Photochemistry of Azide Based Cross-Linkers in Water

描述(由申请人提供)：叠氮类试剂被美国国立卫生研究院支持的研究人员广泛用于光亲和标记和交联性研究。大多数从业者使用一些市面上可买到的叠氮试剂。一般认为，这些试剂在水中的光化学作用类似于简单的苯基叠氮化物在有机溶剂中的光化学作用。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商用试剂，简单的苯叠氮化物、肉桂基、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。我们将用微微、纳和微秒时间分辨的UV-Vis和IR光谱来研究这些试剂，这将揭示光生反应中间体的寿命以及中间体与核苷和氨基酸的绝对反应活性。活性中间体将借助CASSCF、CASPT2和DFT计算以及反应混合物的化学分析进行鉴定。这项工作将使用非NIH的资源来完成。在NIH的支持下，将合成一系列新的叠氮化物前驱体，它们在330 nm以上有很强的吸收，在水中可靠地产生活性中间体，寿命为0.1-100 ns。我们将与俄亥俄州立大学的蔡明道教授合作，重新研究一个经典的系统，即以前用传统的商业试剂(8-叠氮dATP)和新开发的试剂研究过的E.ColiKlenow片段I，以证明商业试剂产生的结果是不可信的。与俄亥俄州立大学的另一位同事Mike Freitas教授合作，我们将用高通量质谱学技术研究标记的Klenow片段。俄亥俄州立大学(OSU)的贝教授和达尔贝教授也将评估这些新试剂作为交联剂，分别使PNAG和YidG蛋白质交联。俄亥俄州立大学将成立一个光探测小组，将合成有机化学家、物理有机化学家、质谱学家和生物化学家聚集在一起。

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.