化作用共同造成的(即:在离子化的M^z+AT系列中电荷迁移的方向是M^z+←A→T,而在离子化的M^z+GC系列中则是M^z+←G←C)。这里所预测的阳离子所引起的W-C碱基对中N-H...N氢键单元^2hJ_NN自旋偶合以及δ（¹H）/Δδ(¹⁵N)化学位移的变化可以为W-C碱基对中金属离子偶合情况的测定提供和一个新的可靠途径。2.我们还探讨了错配,氧化以及去质子等损伤碱基对间N-H...N氢键单元中NMR参数的变化。所用到的计算方法是B3LYP/6-311+G^*水平上的密度泛函方法。NMR参数计算结果表明碱基损伤会对碱基间N-H...N氢键的^2hJ_NN偶合常数以及相应的化学位移(δ（¹H）和Δδ(¹⁵N)产生很大的影响。在几类错配碱基中,有一部分的^2hJ_NN偶合作用会偏离天然碱基1-2 Hz,还有一部分由于构型的变化会造成碱基间N-H...N氢键的断裂,因此不存在^2hJ_NN偶合作用。如果一个氢原子从G-C碱基对的C碱基上或者一个氢质子从G碱基部分脱离,^2hJ_NN偶合常数会降低大约0.3-4.15 Hz。而如果一个氢原子从G碱基或者一个质子从C碱基上离去,那么^2hJ_NN偶合常数将会从原来的4.73 Hz增加到5.0-7.4 Hz。相类似的,GC^·-和GC^·+自由基中的^2hJ_NN偶合常数,即G-C碱基对直接得到或者失去一个单电子,比天然G-C碱基中的^2hJ_NN偶合常数要大3.28-6.45 Hz。结合同样受到干扰的δ（¹H）化学位移(它们的变化趋势与^2hJ_NN偶合常数相似),我们得出可以利用NMR参数的变化来判断碱基是否受到损伤。另外与前边所得到的分析结果类似,核苷间^2hJ_NN偶合作用与相应的轨道能隙以及。σ^*（N-H）轨道上的电子分布有着很好的相关性,这也表明了跨N-H...N氢键的偶合常数是受电荷迁移所控制的。也就是说,当损伤造成电荷从G碱基迁移到C碱基位置的时候,碱基间的^2hJ_NN偶合常数将会减弱。相反的,则会增大。所得到的静电势变化示意图以及G和C碱基间的电荷迁移量同样很好的证实了这个结论。我们对损伤碱基中N-H...N氢键NMR参数的变化可以很好的将损伤与天然的碱基区别开来。3.考虑到苯与杂环扩环修饰是可以提高DNA物理性质以及生物功能的合理修饰方案,我们讨论了此类非常态碱基在外界损伤环境中（即H原子离去反应,质子化和H·自由基攻击反应）所表现出来的稳定性的变化。所用到的依然是密度泛函计算方法。由于C8-G氧化物是DNA破坏中最主要的产物,并且C8位距离引入环的位置也很近,因此,我们重点讨论了扩环对C8位活性的影响。计算的结果表明,芳香环的引入会降低C8位上的电子云密度分布,从而使得C8位的质子H⁺或者H原子变得容易失去,而H·自由基的攻击反应则较天然碱基更难进行。而非芳香性环的引入则会造成不同的影响,即:C8位的去质子化反应以及H原子脱去反应会较难进行,而H·自由基的攻击则会较容易进行。为了更好的解释这个现象,我们对所考察碱基做了详细的NBO分析,并且利用可靠的NICS方法对其芳香性也进行分析。分析结果表明,由于芳香六元环的电子云密度分布小于五元芳香环,因此它的引入会引起五元咪唑环电子云密度的降低;而非芳香环由于带有两个未成对的单电子,因此起到的是电子供体特性,从而会造成G碱基中咪唑环芳香性的增大。并且引入环的芳香/非芳香性越大,影响就会越大,这也是C8位活性会受到引入环不同影响的根本原因。这里所讨论到的扩环碱基在不同DNA破坏环境中稳定性的研究可以鼓励其在进一步的实践应用方面的研究。4.最后我们描述了修饰sDNA（前缀“s”意味着扩环修饰）中空穴（正电荷）迁移的情况,即:双质子偶合空穴迁移。结合经典动力学模拟的大规模密度泛函量化计算结果表明扩环修饰DNA中最高占据轨道（HOMO）轨道的分布与天然DNA中HOMO轨道的分布一致,即,5’-sGTsGsGsG-3′序列中的HOMO最高占据轨道主要分布在sGsGsG三体的5′-sG位置,而孤立sG碱基位置则分布的很少。因此与天然DNA类似,单电子氧化和亲核攻击反应容易发生在sGsGsG三体的5′-sG位置。另外我们证实了通过孤立sG单体结构的改变可以使电荷空穴从原来的sGsGsG三体位置迁移到孤立sG单体位置（即:孤立sGC上的双质子转移现象伴随电荷沿着sDNA双螺旋结构迁移,从而导致空穴分布发生相应的变化）。这是由于双质子转移产物的电离势较原来低,使得其成为一个有效的空穴陷阱位置。这种修饰DNA中空穴迁移的现象与理论以及实验上所测得的天然DNA中双质子偶合空穴迁移的现象一致,表明了电荷也可以在修饰DNAs中的扩环鸟嘌呤之间相互跳跃。而进一步的单体计算结果表明质子转移活化能能垒以及双质子转移产物的电离势要比天然G-C碱基低。因此我们可以预测扩环修饰DNA中的双质子偶合空穴迁移要比天然DNA容易。结合扩环DNAs有着较天然DNA更强的π-π堆积力以及更低HOMO-LUMO能隙等有助于其导电的特性,扩环修饰双螺旋结构中的双质子偶合空穴迁移现象的讨论为其可以成为分子导线应用的候选材料提供了一个新的重要依据。更多还原

【Abstract】 The functions of DNA are vital for inheritance,coding for proteins and the genetic blueprint of life.With the self-assembled structure,the DNA double helix is very stable and provides potential for nanotechnological molecular-wires. However,oxidative and radiation damage from the surrounding environment may bring about significant changes in the geometry of the base pair and overall shape of the DNA strand.Then cell death or even human lesions may happen if these damaged bases are unrepaired or incorrectly repaired.Moreover,metal counter-ions may also have an impact on DNA conformation by neutralizing the charged phosphate backbone or artificial modification for special applications. My thesis addresses the effects of the metal counter-ions,oxidation and radiation damage,size-expanded modification of DNA properties,and discusses possible practical implications of our findings.Some significant progresses have been made,which can be described as follows:1.The effects of metal ion binding on the ^2hJ_NN-coupling andδ（¹H）/Δδ(¹⁵N) chemical shifts of N-H...N H-bond units in internucleotide base pairs were explored by a combination of density functional theory calculations and molecular dynamics（MD） simulations.Results indicate that the NMR parameters vary