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Application of anisotropic NMR parameters to the confirmation of molecular structure

Nature Protocols volume 14, pages 217–247 (2019)Cite this article

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Abstract

The use of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique for structural characterization of organic small molecules. RDCs typically report the relative orientations of different 1H–13C bonds; RCSAs report the relative orientations of different carbon chemical shielding tensors and hence are more useful for proton-deficient molecules. This information is complementary to that obtained from conventional NMR data such as J couplings, isotropic chemical shifts, and nuclear Overhauser effects (NOEs)/rotational frame nuclear Overhauser effects (ROEs). Obtaining anisotropic NMR data requires the creation of an anisotropic sample environment through an alignment medium. Here, we focus on the use of compressed or stretched polymeric gels as two different but fundamentally equivalent methods for introducing sample anisotropy. Protocols are provided for the synthesis of the chloroform-compatible poly(methyl methacrylate) and dimethyl sulfoxide (DMSO)-compatible poly(2-hydroxyethyl methacrylate) gels and sample setup with a preparation time of 2–3 d. The bond-specific RDC data and the atom-specific RCSA data are extracted as changes in 1H–13C couplings and 13C chemical shifts, respectively, between two measurements under different alignment conditions, with a total experimental time of 0.5–4 d. NMR data acquisition and important considerations are described in detail. We also provide step-by-step procedures for the density functional theory (DFT) calculations involved and data analysis using the commercial software MSpin. We use three example compounds, namely cryptospirolepine (505 Da), retrorsine (351 Da), and estrone (270 Da), to demonstrate some important aspects of the workflow, such as input data preparation, handling of structural flexibility, and RCSA data correction when necessary.

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Fig. 1
Fig. 2: Making the gel transfer funnel and the stopper for use with the stretching device.
Fig. 3: NMR sample preparation with the compression device.
Fig. 4: NMR sample preparation with the stretching device.
Fig. 5
Fig. 6: Excerpt from the Mspin input and output files for cryptospirolepine (2).
Fig. 7: Single-tensor RCSA computation for retrorsine.
Fig. 8: Flowchart of the structural verification process using retrorsine as an example.

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Acknowledgements

Y.L., G.E.M., and R.T.W. thank I.E. Ndukwe for work on polymeric gel preparation. A.N.-V. thanks FACEPE (APQ-0507-1.06/15) for financial support. R.R.G. thanks the National Science Foundation for financial support (CHE-1111684). C.G. acknowledges support by the Max Planck Society and the DFG Forschergruppe (DFG FOR 934), as well as the extremely fruitful interactions with the members of this Forschergruppe: M. Reggelin (Darmstadt), M. Köck (Bremerhaven), C. Thiele (Darmstadt), B. Luy (Karlsruhe), and M. Zweckstetter and U. Reinscheid (both from Göttingen). A.N.-V., C.G., and R.R.G. also acknowledge the PhD students, postdoctoral fellows, and colleagues involved in small-molecule anisotropic NMR over the years in their laboratories: L. Verdier, P. Haberz, M. Schmidt, P. Trigo-Mouriño, E. d’Auvergne, H. Sun, N. Nath, J. C. Fuentes, S. B. P. Vemulapalli, N. Karschin, R. Santamaría-Fernández, E. Troche-Pesqueira, L. Gil-Silva, N. V. Tsarevsky, and V. Sánchez-Pedregal.

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Author notes

  1. Yizhou Liu

    Present address: Analytical Research and Development, Pfizer Worldwide Research and Development, Groton, CT, USA

  2. Gary E. Martin

    Present address: Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ, USA

  3. R. Thomas Williamson

    Present address: Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, NC, USA

Authors and Affiliations

  1. Structure Elucidation Group, Process and Analytical Research and Development, Merck Research Laboratories, Rahway, NJ, USA

    Yizhou Liu, Gary E. Martin & R. Thomas Williamson

  2. Departamento de Química Fundamental, CCEN, Universidade Federal de Pernambuco, Cidade Universitária, Recife, Brazil

    Armando Navarro-Vázquez

  3. Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA

    Roberto R. Gil

  4. Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Christian Griesinger

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  1. Yizhou Liu
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Contributions

G.E.M. and R.T.W. conceived the idea of writing this protocol; Y.L. and A.N.-V. contributed equally to manuscript writing. G.E.M. contributed the introduction and proposed the organization of the manuscript. R.R.G. contributed backgrounds and methods in gel preparation and gel compression. C.G. contributed theoretical background. All authors contributed to manuscript editing. G.E.M. coordinated the writing of the manuscript.

Corresponding authors

Correspondence to Yizhou Liu or Armando Navarro-Vázquez.

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Competing interests

A.N.-V. is the author of MSpin and StereoFitter, which are mentioned in the article, and receives royalties from sales of these products. The other authors declare no competing interests.

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Related links

Key references using this protocol

Nath, N. et al. J. Am. Chem. Soc. 138, 9548–9556 (2016): https://pubs.acs.org/doi/abs/10.1021/jacs.6b04082

Liu, Y. et al. Science 356, eaam5349 (2017): http://science.sciencemag.org/content/356/6333/eaam5349

Troche-Pesqueira, E., Anklin, C., Gil, R. R. & Navarro-Vázquez, A. Angew. Chem. Int. Ed. Engl. 56, 3660–3664 (2017): https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201612454

Supplementary information

Supplementary Methods

Input and output files from MSpin for all of the examples used in this protocol, as well as a step-by-step description of the estrone case

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Liu, Y., Navarro-Vázquez, A., Gil, R.R. et al. Application of anisotropic NMR parameters to the confirmation of molecular structure. Nat Protoc 14, 217–247 (2019). https://doi.org/10.1038/s41596-018-0091-9

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  • DOI: https://doi.org/10.1038/s41596-018-0091-9

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