基于多肽邻位氨基与羧基的荧光激活型探针的构建及应用研究

Organic dyes with the advantages of multicolor, high brightness and high photo-stability have been an indispensable and important tool for protein labeling. Aiming at the defect of the traditional peptide-tag and organic dyes with weak binding ability, unspecific labeling of proteins and turn-on, this program proposes to develop a new kind of fluorogenic probes in which the probes covalently bond to target peptide-tag’s proximity amino and carboxyl as well as fluorescent turn-on. The fluorogenic probe are constructed by conjugating the aldehyde functionalized fluorophore and peptide-tag ligand and the aldehyde group acts as both a fluorescence quencher and reactive group which could be form imine group with proximity amino group. The proximity carboxyl group not only catalyze the formation of imines, but also protonated imide in situ formed cyanine dye accompanying with the fluorescence turn-on. Compared with the current techniques, this method is not only suitable for labeling N-terminal and N-terminal, but also suitable for protein domain, as well as suitable for reducing environment and oxidizing environment. In addition, it could be realized the modular selection of fluorophores and ligands. Furthermore, these fluorogenic probes would have rapid labeling rate and high specificity and without any leaving group liberation during the labeling process, which are expected to realized proteins trace, quantification and detection. These probes also have the potential application in protein-protein interaction, discover new target protein and drug-assisted screening and would provide a new tool for understanding the life process of proteins and explicating the related major diseases and pathogenesis.

有机染料具有多色、亮度高及稳定性强等优势，成为研究蛋白不可或缺的工具。本项目针对传统多肽标签与染料对之间结合能力弱，以及特异性点亮缺失的瓶颈，提出发展基于多肽“邻位氨基与羧基”共价标记并同步荧光激活的荧光探针。拟用醛基功能化染料连接配体作为荧光生成型探针，其中，醛基不但作为染料的荧光淬灭基团，还作为与邻位氨基共价反应为亚胺的基团，邻位羧基不但催化亚胺形成，还质子化亚胺形成半菁染料，实现共价标记并同步荧光激活。与当前相关技术相比，该方法不但适用于蛋白C端和N端的标记，还适用于蛋白结构域的标记，不但适用于还原性环境，还适用于氧化性环境；能够实现配体和荧光团的模块化构建，另外，该类探针标记速度快、特异性强，标记过程无游离基团释放；因此，该探针有望实现对蛋白的示踪、定量、检测，并适用于蛋白与蛋白相互作用、发现新的靶标蛋白和辅助药物筛选，为认识蛋白的生命过程、阐述相关重大疾病的发病机理提供新的工具。

荧光标记技术已成为蛋白质研究的重要工具。本项目旨在设计合成特异性强、标记 快、生物相容性好以及背景低的荧光探针，实现蛋白和生物活性分子的特异性标记、示踪以及定量检测，为认识蛋白质的生命过程，阐述相关重大疾病和发病机理提供新的工具。通过该项目的实施，目前取得如下主要成果：1）针对现有光反应活性基团光反应活性低、中间体不稳定、没有选择性等问题，利用邻硝基苄醇 (NB) 作为光反应活性基团，实现了复杂体系中的蛋白时空可控性的共价结合。2）基于TICT的机制构建了一系列光学性质可控调节（波长从蓝光至红光谱系）的荧光探针分子，结合蛋白特异性标签后，所得探针分子可同时实现细胞内外特异性的蛋白标记、示踪及定量检测；3）基于激活荧光机制构建了一系列光学性质可调（波长从绿光到近红外光）的荧光探针，在与目标蛋白结合前几乎没有荧光，而当与目标蛋白结合时，探针因结构的变换发出荧光，从而实现对目标蛋白的共价荧光标记；