Advance neo

Reagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatography was performed on silica gel, spots were visualised with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Bruker Advance Neo (400 MHz) spectrometer with chemical shifts values (δ) in ppm relative to TMS using the residual DMSO-d₆ signal (¹H 2.50; ¹³C 39.52) or CDCl₃ signal (¹H 7.26; ¹³C 77.16) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique. Elemental analyses were measured using Carlo Erba (EA1108) apparatus (Milan, Italy).

¹⁵N solid-state NMR experiments were carried out on a Bruker Avance III spectrometer operating at 9.4 T (Larmor frequency of 40.5 MHz) using a 4 mm magic-angle spinning (MAS) double resonance probe head. Around 10 mg of cuticle per treatment (five individuals) was packed in a 50 µL HRMAS rotor to ensure a proper centering of the sample. ¹H–¹⁵N cross-polarization (CP-MAS) experiments were performed at a MAS frequency of 14,286 Hz with a contact time of 1 ms (¹H and ¹⁵N radiofrequency fields during CP of ca. 55 and 40 kHz, respectively), and with a recycling delay of 1 s. The experimental time was about 2–2.5 days for each spectrum. The ¹⁵N spectra were referenced using L-tyrosine (δ = 40.4 ppm). The ¹H–¹⁵N–¹³C double CP-MAS experiment was carried out on a Bruker Advance Neo spectrometer operating at 14.1 T (¹³C Larmor frequency of 150.9 MHz) using a 3.2 mm Bruker CP-MAS CryoProbe^{TM32 (link)}. The MAS frequency was 11 kHz, and the ¹H–¹⁵N and ¹⁵N–¹³C contact times were 1.5 and 7 ms, respectively. The recycle delay was 3.5 s, and the 65,536 scans were accumulated.