Biradical Polarizing Agents at High Fields

Abstract

The sensitivity enhancements available from dynamic nuclear polarization (DNP) are rapidly reshaping the research landscape and expanding the field of nuclear magnetic resonance (NMR) spectroscopy as a tool for solving complex chemical and structural problems. The past decade has seen considerable advances in this burgeoning method, while efforts to further improve its capabilities continue along many avenues. In this report, we examine the influence of static magnetic field strength and temperature on the reported ¹H DNP enhancements from three conventional organic biradicals: TOTAPOL, AMUPol, and SPIROPOL. In contrast to the conventional wisdom, our findings show that at liquid nitrogen temperatures and 700 MHz/460.5 GHz, these three bisnitroxides all provide similar ¹H DNP enhancements, ε ≈ 60. Furthermore, we investigate the influence of temperature, microwave power, magnetic field strength, and protein sample deuteration on the NMR experimental results.

Figure 1.

Molecular structures of TOTAPOL, AMUPol, and SPIROPOL nitroxide biradicals. The red dots are the unpaired electrons. Only TOTAPOL has flexible methyl groups (8 in total), whereas AMUPol and SPIROPOL are rigid.

Figure 2.

1 M ¹³C-urea in d₈-glycerol/D₂O/H₂O (60/30/10, v/v/v) with 10 mM biradical at 82 ± 2 K, 7 W microwave power, spinning frequency of 5 kHz at 211.96 MHz/140 GHz. (a) ¹H enhancement field profiles demonstrating the external magnetic field dependence of TOTAPOL (red triangles) and AMUPol (cyan squares). (b) DNP-enhanced ¹³C(¹H) CPMAS NMR (μw on) and ¹³C(¹H) CPMAS NMR (μw off) with 10 mM TOTAPOL (red, top) and 10 mM AMUPol (cyan, bottom). Enhancements were recorded at the maximum positive side of the DNP profile.

Figure 3.

1 M ¹³C-urea in d₈-glycerol/D₂O/H₂O (60/30/10, v/v/v) with 10 mM biradical at 82 ± 2 K, spinning frequency of 5.5 kHz at 699.3 MHz/460.5 GHz. (a) DNP-enhanced ¹³C(¹H) CPMAS NMR (μw on) and ¹³C(¹H) CPMAS NMR (μw off) spectra for 1 M ¹³C-urea in 10 mM TOTAPOL. (b) Measured ¹H DNP enhancement (ε) for the same (TOTAPOL) sample (red triangles) or 10 mM AMUPol (cyan squares) between 1.5 to 7 W of continuous microwave power at 460.5 GHz. The line structures of TOTAPOL and AMUPol are shown in the inset.

Figure 4.

One-dimensional ¹³C(¹H) CPMAS DNP NMR of proline at 699.3 MHz/460.5 GHz (a) and 211.9 MHz/140 GHz (b). Two-dimensional ¹³C−¹³C DARR of proline at 699.3 MHz/460.5 GHz (c). The sample is 0.5 M U−¹³C−¹⁵N-proline in 60/30/10 d_8-glycerol/D₂O/H₂O with 10 mM TOTAPOL at 82 ± 2 K and ω_r/2π = 9 kHz.

Figure 5.

Two-dimensional ¹³C−¹³C DNP NMR correlation spectra of ¹H−¹³C−¹⁵N-GB1 (a,c,e) and ²H−¹³C−¹⁵N-GB1 (b,d,f) at 699 MHz/460 GHz. Microcrystals were placed in 60/30/10 d₈-glycerol/D₂O/H₂O with 10 mM TOTAPOL at 82 ± 2 K and a spinning frequency of 7.5 kHz.