核酸（DNA和RNA）是生物体重要的遗传物质及基因信息表达、存储和传递的载体。五种天然碱基是核酸中的主要生色团分子，其对紫外光的吸收主要在UVB和UVC波段。因此，尽管核酸具有相当高的光稳定性，但短波长紫外光仍会导致核酸的直接损伤，形成多种损伤产物，造成细胞癌变、死亡，影响生命体正常的发育、遗传、繁殖等活动。不过从另一方面考虑，这些光损伤产物在消灭病菌和治疗癌症中可发挥举足轻重的作用，常用于一些疾病的光学治疗中。对天然碱基进行修饰所形成的取代碱基对紫外光的吸收会发生红移（甚至可至UVA波段），并且其激发态反应过程不同于天然碱基，造成的光损伤也不易被生物体内酶修复且毒性较强，这些独特的性质使得取代碱基更适合作为潜在的抗癌药物分子。因此，研究取代碱基的光化学和光物理过程，是激发态化学反应动力学的前沿课题，引起诸多实验和理论工作者的广泛关注。

以尿嘧啶的取代碱基为例，卤代尿嘧啶中引入了键能较弱的碳-卤键，有可能导致光解断裂生成自由基，造成链断裂、链交联等损伤。但卤代尿嘧啶的吸收光范围仍比较有限（仅在UVB、UVC区域），且跃迁至激发三重态的可能性较小，减少了很多光化学反应的几率，因此在光化学治疗中的应用前景并不乐观。为了使碱基可吸收波长更长、组织穿透性更强的UVA波段紫外光，研究人员进一步将硫原子引入到碱基上，形成了硫代碱基。硫代碱基的系间窜越效率更高，可生成高量子产率的三重态，并继而敏化氧气产生活性氧物质单态氧（¹O₂），造成一定程度的光损伤。然而，硫代碱基仅可通过普通的光敏机制产生三重态和¹O₂来发生光化学反应，带来的光毒性仍比较有限。

为了开发新型抗癌药物，科研人员合成了一类同时具备上述两种光化学活性基团修饰的双取代碱基药物分子：5-卤代-4-硫代尿嘧啶及其衍生物。生物学的实验表明在UVA光照射下该类分子会产生DNA链断裂、DNA链间交联、DNA链与蛋白质交联等多种损伤产物并杀死癌细胞，展现出比单原子取代的尿嘧啶分子更强的光毒性，具有更优越的光化学治疗前景。然而，引发这类分子如此强的光毒性的深层机制以及涉及的关键反应中间体尚不明确。

本论文利用飞秒和纳秒时间分辨的多种瞬态吸收及发射光谱技术，结合量子化学计算，对5-溴代-4-硫代尿苷和5-碘代-4-硫代尿苷两种新型的修饰核苷分子展开其自身激发态动力学的研究，并在此基础之上研究了5-溴代-4-硫代尿苷与不同种类氨基酸之间光致交联反应的机理。主要工作包括以下三部分：

一、5-卤代-4-硫代尿苷激发态动力学研究

通过飞秒和纳秒时间分辨吸收及发射光谱实验结合量子化学计算，我们首次系统深入地研究了5-溴代-4-硫代尿苷（5Br-4TUrd）在不同波长UVA激光光照下的激发态反应过程。利用飞秒瞬态吸收光谱技术及纳秒闪光光解技术，我们直接探测到了350 nm激发下5Br-4TUrd在亚皮秒内（在水中~0.5 ps，在乙腈中~1.6 ps）生成三重态的过程，以及高量子产率的单态氧（¹O₂），并且在用5Br-4TUrd培养的细胞中检测到了¹O₂信号。其中，三重态和单态氧都是引发DNA发生链断裂等光化学损伤的主要反应活性物种。有趣的是，我们还观测到较高光子能量的UVA光（< 325 nm）可导致5Br-4TUrd在单重态上发生C-Br键解离，生成的关键中间体硫氧嘧啶自由基（4TU?）可引发链断裂或链间交联，这揭示了产生细胞光毒性的另一种涉及自由基光化学反应的机制。通过改变激发波长测量4TU?自由基信号，我们得到光解反应的能量阈值为3.6 eV，这远远低于单卤代尿嘧啶的解离所需最低能量（5 eV）。进一步通过时间分辨红外光谱实验，我们确定了4TU?自由基是一个以硫原子为中心的自由基。结合量子化学计算，我们阐述了硫原子和卤素原子调控激发态反应路径的协同作用：硫原子会显著降低激发态的能量，这导致C-Br键断裂的能量阈值也被降低，因此打开了常规碱基（核苷）极难进行的光解通道。同时，这两个重原子促进了自旋轨道耦合（Spin-orbit coupling，SOC），使得5Br-4TUrd从单重态系间窜越到三重态的过程可发生在超快时间尺度上。

