Highly Efficient Polarizing Agents for MAS-DNP of Proton-Dense Molecular Solids

Abstract

Efficiently hyperpolarizing proton-dense molecular solids through dynamic nuclear polarization (DNP) solid-state NMR is still an unmet challenge. Polarizing agents (PAs) developed so far do not perform well on proton-rich systems, such as organic microcrystals and biomolecular assemblies. Herein we introduce a new PA, cAsymPol-POK, and report outstanding hyperpolarization efficiency on 12.76 kDa U-¹³ C,¹⁵ N-labeled LecA protein and pharmaceutical drugs at high magnetic fields (up to 18.8 T) and fast magic angle spinning (MAS) frequencies (up to 40 kHz). The performance of cAsymPol-POK is rationalized by MAS-DNP simulations combined with electron paramagnetic resonance (EPR), density functional theory (DFT) and molecular dynamics (MD). This work shows that this new biradical is compatible with challenging biomolecular applications and unlocks the rapid acquisition of ¹³ C-¹³ C and ¹⁵ N-¹³ C correlations of pharmaceutical drugs at natural isotopic abundance, which are key experiments for structure determination.

Keywords: Biomolecules; Dynamic Nuclear Polarization; MAS-DNP; Nuclear Magnetic Resonance; Pharmaceuticals; Polarizing Agents.

Figure 1.

Chemical structure of AsymPol-POK and cAsymPol-POK.

Figure 2.

(a) DFT calculated structure of cAsymPol-OH viewed from the side, showing conformer #1 on the left and conformer #2 on the right. (b) Concatenated X-band, 240 GHz EPR spectra and (c) MAS-DNP field profile at 9.4 T for AsymPol-POK (black) and cAsymPol-POK (blue). The corresponding simulated EPR spectra and MAS-DNP field profiles are given as red dotted lines. For the MAS-DNP field profiles, experimental points are reported as circles for AsymPol-POK and squares for cAsymPol-POK, full for experiments and open for simulations.

Figure 3.

Experimental performance of (a)-(b) ampicillin and (c)-(d) indomethacin powders impregnated with 10 mM (grey) and 40 mM (blue) AMUPol, AsymPol-POK and cAsymPol-POK in the standard deuterated DNP matrix, d₈-glycerol/D₂O/H₂O (60:30:10 v/v), at 105 K, 9.4 T and 8 kHz MAS. The plots show the signal-to-noise ratio per square root of the experimental time $(S/N)/\sqrt{t}$ in (a) and (c), and the optimal build-up time constant $T_{opt}$ in (b) and (d) for ampicillin and indomethacin, respectively.

Figure 4.

Molecular structures (a)-(b) and DNP-enhanced 2D ¹³C-¹³C (c)-(d) and ¹⁵N-¹³C (e)-(f) correlation spectra of ampicillin (a,c,e) and indomethacin (b,d,f) at NA. The DQ-SQ experiments in (c) and (d) were acquired using SR26 for dipolar recoupling, experiments in (e) and (f) were obtained with the zf-TEDOR sequence. All experiments were performed at 8 kHz MAS, 105 K and 9.4 T. Experimental times were 3 h for (b), 4 h for (c) and (d), and 5 h for (f). The ¹³C assignment is mapped out in the 2D spectra, yielding one-bond (purple), two-bonds (blue) and three-bonds (yellow) correlations. Spinning side bands, signals from the carrier frequency and signals from the glycerol solvent are indicated with red asterisks

Figure 5.

(a) ¹H-¹³C CP-MAS spectra with (red) and without (black) μwave irradiation of U-¹³C, ¹⁵N LecA, leading to an enhancement factor ${\epsilon }_{On/Off}$ of 130. (b) DNP-enhanced 2D SQ-SQ ¹³C-¹³C correlation spectrum using the DARR sequence of the U-¹³C, ¹⁵N LecA. All spectra were acquired at a MAS frequency of 40 kHz, 9.4 T and 106 K.

Scheme 1.

Synthesis of cAsymPol-POK. BCDP: bis(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite, BTT: 5-benzylthio-1H-tetrazole.