Enantioseparation of Aromatic b 3 -Amino acid by Precolumn Derivatization with o-Phthaldialdehyde and N-Isobutyryl- L-cysteine Birgit Brucher 1,& , Jens Rudat 1 , Christoph Syldatk 1 , Oliver Vielhauer 2 1 Section II: Technical Biology, Institute of Process Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany; E-Mail: birgit.brucher@kit.edu 2 Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany Received: 11 February 2010 / Revised: 4 March 2010 / Accepted: 16 March 2010 Online publication: 17 April 2010 Abstract In this study we describe the enantioseparation of aromatic b 3 -amino acids by precolumn derivatization with o-phthaldialdehyde and N-isobutyryl-L-cysteine. Derivatization conditions were studied in detail for (R,S)-b-phenylalanine and (R,S)-b-tyrosine revealing a reaction time of 1 min and a molar ratio of the reagents b 3 -amino acid to o-phthaldialdehyde to N-isobutyryl-L-cysteine of 1:25:25 as optimal. The method was validated for (R,S)-b-phen- ylalanine in a bacterial cell extract. The analysis provided excellent specificity and repro- ducibility. The limit of quantification was 25 pmol per 0.5 lL injection. The method could be successfully transferred to the enantioseparation of other b 3 -amino acids. Enantioseparation of all studied compounds could be achieved in 4–11 min. Keywords Column liquid chromatography Enantiomer separation b-Amino acids Cell extract o-Phthaldialdehyde and N-isobutyryl-L-cysteine Introduction There has been an increasing interest in the production of chirally pure b-amino acids in recent years because of their occurrence in a variety of pharmaceuti- cally interesting natural products like the anticancer drug Taxol [1] as well as a wide variety of prospective agents such as enediyne C-1027 [2], tychonamide A+B[3] and pedein A + B [4]. Fur- thermore the preparation of peptidomi- metics with b-amino acids has drawn growing attention [5, 6]. Peptidomimet- ics containing b-amino acids could be shown to possess increased proteolysis resistance [7, 8] while still bearing potent biological activity [9, 10]. Chirally pure b-amino acids can be produced via chemical or enzymatic synthesis from various precursors [11, 12]. In conjunction with these synthesis routes analytical methods are required to monitor the enantiomeric purity of the formed products. Especially for the screening, characterization and optimi- zation of novel biocatalysts a fast and sensitive detection method is needed which is applicable to complex sample matrices. Enantioseparation of b 3 -amino acids by LC has been achieved by direct and indirect methods. Many of the recent studies focus on the direct separation of b 3 -amino acid enantiomers on chiral stationary phases (CSP). Several CSPs have been applied such as macrocyclic antibiotic based phases [13–19], cyclo- dextrin phases [18, 20] and chiral crown ether phases [14, 21, 22]. Indi- rect methods include the derivatization with 2,3,4,6-tetra-O-acetyl-b-D-gluco- pyranosyl isothiocyanate (GITC) [14], N-a-(2,4-dinitro-5-fluorophenyl)-L-alani- neamide (FDAA) [14], (S)-N-(4-nitro- phenoxycarbonyl) phenylalanine meth oxyethyl ester ((S)-NIFE) [15, 16] and (1S,2S)-1,3-diacetoxy-1-(4-nitrophenyl)- 2-propylisothiocyanate ((S,S)-DANI) [15]. 2010, 71, 1063–1067 DOI: 10.1365/s10337-010-1578-x 0009-5893/10/06 Ó 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH Full Short Communication Chromatographia 2010, 71, June (No. 11/12) 1063