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.

Figure 1.

Structures of ligand 1, chiral receptors [Cu^II((R,R)-1)]²⁺ and [Cu^II((S,S)-1)]²⁺, ligand 2, chiral receptors [Cu^II((R,R)-2)]²⁺ and [Cu^II((S,S)-2)]²⁺, and indicator chrome azurol S (CAS).

Figure 2.

(a) Supposing that the ee products from an asymmetric reaction were evenly distributed, with a ±15% error in ee values, approximately 75% could be discarded after screening. (b) In the case of a Gaussian distribution of product ee, an even lower number of samples would need to be further analyzed with a slower, high-accuracy technique.

Figure 3.

The 96-well plate used for making the ee calibration curves. (a) For valine, aspartate, histidine, and isoleucine using receptor [Cu^II((R,R)-1)]²⁺ (top four rows) and six test samples for each of the above amino acids (bottom four rows). (b) For valine, tryptophan, alanine, and serine using receptor [Cu^II((R,R)-2)]²⁺ (top four rows) and six test samples for each of the above amino acids (bottom four rows).

Figure 4.

Enantiomeric excess calibration curves obtained using a 96-well plate reader. (a,b) Absorbance at 602 nm as a function of ee for displacement experiments performed in a solution containing CAS (10 μM), Cu(OTf)₂ (200 μM), and (R,R)-1 (2.5 mM) in 1:1 MeOH:H₂O, 50 mM HEPES buffered to pH 7.5, with the addition of (a) aspartate (302 μM) or (b) histidine (202 μM). (c,d) Absorbance at 602 nm as a function of ee for displacement experiments performed with the addition of amino acid into a solution containing CAS (10 μM), Cu(OTf)₂ (105 μM), and (R,R)-2 (8.8 mM) in 1:1 MeOH:H₂O, 50 mM HEPES buffered to pH 7.5, with the addition of (c) alanine (149 μM) or (d) valine (125 μM).

Figure 5.

Chiral shift reagent, sodium[(R)-1,2-diaminopropane-N,N,N′,N′-tetraacetato]samarate(III) hydrate (8).

Scheme 1.

Asymmetric Reaction Used To Produce a Sample of Valine of Unknown ee^,

Scheme 2.

Equations Used To Determine Enantiomeric Excess Using % l-Amino Acid Determined through ANN Analysis