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