Clindamycin Phosphate-Mediated Enantioseparation of Amino Acids by Capillary Electrophoresis: Molecular Modeling and NMR Insights into the Chiral Recognition Mechanism

In this research paper, chiral separation of three amino acids (AAs), namely, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), was achieved by capillary electrophoresis with clindamycin phosphate as the chiral selector. Factors affecting the chiral separation, such as the BGE pH, concentration of clindamycin, organic modifier concentration, capillary temperature and applied voltage, were optimized and discussed. A BGE consisting of 80 mM clindamycin phosphate and 40 mM borate at pH 7.6 with 30% methanol was found suitable for chiral separation of tryptophan and tyrosine while the same BGE at pH 7.3 was suitable for enantioseparation of phenylalanine. The optimum capillary temperature and applied voltage were 20 °C and 19 kV. Molecular docking (Glide SP/MM-GBSA) and 1H NMR chemical-shift perturbation (CSP) analysis were combined to clarify the chiral recognition of tryptophan, phenylalanine, and tyrosine by clindamycin phosphate. Docking results support clear stereochemical discrimination for Trp, weaker discrimination for Phe, and near-degenerate discrimination for Tyr, and revealed that electrostatic attraction discrimination was the main binding anchor, supported by hydrophobic contacts and hydrogen bonding. The NMR results revealed chemical-shift perturbations and line-broadening patterns consistent with changes in the local chemical environments of clindamycin phosphate regions implicated by the docking models. Pronounced signal broadening in DL-Tyr further revealed dynamic binding behavior that could not be detected by docking alone.