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

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-p...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2003-04, Vol.315 (2), p.262-269
Main Authors: Nimura, Noriyuki, Fujiwara, Takako, Watanabe, Aya, Sekine, Masae, Furuchi, Takemitsu, Yohda, Masafumi, Yamagishi, Akihiko, Oshima, Tairo, Homma, Hiroshi
Format: Article
Language:English
Subjects:
Amino Acid Isomerases - analysis
Amino Acid Isomerases - metabolism
Amino Acids - analysis
Amino Acids - chemistry
Amino Acids - isolation & purification
Aspartate racemase
Automation
Calibration
Chiral derivatization
Chromatography, High Pressure Liquid - methods
d-Amino acid
d-Aspartate
Enantioseparation
Fluorometric detection
Molecular Structure
o-Phthalaldehyde
Stereoisomerism
Sulfhydryl Reagents - chemical synthesis
Sulfhydryl Reagents - chemistry
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/S0003-2697(02)00705-4