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Kenneth Raymond和Robert Bergman的联合指导下，他研究了水溶性超分子复合物中的主客体化学，分子识别和催化。他目前是麻省理工学院斯蒂芬·利帕德实验室的NIH博士后研究员，致力于开发一氧化氮的荧光探针。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，作为一名主要研究者来追求他的研究兴趣。他的独立研究将集中在开发用于生物学小分子检测和成像的新工具。 一氧化氮（NO）和硫化氢（H2S）是目前公认的重要的生物气体递质。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益于血管舒张和免疫活性，但过量产生可导致反应性NO物质的增殖，所述反应性NO物质与致癌作用和几种退行性神经系统疾病（包括阿尔茨海默病（AD）、帕金森病和亨廷顿病以及多发性硬化症）有关。类似地，H2S与AD、唐斯综合征和其他形式的金属缺乏有关。H2S在炎症和血压调节中也起着积极的作用。尽管这两种气体发射器的重要性已得到公认，但目前用于活细胞检测的方法是有限的。 该研究的博士后阶段将专注于开发新的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。所提出的家族的荧光探针将使用顺磁性（S=1/2）金属作为荧光猝灭剂和NO结合位点的双重作用。NO的配位将形成抗磁性（S=0）络合物并恢复悬垂荧光团的荧光。吸附或共价连接的这种复合物溶解的单壁碳纳米管（SWNT）将被用来开发NO选择性探针，在近红外发射。 拟议研究的独立研究阶段将调查H2S选择性荧光探针的设计，用于内源性产生的H2S的成像。目前，缺乏这样的H2S检测方法，并且大多数测量依赖于大量组织测量。新的H2S选择性荧光探针用于活细胞将提供急需的工具，研究H2S的生物功能。H2S独特的物理性质将在H2S选择性荧光探针的设计中得到充分利用。荧光团将用专门设计的保护基团衍生化，这些保护基团只能被H2S除去。去除荧光团保护基团将恢复荧光，从而形成H2S的开启探针。 公共卫生相关性：一氧化氮（NO）和硫化氢（H2S）已被鉴定为人体中重要的内源性气体递质，并且与致癌、高血压和包括阿尔茨海默病、帕金森病、唐斯综合征和多发性硬化症在内的几种神经系统疾病有关。尽管有这种兴趣，目前很少有方法来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的荧光探针的设计，这将允许活细胞中这些内源性气体的选择性检测和成像。

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.