Crystallisation studies of glycine-based dipeptides: sequence-dependent solubility, cocrystal formation, and kinetics

Peptides, composed of amino acids, are vital in biological processes and promising for drug development due to their precision in targeting diseases. However, their high production costs, delivery challenges, and variable efficacy hinder widespread use. This study aims to improve therapeutic peptide availability by optimising purification and formulation through crystallisation techniques, focusing on model peptide behaviour in processes like dissolution and cocrystal formation. Using glycine and glycine-based dipeptides as models, the research examines the impact of peptide sequence on solubility, with alanine (-CH₃ side chain) introduced to study sequence effects. Solubility at 298.15 K follows the order: glycyl-L-alanine (gly-ala) > L-alanyl-L-alanine (ala-ala) > glycylglycine (gly-gly) > L-alanylglycine (ala-gly). Molecular dynamics simulations attribute this trend to intramolecular hydrogen bonding, with temperature dependency also observed. Antisolvent effects of ethanol and DMSO on crystallisation were evaluated. A novel cocrystal, Glycine Diglycine Hydrate (GDH), was discovered, characterised by X-ray diffraction and other methods. GDH exhibited lower thermal stability than glycine and gly-gly, with Hirshfeld surface analysis identifying intermolecular hydrogen bonds as stabilising factors. A ternary phase diagram clarified its phase behaviours. Cocrystallisation process of GDH were explored using supersaturated solutions with silica spheres to promote nucleation. Induction time variations, influenced by thermodynamics and kinetics, were analysed using classical nucleation theory (CNT). Results showed nucleation rate sensitivity to concentration changes, driven by hydrogen bonding on silica surfaces. This work highlights the role of hydrogen bonding in peptide solubility and cocrystallisation, offering insights into dipeptide thermodynamics and kinetics. It provides a foundation for multicomponent crystallisation and improved pharmaceutical process control.