High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening

Abstract

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4–6 h and requires 10–20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.

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Fig. 1: Schematic of the enantiomeric excess determination process and experimental workflow.
Fig. 2: Components and self-assembly mechanisms used for enantiomeric excess determination in chiral amines and amine derivatives.
Fig. 3: Tracking complexes by shifts in fluorescence maxima.
Fig. 4: Components and self-assembly mechanisms used for enantiomeric excess determination in chiral diols and sugars.
Fig. 5: Examples of the use of the protocol for determination of enantiomeric purity of diols and their absolute configuration.
Fig. 6: Output of LDA of the expanded calibration dataset using the l-TrpOH/2-FPBA sensor assembly.
Fig. 7: Examples of small and large differences between fluorescence intensities of diastereomeric complexes assembled with opposite enantiomers of chiral analyte at the same final concentration.
Fig. 8: Example of pipetting plate layout in a simple assay for determination of enantiomeric purity of chiral amines, amino alcohols, amino-acid esters, diols, and sugars using one receptor premix.
Fig. 9: Example of pipetting plate layouts in an assay using one receptor premix for simultaneous determination of e.e. and total concentration (yield) with an ANN.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Source data for all figures are provided with the paper.

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Acknowledgements

We are thankful to our colleague T. Minami, who provided expertise that greatly assisted the research. We are also grateful to V. Brega, for her assistance with parallel asymmetric synthesis, and S. Gozem, for his help with computational modeling of BINOL/VAPOL self-assemblies with analytes.

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Authors

Contributions

E.G.S. and P.A. designed the protocol. E.G.S. performed the experiments. P.A. and T.D.J. supervised the project. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Pavel Anzenbacher Jr..

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Key reference(s) using this protocol

Shcherbakova, E. G., Minami, T., Brega, V., James, T. D. & Anzenbacher Jr., P. Angew. Chem. Int. Ed. 54, 7130–7133 (2015): https://doi.org/10.1002/anie.201501736

Shcherbakova, E. G. et al. Chemistry 22, 10074–10080 (2016): https://doi.org/10.1002/chem.201601614

Shcherbakova, E. G., Brega, V., Lynch, V. M., James, T. D. & Anzenbacher Jr., P. Chemistry 23, 10222–10229 (2017): https://doi.org/10.1002/chem.201701923

Integrated supplementary information

Supplementary Figure 1 Obtained fluorescence intensity data for valine methyl ester standards.

Left: Calibration and residual curve obtained using VANOL receptor-analyte complex with standard deviation of the residuals = 2.3%. Right: Calibration and residual curve obtained using BINOL receptor-analyte complex with standard deviation of the residuals = 1.4%. Each data point consists of 20 technical replicates.

Supplementary Figure 2 Obtained fluorescence intensity data for data for cis-1-amino-2-indanol standards.

Left: BINOL receptor-analyte complex with standard deviation of the residuals = 1.9%. Right: VANOL receptor-analyte complex with standard deviation of the residuals = 2.0%. Each data point consists of 20 technical replicates.

Supplementary Figure 3 Obtained fluorescence intensity data for atorvastatin standards.

Standard curve obtained from L-tryptophanol-2-FPBA assembly (1:1, 40 µM) with atorvastatin enantiomers at various ee with standard deviation of the residuals = 0.7%. Each data point consists of 20 technical replicates.

Supplementary Figure 4 Obtained fluorescence intensity data for hydrobenzoin standards.

Standard curve obtained from L-tryptophanol-2-FPBA assembly (1:1, 40 µM) with atorvastatin enantiomers at various ee with standard deviation of the residuals = 0.8%. Each data point consists of 20 technical replicates.

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Supplementary Figs. 1–4 and Supplementary Information Text and Data.

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Shcherbakova, E.G., James, T.D. & Anzenbacher, P. High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening. Nat Protoc 15, 2203–2229 (2020). https://doi.org/10.1038/s41596-020-0329-1

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