Structural Evolution of Protonated Phenylalanine Induced by Stepwise Hydration

We investigated the structural evolution of protonated phenylalanine ions (H⁺Phe) upon stepwise hydration using cryogenic ion spectroscopy. H⁺Phe ions were generated by electrospray ionization and introduced into a reaction trap containing water vapor at 130–170 K to form hydrated complexes, H⁺Phe(H₂O)_n (n = 1–5). Subsequently, these complexes were cooled in a cryogenic ion trap at 4 K and analyzed by ultraviolet photodissociation (UVPD) spectroscopy. The UVPD spectra exhibited well-resolved vibronic bands near the origin bands of the S₀–S₁ transitions. To determine the number and structures of distinct conformers, we employed IR photodissociation and IR ion-dip spectroscopy in conjunction with quantum chemical calculations. The first water molecule binds exclusively to an NH bond of the protonated NH₃⁺ group, while the second and third water molecules bind either to the remaining NH bonds or to the OH group of the carboxyl terminus. In particular, H⁺Phe(H₂O)₄ exists as a single conformer in which all available hydrogen-bonding sites are occupied, completing the first hydration shell. Water molecules preferentially bind to a specific conformer of bare H⁺Phe, indicating hydration-induced conformational selection. These findings provide detailed insights into site-specific hydration and reveal the progressive conformational changes in H⁺Phe induced by sequential water attachment.