Developing fluorescent probes for the endogenous gaseous transmitters NO and H2S

描述（由申请人提供）：候选人获得博士学位。来自加州大学伯克利分校，在肯尼思·雷蒙德和罗伯特·伯格曼的共同指导下，他研究了水溶性超分子复合物的主客体化学、分子识别和催化。他目前是美国国立卫生研究院 (NIH) 博士后研究员，在麻省理工学院史蒂芬·利帕德 (Stephen Lippard) 实验室工作，致力于开发一氧化氮荧光探针。该候选人的研究兴趣涵盖分子识别领域，特别关注微观过程如何导致单个原子、官能团和分子的识别。候选人将利用他在机械研究和分子识别方面的背景来追求他作为首席研究员的研究兴趣。他的独立研究将侧重于开发用于生物学中小分子检测和成像的新工具。 一氧化氮 (NO) 和硫化氢 (H2S) 现在被认为是生物学上重要的气体传送器。 NO 和 H2S 都是内源性产生的，并受到身体的精细调节。一氧化氮在低细胞浓度下有利于血管舒张和免疫活性，但过量产生会导致反应性一氧化氮种类的增殖，这些活性一氧化氮与致癌和多种退行性神经系统疾病有关，包括阿尔茨海默病（AD）、帕金森病、亨廷顿病以及多发性硬化症。同样，H2S 也与 AD、唐氏综合症和其他形式的金属缺乏症有关。 H2S 在炎症和血压调节中也发挥着积极作用。尽管这两种气体发射器的重要性已得到公认，但目前在活细胞中进行检测的方法仍然有限。 拟议研究的博士后阶段将重点开发新型 NO 选择性荧光探针，以解决目前 NO 检测的局限性。基于过渡金属的 NO 结合位点将用于开发可以可逆地结合 NO 的探针和在 NIR 中发射的探针。所提出的荧光探针系列将使用顺磁性 (S=1/2) 金属，充当荧光猝灭剂和 NO 结合位点的双重作用。 NO 的配位将形成抗磁性 (S=0) 复合物并恢复悬垂荧光团的荧光。此类复合物与溶解的单壁碳纳米管 (SWNT) 的吸附或共价连接将用于开发在 NIR 中发射的 NO 选择性探针。 拟议研究的独立研究阶段将研究用于内源产生的 H2S 成像的 H2S 选择性荧光探针的设计。目前，缺乏此类 H2S 检测方法，并且大多数测量依赖于大块组织测量。用于活细胞的新型 H2S 选择性荧光探针将为研究 H2S 的生物学功能提供急需的工具。 H2S 独特的物理性质将在 H2S 选择性荧光探针的设计中得到利用。荧光团将用专门设计的保护基团进行衍生，这些保护基团只能通过 H2S 去除。去除荧光团保护基团将恢复荧光，从而形成 H2S 的开启探针。 公共健康相关性：一氧化氮 (NO) 和硫化氢 (H2S) 均已被确定为人体内重要的内源性气体递质，并与致癌、高血压和多种神经系统疾病（包括阿尔茨海默病、帕金森病、唐氏综合症和多发性硬化症）有关。尽管存在这种兴趣，但目前很少有方法可以检测或成像这些小分子递质的细胞内水平。该提案提出了 NO 和 H2S 荧光探针的设计，可以对活细胞中的这些内源性气体进行选择性检测和成像。

DESCRIPTION (provided by applicant): The candidate received his Ph.D. from the UC Berkeley, under the joint direction of Kenneth Raymond and Robert Bergman where he studied host-guest chemistry, molecular recognition, and catalysis in water-soluble supramolecular complexes. He is currently an NIH postdoctoral fellow in Stephen Lippard's laboratory at MIT working on developing fluorescent probes for nitric oxide. The candidate's research interests span the field of molecular recognition with a specific focus on how microscopic processes lead to the recognition of individual atoms, functional groups and molecules. The candidate will use his background in mechanistic studies and molecular recognition to pursue his research interests as a principle investigator. His independent research will focus on the development of new tools for the detection and imaging of small molecules in biology. Nitric oxide (NO) and hydrogen sulfide (H2S) are now accepted as biologically important gaseous transmitters. Both NO and H2S are produced endogenously and are finely regulated by the body. Nitric oxide is beneficial for vasodilation and immune activity at low cellular concentrations but overproduction can lead to the proliferation of reactive NO species that have been implicated in carcinogenesis and several degenerative neurological disorders, including Alzheimer's (AD), Parkinson's, and Huntington's disease, as well as multiple sclerosis. Similarly, H2S has been implicated in AD, Downs syndrome and other forms of metal deficiency. H2S also plays an active role in inflammation and in blood pressure regulation. Despite the recognized importance of both of these gaseous transmitters, the current methods for detection in live cells are limited. The postdoctoral phase of the proposed research will focus on the development of new NO-selective fluorescent probes that address current limitations of NO detection. Transition metal based NO binding sites will be used to develop probes that can reversibly bind NO and probes that emit in the NIR. The proposed family of fluorescent probes will use paramagnetic (S=1/2) metals serving the dual role as both fluorescence quencher and NO binding site. Coordination of NO will form a diamagnetic (S=0) complex and restore the fluorescence of the pendant fluorophore. Adsorption or covalent attachment of such complexes to solubilized single-walled carbon nanotubes (SWNTs) will be used to develop NO-selective probes that emit in the NIR. The independent research phase of the proposed research will investigate the design of H2S-selective fluorescent probes for the imaging of endogenously produced H2S. Currently, such H2S detection methods are lacking and most measurements rely on bulk tissue measurements. The new H2S-selective fluorescent probes for use in live cells will provide much needed tools for the study of the biological functions of H2S. The unique physical properties of H2S will all be exploited in the design of H2S-selective fluorescent probes. Fluorophores will be derivatized with specially designed protecting groups that can only be removed by H2S. Removal of the fluorophore protecting group will restore the fluorescence, thus forming a turn-on probe for H2S. PUBLIC HEALTH RELEVANCE: Both nitric oxide (NO) and hydrogen sulfide (H2S) have been identified as important endogenous gaseous transmitters in the human body and have been implicated in carcinogenesis, hypertension, and several neurological disorders including Alzheimer's disease, Parkinson's disease, Downs syndrome, and multiple sclerosis. Despite this interest, there are currently few methods to detect or image intracellular levels of these small molecule transmitters. This proposal presents the design of fluorescent probes for NO and H2S, which would allow for the selective detection and imaging of these endogenous gasses in live cells.