交换相互作用和弛豫时间可调的杂化双自由基的设计合成与高场动态核极化性能研究

Dynamic nuclear polarization (DNP) is a powerful technique to increase sensitivity of solid-state nuclear magnetic resonance and have found wide applications in the fields of structural biology and material science. Development of new efficient polarizing agents is of crucial importance for high-field DNP. However, development of the polarizing agents based on the most commonly used homogeneous biradicals has confronted with a bottleneck. Our previous studies demonstrated that heterogeneous trityl-nitroxide (TN) biradicals are the most promising high-field polarizing agents. However, the factors controlling their DNP properties are not clear to date and thus their DNP properties are still far away from optimization. Therefore, this project will start with design and synthesis of both trityl and nitroxide monoradicals as well as modulation of the linker nature to construct a series of TN biradicals with tunable relaxation times and spin-spin exchange interactions. Then, the correlation among molecular structure, physiochemical properties and DNP properties of TN biradicals will be comprehensively explored by electron paramagnetic resonance spectroscopy and high-field DNP technique. The biradicals with the optimal DNP properties will be opted out at this stage. Finally, based on the molecular structure of the optimal biradicals, new biradicals with specific reactivity to cysteine in proteins or targeting to lipids will be further synthesized as site-specific polarizing agents. The application potential of these polarizing agents in studying structure and dynamics of membrane proteins will be explored using KcsA and ASR as protein models. The present project will shed new lights on synthesis of highly efficient high-field DNP polarizing agents and thus promote development of the high-field solid-state nuclear magnetic resonance technique.

动态核极化（DNP）技术是增强固体核磁共振灵敏性的强有力手段，在结构生物学和材料学等领域应用广泛。极化剂是高场DNP技术发展的关键要素，但现有基于同源双自由基的极化剂研发已面临瓶颈。申请人前期工作证实，trityl-氮氧（TN）杂化双自由基是目前最有前景的高场极化剂，但其DNP调控机制尚未明确，且性能远未最优化。为此，本项目拟通过合成结构新颖的trityl和氮氧单自由基，并调节二者间的连接链属性，以实现弛豫时间和交换相互作用等性质可调的TN双自由基的构建；采用电子顺磁共振波谱和高场DNP技术等揭示双自由基的分子结构、理化性质与DNP性能间的内在联系，筛选出性能最佳的极化剂；基于此，合成靶向半胱氨酸和脂质体的位点特异性极化剂，并以KcsA和ASR为蛋白模型探索新型极化剂在膜蛋白结构与动力学研究方面的应用潜质。本项目的有效实施将为高场极化剂研发提供理论指导,势必助力高场固体核磁共振技术的发展。

高场固体核磁共振（ssNMR）的内在低灵敏性是限制其结构生物学应用的重要原因，而动态核极化(DNP)技术是增强高场ssNMR灵敏性的有效手段。极化剂研发是促进DNP技术发展的关键，也是该领域近十年的研究热点。本项目以极化剂研发领域最有前景的trityl-nitroxide（TN）双自由基为研究对象，通过调节双自由基的理化性质（特别是交换相互作用和弛豫时间）实现高效极化剂的构建，取得的主要标志性研究成果如下：（1）证实了亲水性、交换相互作用以及弛豫时间是调节TN双自由基的高场DNP性能的关键因素。（2）得到了高亲水性以及交换相互作用和弛豫时间可控的三个系列极化剂（NATriPols、SNAPols和StaPols），可作为高性能的“分散型”极化剂。（3）极化剂SNAPol-1和StaPol-1对蛋白质的高场DNP增强倍数超过了100，刷新了现有极化剂的DNP增强记录，是极化剂研发领域的新突破。（4）采用不同的侧链以及对位取代基实现trityl自由基的螺旋构型、水溶性以及稳定性的可控调节，为trityl自由基研发提供了新策略。除了项目的既定研究目标，本项目还探索了SNAPol-1和StaPol-1在细胞内DNP领域的应用前景。本项目所取得的研究成果不但为理化性质可调的trityl自由基研发提供了很好的思路，可有效推动trityl自由基在磁共振领域的广泛应用，同时也为高性能高场极化剂研发提供了借鉴，为推动了ssNMR技术在细胞内蛋白质结构与动力学方面的应用奠定了坚实基础。

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