Diastereo- and enantioseparation of a N(α)-Boc amino acid with a zwitterionic quinine-based stationary phase: focus on the stereorecognition mechanism

Abstract

A chiral chromatography method enabling the simultaneous diastereo- and enantioseparation of N(α)-Boc-N(4)-(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 (4.0mM) and diethylamine (2.5mM), 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 kcalmol(-1), encoding for the interaction energy between the selector SO unit and the whole system), INTER_SA (in kcalmol(-1), encoding for the interaction energy between SO and the sole selectand SA), and SELF (in kcalmol(-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.

Keywords: Chiral recognition mechanism; Cinchona alkaloid-derived zwitterionic chiral stationary phases; Diastereo- and enantioseparation; Electronic circular dichroism; Pharmaceutically relevant N(α)-Boc amino acid; Polar-ionic mode.