Sensitivity Enhanced NMR

Abstract

The topic of this thesis is the investigation of how sensitivity enhanced Nuclear Magnetic Resonance (NMR) spectra of biological systems could be obtained by dynamic nuclear polarization (DNP) under conditions close to their physiological environment in solution. Here, the Overhauser effect is the driving mechanism for DNP, which depends on the diffusion kinetics of the polarizer molecule with an unpaired electron and the nuclei of the target molecule. For this thesis, a liquid state shuttle DNP spectrometer was chosen, where the nuclei are polarized in a field of 0.34 T and transferred to a field of 14.09 T for high resolution NMR detection. Several technical modifications were successively implemented, which increased stability and improved reproducibility of the measurements. High-field DNP enhancements εhf up to -2.4 (Hη2 ) were measured for the proton signals of L-tryptophan. A general global enhancement factor εglobal was introduced which includes advantages and disadvantages of the shuttle DNP approach, such as an increased acquisition rate of the DNP experiments and line broadening due to the presence of the radical, respectively. Subsequently, it was applied and adapted to one-dimensional NMR measurements. For this purpose the polarization build-up time and the acquisition time was optimized for the Boltzmann polarization and the DNP measurement to gain a maximal signal-to-noise ratio per unit measurement time based on T1 and T2∗, respectively. With this, an applied global enhancement factor εapp of -4.0 for the Hδ1 of L-tryptophan was measured. Furthermore, the DNP spin properties of the protons, such as relaxation rates, were measured and compared with each other. The thereby obtained coupling factors implied that the proton accessibility for the polarizer molecule had an important influence on the intermolecular dipolar interaction between the nuclear spins of the target molecule and the electron of the polarizer. It was shown, that this interaction is described best by a model based on translational diffusion. With this model, the distance of closest approach was determined for the protons of L-tryptophan. These distances range from 3 to 5 Å corresponding to the accessibility of each respective proton. In addition, carbon DNP enhancements between -0.3 and -2.5 were measured for deuterated L-tryptophan-d8,15N2,13C11. Calculations demonstrated that these carbon enhancements were in agreement with the previously calculated distance of closest approach of the proton spins and therefore confirmed the approach of the translational diffusion model, too. In further measurements on protonated L-tryptophan-15N2,13C11, the three-spin effect was observed for the first time for a solute molecule. This effect, based on a dipolar interaction between the proton, the carbon and the electron spin, caused positive enhancements for all carbons up to 2.3, but the carbonyl carbon with an enhancement of -2.5. These findings are in agreement with an expanded coupling factor, which includes the intramolecular carbon-proton interaction alongside the intermolecular carbon-electron interaction. In a concluding step, shuttle DNP experiments were conducted on a protein (Ubiquitin-U-15N,U-13C). For this purpose, a two dimensional shuttle DNP 1H-13C-HSQC spectrum was recorded. For the first time, a DNP transfer to the surface of a protein was demonstrated in the liquid state.