此外，我们对5-碘代-4-硫代尿苷（5I-4TUrd）的激发态反应过程进行了类似的研究，发现二者的激发态反应动力学略有差异。5I-4TUrd三重态生成过程较5Br-4TUrd而言变慢，而三重态的衰减加快。并且，355 nm的光子能量即可引发5I-4TUrd的C-I键裂解，其光解能量阈值较5Br-4TUrd更低。这说明在UVA更长波段下就可导致5I-4TUrd的光解，因此在光化学治疗中，5I-4TUrd比5Br-4TUrd更适用于作为由自由基引发DNA/RNA损伤的药物分子。

通过对5Br-4TUrd、5I-4TUrd激发态动力学的研究，我们为这两类双取代核苷优越的UVA光毒性提供了深入的机理理解，并阐明了此类修饰核苷作为潜在光化学治疗药物的合理性。

二、5-卤代-4-硫代尿苷与典型氨基酸光致交联反应的机理研究

核酸与蛋白质的相互作用是生物体进行生命活动的基础, 研究二者相互作用的机制对解释生命活动具有重要的指导意义。在各种研究手段中，紫外交联免疫共沉淀技术（CLIP）和光活化核苷酸增强紫外交联免疫共沉淀技术（PAR-CLIP）被广泛应用，其中光致交联反应是这两类技术的核心。常规的核酸与蛋白质交联需在短波长紫外光下才可进行，这会导致大量副产物生成并降低交联产物的产率。为了提高光致交联反应的效率，研究人员尝试使用能够吸收长波长紫外光的修饰碱基作为可以被选择性激发的光敏剂。其中，卤代尿嘧啶生物反应活性高，常被用于引入到DNA或RNA链中与特定蛋白质发生光致交联反应，然而它的吸收波长仍较短，依然不能避免同时激发常规碱基带来的副产物。为此，研究人员寻找到对皮肤组织穿透力更强且能量更低的UVA波段有主要吸收的硫代碱基作为PAR-CLIP技术中的主要光敏剂。本论文研究的5-卤代-4-硫代尿苷相对于单取代的4-硫代尿苷来说，吸收光谱会进一步红移，且其具有硫原子和卤素原子两种官能团的性质，可能会带来更加新颖和高效的光致交联反应。

我们选择5-溴代-4-硫代尿苷（5Br-4TUrd）与两种典型的氨基酸：芳香族氨基酸色氨酸（TrpH）和直链碱性氨基酸赖氨酸（Lys）作为模型体系, 通过飞秒和纳秒时间分辨的多种瞬态吸收光谱技术探测到关键瞬态中间体，并同时结合高效液相色谱-质谱联用技术探测到最终交联产物，揭示了该核苷与氨基酸光致交联的基元反应机理。

首先，我们研究了5Br-4TUrd与氧化电势最低的芳香族氨基酸TrpH之间的光致交联反应机理。当5Br-4TUrd为中性分子时，二者反应是激发三重态机理，初始反应为5Br-4TUrd三重态与TrpH的光致电子转移反应。为了提高反应效率，我们调控二者混合溶液的pH至微碱性（~8.0），使5Br-4TUrd脱去3号位质子为阴离子形式（5Br-4TUrd^-），可与TrpH主链上氨基端的NH₃⁺通过静电作用形成复合物从而距离被拉近。结合飞秒瞬态紫外-可见吸收光谱和纳秒瞬态红外吸收光谱，我们观测到了几种关键中间体：TrpH?⁺、5Br-4TUrd?^-、5Br-4TUH?、S-4TU?及Trp?，并且通过高效液相色谱-质谱联用技术检测到了最终的交联产物有两种，交联位点在TrpH上为主链的N原子、在5Br-4TUrd上分别为S原子及嘧啶环上4号位C原子。通过对各关键中间体的时间演变过程及最终产物结构的分析，我们得出了5Br-4TUrd^-与TrpH发生光致交联反应为激发单重态机理： = 1 \* GB3 ① 5Br-4TUrd^-被UVA光激发至其单重态，并在单重态上与TrpH发生光致电子转移反应，形成5Br-4TUrd?^2-和TrpH?⁺； = 2 \* GB3 ②随后两个自由基之间发生质子转移反应，生成5Br-4TUrd?^-和Trp?； = 3 \* GB3 ③这两个自由基会继续发生一步质子转移反应，一个分支是Trp?将质子转移到S原子上，生成了5Br-4TUH?自由基；另一个分支是Trp?将质子转移到Br原子上，脱去一分子HBr，并生成S中心的4TU?自由基（S-4TU?）； = 4 \* GB3 ④随后两个自由基分别与Trp(-H)?^-发生复合，5Br-4TUH?与Trp(-H)?^-复合并脱去一分子H₂S，形成交联位点在C4-N位的交联产物；而S-4TU?直接与Trp(-H)?^-发生复合，形成交联位点在S-N位的交联产物。

