Development of Laser-Mediated Hyper-Sensitive NMR in Liquids

描述(由申请人提供)：在许多生物医学研究领域取得进展的一个重要障碍是缺乏对溶液中感兴趣的生物分子的原子分辨率、结构和动态表征。主要的障碍往往是可用的材料数量不足，或者需要保持目标分子的稀释度以防止其聚集。这项研究的目标是开发新的工具，通过激光驱动的光化学诱导动态核极化(PHOTO-CIDNP)来显著提高溶液中的核磁共振光谱的灵敏度。这项工作涉及的仪器包括高功率激光器(包括连续波和脉冲)，通过光纤耦合到配备四重共振(HFCN)低温探头的商用600 MHz核磁共振光谱仪。这项工作所取得的进展将有助于许多研究人员解决具有医学意义的重要生物学问题，并将使在稀溶液中温和的生理相关条件下进行高分辨率核磁共振分析成为可能。所提出的研究涉及广泛的异核相关光敏CIDNP核磁共振方法的开发，包括产生新的脉冲序列，以实现1，2和3D光敏CIDNP敏感性增强的数据收集。非均匀采样(NUS)将作为脉冲序列设计和后续数据重建的一部分，用于高维实验。对于各种自旋1/2核，主要是1H、15N、13C和19F，通过光致CIDNP效应将增强纵向平衡和非平衡磁化强度。在13C和19F的情况下，感兴趣的生物分子(作为PhotoCIDNP的一部分产生)的瞬时自由基阳离子的特别大的超精细耦合有望产生极大的增强。关于13C PHOTO-CIDNP的初步数据非常令人鼓舞，并支持拟议实验的可行性。我们还将开发新型的光-CIDNP光敏剂染料并进行匹配实验。此外，我们将协同结合实验和合作努力，涉及从头计算电子密度和超精细耦合常数计算，以识别和预测性能最好的染料。最后，我们将开发采用双激光照射方案的试验性方法，其中包括协同使用能够在可见光范围内激发光-CIDNP染料的激光和在�AE�*跃迁范围内激发感兴趣的分子的激光。后一范围对应于所有氨基酸的主链和侧链羰基的激发，使它们更容易被瞬时氧化，并在具有生物医学重要性的多肽和蛋白质的背景下产生更大的敏感性增强因子。

DESCRIPTION (provided by applicant): A substantial barrier to progress in many areas of biomedical research is the lack of atomic-resolution structural and dynamic characterization of the biomolecule of interest in solution. The main obstacle is often the insufficient amount of available material or, alternatively, the need to keep the target molecule dilute to prevent its aggregation. The goal of this research is to develop novel tools to significantly increase the sensitivity of NMR spectroscopy in solution via laser-driven photo-chemically induced dynamic nuclear polarization (photo-CIDNP). The instrumentation involved in this work comprises high-power lasers (both continuous-wave and pulsed) coupled, via fiber optic, to a commercial 600 MHz NMR spectrometer equipped with a quadruple-resonance (HFCN) cryogenic probe. The advances enabled by this work will facilitate the tackling of important biological questions of medical significance by many investigators, and will enable high- resolution NMR analysis under mild, physiologically relevant conditions in dilute solution. The proposed research involves extensive method development in heteronuclear correlation photo-CIDNP NMR, including the generation of novel pulse sequences to enable 1, 2 and 3D photo-CIDNP sensitivity-enhanced data collection. Non-uniform sampling (NUS) will be implemented as part of the pulse sequence design and subsequent data reconstruction, for the higher-dimensionality experiments. Longitudinal equilibrium and non- equilibrium magnetization will be enhanced via the photo-CIDNP effect for a variety of spin-1/2 nuclei, primarily 1H, 15N, 13C and 19F. In the case of 13C and 19F, the particularly large hyperfine coupling of the transient radical cations of the biomolecules of interest (which is generated as part of photo-CIDNP) promise to yield extremely large enhancements. Preliminary data on 13C photo-CIDNP are extremely encouraging and support the feasibility of the proposed experiments. We will also develop novel photo-CIDNP photosensitizer dyes and matching experiments to use them. In addition, we will synergistically combine experiments with collaborative efforts involving ab initio electron density and hyperfine-coupling-constant calculations to identify and predict the best-performing dyes. Finally, we will develop pilot approaches employing double laser irradiation schemes, which involve the concerted use of a laser capable of exciting the photo-CIDNP dye in the visible range, and another laser exciting the molecule of interest in the � AE�* transition range. The latter range corresponds to excitation of backbone and side-chain carbonyls of all amino acids, to render them more susceptible to transient oxidation and develop larger sensitivity-enhancement factors in the context of biomedically important polypeptides and proteins.