High frequency dynamic nuclear polarization

Abstract

During the three decades 1980-2010, magic angle spinning (MAS) NMR developed into the method of choice to examine many chemical, physical, and biological problems. In particular, a variety of dipolar recoupling methods to measure distances and torsion angles can now constrain molecular structures to high resolution. However, applications are often limited by the low sensitivity of the experiments, due in large part to the necessity of observing spectra of low-γ nuclei such as the I = 1/2 species (13)C or (15)N. The difficulty is still greater when quadrupolar nuclei, such as (17)O or (27)Al, are involved. This problem has stimulated efforts to increase the sensitivity of MAS experiments. A particularly powerful approach is dynamic nuclear polarization (DNP) which takes advantage of the higher equilibrium polarization of electrons (which conventionally manifests in the great sensitivity advantage of EPR over NMR). In DNP, the sample is doped with a stable paramagnetic polarizing agent and irradiated with microwaves to transfer the high polarization in the electron spin reservoir to the nuclei of interest. The idea was first explored by Overhauser and Slichter in 1953. However, these experiments were carried out on static samples, at magnetic fields that are low by current standards. To be implemented in contemporary MAS NMR experiments, DNP requires microwave sources operating in the subterahertz regime, roughly 150-660 GHz, and cryogenic MAS probes. In addition, improvements were required in the polarizing agents, because the high concentrations of conventional radicals that are required to produce significant enhancements compromise spectral resolution. In the last two decades, scientific and technical advances have addressed these problems and brought DNP to the point where it is achieving wide applicability. These advances include the development of high frequency gyrotron microwave sources operating in the subterahertz frequency range. In addition, low temperature MAS probes were developed that permit in situ microwave irradiation of the samples. And, finally, biradical polarizing agents were developed that increased the efficiency of DNP experiments by factors of ∼4 at considerably lower paramagnet concentrations. Collectively, these developments have made it possible to apply DNP on a routine basis to a number of different scientific endeavors, most prominently in the biological and material sciences. This Account reviews these developments, including the primary mechanisms used to transfer polarization in high frequency DNP, and the current choice of microwave sources and biradical polarizing agents. In addition, we illustrate the utility of the technique with a description of applications to membrane and amyloid proteins that emphasizes the unique structural information that is available in these two cases.

Figure 1

Polarizing agents commonly used for high field DNP experiments. (a) narrow line radicals trityl and BDPA used for the SE; (b) TEMPO based biradicals TOTAPOL and bis-TEMPO-bis-ketal (bTbk) used for the CE.

Figure 2

(top) Energy level diagram illustrating DNP via the solid effect (SE). At thermal equilibrium (left), populations of the same electron spin subspaces are governed by the Boltzmann distribution. Mixing of states in the nuclear and electron spin subspaces (right), leads to partially allowed double quantum (DQ) and zero quantum (ZQ) transitions, and positive and negative enhancements, , respectively. The mixing of states is proportional to a constant q, which is inversely proportional to B₀. Therefore, the enhancement in the Solid Effect DNP scales as $B_0^{-2}$. (bottom) A plot of the enhancement from SA-BDPA as a function of magnetic field (¹H frequency) showing the positive and negative enhancements. ω_NMR and ω_EPR are the NMR and EPR frequencies and ω_e ±ω_n are the sum and difference of the EPR and NMR frequencies.

Figure 3

(top) Energy diagram illustrating DNP via the CE. At equilibrium (left), under the matching condition, there is degeneracy and 1:1 population of the two shaded levels. The EPR spectrum of an ideal biradical for CE (middle) has two narrow lines separated by the nuclear Larmor frequency. Saturation of transitions near the first (second) EPR line gives rise to a positive (negative) DNP enhancement (right). (bottom) Field profile for bTbk with an enhancement ε= 230

Figure 4

¹³C CP MAS NMR spectra of (A) 1M U-[¹³C-¹⁵N] urea and (B) 0.5 M U-[¹³C-¹⁵N] proline at 80 K with and without microwave irradiation. The DNP enhancements are ε=181 and ε=134, respectively. Both samples contained 10 mM TOTAPOL in a 60/30/10 ratio of d₈-glycerol/D₂O/H₂O. Experimental parameters are: 4 scans, recycle delay 4 s, microwave power ~12.5 W, γB₁(¹H)=83 kHz, γB₁(¹³C)=71 kHz, ω_r/2π= 5 kHz.

Figure 5

¹³C CP DNP enhancements of U-[¹³C ,¹⁵N] urea with 10 mM TOTAPOL plotted as a function of μw power at 80 K (left) and as a function of temperature at 12.5 W μw (right). ω_r/2π= 7 kHz.

Figure 6

(left) The ion-motive photocycle of bR. The subscript for each intermediate represents the wavelength (in nm) of maximum visible absorption. (middle) ¹⁵N CP DNP spectra [ζ-¹⁵N-Lys] bR prepared with 15 mM TOTAPOL in 60/30/10 ratio of d₈-glycerol/D₂O/H₂O in 0.3 M guanidinium hydrochloride at pH 10. (A) the dark adapted (DA) state comprises a thermal equilibrium mixture of bR₅₅₅ and bR₅₆₈ (B) LA (bR₅₆₈) accumulated by 532 nm irradiation of the rotating sample for 4 hours 273 K (C) the Mo intermediate created by 532 nm irradiation of rotating LA at 230 K. The spectra of all three intermediates were obtained in roughly 2 hours with a spinning frequency of 7 kHz. (right) 2D spectrum obtained from DA bR illustrating the splittings observed at low temperature due to inequivalent sites.

Figure 7

¹⁵N-¹³C spectrum obtained from dark adapted U-[¹³C,¹⁵N]-bR after selective excitation of the ¹⁵N Schiff base, CP to the ¹³C-15 of the retinal and ¹³Cε of Lys216, followed by RFDR mixing. The spectrum shows cross-peaks between the Schiff base ¹⁵N and ¹³C-12,13,14,15,20 on the retinal chromophore and ¹³Cε Lys216 ^,. The arrow indicates the trans-cis isomerization of the C13=C14 bond that occurs during the photocycle.

Figure 8

Comparison between room temperature and DNP enhanced, low temperature correlation spectra of PI3-SH3. The spectra were obtained with ZF-TEDOR recoupling (τ_mix = 16 ms) from sample prepared from partially labeled fibrils [¹⁵N, ¹²C] PI3-SH3 /[¹⁴N,¹³C] PI3-SH3 (50:50 molar ratio). (a) ¹⁵N-¹³C intermolecular correlations in PI3-SH3 fibrils at 300 K obtained at 750 MHz in 16 days of acquisition time. (b). Same sample and identical spectral regions were recorded at 100 K and 400 MHz with DNP enhancement in 32 h. (c). Illustration of the 23 interstrand contacts established from ¹³C -¹⁵N peaks in the 750 MHz spectra acquired at 300 K in a. (d) the 52 interstrand contacts established from the 400 MHz DNP enhanced spectra recorded at 100 K shown in (d).