Dynamic Nuclear Polarization Magic Angle Spinning Nuclear Magnetic Resonance: Method Development and Applications

Abstract

The signal-to-noise ratio of MAS NMR signals can be enhanced via dynamic nuclear polarization (DNP) by several orders of magnitude. DNP can provide new perspectives in structural biology, metabolomics studies and material science. At cryogenic temperatures (100 K) stable water-soluble nitroxide biradicals are added to the investigated samples as polarizing agents. The large polarization of the biradical electrons can be transferred to the analytes under continuous microwave irradiation via the so-called cross effect (CE). Hence, the use of DNP enables MAS NMR experiments that suffer from inherent low sensitivity and would not have been executed normally due to the excessively long experimental time required for signal averaging. The current challenge in DNP MAS NMR is the homogeneous and heterogeneous line broadening and the temperature dependence of the CE. In this work, experimental conditions, parameters and aspects that determine the CE efficiency were investigated. Novel polarizing agents were tested for the first time, the methodology further developed and the new insights applied to biomolecular projects. The amino acid proline and the 62 amino acid long protein SH3 in microcrystalline form were used as model systems. Four CD3-TOTAPOL isotopologues were investigated and compared to already established biradicals such as TOTAPOL and AMUPol in the course of this work. Their CE efficiency was assessed by comparing enhancement values and the signal-to-noise ratio per 10 min (10minSNR) for 1D hC (1H- 13C CP) experiments in a temperature range between 100 and 200 K at 9.4 T. Signal-to-noise ratio were analyzed and compared with a newly devised procedure. The deuteration of the methyl groups of 1H-TOTAPOL led to larger signal enhancements compared to 1H-TOTAPOL. The effect of deuteration of 1H-TOTAPOL did not influence the electron relaxation parameter as initially hypothesized and are not the reason for increased CE efficiency of the polarizing agent. Highest signal enhancements were obtained for the isotopologue CD3-TOTAPOL-0 that has deuterated methyl groups but protonated 3 and 5 positions of the TEMPO ring. The results highlight the importance of protons that are at close proximity to the radical center, presumably involved in the initial polarization buildup. In addition, the new polarizing agent bcTol, designed for biological applications was tested for the first time in the course of this work. The maximum signal enhancement that could be obtained was e ~ 248 for a sample containing microcrystalline SH3 at 110 K. The bcTol biradical showed unprecedented solubility in water, GDH and [D8]-glycerol. Measurements of signal-to-noise per unit time suggest a comparable DNP performance of bcTol at 110 K to that of AMUPol.Furthermore, a 10minSNR study was conducted to investigate the influence of electron relaxation parameters and the radical linker. Therefore, the novel polarizing agents and cyolyl-TOTAPOL and bcTol-M were investigated for the first time. bcTol-M is similar to bcTol but with methyl groups on the nitrogen atoms showed greater 10minSNR ratio than bcTol. The maximum signal enhancement that could be measured with this radical was e ~ 302 on a proline sample in a 3.2 mm sapphire rotor at 110 K. The superior performance compared to bcTol and AMUPol can be mainly attributed to the shorter 1H-T1 times. The presence of the methyl groups on the linkage presumably promotes nuclear relaxation. Given the simplified handling of the radical and its good performance in hC cross polarization experiments, bcTol-M constitutes an ideal polarizing agent for biomolecular DNP MAS NMR studies. The three urea-based radicals AMUPol, bcTol und bcTol-M show a considerable higher enhancement and SNR compared to 1H-TOTAPol and cyolyl-TOTAPol. Among the urea based radicals, the enhancement values do not differ to a large extent but the values for the SNR do. Furthermore, the electron relaxation times that were measured at the same field as the NMR spectra (9.4 T) for the radicals 1H- TOTAPOL, cyolyl-TOTAPOL, bcTol, AMUPol, and bcTol-M do not dominate the performance of the radicals at 110 K, 8 kHz MAS and 9.4 T. The different performance correlates with the chosen type of linker and the degree of alkylation, affecting the size of the effective electron-electron dipolar coupling and the nuclear relaxation times. A sample preparation protocol for the SH3 samples was developed in order to get reliable and reproducible results. 2D 13C-13C DARR spectra were recorded under DNP conditions for each radical. The best resolution was obtained for the samples containing AMUPol as polarizing agent. The signal-to-noise ratio at 200 K on SH3 standard samples is 15 times larger compared to a sample without polarizing agent. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours respectively. The obtained resolution allows for some spectral assignment of amino acid side chains. For the novel nitroxide biradical bcTol the signal-to-noise per unit time were higher compared to AMUPol at 181 K. 2D spectra of the SH3 domain sample recorded at 181 K gave a signal enhancement of e ~ 40 and show sufficient resolution for structural studies. In the final sections of this thesis, the power of these new insights and developments were demonstrated on diverse biological problems.