Transitioning Enantioselective Indicator Displacement Assays for α-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.

Chiral Recognition by Fluorescence: One Measurement for Two Parameters

“…To date, a number of techniques are under development for the high-throughput enantiomeric composition determination [13][14][15][16][17], including time resolved IR-thermographic method [18][19][20][21], electron spray mass spectrometry [22][23][24][25][26], capillary electrophoresis [27], UV/Vis [28][29][30], circular dichroism [31,32], and fluorescence [33,34]. Among these approaches, optical methods have been attracting increasing attention due to their ability for quick data collection and compatibility with high-throughput screening system.…”

Array‐Based Sensors

“…Also, a similar method was then applied to enantioselective indicator displacement assays for R-amino acids to achieve high-throughput screening of enantiomeric excess (ee) values. Complexes of chiral diamines with Cu(II) were incubated with the color indicator chrome azurol and the dye displacement as a function of ee was analyzed using ANN as well as using calibration plots [111]. Similar methods aimed at simplification of enantioselective indicator displacement assays have also been presented in the literature [112].…”

Colorimetric sensing of anions in water using ratiometric indicator-displacement assay

“…In recent years, indicator-displacement assay (IDA) has been widely investigated [25][26][27][28]. This method is largely derived from the color change of colorimetric or fluorescent ligands, which compete with analytes for binding to the receptors.…”