Selective (1)H-(14)N Distance Measurements by (14)N 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 (1)H-(14)N distances with high accuracy. However, complications may arise when applying this sequence to systems with multiple inequivalent (14)N nuclei, especially a single (1)H sitting close to multiple (14)N atoms. Due to its broadband characteristics, the PM pulse saturates all (14)N atoms; hence, the single (1)H simultaneously experiences the RESPDOR effect from multiple (1)H-(14)N couplings. Consequently, no reliable H-N distances are obtained. To overcome the problem, selective (14)N saturation is desired, but it is difficult because (14)N is an integer quadrupolar nucleus. Alternatively, (14)N 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 (14)N 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 (14)N 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 (14)N OT, the working condition is usually an intermediate between REDOR and RESPDOR. The degree of (14)N OT population transfer can be determined from the results of protons with short distances to (14)N and then can be used to obtain long-distance determination of other protons to the same (14)N site. Herein, we combine the (14)N OT and REDOR/RESPDOR to explore the feasibility of selective (1)H-(14)N 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 (14)N site, whereas the latter consists of two (14)N sites. The experimental optimizations and reliable fittings by the universal curves are described. The extracted (1)H-(14)N distances by OT-REDOR are in good agreement with those determined by PM-RESPDOR and diffraction techniques.