Solid state nuclear magnetic resonance with magic-angle spinning and dynamic nuclear polarization below 25 K

Abstract

We describe an apparatus for solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS) at 20–25 K and 9.4 Tesla. The MAS NMR probe uses helium to cool the sample space and nitrogen gas for MAS drive and bearings, as described earlier [1], but also includes a corrugated waveguide for transmission of microwaves from below the probe to the sample. With a 30 mW circularly polarized microwave source at 264 GHz, MAS at 6.8 kHz, and 21 K sample temperature, greater than 25-fold enhancements of cross-polarized ¹³C NMR signals are observed in spectra of frozen glycerol/water solutions containing the triradical dopant DOTOPA-TEMPO when microwaves are applied. As demonstrations, we present DNP-enhanced one-dimensional and two-dimensional ¹³C MAS NMR spectra of frozen solutions of uniformly ¹³C-labeled l-alanine and melittin, a 26-residue helical peptide that we have synthesized with four uniformly ¹³C-labeled amino acids.

Introduction

Demonstrations by Griffin and coworkers [2] of large nuclear magnetic resonance (NMR) signal enhancements in frozen solutions at high magnetic fields and under magic-angle spinning (MAS) through dynamic nuclear polarization (DNP) have stimulated much interest in DNP for solid state NMR studies of biological [3], [4], [5], [6], [7], [8], [9] and non-biological [10], [11], [12], [13], [14] systems in recent years. A popular approach is to use continuous-wave gyrotron microwave sources, capable of producing many watts at frequencies above 200 GHz [15], [16], [17], [18], and to operate at sample temperatures in the 80–100 K range [19], [20]. In these studies, samples are typically doped with nitroxide-based biradical dopants in frozen solutions [21], [22], although other dopants have also been described [23], [24], [25].

Our laboratory has shown that double- and triple-resonance MAS NMR can be performed at sample temperatures below 30 K (which we call “ultra-low” temperatures, in contrast to more conventional low-temperature MAS NMR experiments that use liquid nitrogen for cooling) using a novel probe design in which the sample space is cooled with helium, but nitrogen gas is used for MAS drive and bearings [1]. This design permits stable, long-term operation without excessive consumption of liquid helium. As we have demonstrated, this design can also provide the radio-frequency (rf) power-handling capabilities, NMR signal detection efficiencies, NMR linewidths (i.e., field homogeneity), and MAS frequencies that are required for many common solid state NMR measurements on biomolecular systems. After developing this probe design, we decided to investigate its utility in DNP experiments, motivated by the idea that operation at very low sample temperatures might permit large absolute NMR signals to be obtained with relatively low microwave powers, principally because the partial saturation of electron spin transitions required for DNP [26] can be achieved with lower microwave fields at lower temperatures due to the smaller nitroxide electron spin–lattice relaxation rates [27]. In subsequent ¹H NMR and cross-polarized ¹³C NMR experiments on nitroxide-doped frozen solutions without MAS [28], [29], [30], we have shown that large DNP effects can indeed be observed at temperatures below 30 K. In this paper, we describe our initial results from the combination of ultra-low-temperature MAS NMR with microwave irradiation for DNP.