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

Nature Protocols volume 15, pages 2203–2229 (2020)Cite this article

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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.

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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.

References

  1. Parker, D. NMR determination of enantiomeric purity. Chem. Rev. 91, 1441–1457 (1991).

    Article  CAS  Google Scholar 

  2. Kelly, A. M. et al. Simple protocols for NMR analysis of the enantiomeric purity of chiral diols. Nat. Protoc. 3, 215–219 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Kelly, A. M., Pérez-Fuertes, Y., Arimori, S., Bull, S. D. & James, T. D. Simple protocol for NMR analysis of the enantiomeric purity of diols. Org. Lett. 8, 1971–1974 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Yeste, S. L., Powell, M. E., Bull, S. D. & James, T. D. Simple chiral derivatization protocols for 1 H NMR and 19 F NMR spectroscopic analysis of the enantiopurity of chiral diols. J. Org. Chem. 74, 427–430 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Bentley, K. W., Nam, Y. G., Murphy, J. M. & Wolf, C. Chirality sensing of amines, diamines, amino acids, amino alcohols, and α-hydroxy acids with a single probe. J. Am. Chem. Soc. 135, 18052–18055 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Jo, H. H., Lin, C.-Y. & Anslyn, E. V. Rapid optical methods for enantiomeric excess analysis: from enantioselective indicator displacement assays to exciton-coupled circular dichroism. Acc. Chem. Res. 47, 2212–2221 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Dragu, E. A., Naubron, J.-V., Hanganu, A., Razus, A. C. & Nica, S. Absolute configuration determination of azulenyl diols isolated from asymmetric pinacol coupling: chiral azulene-containing diols. Chirality 27, 826–834 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Welch, C. J. Microscale chiral HPLC in support of pharmaceutical process research. Chirality 21, 114–118 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Bobbitt, D. R. & Linder, S. W. Recent advances in chiral detection for high performance liquid chromatography. TrAC Trends Anal. Chem. 20, 111–123 (2001).

    Article  CAS  Google Scholar 

  10. Roussel, C., Rio, A. D., Pierrot-Sanders, J., Piras, P. & Vanthuyne, N. Chiral liquid chromatography contribution to the determination of the absolute configuration of enantiomers. J. Chromatogr. A 1037, 311–328 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Berova, N., Bari, L. D. & Pescitelli, G. Application of electronic circular dichroism in configurational and conformational analysis of organic compounds. Chem. Soc. Rev. 36, 914 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Pirkle, W. H. & Pochapsky, T. C. Considerations of chiral recognition relevant to the liquid chromatography separation of enantiomers. Chem. Rev. 89, 347–362 (1989).

    Article  CAS  Google Scholar 

  13. Reetz, M. T. et al. A GC-based method for high-throughput screening of enantioselective catalysts. Catal. Today 67, 389–396 (2001).

    Article  CAS  Google Scholar 

  14. Leung, D., Kang, S. O. & Anslyn, E. V. Rapid determination of enantiomeric excess: a focus on optical approaches. Chem. Soc. Rev. 41, 448–479 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Tsukamoto, M. & Kagan, H. B. Recent advances in the measurement of enantiomeric excesses. Adv. Synth. Catal. 344, 453 (2002).

    Article  CAS  Google Scholar 

  16. Finn, M. G. Emerging methods for the rapid determination of enantiomeric excess. Chirality 14, 534–540 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Pu, L. Fluorescence of organic molecules in chiral recognition. Chem. Rev. 104, 1687–1716 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Kubo, Y., Maeda, S., Tokita, S. & Kubo, M. Colorimetric chiral recognition by a molecular sensor. Nature 382, 522–524 (1996).

    Article  CAS  Google Scholar 

  19. Kubo, Y., Hirota, N., Maeda, S. & Tokita, S. Naked-eye detectable chiral recognition using a chromogenic receptor. Anal. Sci. 14, 183–189 (1998).

