可逆、比率型硒代半胱氨酸荧光探针的构建及生物应用

Selenocysteine (Sec) is the 21st proteinogenic amino acid in the organism, and both its deficiency and excess would increase the incidence of diseases such as cancer and type 2 diabetes. Real-time detection of Sec in situ in living cells will greatly promote its pathological roles study in the development and progression of cancer. However, the irreversible reactions of the present reported fluorescent probes with Sec blocked their application in Sec dynamic imaging in cells. At the same time, barely ratiometric fluorescent probes was reported. These fluorescent probes with “turn-on” fluorescent responses might present "false result" caused by the distributional heterogeneity of the probe in cells. In view of the above shortcomings, this project intends to proceed from the following two aspects: Firstly, fluorophores such as coumarin conjugated α, β-unsaturated ketones are used as recognition sites to construct ICT regulated ratiometric fluorescent probes. The reversible reaction between α, β-unsaturated ketones and -SeH promotes the real-time Sec detection. Secondly, replacing the diethylamine group in the fluorophores with azetidine group to improve their quantum yield, photostability and sensitivity toward Sec. These features allow the probes to detect the inherent Sec in living cells. The relationship between probe structures and their Sec detection performance will be evaluated systematically to provide powerful fluorescent tools for the pathology study of selenium in cancer.

硒代半胱氨酸（Sec）是生物体内基因编码的第21种蛋白原氨基酸，其缺乏和过剩均会增加癌症、二型糖尿病等疾病的发病率。对细胞中Sec浓度的实时、原位检测将极大地促进其在癌症发病、发展过程中的作用研究。但是，现有荧光探针与Sec的反应不可逆，无法检测细胞中Sec浓度的动态变化。同时，比率型Sec荧光探针匮乏，难以克服探针在细胞内的分布差异而造成的“假性结果”。针对上述不足，本项目拟从以下两方面着手：第一，以香豆素等染料为荧光团，以与之共轭的α，β-不饱和酮为识别位点，构建ICT调控的比率型荧光探针，利用α，β-不饱和酮与-SeH的可逆反应实现对Sec的实时检测。第二，以氮杂环丁烷取代传统染料中的二乙胺基等基团，提高探针的量子产率、光稳定性和对Sec检测的灵敏性，实现对细胞中固有Sec的检测。系统地评估探针结构与对Sec检测性能的关系，为硒的癌症病理学作用研究提供有力的可视化工具。

本项目针对硒代半胱氨酸的特异性比率型荧光检测，以α,β-不饱和酮为反应位点，香豆素、花菁等荧光染料作为荧光团构建荧光探针。项目按照拟定的研究工作计划开展，合成了系列荧光探针，并对其光物理性质、化学性质进行了系统测试分析。我们发现，α,β-不饱和酮不能很好地在半胱氨酸共存的条件下实现对硒代半胱氨酸的高特异性响应。这是由于，尽管硒代半胱氨酸的活性基团硒原子的亲核加成反应活性较半胱氨酸中的硫原子高，但其显著增大的原子半径受到反应位点空间位阻的影响而无法优先与荧光探针反应而表现特异的荧光信号。因此，我们基于本项目中制备得到的系列荧光探针，对其用于荧光传感的构效关系进行了更有意义的探索。具体研究成果包括：利用α,β-不饱和酮的亲电反应活性调控，实现了对血清中结构极为相似的半胱氨酸和同型半胱氨酸的定量荧光分析；得到了对细胞、组织等生物样本中半胱氨酸大幅度浓度变化的特异性荧光监测探针；利用α,β-不饱和酮与赖氨酸中氨基的缩合反应用于对人血清白蛋白的共价荧光化修饰，并实现了对常用临床药物血药浓度的非破坏性原位监测；对常规花菁染料进行工程化改造，将其Stokes位移扩大了10倍，并用于高信噪比活体荧光成像。