considerably upon cation binding to the natural G-C or A-T base pairs,and thus can be used to identify the status of the base pairs,if cation-perturbed.The basic trend is that cation perturbation causes ^2hJ_NN to increase,Δδ(¹⁵N) to decrease,andδ（¹H） to shift upfield for G-C,and in the opposite directions for A-T.The magnitudes of variation are closely related to the Lewis acidity of the metal ions. For both base pair series(M^z+GC and M^z+AT),these NMR parameters are linearly correlated among themselves.Their values depend strongly on the energy gaps (ΔE_LP→σ^*) and the second-order interaction energies（E（2）） between the donor N lone-pair（LP_N） and the acceptorσ_N-H^* localized NBO orbitals.In addition,the ^2hJ_NN changes are also sensitive to the amount of charge transfer from LP_N toσ_N-H^* NBOs or from the purine to the pyrimidine moieties.The different trends are a consequence of the different H-bond patterns,and thus the different charge transfer directions in the cationized M^z+ AT series,M^z+←A→T,and the cationized M^z+GC series,M^z+←G←C.The predicted cation-induced systematic trends of ^2hJ_NN andδ(¹⁵N,¹H) in N-H...N H-bond units may provide a new approach to the determination of H-bond structure and strength in Watson-Crick base pairs,and provide an alternative probe of the heterogeneity of DNA sequences.2.On the basis of the ¹⁵N NMR HN-COSY method,NMR parameters including intemucleotide ^2hJ_NN spin-spin couplings and chemical shifts(δ（¹H） andΔδ(¹⁵N)) of N-H...N H-bond units in natural and damaged base pairs(viz., mismatched pairs,GC^·- and GC^·+ radicals,dehydrogenated and deprotonated G-C pairs) were predicted using the appropriated density functional theory calculations with a large basis set.For those damaged base pairs,their ^2hJ_NN,δ（¹H） andΔδ(¹⁵N) associated the N-H...N H-bond pattern are considerably different from those of the natural canonical G-C and A-T,and may be taken as the important indexed for prejudging if G-C and A-T are damaged.Similar with the results in above work,detailed NBO analysis shows that ^2hJ_NN couplings are strongly interrelated with the energy gaps(ΔE_LP→σ^*) between the donor N lone-pair（LP_N） and the acceptorσ_N-H^* localized NBO orbitals,and also are sensitive to the electron density distributions over theσ_（N-H）^* orbital,indicating that ^2hJ_NN couplings across the N-H...N H-bonds are charge-transfer-controlled.This is well supported by variation of the electrostatic potential surfaces and corresponding charge transfer amount between G and C moieties.The present data indicate that measurements of NMR parameters associated with the N-H...N H-bond may be used to discriminate between natural G-C pair and the radiation-damaged G-C pairs.3.Considering homo/hetero ring-expanded DNA analogues are rationally-modified DNA motifs with improved physical or biological properties, the stability of these artificial DNA base pairs was examined with regard to three aspects associated with DNA damage;namely deprotonation,H-abstraction and H^·-radical addition using density functional theory.The effect of size-expansion on C8 activity was investigated because C8-oxidative guanine（G） is one of the most important products of DNA damage.Computational results indicate that the insertion of an aromatic spacer ring in G considerably decreases the electron density over the C8 site,leading to easier deprotonation or H-abstraction from the C8 