Reversible Nitric Oxide Detention in Aqueous Solution

说明(申请人提供)：氧化镍(NO)作为一种生物信号剂发挥作用，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。一种可以直接在活细胞中检测到NO分子的探针将大大有助于阐明NO的生理作用。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，通常情况下，到目前为止已经研究过的这种类型的生物兼容系统在性质上是不可逆的，使得在活体样品中进行NO通量的时空成像是不可行的。这项拟议的研究集中在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。这种系统是为活体生物样本中NO的生物成像而设计的。我们将使用的传感策略来解决这个问题，将依赖于二硫代氨基甲酸铜(11)和二硫杂环烯(NO)络合物(NO受体)的构建，这些络合物与给电子体BODIPY荧光染料(RECTORER)连接，通过光诱导电子转移(PET)取消机制，在NO与铜(11)位结合时显示“开启”发射。最初的工作将包括基于生理上相容的L-半胱酸骨架构建不同的NO受体复合体家族。利用包括UV-Vis、FT-IR和核磁共振光谱在内的各种物理技术，将在模拟生物条件的缓冲水溶液中筛选这一系列铜(11)络合物的可逆NO结合。最适合体内检测NO的铜(11)络合物随后将被结合到BODIPY-NO受体结合物中，在没有NO的情况下，BODIPY-NO受体结合物将不发射。这些偶联物的可逆“开启”荧光反应将在生理条件下被监测。最有效的NO检测结合物将用于内源性和人工引入的NO对巨噬细胞和神经母细胞瘤细胞类型的可逆生物成像。不同外界刺激对细胞NO通量的影响也将是一个感兴趣的点，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问题。最后，使用活体海马脑片的实验将被用于绘制NO释放图，并跟踪在引入外部刺激(即嗅觉反应)时的神经信号和网络形成。这项工作与公众健康相关，因为活体样本中NO的生物成像无疑将揭示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，与心血管健康和普通肿瘤学相关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.