基于“激发态扭转引起的分子内质子转移（ESTIPT）”机理构建通用型蛋白比率荧光探针

Fluorescent environment-sensitive probes are specially designed dyes that change their fluorescence intensity or emission wavelength in response to change in their microenvironment polarity, viscosity, or molecular order. These probes are widely used to study protein-small molecule interactions, such as protein sensing and high throughput drug screening, by covalently linking small molecule to these dyes which are sensitive to the microenvironment around the binding site of Protein-small molecule interactions. These environmental parameters, including polarity, viscosity and molecular order, are not always responsive to changes in the response of protein-small molecule interactions. As a result, the design of environment-sensitive protein probe needs to be screened by the combination of environmental physical parameter sensitive dye and response mechanisms. Recently, the applicant discovered an excited state torsion controlled by water in the formation of intramolecular hydrogen bonds. The intramolecular proton transfer process ESTIPT, It is shown that the ratio fluorescence changes of ESTIPT are controlled in water and non-aqueous environment. We have successfully realized the ratio recognition and imaging of carbonic anhydrase and the screening of drugs in living cell by using ESTIPT fluorescent dyes which are sensitive to microenvironment "water parameter". The "water parameter" is ubiquitous in the microenvironment of the action of protein-small molecule interactions. In this project, we constructed ratiometric fluorescent probes for proteins based on ESTIPT sensitive dyes by connecting different targeted small molecules on these dyes.

环境敏感荧光染料在共价连接的靶向小分子与目标蛋白结合前后识别所处微环境的改变，以荧光强度或波长改变作为荧光信号响应蛋白与小分子的相互作用，用于蛋白识别和药物筛选等领域。这类染料所识别的微环境指的是极性、粘度或分子构型的改变，而这些环境参数并不是每一种蛋白小分子作用都会引起响应变化，造成了设计环境敏感型蛋白探针需要采用“环境物理参数敏感染料-响应机制”组合方式筛选获得。近期申请人发现了一种水控制分子内氢键形成的激发态扭转引起的分子内质子转移（ESTIPT）过程，表现为在水与非水环境中受控于ESTIPT发生比率荧光变化。我们利用微环境“水参数”敏感的ESTIPT荧光染料成功实现了对碳酸酐酶的比率识别、荧光成像和活细胞药物筛选。考虑到“水参数”在蛋白小分子作用微环境变化中普遍存在，本项目我们通过在染料上连接不同的靶向小分子，基于ESTIPT敏感染料构建通用型蛋白比率荧光探针。

蛋白质与小分子相互作用的研究对于药物开发、蛋白结构与功能研究、蛋白的示踪、浓度检测具有重要意义。因此，在分子水平上监测蛋白质与小分子相互作用也逐步成为了毒理学、化学、分子生物学及临床医学的重要研究领域。环境敏感型荧光染料能够在结合目标蛋白前后识别所处微环境的变化，并将荧光强度或波长的改变作为荧光信号来响应蛋白与小分子之间的相互作用。此类蛋白荧光探针是当前化学生物学的研究热点之一，已成功应用在蛋白识别、药物筛选、蛋白折叠和蛋白蛋白相互作用等多个领域。然而，这类环境敏感型荧光探针仍然面临两点巨大挑战：一是单个环境敏感型荧光染料难以做到对多种不同蛋白的识别，缺少通用性，使得设计环境敏感型蛋白探针只能依赖 “环境物理参数敏感染料-响应机制”组合来进行筛选；二是目前环境敏感型染料以荧光增强为主要的响应模式，而荧光比率变化的蛋白探针报道极少，难以实现活细胞内对目标蛋白实时、定量识别与原位成像。因此，如何构建通用型的蛋白比率荧光探针依然是目前的一个挑战。本项目提出水控制分子内氢键形成的激发态扭转引起的分子内质子转移（ESTIPT）过程，这类染料在水与非水环境中受控于ESTIPT发生比率荧光变化，从而达到对目标蛋白的比率识别。我们首先基于萘酰亚胺母体连接了多种靶向基团实现了对不同目标蛋白的比率识别与荧光比率成像，首次实现了对碳酸酐酶、Halo-tag、SNAP-tag等蛋白的比率成像。同时，在活细胞内对不同碳酸酐酶抑制剂的活性进行了解析，有望实现抑制剂的快速筛选。最终，将ESTIPT响应基团引入多种荧光团中，均实现了荧光的比率变化，建立了多色比率荧光探针库用于通用型蛋白探针的构建。