其次，我们研究了5Br-4TUrd与直链型碱性氨基酸Lys之间的光致交联反应机理。通过稳态红外吸收光谱，我们同样观测到了两种交联产物，但是二者反应的机制与5Br-4TUrd和TrpH的反应有所不同。由于Lys为碱性氨基酸，所以二者混合后5Br-4TUrd即可脱质子形成阴离子形式的5Br-4TUrd^-，并与Lys中侧链氨基端NH₃⁺通过静电作用形成复合物而距离拉近。由于阴离子形式的5Br-4TUrd^-不易得电子，且Lys的氧化电势较高不易给出电子，而二者之间更容易发生质子转移，因此我们在飞秒瞬态吸收光谱上仅观测到5Br-4TUrd^-被UVA光激发至其单重态后，¹(5Br-4TUrd^-)^*经过系间窜越并得质子形成中性分子³(5Br-4TUrd)^*的过程，但未检测到二者单重态上电子转移反应生成的自由基信号。不过一旦在激发态得质子变成中性形式，生成的³(5Br-4TUrd)^*得电子能力加强、并且三重态寿命较长，可与Lys发生电子转移反应，因此我们在纳秒时间分辨红外吸收光谱中同样捕捉到了5Br-4TUrd?^-、5Br-4TUH?及S-4TU?这几种关键自由基，说明二者发生了激发三重态上的电子转移反应，并引发了后续的质子转移反应和自由基复合过程。最终两种产物的交联位点依旧在5Br-4TUrd的S原子及4号位C原子上。

综上所述，我们发现5Br-4TUrd无论是在单重态还是在三重态上，光致电子转移反应都是引发其与氨基酸发生共价交联的关键步骤。并且在后续质子转移及自由基复合过程中，5Br-4TUrd存在两个交联位点的中间体，最终生成了两种共价交联加合物。以上结果说明5Br-4TUrd在CLIP和PAR-CLIP技术中比单取代修饰核苷更具有优势：一方面，它与氨基酸的光致交联反应既可发生在单重态也可发生在三重态，这大大增加了反应几率；另一方面，它与氨基酸可形成更丰富的交联产物。本研究为5Br-4TUrd与不同类型氨基酸之间的光致共价交联反应提供了结构和机理信息，为揭示DNA/RNA与蛋白质之间光致交联反应的确切位点及涉及电子转移、质子转移的反应本质提供了直接的实验证据，为发展新型的修饰核苷作为光交联探针用于研究DNA/RNA与蛋白质之间相互作用提供了基础。

三、硫代碱基与单态氧反应的动力学研究

硫代碱基衍生物作为光敏化剂及免疫抑制剂被广泛应用。硫原子的取代使硫代碱基可吸收UVA波段紫外光，并通过系间窜越至其三重激发态，随后敏化氧气产生广泛应用于光动力治疗（Photodynamics Therapy，PDT）的活性氧物质单态氧（¹O₂）。然而，硫代碱基易与自身敏化产生的¹O₂发生氧化反应，导致光毒性。在已有关于硫代碱基与¹O₂反应机理的研究基础上，我们采用瞬态光谱技术直接测得¹O₂在1270 nm处磷光信号的衰减动力学，得到在不同溶剂中几种硫代碱基（6-硫代鸟嘌呤，4-硫代尿嘧啶，2,4-双硫代尿嘧啶，及5-溴代-4-硫代尿嘧啶）分别与¹O₂反应的速率常数。结果表明硫代碱基与¹O₂反应速率常数受溶剂的极性影响，随着溶剂极性增大，反应速率常数减小，这有利于硫代碱基在PDT中的应用。此外，通过对比研究，我们发现¹O₂与硫代嘧啶类碱基的反应速率常数比其与硫代嘌呤类碱基的速率常数小5倍左右，这说明在PDT中以硫代嘧啶类药物作为光敏剂是较为合适的选择。此外，2,4-双硫代尿嘧啶及5-溴代-4-硫代尿嘧啶比4-硫代尿嘧啶生成的¹O₂量子产率更高，且它们三者与¹O₂反应的速率常数相当，从这个角度考虑双取代嘧啶类碱基更适合在PDT中使用。这些研究结果可为PDT中选择合适的光敏药物分子提供反应动力学方面的基础信息。