    Article  CAS  Google Scholar 

  20. Chen, X.-X., Jiang, Y.-B. & Anslyn, E. V. A racemate-rules effect supramolecular polymer for ee determination of malic acid in the high ee region. Chem. Commun. 52, 12669–12671 (2016).

    Article  CAS  Google Scholar 

  21. Fossey, J. S. et al. Rapid determination of enantiomeric excess via NMR spectroscopy: a research-informed experiment. J. Chem. Educ. 94, 79–84 (2017).

    Article  CAS  Google Scholar 

  22. Brittain, W. D. G. et al. The Bull–James assembly as a chiral auxiliary and shift reagent in kinetic resolution of alkyne amines by the CuAAC reaction. Org. Biomol. Chem. 14, 10778–10782 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shabbir, S. H., Regan, C. J. & Anslyn, E. V. A general protocol for creating high-throughput screening assays for reaction yield and enantiomeric excess applied to hydrobenzoin. Proc. Natl Acad. Sci. 106, 10487–10492 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Thanzeel, F. Y. & Wolf, C. Substrate-specific amino acid sensing using a molecular D/ l-cysteine probe for comprehensive stereochemical analysis in aqueous solution. Angew. Chem. Int. Ed. 56, 7276–7281 (2017).

    Article  CAS  Google Scholar 

  25. Bentley, K. W., Proano, D. & Wolf, C. Chirality imprinting and direct asymmetric reaction screening using a stereodynamic Brønsted/Lewis acid receptor. Nat. Commun. 7, 12539 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. De Los Santos, Z. A. & Wolf, C. Chiroptical asymmetric reaction screening via multicomponent self-assembly. J. Am. Chem. Soc. 138, 13517–13520 (2016).

    Article  PubMed  CAS  Google Scholar 

  27. Bentley, K. W., Zhang, P. & Wolf, C. Miniature high-throughput chemosensing of yield, ee, and absolute configuration from crude reaction mixtures. Sci. Adv. 2, e1501162 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Tickell, D. A., Lampard, E. V., Lowe, J. P., James, T. D. & Bull, S. D. A protocol for NMR analysis of the enantiomeric excess of chiral diols using an achiral diboronic acid template. J. Org. Chem. 81, 6795–6799 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Wu, X. et al. Induced helical chirality of perylenebisimide aggregates allows for enantiopurity determination and differentiation of α-hydroxy carboxylates by using circular dichroism. Chemistry 20, 11793–11799 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Tickell, D. A., Mahon, M. F., Bull, S. D. & James, T. D. A simple protocol for NMR analysis of the enantiomeric purity of chiral hydroxylamines. Org. Lett. 15, 860–863 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Shcherbakova, E. G., Minami, T., Brega, V., James, T. D. & Anzenbacher, P. Determination of enantiomeric excess in amine derivatives with molecular self-assemblies. Angew. Chem. Int. Ed. 54, 7130–7133 (2015).

    Article  CAS  Google Scholar 

  32. Shcherbakova, E. G. et al. Toward fluorescence-based high-throughput screening for enantiomeric excess in amines and amino acid derivatives. Chemistry 22, 10074–10080 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Shcherbakova, E. G., Brega, V., Lynch, V. M., James, T. D. & Anzenbacher, P. High-throughput assay for enantiomeric excess determination in 1,2- and 1,3-diols and direct asymmetric reaction screening. Chemistry 23, 10222–10229 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Pérez-Fuertes, Y. et al. Simple protocols for NMR analysis of the enantiomeric purity of chiral primary amines. Nat. Protoc. 3, 210–214 (2008).

    Article  PubMed  CAS  Google Scholar 

  35. Pérez-Fuertes, Y. et al. Simple protocol for NMR analysis of the enantiomeric purity of primary amines. Org. Lett. 8, 609–612 (2006).

