Topical Developments in High-Field Dynamic Nuclear Polarization

Abstract

We report our recent efforts directed at improving high-field DNP experiments. We investigated a series of thiourea nitroxide radicals and the associated DNP enhancements ranging from ε = 25 to 82 that demonstrate the impact of molecular structure on performance. We directly polarized low-gamma nuclei including ¹³C, ²H, and ¹⁷O using trityl via the cross effect. We discuss a variety of sample preparation techniques for DNP with emphasis on the benefit of methods that do not use a glass-forming cryoprotecting matrix. Lastly, we describe a corrugated waveguide for use in a 700 MHz / 460 GHz DNP system that improves microwave delivery and increases enhancements up to 50%.

Keywords: DNP; NMR; cross-effect; cryoprotection; polarizing agent; radicals.

Figure 1

¹³C{¹H} cross-polarization of ¹³C-urea in a 60/30/10 v/v d₈-glycerol/D₂O/H₂O with 20 mM TOTAPOL (top, ¹H DNP) and 40 mM TEMPO (bottom, ¹H DNP) acquired at 140 GHz / 212 MHz DNP NMR spectrometer with 8 W of microwave power, 4.5 kHz MAS, and 16 scans (on-signal) and 256 scans (off-signal).

Figure 2

¹³C{¹H} cross-polarization spectra of ¹³C-urea in DMSO/D₂O/H₂O (60:30:10, v/v) and 10 mM biradical polarizing agent (20 mM electrons) acquired at 140 GHz / 212 MHz DNP NMR spectrometer with 8 W of microwave power. ¹H DNP enhancements were scaled with respect to TOTAPOL using three thiourea variants. From top to bottom five radicals were studied including TOTAPOL (black), BT-urea (red), BT-thio-1 (thionocarbonate, grey), BT-thio-2 (BT-thionourethane, blue) and BT-thio-3 (BT-thiourea, green). The spectra inset are the on/off ¹³C{¹H} CPMAS spectra scaled to the TOTAPOL enhancement in DMSO/water mixture.

Figure 3

bTtereph synthetic process (a) and resulting 140 GHz EPR spectrum (b) and ¹H DNP field (c) profile of 10 mM bTtereph incorporated in 95% deuterated amorphous ortho-terphenyl.

Figure 4

Chemical structures and 140 GHz EPR spectra of three narrow-line radicals: (a) Trityl (OX063), (b) TMT, and (c) SA-BDPA.

Figure 5

Direct polarization of ¹³C (circle, blue), ²H (diamond, red) and ¹⁷O (triangle, grey) field profiles acquired at 5 T using 40 mM Trityl radical. 140 GHz EPR spectrum of trityl (black, top) with the appropriate SE matching conditions illustrated with the corresponding colored dashed lines.

Figure 6

Direct polarization of low-gamma nuclei using 40 mM trityl on (a) ¹³C (ν_L = 53 MHz), (b) ²H (ν_L = 32 MHz) and (c) ¹⁷O (28 MHz) in a glycerol/water cryoprotectant. DNP enhanced signals were acquired using 8 W of CW microwave power with the magnetic field set to the optimum field position (positive) shown in Figure 5.

Figure 7

MAS DNP sample preparation protocols for biophysical systems. Without cryoprotecting solvents (sans) include distributing a polarizing agent within the organic solid: amorphous (a) or crystalline (b) 95% deuterated ortho-Terphenyl (OTP) with 0.5 mol% bTtereph or using the SedDNP approach, U-¹³C,¹⁵N-Apoferritin (2 mM TOTAPOL) (c). Alternative is distributing the radical in a cryoprotecting solvent (avec) homogenously, U-¹³C,¹⁵N-Apoferritin in d₈-glycerol/D₂O/H₂O (v/v 60/35/5) and 15 mM TOTAPOL (d) or heterogeneously using microcrystals, [U-¹³C,¹⁵N GNNQ]QNY in d₈-glycerol/D₂O/H₂O (w/w 70/23/7) and 35 mM TOTAPOL (e).

Figure 8

Artistic rendering of the new waveguide designed for the 460 GHz / 700 MHz DNP NMR spectrometer (FBML-MIT). The inset is an ¹³C{¹H} cross-polarization MAS spectrum (mw on/off) of 1M ¹³C-Urea in d₈-glycerol/D₂O/H₂O (v/v 60/30/10) with 10 mM TOTAPOL and packed into a 3.2 mm sapphire rotor, acquired at 80 K and a spinning frequency of 5.2 kHz. Abbreviations: copper (Cu), aluminum (Al) and stainless steel (SS).

Figure 9

(A) ¹³C-¹³C DARR spectrum of U-¹³C-Proline (0.5 M) in d₈-glycerol/D₂O/H₂O (v/v 60/30/10) with 10 mM TOTAPOL (¹H enhancement of 33 (3)) using a 20 ms DARR mixing period. (B) An enlarged aliphatic and carbonyl region illustrating the connectivity of U-¹³C-Proline. Sample was packed into a 3.2 mm sapphire rotor, data was acquired with 8 scans, rd = 20 s, 64 increments, 11 W of microwave power, sample temperature 82 (2) K and a spinning frequency of 9.2 kHz.

Figure 10

¹³C-¹³C correlation spectrum of U-¹³C,¹⁵N-apoferritin at 5 T (green, ω_r/2π = 4.8 kHz, T = 82 K, mw = 9 W) and 16.4 T (blue, ω_r/2π = 8.8 kHz, T = 87 K, mw = 10 W) using DNP MAS NMR.

Scheme 1

Spin population distribution for a two-spin (1 electron and 1 nucleus) system at thermal equilibrium (A). SE conditions for the positive, ω_0S − ω_0I (B) and negative enhancement, ω_0S + ω_0I (C).

Scheme 2

Spin population distribution for a three-spin (2 electrons and 1 nucleus) system at thermal equilibrium with the NMR transitions marked (A). The CE condition for the negative (B) and positive (C) enhancement. Microwave saturation of the electron transition (ω_0S1 or ω_0S2) leads to a three-spin flip-flop-flip process that distributes the population (ω_CE), thus increasing the net nuclear polarization.