Nucleic acids (DNA and RNA) are the most important genetic material of organisms in the process of development, inheritance, reproduction, etc. The five canonical bases in nucleic acids are the main chromophore molecules, which can widely absorb UVB and UVC light. Although nucleic acid has a fairly high photostability, the direct photo-damages to nucleic acid were inevitable, leading to the formation of a variety of damage products, causing carcinogenesis and apoptosis consequently. On the other hand, these photo-induced damages could be applied to eradicating germs and curing cancers, playing vital role in phototherapies of some diseases. Interestingly, modification of canonical nucleosides as photosensitizers offers a promising strategy to introduce a longer-wavelength-absorbing chromophore into DNA/RNA, exhibiting enhanced photo-reactivity exploited as antiviral, anticancer and immune-suppression drugs.

Inspired by the substitution of halogen atoms and sulfur atom, some researchers synthesized various double-substituted halogen-thio-uracil and their derivates, bearing both photochemical active functionalities of 5-halogen-uridies and 4-thiouridine. Biological experiments have demonstrated that these modified nucleobases can produce various photo-damages such as chain breakages, crosslinks between DNA/RNA chains, and crosslinks between nucleic acids and proteins under UVA light irradiation, showing stong phototoxicity. However, the intrinsic molecular mechanisms that initiate cytotoxicity of these double-substituted molecules and the key reaction intermediates involved are yet unexplored.

In this thesis, we employed femtosecond and nanosecond time-resolved transient absorption and emission spectroscopies, combined with theoretical calculations, to demonstrate the excited-state dynamics of two modified nucleosides, 5-bromo-4-thiouridine (5Br-4TUrd) and 5-iodo-4-thiouridine (5I-4TUrd), and reveal the mechanisms of photo-induced crosslinking reaction between them and different kinds of amino acids. The main results were discussed in the following three parts:

Part 1. Double Substitution Synergistically Tunes the Excited State Dynamics of 5-halogen-4-thiouridine to Enrich the Nucleoside Photochemistry

Herein, we have systematically studied the dynamics characterization of 5Br-4TUrd through femtosecond and nanosecond time-resolved spectroscopies combined with high-level ab initio calculations, for the first time.

Using femtosecond transient absorption spectroscopy and nanosecond flash photolysis, we directly detected the triplet formation process of 5Br-4TUrd in sub-picoseconds (~0.5 ps in water and ~1.6 ps in acetonitrile) under 350 nm excitation, engendering efficient formation of the long-lived reactive triplet state T₁, which subsequently sensitizes oxygen to ¹O₂ in solution and in biological cellular environments. Both triplet state and ¹O₂ are reactive species that trigger photochemical damages such as strand breaks in DNA. Surprisingly, we observed that higher photon energy UVA light (< 325 nm) can lead to C-Br bond cleavage in the singlet state of 5Br-4TUrd, resulting in the key intermediate thiouracil radical (4TU?), which can initiate cross-links or strand-beak DNA damages, revealing another mechanism that produce cellular phototoxicity. Combined with quantum-chemical calculations, we illustrate the synergistic effect of sulfur atom and halogen atom in regulating the excited state reaction pathway of 5Br-4TUrd: the sulfur atom significantly reduces the energy of the excited states, which can also lead to a lower energy threshold for C-Br bond cleavage, thus opening a unique photodissociation patyway, which is extremely difficult for the conventional nucleobases or nucleosides. As well, the two heavy atoms promote the spin-orbit coupling (SOC), so that the intersystem crossing (ISC) from the singlet state to the triplet state of 5Br-4TUrd occurs on an ultrafast time scale.

In addition, we found that the C-I bond of 5I-4TUrd can cleavage under UVA of lower energy, that its photodissociation energy threshold is lower than that of 5Br-4TUrd, impying that 5I-4TUrd is more favorable to produce 4TU? radical and concomitant photodamages.

