Diastereo- and enantioseparation of a Nα-Boc amino acid with a zwitterionic quinine-based stationary phase: Focus on the stereorecognition mechanism

•The ZWIX(+) column allowed getting the Boc-Aph(Hor)-OH (1) isomeric peaks resolved.•ECD studies and molecular dynamic simulations allowed to assign the elution order.•Molecular descriptors revealed the active role of achiral elements of the CSP.

A chiral chromatography method enabling the simultaneous diastereo- and enantioseparation of Nα-Boc-N4-(hydroorotyl)-4-aminophenylalanine [Boc-Aph(Hor)-OH, 1] was optimized with a quinine-based zwitterionic stationary phase. The polar-ionic eluent system consisting of ACN:MeOH:water—49.7:49.7:0.6 (v/v/v) with formic acid (4.0 mM) and diethylamine (2.5 mM), allowed the successful separation of the four acid stereoisomers: αd,d-/d,l-1 = 1.08; αd,l-/l,d-1 = 1.08; αl,d-/l,l-1 = 1.40.According to the in-house developed synthetic procedure and the recorded electronic circular dichroism spectra, the following stereoisomeric elution order was readily established in the optimal chromatographic conditions: d,d-1 < d,l-1 < l,d-1 < l,l-1.With the aim of better understanding the molecular basis of the retention behaviour of the four stereoisomers in the employed chromatographic system and conditions, a computational protocol consisting in molecular dynamics simulations was applied. The use of the three descriptors INTER (in kcal mol−1, encoding for the interaction energy between the selector SO unit and the whole system), INTER_SA (in kcal mol−1, encoding for the interaction energy between SO and the sole selectand SA), and SELF (in kcal mol−1, encoding for the conformational energy of SA relative to its minimum energy registered by the collected snapshots) revealed the active role of achiral sub-structural elements of the chiral stationary phase and eluent components in the overall stereorecognition mechanism.

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