This study aimed to develop a technique to tackle inhomogeneous broadening, a major cause in the loss of site-specific information in solid-state NMR, using a technique termed saturation exchange spectroscopy (SEXY).
Figure 1. Saturation exchange spectroscopy’s application in inhomogeneous broadening. NMR requires the absorbance of RF radiation to cause ‘flipping’ of nuclear spins between two spin states (in the case of 13C) with a population imbalance. Frequency-selective RF pulses, of sufficiently high energy, irradiate single peaks within a broadened region resulting in equalisation of populations within both spin states. Thus, the NMR signal sharply decreases (saturates). Saturation transfer subsequently occurs via spin diffusion, a process driven by dipolar coupling (through space interactions), resulting in the loss of signal of coupled peaks. Spin diffusion arises among nuclear spins of the same kind (like-spins) within a few to several Angstroms in space.11 Therefore, previously unattainable structural information, from broadened peaks, is now accessible. Spin diffusion occurs due to the presence of Ii+Ij- and Ii-Ij+ terms within the dipolar spin Hamiltonian between two like-spins.11,12 These terms cause mutual flipping of two nearby spins resulting in a transfer of polarisation from the first spin to the second. This mechanism causes propagation of spin polarisation until it is lost by longitudinal relaxation (T1) processes.
Proof-of-concept was obtained using N-formyl-Met-Leu-Phe-OH (MLF), a simple tripeptide, whereby sites on the amino acid residues were selectively saturated and saturation transfer was observed.
Figure 4. Two-dimensional 13C SEXY spectra of fMLF. Each row in the indirect dimension of the spectra consists of a saturation pulse at a specific frequency. (b) An easy to interpret difference spectrum was obtained by subtracting each row of (a) the original data by a 1D unsaturated spectrum. (c) The aliphatic region was of most interest as extensive saturation transfer is visible. In total, 17 saturation pulses were applied, two of which were off-resonance, at the following frequencies (ppm): 188.9 (1), 174.8, 173.2, 172.0, 135.6, 130.0, 127.8, 56.8, 54.3, 52.1, 40.6, 37.6, 28.8, 24.8, 19.7, 14.1 and -9.9 (17).
Two-dimensional 13C SEXY spectra obtained at fast and slow magic angle spinning frequencies were comparable to 2D 13C-13C Proton-driven spin diffusion (PDSD) spectra suggesting that SEXY was well optimised and could also be used for structural assignment.
Figure 8. Two-dimensional 13C spectra obtained using (a) PDSD and (b) SEXY. A mixing/ saturation time of 0.300 s was utilised. The total analysis time for the PDSD experiment was ~3.5 hours and ~1.5 hours for the SEXY experiment.
Directing future studies on the analysis of complex inhomogenous systems would determine if SEXY is a suitable approach for tackling inhomogeneous broadening, one of ssNMR's greatest issues.