Fluorescent Zinc Sensors for Cellular Imaging

描述（由申请人提供）：锌作为多种金属酶的辅助因子，参与广泛的生物过程，如神经传递、基因表达、细胞凋亡和免疫系统功能。细胞内的锌大部分与蛋白质结合，胞质中游离锌的浓度极低；然而，总锌的一小部分被定位在细胞质囊泡和自由交换。有强有力的证据表明，这些囊泡在锌（ll）的细胞内调节和可能的其他重要生物过程中起着核心作用。然而，这些过程的性质和囊泡的生物学功能是难以捉摸的。由于缺乏适合于活细胞研究的锌（ll）特异性荧光探针，对囊泡锌动力学的研究一直受到阻碍。本文描述了锌（ll）特异性荧光探针的设计、合成和表征，该探针将克服现有探针的问题，使研究人员能够阐明细胞锌稳态的机制。所提出的传感器是专门为双光子激发显微镜设计的，这是一种非侵入性活细胞成像技术，可大大降低光毒性。该传感机制基于锌诱导的激发态质子转移抑制（ESIPT），这是一种新的设计概念，可提供适合定量发射比测量的大光谱位移。提出的研究填补了金属离子形态的生物化学的一个关键的利基。目前，缺乏一种非侵入性的锌特异性荧光传感器是研究细胞内锌调节和运输的一个限制因素。

DESCRIPTION (provided by applicant): Zinc serves as a cofactor in various metallo enzymes and is involved in a broad range of biological processes, such as neurotransmission, gene expression, apoptosis, and immune system functions. Much of the intracellular zinc is bound to proteins and the concentration of free zinc in the cytoplasm is extremely low; however, a fraction of the total zinc is localized in cytoplasmic vesicles and freely exchangeable. There is strong evidence, that these vesicles play a central role in the intracellular regulation of Zn(ll) and possibly other vital biological processes. However, the nature of these processes and the biological function of the vesicles are elusive. The investigation of the dynamics of vesicular zinc has been hampered in particular by the lack of Zn(ll)-specific fluorescent probes, that are suitable for live cell studies. This proposal describes the design, synthesis and characterization of Zn(ll)-specific fluorescent probes, that should overcome problems of existing probes and enable researchers to elucidate the mechanisms of cellular zinc homeostasis. The proposed sensors are specifically designed for two-photon excitation microscopy, a noninvasive live cell imaging technique that drastically reduces photoxicity. The sensing mechanism is based on zinc-induced inhibition of excited state proton transfer (ESIPT), a new design concept that provides large spectral shifts suitable for quantitative emission ratiometric measurements. The proposed research fills a critical niche in the biological chemistry of metal ion speciation. The lack of a non-invasive zinc specific fluorescent sensor is currently a limiting factor in the study of intracellular zinc regulation and trafficking.