Raman spectroscopic investigation of pressure-induced transitions in 5-methyluridine hemihydrate crystal

The structural flexibility of modified nucleosides is closely related to the stability and adaptability of RNA architectures. High pressure introduces a unique thermodynamic perturbation that can reveal hidden conformational states and subtle intermolecular interactions in these molecular systems. This work presents a comprehensive investigation of the structural and vibrational properties of 5-methyluridine hemihydrate (5 mU) under high hydrostatic pressure up to 7.1 GPa using Raman spectroscopy. Rietveld refinement at ambient conditions confirms an orthorhombic structure with space group P2₁2₁2₁. High-pressure Raman spectra exhibit significant changes over the entire spectral range (100-3500 cm^-1), providing clear evidence of two pressure-induced conformational transition regions centered near ∼1 and ∼ 3 GPa. The first transition region is predominantly associated with CH and CH₃ vibrations, ribose banding modes, and the emergence of a new band at the lattice modes. The second transition region involves distortions of ribose and uracil moieties and changes in CO, NH, and OH modes, as well as the appearance of a new band in the lattice region, reflecting hydrogen-bond rearrangement. Principal Component Analysis applied to selected spectral regions provides quantitative support for these findings, revealing distinct clustering across the corresponding pressure ranges. A comparative analysis with unmodified uridine demonstrates that methylation, together with the hydrogen-bonding network involving water molecules, plays a key role in modulating the crystal response to pressure by tuning intermolecular interactions, highlighting the potential of 5 mU as a promising system for investigating molecular adaptability under extreme conditions.