In summary, our reslts show that thionation could tune excited states to much lower energies, acting in synergy with halogenation to tailor the excited state reactivity to the reaction of interest (e.g. UVA-induced radical production), thus potentiating significantly the photo-reactivity of the nucleosides toward various applications. These findings provide in-depth mechanistic understandings of the superior UVA phototoxicity of 5Br-4TUrd and 5I-4TUrd from dynamics perspective and shed light on the rational design of double-substituted nucleosides as prospective photochemotherapy drugs. 

Part 2. Study on the Mechanism of UVA-induced Crosslinking between 5-bromo-4-thio-uridine and Tryptophan or Lysine Using Time-resolved Spectroscopies

DNA/RNA-protein interactions are inevitably existing in many fundamental biological processes of organisms, and to study the mechanism of the interaction between nucleic acid and protein is significant for the interpretation of life activities. Among various research methods, cross-linking with immunorecipitation (CLIP) and photoactivatable ribonucleoside-enhanced cross-linking with immunoprecipitation (PAR-CLIP) are widely used, among which UV-induced cross-linking reaction is the key issue. The cross-linking reaction of conventional nucleic acids and proteins requires short-wavelength ultraviolet light, which results in the formation of a large number of by-products and reduces the yield of cross-linked products. To improve the efficiency of the photo-crosslinking reaction, the researchers tried to use modified nucleobases that absorb longer-wavelength ultraviolet light as photosensitizers that can be selectively excited. Among them, 5-halo-4-thio-uridine studied in this paper can absorb further red-shifted compared with single-substituted nuceosides, and it has the properties of two functional groups of sulfur atom and halogen atom, leading them as promising photosensitizers in PAR-CLIP technology.

In this part, we choose 5-bromo-4-thiouridine (5Br-4TUrd) and two typical amino acids: the aromatic amino acid tryptophan (TrpH) and the linear basic amino acid lysine (Lys) as model systems, to clarify the mechanism of the photo-crosslinking reaction between them. By femtosecond and nanosecond time-resolved transient absorption spectroscopies to moniter the key transient intermediates, and by HPLC-MS spectrometry to detect the final cross-linking products, we reveal the elementary reaction mechanism of the photo-crosslinking reaction between the nucleoside and amino acid.

We found that the photoinduced electron transfer (PET) reaction initiates covalent crosslinking between the singlet or triplet 5Br-4TUrd and amino acids. Tryptophan has a low oxidation potential, so it can react with both singlet and triplet 5Br-4TUrd , while Lys can only undergo electron transfer reaction with the long-lived 5Br-4TUrd triplet state. Besides, we detected two intermediates (5Br-4TUH? and S-4TU?) during the subsequent proton transfer and free radical recombination process, leading to two different covalent crosslink products, indicating that 5Br-4TUrd has higher crosslinking activity than monosubstituted 5-bomouridine or 4-thiouridine.

The results provide key information about RNA-protein photo-crosslinking specific sites and mechanism, which has advanced our knowledge on RNA-protein interactions of RBPs and RNPs in neurologic diseases, RNA-binding enzymes of intermediary metabolism and RNA-binding architectures.

Part 3. Reaction Kinetics between Thiobases and Singlet Oxygen Studied by Direct Detection of the ¹O₂ Luminescence Decay

Thiobase derivatives have received important investigations due to their wide usage as phototherapeutic agents and their potential carcinogenic side effects as immunosuppressants. The substitution of oxygen atom by the sulfur atom makes the ultraviolet absorption of thiobases redshifted and absorbs UVA light (>300 nm), resulting in unusual high quantum yield of triplet state to generate the singlet oxygen (¹O₂) through photosensitization. As a type of reactive oxygen species, ¹O₂ is highly reactive toward thiobases. Herein, we report the measurements of reaction rate constants between different thiobases and ¹O₂ in different solvents through the direct detection of ¹O₂ luminescence decay kinetics at 1270 nm. The rate constants of thiouracils with ¹O₂ are five times smaller than that of thioguanine with ¹O₂, which suggests that thiopurines are more reactive than thiopyrimidines and thus less suitable to be a photosensitive drug on the application of photodynamic therapy. Additionally, the rate constants of thiobases and ¹O₂ were found to be obviously influenced by the solvent polarity. With the increase of solvent polarity, the rate constants of thiobases and ¹O₂ decrease. Overall, these results provide a basic information on reaction kinetics for selecting suitable photosensitive drugs in photodynamic therapy (PDT).