Transitioning enantioselective indicator displacement assays for alpha-amino acids to protocols amenable to high-throughput screening.

ABSTRACT: Enantioselective indicator displacement assays (eIDAs) for alpha-amino acids were conducted in a 96-well plate format to demonstrate the viability of the technique for the high-throughput screening (HTS) of enantiomeric excess (ee) values. Chiral receptors [Cu(II)(1)](2+) and [Cu(II)(2)](2+) with the indicator chrome azurol S were implemented for the eIDAs. Enantiomeric excess calibration curves were made using both receptors and then used to analyze true test samples. These results were compared to those previously obtained with a conventional UV-vis spectrophotometer, and they showed little to no loss of accuracy, while the speed of analysis was increased. A sample of valine of unknown ee was synthesized through an asymmetric reaction to produce a realistic reaction sample, which was analyzed using receptor [Cu(II)(1)](2+). The experimentally determined ee using our eIDA was compared to that obtained by chiral HPLC and (1)H NMR chiral shift reagent analysis. This gave errors of 4.7% and 12.0%, respectively. In addition to the use of ee calibration curves, an artificial neural network (ANN) was used to determine the % L-amino acid of the test samples and of the sample of valine of unknown ee from the asymmetric reaction. This method obtained errors of 5.9% and 2.2% compared to chiral HPLC and (1)H NMR chiral shift reagent analysis, respectively. The technique using calibration curves for the determination of ee on a 96-well plate allows one to determine 96 ee values in under a minute, enabling its use for HTS of asymmetric reactions with acceptable accuracy.

SUBMITTER: Leung D 

PROVIDER: S-EPMC7815441 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature