Polarizing agents and mechanisms for high-field dynamic nuclear polarization of frozen dielectric solids

Abstract

This article provides an overview of polarizing mechanisms involved in high-frequency dynamic nuclear polarization (DNP) of frozen biological samples at temperatures maintained using liquid nitrogen, compatible with contemporary magic-angle spinning (MAS) nuclear magnetic resonance (NMR). Typical DNP experiments require unpaired electrons that are usually exogenous in samples via paramagnetic doping with polarizing agents. Thus, the resulting nuclear polarization mechanism depends on the electron and nuclear spin interactions induced by the paramagnetic species. The Overhauser Effect (OE) DNP, which relies on time-dependent spin-spin interactions, is excluded from our discussion due the lack of conducting electrons in frozen aqueous solutions containing biological entities. DNP of particular interest to us relies primarily on time-independent, spin-spin interactions for significant electron-nucleus polarization transfer through mechanisms such as the Solid Effect (SE), the Cross Effect (CE) or Thermal Mixing (TM), involving one, two or multiple electron spins, respectively. Derived from monomeric radicals initially used in high-field DNP experiments, bi- or multiple-radical polarizing agents facilitate CE/TM to generate significant NMR signal enhancements in dielectric solids at low temperatures (<100 K). For example, large DNP enhancements (∼300 times at 5 T) from a biologically compatible biradical, 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL), have enabled high-resolution MAS NMR in sample systems existing in submicron domains or embedded in larger biomolecular complexes. The scope of this review is focused on recently developed DNP polarizing agents for high-field applications and leads up to future developments per the CE DNP mechanism. Because DNP experiments are feasible with a solid-state microwave source when performed at <20K, nuclear polarization using lower microwave power (<100 mW) is possible by forcing a high proportion of biradicals to fulfill the frequency matching condition of CE (two EPR frequencies separated by the NMR frequency) using the strategies involving hetero-radical moieties and/or molecular alignment. In addition, the combination of an excited triplet and a stable radical might provide alternative DNP mechanisms without the microwave requirement.

Figure 1

Polarizing agents: (1) 4-hydroxy-TEMPO, (2) 4-amino-TEMPO, (3) trityl [154], (4) BDPA [104], (5) BTnE, n=2, 3 or 4 [49*], (6) TOTAPOL [50], (7) DOTOPA-TEMPO [52*], (8) BTOXA [22*], (9) BTOX [22*], (10) BTurea [22*], (11) bTbk [48], (12) BDPA-TEMPO [81], (13) BTcholesterol [99], (14) pyrelene-TEMPO. What is illustrated includes the commonly used radicals for high-field DNP (1–12) and future designer polarizing agents for aligned membranes (13) and photoexcited DNP (14).

Figure 2

(a) Illustrative pulse sequence for a general DNP-NMR SSNMR experiment. The saturation prior to the experiment ensures the same initial NMR signals regardless of the microwave irradiation mode, which is either continuous or intermittent for NMR pulsing and detection periods. (b) Typical DNP buildup under MAS conditions, such as the results shown for polarized ¹³C-urea signals from DNP using BT2E in d₆- DMSO/D₂O/H₂O (6:3:1 w/w/w) at 90 K and 5 T. The enhancement factor was assessed by comparing NMR signals with and without microwave irradiation and showed little dependence on microwave irradiation time under MAS. (c) The DNP buildup time constant was similar to T_1n because the polarizing agents were well-diluted against the concentration of bulk protons. Figures are reprinted from [22*].

Figure 3

High-field DNP polarizing mechanisms depend on the total EPR line shape of the utilized polarizing agents. (a) SE driven by 40 mM trityl radicals; (b) CE/TM driven by 40 mM monomeric 4-hydroxy-TEMPO; (c) CE/TM driven by radical mixture with 20 mM 4-hydroxy-TEMPO and 20 mM trityl radicals. The experiments were performed in solutions with 2 M ¹³C-urea in 6:3:1 w/w/w d₆-DMSO/D₂O/H₂O doped with TEMPO and/or trityl radicals. Note that the CE/TM mechanism was enhanced by the appropriate g-value separation between TEMPO and trityl (~80 G or 224 MHz) as shown by the dashed EPR line shape for trityl in (b). The experimental data points are shown in open circles, and theoretical curves are drawn in red lines. Figures are reprinted from [33].

Figure 4

(a) Illustration of the correct EPR frequency separation for efficient CE that requires that the |β_S1α_S2α_I> and |α_S1β_S2β_I> product spin states are degenerate, where S1 and S2 denote two electron spins and I denotes the nuclear spin. Without the degeneracy, the level mixing becomes ineffective due to level separation, as indicated by the yellow shading. (b) The EPR spectrum (shown in MHz) of TEMPO at 5 T with the corresponding TEMPO orientations shown roughly below the EPR frequencies. The anisotropic g-tensor of nitroxide permits the required EPR frequency matching (ω_0e2−ω_0e1=ω_0n) via appropriate nitroxide molecular orientations with respect to the external magnetic field (pointing up in the figure).

Figure 5

DNP enhancements in standard solutions (2 M ¹³C-urea, 6:3:1 w/w/w d₆- DMSO/D₂O/H₂O in a 4 mm o.d. sapphire rotor) doped with electron spins totaled at 10 mM and measured at 90 K and 5 T. Considering uncertainty, the best-performing polarizing agents are BT2E and TOTAPOL, the latter of which is biologically compatible and yields higher enhancement with sufficient microwave power (i.e., irradiated in a 2.5 o.d. mm sapphire rotor). Also interestingly, the worst DNP enhancement from BTOXA manifested the requirement of EPR frequency matching for CE. The figure is reprinted from [22*].

Figure 6

Line shape fitting of the EPR spectra (simulations in solid lines and experimental data in dots) of BTOXA measured with 9 and 140 GHz microwave frequency. The multi-frequency spectra were simultaneously fitted with a set of spectral parameters that helped constrain the underlying biradical conformations. For example, possible molecular structures of BTOXA refined from the fitting are shown in the inset. A planar BTOXA molecule yields close EPR frequencies of the tethered nitroxides, regardless of the molecular orientation and thus impedes the efficiency of CE. Figures are reprinted from [22*].