Reversible Nitric Oxide Detention in Aqueous Solution

描述（由申请人提供）：一氧化氮（NO）作为一种生物信号剂，根据各种尚未完全描述的因素，调节有益和有害的生物过程。阐明一氧化氮的生理作用将大大受益于一种可以直接在活细胞中检测到分子的探针。基于荧光的方法提供了一种可能的方法来满足这些要求，然而，一般来说，迄今为止所研究的这种类型的生物相容系统本质上是不可逆的，这使得活样品中NO通量的时空成像不可行。提出的研究重点是开发一种基于分子荧光的一氧化氮（NO）传感器，该传感器能够在生理条件下可逆地发挥作用。这种系统被设计用于活体生物样品中NO的生物成像。我们将采用传感策略来解决这一问题，将依赖于铜（ll）二硫代氨基甲酸酯和二硫代烯配合物（NO受体）的构建，这些配合物（NO受体）连接到供电子的BODIPY荧光染料（报告者）上，当NO结合到铜（ll）位点时，通过光诱导电子转移（PET）消除机制显示“开启”发射。最初的工作将涉及基于生理上兼容的l -半胱酸骨架的多种NO受体复合物家族的构建。该铜（ll）配合物阵列将在复制生物条件的缓冲水溶液中筛选可逆NO结合，使用各种物理技术，包括UV-Vis， FT-IR和NMR光谱。随后，最适合体内NO检测的铜（ll）配合物将被纳入BODIPY-NO受体偶联物中，该偶联物在没有NO的情况下将是非发射的。这些偶联物的可逆“开启”荧光反应将在生理条件下进行监测。最有效的NO检测偶联物将用于巨噬细胞和神经母细胞瘤细胞类型的内源性和人工引入NO的可逆生物成像。各种外部刺激对细胞NO通量的影响也将是一个有趣的点，健康细胞和癌细胞中NO通量的时空特征的比较也将是一个有趣的点。最后，使用活海马脑切片的实验将用于绘制NO释放图，并跟踪引入外部刺激（即嗅觉反应）时的神经信号和网络形成。这项工作与公共卫生有关，因为活样本中NO的生物成像无疑将阐明该分子作为调节和致病因子的作用。除了与神经功能相关外，对心血管健康和一般肿瘤学的影响也非常令人感兴趣。

DESCRIPTION (provided by applicant): Nitic oxide (NO) operates as a biological signalling agent, which regulates both beneficial and harmful biological processes, depending on a variety of not yet fully delineated factors. Elucidation of the physiological roles of NO would benefit substantially from a probe that can detect the molecule directly in living cells. Fluorescence-based methodologies offer one possible approach to satisfy these requirements, however, in general, biologically compatible systems of this type, which have been studied to date are irreversible in nature, making the spatiotemporal imaging of NO flux in live samples unfeasible. The proposed research is centered on the development of a molecular fluorescence-based sensors for nitric oxide (NO) that are capable of functioning reversibly under physiological conditions. Such systems are being designed for the bioimaging of NO in living biological samples. The sensing strategy we will employ to address this issue, will rely on the construction of copper(ll) dithiocarbamate and dithiolene complexes (NO receptors) tethered to electron donating BODIPY fluorescent dyes (reporters), which display "turn-on" emission upon NO binding to the copper(ll) site via a photoinduced electron-transfer (PET) abolition mechanism. Initial work will involve the construction of a diverse family of NO receptor complexes based on a physiologically compatible L-cysteic acid backbone. This array of copper(ll) complexes will be screened for reversible NO binding in buffered aqueous solutions that replicate biological conditions, using various physical techniques including UV-Vis, FT-IR and NMR spectroscopies. The copper(ll) complexes best suited for NO detection in-vivo will subsequently be incorporated into BODIPY-NO receptor conjugates, which will be nonemissive in the absence of NO. The reversible "turn-on" fluorescence response of these conjugates will be monitored under physiological conditions. The most efficacious NO-detection conjugates will be employed in the reversible bioimaging of both endogenous and artificially introduced NO for both macrophage and neuroblastoma cell types. The effect of various external stimuli on cellular NO flux will also be a point of interest as will the comparison of the spatiotemporal characteristics of NO flux in healthy and cancer cells. Finally, experiments using live hippocampal brain slices will be used to map NO release and track neural signaling and network formation upon introduction of external stimuli (i.e. olfactory response). This work is relevant to public health, as the bioimaging of NO in living samples will undoubtedly shed light on the molecule's role as a regulatory and pathogenic agent. In addition to being pertinent to neurological function, implications with respect to cardiovascular health and general oncology are also of extreme interest.