Photochemistry of Azide Based Cross-Linkers in Water

叠氮化物交联剂在水中的光化学

• 批准号: 7426870
• 负责人: MATTHEW S PLATZ
• 金额: $ 21.7万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7426870
• 关键词: Amino Acids; Arts; Azides; Bacteria; Benzophenones; Biological; Buffers; Cell Adhesion; Cell surface; Chemical Structure; Chemicals; Chemistry; Collaborations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cross-Linking Reagents; DNA; DNA Polymerase I; Databases; Environment; Escherichia coli; Family; Foundations; Goals; Halogens; Higher Order Chromatin Structure; Ketones; Label; Laboratories; Learning; Light; Literature; Maps; Mediating; Methodology; Methods; Microbial Biofilms; Nitrogen; Nucleic Acids; Nucleoproteins; Nucleosides; Ohio; Organic Chemicals; Organic Chemistry; Organic solvent product; Photoaffinity Labels; Photochemistry; Principal Investigator; Property; Protein Family; Proteins; Publications; Publishing; RNA; Reaction; Reagent; Renaissance; Reporting; Research; Research Personnel; Resolution; Resources; Scientist; Series; Site; Solutions; Spectrum Analysis; Standards of Weights and Measures; Structural Protein; Structure; Students; Sulfur; System; Techniques; Time; Triplet Multiple Birth; United States National Institutes of Health; Universities; Water; Work; absorption; adduct; aqueous; base; benzophenone; chromophore; covalent bond; crosslink; design; experience; functional group; insight; macromolecule; member; nanosecond; photolysis; professor; programs; quantum; reaction rate; research study; two-photon

DESCRIPTION (provided by applicant): Azide based reagents are used extensively by NIH supported investigators in photoaffinity labelling and cross-linking studies. Most practitioners utilize a few commercially available azide reagents. It is generally believed that the photochemistry of these reagents in water is similar to that of simple phenyl azides in organic solvents. Preliminary results indicate that this assumption is not generally correct. Thus, we will systematically study the photochemistry of widely used commercially available reagents, simple phenyl azides, cinnamyl, sulfonyl, phoshoryl and acyl azides in water and in buffer solution. We will study these reagents by pico, nano and microsecond time-resolved UV-Vis and IR spectroscopy, which will reveal the lifetimes of the light generated reactive intermediates and absolute reactivities of the intermediates with nucleosides and amino acids. The reactive intermediates will be identified with the aid of CASSCF, CASPT2 and DFT calculations and chemical analysis of reaction mixtures. This work will be done with non-NIH resources. With NIH support a series of new azide precursors will be synthesized, which absorb strongly above 330 nm and reliably produce reactive intermediates in water with lifetimes of 0.1-100 ns. In collaboration with OSU colleague Professor Ming-Daw Tsai we will re-investigate a classic system, the E. coli Klenow Fragment I which has been previously studied with a traditional, commercial reagent (8-azido dATP) and a newly developed reagent to demonstrate that the commercial reagent generates untrustworthy results. In collaboration with a second OSU colleague, Professor Mike Freitas, we will study the labelled Klenow Fragment with high throughput Mass Spectrometric techniques. These new reagents will also be evaluated as cross-linking agents by Professors Pei and Dalbey of The Ohio State University (OSU) to cross-link the PNAG and YidG proteins, respectively. A photo-probe group will be created at OSU bringing together synthetic organic chemists, physical-organic chemists, mass spectrometrists and biochemists.

描述（由申请方提供）：NIH支持的研究者在光亲和标记和交联研究中广泛使用叠氮化物基试剂。大多数从业者使用一些市售的叠氮化物试剂。一般认为，这些试剂在水中的光化学性质类似于简单的叠氮苯在有机溶剂中的光化学性质。初步结果表明，这一假设并不普遍正确。因此，我们将系统地研究广泛使用的市售试剂，简单的苯基叠氮化物，肉桂酰基，磺酰基，磷酰基和酰基叠氮化物在水和缓冲溶液中的光化学。我们将研究这些试剂的皮科，纳米和微秒时间分辨紫外-可见光谱和红外光谱，这将揭示光产生的反应中间体的寿命和绝对反应的中间体与核苷和氨基酸。反应中间体将借助CASSCF、CASPT 2和DFT计算以及反应混合物的化学分析来鉴定。这项工作将由非NIH资源完成。在NIH的支持下，将合成一系列新的叠氮化物前体，它们在330 nm以上有强烈的吸收，并在水中可靠地产生活性中间体，寿命为0.1-100 ns。在与俄勒冈州立大学的同事蔡明道教授的合作中，我们将重新研究一个经典的系统，E。coli Klenow Fragment I之前已使用传统的商业试剂（8-叠氮基dATP）和新开发的试剂进行了研究，以证明商业试剂产生不可信的结果。我们将与俄勒冈州立大学的第二位同事Mike Freitas教授合作，利用高通量质谱技术研究标记的Klenow片段。俄亥俄州州立大学（OSU）的Pei和Dalbey教授还将评估这些新试剂作为交联剂，分别交联PNAG和YidG蛋白。俄勒冈州立大学将成立一个光探针小组，汇集合成有机化学家、物理有机化学家、质谱仪专家和生物化学家。