Enantiomeric Separation of Monosubstituted Tryptophan Derivatives and Metabolites by HPLC with a Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phase and Its Application to the Evaluation of the Optical Purity of Synthesized 6-Chloro-l-Tryptophan.

Abstract

6-Chlorotryptophan possesses unique bioactivity and can be used as a precursor for several bioactive compounds in medicinal chemistry. It was enantioselectively synthesized by condensing 6-chloroindole with racemic N-acetylserine, followed by enzymatic hydrolysis with l-aminoacylase (EC 3.5.1.14). The optical purity was examined by conducting high-performance liquid chromatography with a Cinchona alkaloid-based zwitterionic chiral stationary phase (CSP) [CHIRALPAK(®) ZWIX(+)], which bears a chiral trans-2-aminocyclohexanesulfonic acid moiety tagged at C-9 of the Cinchona alka-loid. The zwitterionic CSP enabled efficient enantiomeric separations of monosubstituted tryptophan derivatives 1-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-methoxytryptophan, and 6-chlorotryptophan with a methanol/H2O (98/2) mobile phase containing formic acid (FA) and diethylamine (DEA) additives. The mobile phase contains 25-75 mM FA and 20-50 mM DEA, enabling good separation of the enantiomers of each tryptophan derivative (α > 1.25). Thus, the optical purity of the synthesized 6-chloro-l-tryptophan was easily determined (greater than 99.0%) using HPLC with the zwitterionic CSP.