An H/F/X/Y Fast-MAS NMR Probe Particularly for Alzheimer's and Cancer Research

一种用于阿尔茨海默病和癌症研究的H/F/X/Y快速MAS核磁共振探针 摘要 目前超过20%的药物(以及更大比例的研发中药物)是氟化的。 (包括百忧解、立普妥和赛普维等畅销药物)。在过去的十年里，稳步发展 魔角旋转(MAS)固态核磁共振(ss核磁共振)被证明是最强大的分析工具 用于研究大分子结构及其动力学。对于溶液核磁共振，四通道多核 1H/19F/X/2H探头最近变得更容易获得，这种探头已被证明是非常 对鉴定和表征活性物质有价值(使用1H/15N、19F/13C和19F/2H/15N方法) 片段，它们与可溶性蛋白质的结合，以及它们对这种蛋白质-蛋白质相互作用的影响。问题 这类方法不适用于不溶性蛋白质--例如对蛋白质至关重要的聚集体和纤维 阿尔茨海默氏症(AD)、帕金森氏病(PD)，甚至可能是普恩介导性疾病。这个 淀粉样β蛋白(Aβ)级联假说开始为神经退行性疾病领域带来统一， 但这是了解初始种子阶段以外的总体进展和治疗的关键工具 一直不可用。 适用于含氟药物及其相互作用多通道研究的MAS探针 由于现有射频电路在操作上的困难，高场核磁共振仪器无法使用 在高频下接近共振。在第一阶段，我们演示了一种新的单线圈电路 首次允许使用500 MHz的核磁共振数据和台架实验进行高效高场H/F/X/Y MAS 以及800 MHz下的全波详细建模。 该第二阶段提案寻求资金，以继续开发和测试高场H/F/X/Y 基于新型单线圈射频电路的FAST-MAS探头，优化了19F同时检测 任何或所有其他通道上的照射或检测，适用于7-28T范围内的MAS，带转子 直径从0.7-3毫米。分析表明，光谱线展宽的很大一部分 许多MAS实验是从J耦合(不是MAS平均的)到异核和自旋-- 相关效应--温度梯度、轴向振动和磁性。同时脱钩的能力 1H、2H和13C或15N在19F检测期间，使用FAST-MAS在旋转器中进行优化，以便通过电路实现高分辨率 与高达1200 MHz的B0兼容的探头设计将允许大幅提高光谱分辨率和 例如，对淀粉样蛋白组件及其前体聚集体中的19F标记配体的敏感性，或在19F中 标记的DNA致癌加合物。 新的探头将允许强大的核磁共振采集和自动化结构套件 为溶液核磁共振开发的测定协议，主要依赖于间接检测的三重和四重- 共振方案，成功地应用于小于1毫克的刚性氟化样品。探测仪 将兼容自动样品交换和90K至420K的样品温度。此外， 对于所有初级核素(1H、19F、31P、13C、2H， 15N和17O)，它将可调到几乎所有感兴趣的组合，从而使其在 新陈代谢、材料科学、催化和可持续能源等领域。 关键词：氟核磁共振，阿尔茨海默氏症，淀粉样蛋白，MAS，蛋白质聚集体，四共振。

An H/F/X/Y Fast-MAS NMR Probe Particularly for Alzheimer’s and Cancer Research Abstract More than 20% of current drugs (and a much greater fraction of those in development) are fluorinated (including such block-busters as Prozac, Lipitor, and Ciprobay). Steady progress over the past decade has shown magic angle spinning (MAS) solid-state NMR (ssNMR) to be arguably the most powerful analytical tool for studying macro-molecular structures and their dynamics. For solution NMR, four-channel multinuclear 1H/19F/X/2H probes have recently become more readily available, and such have proven to be extremely valuable for identification and characterization (using 1H/15N, 19F/13C, and 19F/2H/15N methods) of active fragments, their binding to soluble proteins, and their effects on such protein-protein interactions. The problem is that such methods don’t work with insoluble proteins – such as the aggregates and fibrils that are central to Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and probably even prion-mediated diseases. The amyloid beta (Aβ) cascade hypothesis is beginning to bring unity to the field of neurodegenerative diseases, but a key tool for understanding aggregate progression and treatment beyond the stages of the initial seeds has not been available. MAS probes suitable for the needed multi-channel studies of fluorinated drugs and their interactions have not been available for high-field NMR instruments because of the difficulties of prior rf circuits in handling close resonances at high frequencies. During the Phase-I, we demonstrated that a novel single-coil circuit permits, for the first time, efficient high-field H/F/X/Y MAS, with NMR data at 500 MHz and bench experiments and full-wave detailed modeling at 800 MHz. This Phase-II proposal seeks funding to continue the development and testing of high-field H/F/X/Y fast-MAS probes based on a novel “single-coil” rf circuit optimized for 19F detection with simultaneous irradiation or detection on any or all of the other channels, and suitable for MAS at fields from 7-28 T, with rotor diameters from 0.7-3 mm. Analysis suggests that a substantial portion of the spectral line broadening seen in many MAS experiments is from J-couplings (which is not averaged by MAS) to heteronuclei and spinner- dependent effects – thermal gradients, axial vibration, and magnetism. The ability to simultaneously decouple 1H, 2H, and 13C or 15N during 19F detection with fast-MAS in a spinner optimized for high resolution with a circuit and probe design compatible with B0 up to 1200 MHz will permit a dramatic increase in spectral resolution and sensitivity on, for example, 19F-labeled ligands in amyloid assemblies and their precursor aggregates, or in 19F labeled DNA-carcinogen adducts. The novel probe would allow the powerful suite of NMR acquisition and automated structure determination protocols developed for solution NMR, which rely mostly on indirect-detected triple- and quad- resonance schemes, to be successfully applied to rigid fluorinated samples smaller than a milligram. The probe will be compatible with automated sample exchange and sample temperatures from 90 K to 420 K. Moreover, it will be essentially devoid of problematic background signals for all the primary nuclides (1H, 19F, 31P, 13C, 2H, 15N, and 17O), and it will be tunable to virtually all combinations of interest, thereby making it also invaluable in such areas as metabolism, materials science, catalysis, and sustainable energy. Key words: fluorine NMR, Alzheimer’s, amyloids, MAS, protein aggregates, quad-resonance.