site and more difficult H^·-radical attack on the C8 site.However,the opposite phenomenon is observed if the spacer ring is antiaromatic,because of the increased electron density over the C8 site.Moreover,these effects are more prominent the larger the aromaticity or antiaromaticity of the spacer ring. Further analyses,using natural bond orbitals and the nucleus-independent chemical shift index of aromaticity,indicate that the changes of the electron distribution over the C8 site arise because the aromatic spacer ring,involved in conjugation structure,increases the electron delocalization from the electron-rich imidazole ring to the diatropic six-membered rings,while the antiaromatic spacer ring acts as an electron-donating group,not only inhibiting the above electron delocalization,but also slightly increasing the electron density over the C8 site. The improved stability of these size-expanded bases pairs in different DNA-damaged environments may encourage their use in practical applications.4.Electron hole（radical cation） migration in rationally-designed size-expanded DNAs（named sDNA here）,where the quantum transport of an injected charge is coupled with the double proton transfer in isolated sGC pair,is described here.Classical molecular dynamics simulations in conjunction with large-scale density functional theory calculations reveal that the distribution of highest occupied molecular orbital（HOMO） in size-expanded duplex sDNA is similar with that in B-form DNA,that is,the HOMO of stacked 5’-sGTsGsGsG-3’ sequence is especially high in energy and largely localized on the 5’-sG of the sGsGsG triplet,with no HOMO at the single sG base.As a consequence, one-electron oxidation and electrophilic attacks are favored at the 5’-sG site of the sGsGsG triplet as that in natural DNA duplex.It is also demonstrated that a charge sink from the sGsGsG triplet can be created by modification of the isolated sG base（viz.,double proton transfer in the isolated sGC pair induces correlated changes in the special distribution of the hole,with concomitant charge transport along the sDNA double helix）.The ion potential（IP） energy of the sG tautomer generated by double proton transfer is lower than that of an isolated sG,thus making the site an effective hole trap.The charge sink phenomenon in sDNA matches very well with theoretical and experimental research for the double proton coupled charge transfer in natural DNA base pairs（Gervasio et al.Angew. Chem.Int.Ed.2006,45,5606）,implying that the charge can hop reversibly between all size-expanded guanines in sDNA sequences containing isolated sGC sites between the source and sink.Moreover,future detailed calculation results show that the proton transfer activity energies（Ea） as well as IPs of the sGC base pairs are lower than those of natural G-C base pair.That means the double proton coupled charge transfer in size-expanded DNAs is easier than that in natural DNA double helix.Double proton coupled proton transfer discussed here provide a new evidence that the size-expanded DNA double helix can also mediates charge transport over a distance.Associated with the fact that the size-expanded DNAs have strongerπ-πstacking interactions and lower HOMO-LUMO gaps than natural DNAs,it can be concluded that size-expanded DNAs could be a good plausible candidate for molecular-wire application,which is not directly accessible to experimental probes.更多还原