title: A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay.
abstract: A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.
authors.affiliation: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nimura
authors.forename: Takako
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Aya
authors.initials: A
authors.lastname: Watanabe
authors.forename: Masae
authors.initials: M
authors.lastname: Sekine
authors.forename: Takemitsu
authors.initials: T
authors.lastname: Furuchi
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
authors.forename: Tairo
authors.initials: T
authors.lastname: Oshima
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Homma
mesh_terms: D000591:Amino Acid Isomerases / Q000032:analysis / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D001331:Automation; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015394:Molecular Structure; D013237:Stereoisomerism; D013439:Sulfhydryl Reagents / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D013439:Sulfhydryl Reagents; D000591:Amino Acid Isomerases; C019749:aspartate racemase