Selective 1H-14N Distance Measurements by 14N Overtone Solid-State NMR Spectroscopy at Fast MAS

Accurate distance measurements between proton and nitrogen can provide detailed information on the structures and dynamics of various molecules. The combination of broadband phase-modulated (PM) pulse and rotational-echo saturation-pulse double-resonance (RESPDOR) sequence at fast magic-angle spinning (MAS) has enabled the measurement of multiple H-1-N-14 distances with high accuracy. However, complications may arise when applying this sequence to systems with multiple inequivalent N-14 nuclei, especially a single H-1 sitting close to multiple N-14 atoms. Due to its broadband characteristics, the PM pulse saturates all N-14 atoms; hence, the single H-1 simultaneously experiences the RESPDOR effect from multiple H-1-N-14 couplings. Consequently, no reliable H-N distances are obtained. To overcome the problem, selective N-14 saturation is desired, but it is difficult because N-14 is an integer quadrupolar nucleus. Alternatively, N-14 overtone (OT) NMR spectroscopy can be employed owing to its narrow bandwidth for selectivity. Moreover, owing to the sole presence of two energy levels (m = +/- 1), the N-14 OT spin dynamics behaves similarly to that of spin-1/2. This allows the interchangeability between RESPDOR and rotational-echo double-resonance (REDOR) since their principles are the same except the degree of N-14 OT population transfer; saturation for the former whereas inversion for the latter. As the ideal saturation/inversion is impractical due to the slow and orientation-dependent effective nutation of N-14 OT, the working condition is usually an intermediate between REDOR and RESPDOR. The degree of N-14 OT population transfer can be determined from the results of protons with short distances to N-14 and then can be used to obtain long-distance determination of other protons to the same N-14 site. Herein, we combine the N-14 OT and REDOR/RESPDOR to explore the feasibility of selective H-1-N-14 distance measurements. Experimental demonstrations on simple biological compounds of L-tyrosine.HCl, N-acetyl-L-alanine, and L-alanyl-L-alanine were performed at 14.1 T and MAS frequency of 62.5 kHz. The former two consist of a single N-14 site, whereas the latter consists of two N-14 sites. The experimental optimizations and reliable fittings by the universal curves are described. The extracted H-1-N-14 distances by OT-REDOR are in good agreement with those determined by PM-RESPDOR and diffraction techniques.