论文总字数：26273字

摘 要

Abstract IV

第一章 综述 1

1.1引言 1

1.2荧光探针简介 1

1.2.1荧光产生机理 1

1.2.2荧光探针设计原理 2

1.2.2.1荧光基团—（连接基团）—识别基团 2

1.2.2.2取代置换法 2

1.2.2.3化学计量器法 3

1.2.2.4荧光探针分子对接（Docking）模拟技术 3

1.2.3荧光探针响应原理 4

1.2.3.1荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET） 4

1.2.3.2分子内电荷转移（Intramolecular Charge Transfer, ICT） 4

1.2.3.3光诱导电子转移（Photoinduced Electron Transfer, PET） 5

1.2.4双光子荧光成像技术 6

1.3 NTR 8

1.3.1 NTR的分类及性质 8

1.3.2 荧光NTR探针研究进展 8

1.4本论文的研究思路和研究方法 12

第二章 实验材料与实验方法 13

2.1实验材料 13

2.2探针设计方法：分子对接模拟技术（Docking） 15

2.3探针合成方法 15

2.4探针表征方法 15

2.5探针性能测试方法 15

2.5.1测试吸收和激发所在波长 16

2.5.2反应时间动力学性能测定 16

2.5.3浓度梯度测试 16

2.5.4选择性测试 16

2.5.5生物成像测试 17

2.5.5.1肝癌细胞培养、着色 17

2.5.5.2细胞毒性测试 17

2.5.5.3共聚焦双光子显微成像测试 17

第三章 结果与分析 18

3.1 Docking结果分析 18

3.2 β-萘酚类荧光NTR探针的合成及表征(见于附录) 19

3.3性能测试结果分析 22

3.3.1荧光探针响应机理 22

3.3.2荧光光谱性质 23

3.3.3时间动力学实验 24

3.3.4浓度梯度测试 25

3.3.5选择性测试 27

3.3.6细胞毒性测试 28

3.3.7共聚焦双光子显微成像 29

第四章 结论与展望 31

4.1结论 31

4.2展望 32

本科期间研究成果 33

参考文献 34

附录 38

致谢 45

近红外双光子荧光NTR探针的设计、合成与应用

摘要

硝基还原酶（Nitroreductase, NTR）是黄素酶类氧化还原酶中的一种，其结构中包含黄素单核苷酸单元或黄素腺嘌呤二核苷酸单元，因其在肿瘤细胞中会因细胞缺氧而过度表达，可作为肿瘤细胞的低氧标志物之一。它能够利用烟酰胺嘌吟二核苷酸（Nicotinamide Adenine Dinucleotide Phosphate, NADPH）烟酰胺嘌吟二核苷酸磷酸（Nicotinamide Adenine Dinucleotide, NADH）作为辅酶，实现对多种硝基化合物的还原代谢，将硝基还原为对应的亚硝酸、羟胺或氨基酸衍生物。依据这种还原行为可以设计出含有硝基官能团的荧光探针用于实体瘤细胞中缺氧状况的检测，对于指示肿瘤细胞的存在与否具有重要的研究意义。

小分子荧光探针是一类能够将分子间的相互作用转化为可以用于检测的信号传递给外界的工具，由于其具备化学稳定性高、生物兼容性好、生物选择性好、灵敏度高、响应快等诸多特点，在化学、医药及生物等领域广泛应用。

目前已有基于NTR而设计的小分子双光子荧光探针报道，但是这些探针均存在双光子吸收界面小，反应活性低，未能在疾病模型中验证探针适用性等缺点。本设计拟在6-（二甲基氨基）-3 - （（4-硝基苄基）氧基）-2-萘甲醛的醛基位接上丙二酸二甲酯，形成推-拉电子体系，合成新型荧光NTR探针，并研究了该荧光探针在体外的光学性能，为进一步开发新一代的近红外双光子荧光NTR探针提供参考。

关键词：NTR 双光子荧光 小分子探针 分子对接

Design, Synthesis and Application of Near-infrared Two-photon Fluorogenic NTR Probes

Abstract

Nitroreductase (NTR) belongs to the family of flavin-containing enzymes, which contain flavin mononucleotide units or flavin adenine dinucleotide units. Since it is overexpressed in hypoxic tumors, it can be used as one of the hypoxic markers of tumor cells. It can use Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Nicotinamide Adenine Dinucleotide (NADH) as a coenzyme to achieve the reduction and metabolism of various nitro compounds.NTR can catalyze the reduction of nitroaromatic compounds to the corresponding nitrous acid, hydroxylamine or amino acid derivatives. On the basis of the reduction behavior of NTR, fluorescent probes with the nitro functional groups can be designed to detect the hypoxic status of cancer cells, which is of great importance on clinical diagnosis of cancers.

Small molecule fluorogenic probes are tools that can convert molecular interactions into signals that can be used for detection. Small molecule fluorescent probes have been widely used in the chemical, medical and biological fields, due to their following characteristics, high chemical stability, good biocompatibility, good selectivity, high sensitivity, and short response time.

At present, an amount of two-photon small molecule fluorogenic probes for nitroreductase have been reported, but most of them have the disadvantage as small two-photon action cross sections, lower reaction activity with NTR and never showing evidence of disease model results.This design is based on the structure of 6-(dimethylamino)-3-((4-nitrobenzyl)oxy)-2-naphthaldehyde. The aldehyde position was connected with dimethyl malonate to form an electron push-pull system to synthesize a nitroreductase fluorescent probe. The optical properties in vitro of the fluorescent probe were studied, which provided a reference for the further development of a new generation of near-infrared two-photon fluorescence NTR probe.

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