    Article  PubMed  CAS  Google Scholar 

  36. Shcherbakova, E. G. et al. Supramolecular sensors for opiates and their metabolites. J. Am. Chem. Soc. 139, 14954–14960 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bao, J. et al. Synthesis, resolution, and determination of absolute configuration of a vaulted 2,2ʹ-binaphthol and a vaulted 3,3ʹ-biphenanthrol (VAPOL). J. Am. Chem. Soc. 118, 3392–3405 (1996).

    Article  CAS  Google Scholar 

  38. Zhang, Y. et al. Highly enantioselective deracemization of linear and vaulted biaryl ligands. Org. Lett. 5, 1813–1816 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Brussee, J. & Jansen, A. C. A. A highly stereoselective synthesis of s(-)-[1,1′-binaphthalene]-2,2′-diol. Tetrahedron Lett. 24, 3261–3262 (1983).

  40. Shcherbakova, E. G., Minami, T., Brega, V., James, T. D. & Anzenbacher, P. Determination of enantiomeric excess in amine derivatives with molecular self-assemblies. Angew. Chem. Int. Ed. 54, 7130–7133 (2015).

    Article  CAS  Google Scholar 

  41. Galbraith, E. et al. Dynamic covalent self-assembled macrocycles prepared from 2-formyl-aryl-boronic acids and 1,2-amino alcohols. N. J. Chem. 33, 181–185 (2009).

    Article  CAS  Google Scholar 

  42. Wilson, A., Gasparini, G. & Matile, S. Functional systems with orthogonal dynamic covalent bonds. Chem. Soc. Rev. 43, 1948–1962 (2014).

    Article  CAS  PubMed  Google Scholar 

  43. Burns, J. A. & Whitesides, G. M. Feed-forward neural networks in chemistry: mathematical systems for classification and pattern recognition. Chem. Rev. 93, 2583–2601 (1993).

    Article  CAS  Google Scholar 

  44. Müller, M. Chemoenzymatic synthesis of building blocks for statin side chains. Angew. Chem. Int. Ed. 44, 362–365 (2005).

    Article  CAS  Google Scholar 

  45. Krueger, A. T. & Imperiali, B. Fluorescent amino acids: modular building blocks for the assembly of new tools for chemical biology. ChemBioChem 14, 788–799 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Berrueta, L. A., Alonso-Salces, R. M. & Héberger, K. Supervised pattern recognition in food analysis. J. Chromatogr. A 1158, 196–214 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Varmuza, K. Introduction to Multivariate Statistical Analysis in Chemometrics (CRC Press, 2009).

  48. Basheer, I. & Hajmeer, M. Artificial neural networks: fundamentals, computing, design, and application. J. Microbiol. Methods 43, 3–31 (2000).

    Article  CAS  PubMed  Google Scholar 

  49. Vapnik, V. N. The Nature of Statistical Learning Theory (Springer, 2000).

  50. Muller, K.-R., Mika, S., Ratsch, G., Tsuda, K. & Scholkopf, B. An introduction to kernel-based learning algorithms. IEEE Trans. Neural Netw. 12, 181–201 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Naguib, I. A. & Darwish, H. W. Support vector regression and artificial neural network models for stability indicating analysis of mebeverine hydrochloride and sulpiride mixtures in pharmaceutical preparation: a comparative study. Spectrochim. Acta Part A 86, 515–526 (2012).

    Article  CAS  Google Scholar 

  52. Schmidhuber, J. Deep learning in neural networks: an overview. Neural Netw. 61, 85–117 (2015).

    Article  PubMed  Google Scholar 

  53. Janzen, W. P. (ed) High Throughput Screening: Methods and Protocols (Humana Press, 2009).

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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 and Affiliations

  1. Department of Chemistry, Bowling Green State University, Bowling Green, OH, USA

    Elena G. Shcherbakova & Pavel Anzenbacher Jr.

  2. Department of Chemistry, University of Bath, Bath, UK

    Tony D. James

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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.

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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.

Supplementary information

Supplementary Information

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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