【作者】 赵欣；

【导师】 赵文杰；

【作者基本信息】 内蒙古大学 ， 化学， 2025， 硕士

【摘要】 肿瘤缺氧是实体瘤微环境的重要特征,与肿瘤进展、转移及治疗抵抗密切相关。肿瘤细胞异常增殖导致氧代谢失衡,引发缺氧微环境中偶氮还原酶（AzoR）等显著上调。AzoR作为缺氧标志物,其表达水平与肿瘤的缺氧程度密切相关,因此,开发高效、灵敏的AzoR检测技术对癌症的早期诊断和治疗监测具有重要价值。然而,现有的AzoR检测方法存在响应时间长、信噪比低等问题,限制了其在临床中的应用。有机小分子荧光探针因其结构可设计、灵敏度高、响应迅速等优势,逐渐成为缺氧检测的研究热点。基于此,本研究旨在通过取代基调控识别位点偶氮键（N=N）键亲电能力的策略,设计并合成一系列新型识别分子（Azos）及荧光探针（DCM-Azo-Rs）,实现对肿瘤缺氧微环境的高灵敏度、快速检测。具体如下:（1）围绕AzoR的底物特异性,通过取代基调控策略设计并合成了四个偶氮衍生物识别分子（Azos系列）。实验表明,在模拟缺氧条件（连二亚硫酸钠,SDT）下,相较于具有给电子取代基的识别分子Azo-p OCH₃（60 s）来说,具有吸电子取代基的识别分子Azo-t Bu与SDT的响应时间仅14 s,且在生理环境（pH=7.4）中稳定性最优。核磁共振氢谱（¹H NMR）与质谱（MS）分析揭示了SDT通过两步还原机制断裂N=N键,生成胺类化合物,为后续探针开发奠定了基础。理论计算（DFT）进一步表明,吸电子取代基（叔丁氧羰基,t Bu）通过诱导效应显著增强N=N键的亲电性,降低其键解离能,从而提升还原反应速率。本章通过系统优化反应条件,阐明了取代基效应对分子识别效率的调控机制,为高灵敏度AzoR探针的构建提供了理论依据。（2）在上述工作基础上,以具有分子内电荷转移（ICT）效应的荧光团DCM-NH₂为母体,基于ICT机制构建了系列“开-关”型荧光探针DCM-Azo-Rs。由于N=N键的高效荧光猝灭效应,探针在490 nm激发下无荧光信号。当探针被AzoR特异性识别后,偶氮键发生还原断裂,ICT通道恢复并在622 nm处产生荧光发射。通过评估探针的光谱性能及表观反应动力学参数,筛选出经t Bu修饰的优化探针DCM-Azo-t Bu。该探针展现出142 nm的大斯托克斯位移和极低的背景荧光,荧光增强315倍,响应时间缩短至10 min,检测限为0.20μM。与其他探针相比,DCM-Azo-t Bu在近生理pH条件下具有优异稳定性,同时兼具快速响应特性、良好的生物相容性和稳定的发光性能,成功应用于活细胞及缺血动物模型的实时、灵敏缺氧成像检测。更多还原

【Abstract】 Tumor hypoxia is an important characteristic of the microenvironment of solid tumors and is closely related to tumor progression,metastasis,and treatment resistance.The abnormal proliferation of tumor cells leads to an imbalance in oxygen metabolism,resulting in a significant upregulation of azoreductase（AzoR）and other factors in the hypoxic microenvironment.As a hypoxia biomarker,the expression level of AzoR is closely related to the degree of tumor hypoxia.Therefore,the development of efficient and sensitive AzoR detection techniques is of great value for the early diagnosis and treatment monitoring of cancer.However,the existing AzoR detection methods have problems such as long response time and low signal-to-noise ratio,which limit their clinical applications.Organic small-molecule fluorescent probes have gradually become a research hotspot in hypoxia detection due to their advantages such as designable structures,high sensitivity,and rapid response.Based on this,the purpose of this study is to design and synthesize a series of novel recognition molecules（Azos）and fluorescent probes（DCM-Azo-Rs）through the strategy of regulating the electrophilic ability of the azo bond（N=N）at the recognition site by substituents,so as to achieve highly sensitive and rapid detection of the hypoxic microenvironment of tumors.The details are as follows:（1）Focusing on the substrate specificity of AzoR,four azo derivative recognition molecules（Azos series）were designed and synthesized through the substituent regulation strategy.Experiments show that under simulated hypoxic conditions（sodium dithionite,SDT）,compared with the recognition molecule Azo-p OCH₃（60 s）with an electron-donating substituent,the recognition molecule Azo-t Bu with an electron-withdrawing substituent has a response time of only14 s to SDT,and it has the best stability in a physiological environment（pH=7.4）.Nuclear magnetic resonance hydrogen spectroscopy（¹H NMR）and mass spectrometry（MS）analysis revealed that SDT breaks the N=N bond through a two-step reduction mechanism to generate amine compounds,laying the foundation for the subsequent development of probes.Theoretical calculations（DFT）further show that the electron-withdrawing substituent（tert-butoxycarbonyl,t Bu）significantly enhances the electrophilicity of the N=N bond through the inductive effect and reduces its bond dissociation energy,thus increasing the rate of the reduction reaction.In this chapter,through systematic optimization of the reaction conditions,the regulatory mechanism of the substituent effect on the molecular recognition efficiency was clarified,providing a theoretical basis for the construction of highly sensitive AzoR probes.（2）Based on the above work,using the fluorophore DCM-NH₂with the intramolecular charge transfer（ICT）effect as the parent,a series of"on-off"type fluorescent probes DCM-Azo-Rs were constructed based on the ICT mechanism.Due to the highly efficient fluorescence quenching effect of the N=N bond,the probe has no fluorescence signal under excitation at 490 nm.When the probe is specifically recognized by AzoR,the azo bond is reductively cleaved,the ICT channel is restored,and fluorescence emission occurs at 622 nm.By evaluating the spectral properties and apparent reaction kinetic parameters of the probes,the optimized probe DCM-Azo-t Bu modified with t Bu was screened out.This probe exhibits a large Stokes shift of 142 nm,extremely low background fluorescence,a 315-fold fluorescence enhancement,a shortened response time to 10 min,and a detection limit of 0.20μM.Compared with other probes,DCM-Azo-t Bu has excellent stability under near-physiological pH conditions,and at the same time has the characteristics of rapid response,good biocompatibility,and stable luminescence performance.It has been successfully applied to the real-time and sensitive hypoxia imaging detection of living cells and ischemic animal models.更多还原