Sample Preparation and DNP-Enhanced CPMAS of 13C-Proline

The DNP samples are prepared by adding a polarizing agent (such as e.g. TOTAPOL biradical) to a shared solvent or alternatively by utilizing a native radical on the sample of interest. The samples are measured under MAS while at low temperatures, typically 100–120 K. Unmodified NMR experiments are performed while benefitting from continuously DNP-enhanced signal intensity through CW microwave irradiation.

DNP-Enhanced CPMAS of 13C-Proline
DNP-Enhanced CPMAS of 13C-Proline in Glycerol/Water with 10 mM AMUPOL at 395 GHz/600 MHz: 25 µl sample, 1.5 mg U-13C-15N Proline, 8 kHz MAS, CPMAS with Spinal 64 decoupling, 100 k sample temperature, 8 scans and 10 s recycle delay for both microwaves on and off spectra.

Improved sensitivity from DNP allows the characterization of expansin protein binding to plant cell walls

DNP experiments allow rapid detection of ~1% U-13C,15N expansin mixed with plant cell walls. A REDOR filter selects only signals from the expansin 13C signals and spin diffusion mixing following the REDOR filter reveals correlations between expansin and the cell-wall polysaccharides. Comparison of the wild-type protein with two mutants indicate that site-specific cellulose binding is correlated with strong wall-loosening activity.

DNP fig3 improved
Fig. 3. (A) Graphic depicting expansin binding to plant cell walls. WT (B) and RKK mutant (C) spin diffusion spectra showing the buildup of signals from the protein to the cell-wall polysaccharides. (D) Comparison of signals transferred from the WT and two mutants to the plant cell walls. (E) 2D 13C-13C spectrum showing direct evidence of protein-polysaccharide correlations through spin diffusion.
T. Wang et al. PNAS 2013, 110, 16444-16449

DNP-enhanced NMR in biological solids

A wide range of biological samples have been successfully enhanced on the Bruker DNP-NMR spectrometer including small peptides, soluble proteins, membrane proteins, and large biological complexes.

DNP-enhanced NMR in biological solids
z-filtered (ZF) TEDOR experiment comparing sensitivity and spectral content at 300 K, 700 MHz without DNP and 100 K, 400 MHz with DNP.
M. J. Bayro, et al. J. Am. Chem. Soc., 2011, 133, 13967

Material Science

DNP NMR allows the characterization, at the molecular level, of hybrid organic silica material. These materials are key compounds for applications in catalysis, drug delivery, separation and purification devices.

DNP in material science
DNP experiments on natural abundance Mat-PrIm: (a) Structure of bCTbK polarizing agent; DNP-enhanced 13C, (b) and 15N (c) 1D spectra and 1H- 13C (d), 1H- 15N (e), 1H-29Si, (f) heteronuclear correlation spectra. 1H DNP signal enhancement = 78 at 100.
K. A. Zagdoun et al. J. Am. Chem. Soc., 2012, 134, 2284

DNP with Fast MAS: Rapid Structural Characterization

25 kHz MAS improves the resolution of DNP spectra, enables long-range inter-residue polarization transfers, and is helpful for extending assignments. DNP experiments were performed on Pf1 bacteriophage with non-uniform sampling (NUS) for rapid acquisition of multidimensional experiments.

DNP with Fast MAS
Fast spinning leads to longer relaxation times and assignments are simplified by use of long-range transfer sequences (e.g. CANCOCA, sequential sidechain correlation). Full assignment of intact virus particle was possible in under 1 week of experiment time
Reference: Sergeyev I. et al. (2017) Proc Natl Acad Sci USA, 114: 5171