Vibrational spectroscopy and molecular modeling reveal thermorotameric lipid-tail dynamics of AM404.

AM404, a bioactive metabolite of acetaminophen/paracetamol, is a lipid-like signaling molecule whose activity may depend not only on its aromatic-amide head group but also on the conformational behavior of its arachidonoyl tail. We combined temperature-dependent attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) of an air-dried film, a complementary room-temperature Fourier-transform Raman (FT-Raman) fingerprint, molecular dynamics (MD) simulations of a single AM404 molecule in water, and MD-informed gas-phase density functional theory (DFT) calculations. Between 37 and 45 °C, the ATR-FTIR I(2872)/I(2855) chain-sensitive ratio decreased monotonically from 0.681 ± 0.006 to 0.651 ± 0.008 in technical replicate measurements, while the aromatic-amide region remained spectroscopically stable. The aqueous MD model localized the principal conformational variability to the arachidonoyl tail while preserving broadly similar global compactness across the simulated temperatures. DFT calculations captured the corresponding broad vibrational regions and supported a regional assignment of conformer-sensitive changes to tail-related C-H stretching and skeletal modes; they were not used as a quantitative model of the dried film. The four selected MD-derived conformers occupied a shallow, method-dependent energetic range, and the relative Boltzmann factors calculated from their DFT Gibbs energies are comparative descriptors rather than equilibrium populations. Together, these observations indicate that a modest temperature increase within the physiological-to-fever range is accompanied by detectable changes in the lipid-tail-sensitive response of AM404, while the aromatic-amide region remains comparatively stable. The study provides reference vibrational data for AM404 and identifies the conformationally flexible arachidonoyl tail as the principal molecular region associated with the observed temperature-associated spectral response.