Fluorescent Sensors to Investigate Zinc Neurochemistry

描述(申请人提供)：本研究的长期目标是 为锌提供明亮的荧光传感器，以研究其神经化学。 锌以高浓度存在于突触前神经元的小泡中 并被释放到突触裂隙中以响应 生理信号。我们假设这种锌的释放可以用来测绘 通过遵循时间和位置模式的神经网络 刺激后发生的荧光变化。无节制地释放 例如，神经元锌对缺血的反应会导致锌诱导的死亡 大脑皮层神经元。这里设计的传感器将提供一个强大的工具 追踪被怀疑与此类事件相关的锌水平以及 神经系统疾病，包括家族性肌萎缩侧索硬化症和 阿尔茨海泽默氏病。该提案的重点是设计和合成 用于选择性锌结合的三类配体，每一类都产生一个 荧光反应。传感器都是荧光素的衍生物，选择 由于它具有高量子产额，长波长激发和发射特性， 以及被化学操纵的能力。第一类配体的改进 锌离子结合时的荧光亮度，它被猝灭 光诱导电子转移(PET)，直到锌结合恢复它。这一种 传感器的代表是与“Zinpyr”家族的初步工作 4‘和5’位上含有荧光素官能化的分子 具有双(2-吡啶甲基)氨基甲基锌结合基团。第二种方法 通过锌与氮原子的配位提供有比例的荧光团 一种罗丹明/荧光素混合骨架，我们将其命名为“罗丹明” 配基。在这里，未绑定和绑定的传感器都会发出荧光，但在 不同的波长。要合成的第三类分子和 已研究将锌结合部分定位为悬垂之间的间隔物 发生共振能量转移(ET)的荧光给体/受体对 锌结合时效率更高。所有的合成路线都是模块化的 收敛，允许锌离子结合单位的系统变化以 可获得广泛的离解常数和溶解度性质。这个 结构、生成常数、生成速率和解离速率， 锌的溶解度、溶液稳定性和荧光寿命 这些传感器的复合体将被研究。它们的细胞定位 将通过单光子和双光子显微镜方法进行研究。一种战略 将传感器连接到突触后神经元的细胞外表面以 在突触激发后监测锌的到达将被追踪。

DESCRIPTION (provided by applicant): The long-term goal of this research is to provide bright fluorescent sensors for zinc to investigate its neurochemistry. Zinc occurs at high concentrations in vesicles located in presynaptic neurons of the hippocampus and is released into the synaptic cleft in response to a physiological signal. We hypothesize that such zinc release can be used to map neural networks by following the temporal and positional pattern of fluorescence changes that occur following stimulation. Uncontrolled release of neuronal zinc, for example in response to ischemia, leads to Zn-induced death of cortical neurons. The sensors devised here will provide a powerful tool for tracking zinc levels suspected to correlate with such events as well as neurological diseases, including familial amyotrophic lateral sclerosis and Alzheizemer's disease. The proposal focuses on the design and synthesis of three classes of ligands for selective zinc binding, each giving rise to a fluorescent response. The sensors are all derivatives of fluorescein, chosen for its high quantum yield, long wavelength excitation and emission properties, and ability to be manipulated chemically. The first class of ligands improves the brightness of the fluorescence upon Zn2+-binding, which is quenched by photoinduced electron transfer (PET) until zinc binding restores it. This kind of sensor is typified by preliminary work with the "Zinpyr" family of molecules, which contain fluorescein functionalized at the 4' and 5' positions with bis(2-pyridylmethyl)-aminomethyl zinc-binding moieties. A second approach affords ratioable fluorophores by coordination of zinc to the nitrogen atom of a hybrid rhodamine/fluorescein skeleton that we designate as "rhodafluor" ligands. Here, both the unbound and bound sensors fluoresce, but emit at different wavelengths. The third class of molecules to be synthesized and investigated positions the zinc-binding moiety as a spacer between pendant fluorescent donor/acceptor pairs that undergo resonance energy transfer (ET) more efficiently upon zinc binding. All the synthetic routes are modular and convergent, allowing for systematic variation of the Zn2+-binding unit to access a wide range of dissociation constants and solubility properties. The structures, formation constants, rates of formation and dissociation, solubility, solution stability, and fluorescence lifetimes of the zinc complexes of these sensors will be investigated. Their cellular localization will be studied by one- and two-photon microscopic methods. A strategy for attaching the sensors to the extracellular surface of post-synaptic neurons to monitor zinc arrival after synaptic firing will be pursued.