Fluorescent Zinc Sensors for Cellular Imaging

描述(申请人提供)：锌在各种金属酶中作为辅因子，参与广泛的生物学过程，如神经传递、基因表达、细胞凋亡和免疫系统功能。细胞内的大部分锌与蛋白质结合，细胞质中的游离锌浓度极低；然而，总锌的一小部分定位于细胞质小泡中，并可自由交换。有强有力的证据表明，这些小泡在锌(11)的细胞内调节以及可能的其他重要生物过程中发挥着核心作用。然而，这些过程的本质和囊泡的生物学功能是难以捉摸的。囊泡锌的动力学研究一直受到阻碍，特别是由于缺乏适合于活细胞研究的锌(11)特异性荧光探针。这项建议描述了锌(II)特异性荧光探针的设计、合成和表征，它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。建议的传感器是专门为双光子激发显微镜设计的，这是一种显著降低光毒性的非侵入性活细胞成像技术。该传感机制是基于锌诱导抑制激发态质子转移(ESIPT)的，这是一种新的设计概念，提供了适合于定量发射比率测量的大光谱移位。这项拟议的研究填补了金属离子形态生物化学中的一个关键领域。目前，缺乏一种非侵入性的锌特异性荧光传感器是细胞内锌调控和转运研究的一个限制因素。

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.