Investigation of Ethanol and Isopropanol as Greener Alternatives to Acetonitrile in the RP-HPLC Purification of Histone Tail Peptides Bearing Acylation-Type Post-Translational Modifications

Background: Histone post-translational modifications (PTMs) play a pivotal role in the regulation of chromatin structure and gene expression, making them key targets in structural and epigenetic research. Synthetic histone peptides bearing specific PTMs are essential tools for elucidating the molecular mechanisms of histone function and protein–histone interactions. Methods: We synthesized histone H4 tail peptides containing site-specific lysine modifications using solid-phase peptide synthesis (SPPS). The correct synthesis of the peptides was confirmed by their molecular weights using a mass spectrometer. Results: An improved high-performance liquid chromatography (HPLC) method was developed to efficiently separate peptides with one modification difference. In alignment with green chemistry principles, we evaluated ethanol and isopropanol as an alternative organic solvent to acetonitrile in the mobile phase. The optimized HPLC method using acetonitrile enabled effective resolution of closely related peptide species, providing peptides suitable for downstream applications requiring high purities such as structural biology. Conclusions: This study presents a strategy for the purification of histone PTM peptides, emphasizing both analytical performance and sustainability. Further investigation must be undergone to develop high-precision purification using green chemicals.