Developing fluorescent probes for the endogenous gaseous transmitters NO and H2S

描述(由申请人提供)：候选人在加州大学伯克利分校获得博士学位，在Kenneth Raymond和Robert Bergman的共同指导下，他在那里学习主客体化学、分子识别和水溶性超分子化合物中的催化。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荧光探针。该候选人的研究兴趣横跨分子识别领域，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，作为一名主要研究员来追求他的研究兴趣。他的独立研究将专注于开发新的工具，用于检测和成像生物学中的小分子。一氧化氮(NO)和硫化氢(H2S)现在被认为是重要的生物气体递质。一氧化氮和硫化氢都是由内源性产生的，并受到身体的精细调节。在低细胞浓度下，一氧化氮有利于血管扩张和免疫活性，但过量生产会导致反应性一氧化氮物种的增殖，这些活性一氧化氮与癌症和几种退行性神经疾病有关，包括阿尔茨海默氏症(AD)、帕金森氏症、亨廷顿病以及多发性硬化症。同样，硫化氢也与阿尔茨海默病、唐氏综合症和其他形式的金属缺乏症有关。硫化氢在炎症和血压调节中也起着积极的作用。尽管这两种气体传递器的重要性得到了公认，但目前在活细胞中检测的方法是有限的。拟议研究的博士后阶段将集中于开发新的非选择性荧光探针，以解决目前NO检测的局限性。基于过渡金属的NO结合位点将被用于开发可可逆地结合NO的探针和在近红外光谱中发射的探针。建议的荧光探针家族将使用顺磁性(S=1/2)金属，兼有荧光猝灭和无结合位点的双重作用。NO的配位将形成一个抗磁性(S=0)的络合物，并恢复悬挂的荧光团的荧光。将此类络合物吸附或共价连接到溶解的单壁碳纳米管(SWNTs)上，将用于开发在近红外光谱中发射的非选择性探针。拟议研究的独立研究阶段将调查用于内源产生的硫化氢成像的硫化氢选择性荧光探针的设计。目前，这样的硫化氢检测方法还很缺乏，大多数测量依赖于大块组织的测量。用于活细胞的新的硫化氢选择性荧光探针将为研究硫化氢的生物学功能提供急需的工具。硫化氢独特的物理性质将在硫化氢选择性荧光探针的设计中得到充分利用。荧光团将通过特殊设计的保护基团进行衍生化，这些保护基团只能被硫化氢去除。去除荧光团保护基团将恢复荧光，从而形成对硫化氢的开启探针。 与公共健康相关：一氧化氮(NO)和硫化氢(H2S)都被确认为人体内重要的内源性气体传递体，与癌症、高血压和几种神经疾病有关，包括阿尔茨海默病、帕金森氏病、唐氏综合症和多发性硬化症。尽管人们对此感兴趣，但目前几乎没有方法来检测或成像这些小分子递质的细胞内水平。这项建议提出了NO和H_2S的荧光探针的设计，这将允许在活细胞中选择性地检测和成像这些内源性气体。

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.