Solid-state NMR observation of chitin in whole cells by indirect 15N detection with NC, NCC, CNC and CNCC polarization transfers

Chitin is the most important nitrogen-containing polysaccharide found on Earth. This polysaccharide is a polymer of an N-acetylglucosamine and it is a crucial structural component of fungal cell walls and crustaceans. Magic-angle spinning solid-state NMR is emerging as a powerful analytical approach to study polysaccharides in the context of intact cell walls and whole cells. The presence of an acetamido group in chitin is attractive for ¹⁵N solid-state NMR. Here we investigate the use of various multi-step polarization transfer experiments incorporating indirect ¹⁵N detection at moderate spinning frequency, adapted from pulse sequences commonly employed for residue resonance assignment in biosolid proteins. The ¹³C,¹⁵N chitin spin topology slightly differs from amino acids, and we discussed the use of frequency-selective ¹⁵N-¹³C cross-polarization transfers followed by broadband or frequency-selective homonuclear ¹³C-¹³C transfers to detect chitin resonances. Demonstrated here for chitin found in the cell wall of the fungus Aspergillus fumigatus, the use of indirect ¹⁵N detection through multi-step polarization transfers could be advantageous to investigate more complex nitrogen-containing polysaccharides found in whole cells and peptidoglycan samples.