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Aromatic pentaamide macrocycles bind anions with high affinity for transport across biomembranes

Nature Chemistry volume 15, pages 1559–1568 (2023)Cite this article

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Abstract

The convergent positioning of functional groups in biomacromolecules leads to good binding, catalytic and transport capabilities. Synthetic frameworks capable of convergently locking functional groups with minimized conformational uncertainty—leading to similar properties—are highly desirable but rare. Here we report C5-symmetric aromatic pentaamide macrocycles synthesized in one pot from the corresponding monomers. Their crystal structures reveal a star-shaped, fully constrained backbone that causes ten alternating NH/CH hydrogen-bond donors and five large amide dipoles to orient towards the centre of the macrocycle. With a highly electropositive cavity in a high-energy unbound state, the macrocycles bind anions in a 1:1 stoichiometry in solution, with high affinity for halides and very high affinity for oxoanions. We demonstrate that such macrocycles are able to transport anions across lipid bilayers with a high chloride selectivity and restore the depleted airway surface liquid of cystic fibrosis airway cell cultures.

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Fig. 1: Linear oligoamide 1 and macrocycles c5 consisting of basic residues derived from 5-amino-N-acylanthranilic acid.
Fig. 2: Binding stoichiometry of macrocycles c5 with anions.
Fig. 3: 1H NMR spectra and association constants (log Ka) of c5a.
Fig. 4: Transmembrane transport of halide ions promoted by macrocycle c5a.
Fig. 5: Macrocycle c5a restores ASL volume haemostasis in CF airway epithelial cultures.

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Data availability

Single-crystal X-ray structure data for macrocycle c5c (CCDC reference number: 2124841) can be obtained free of charge from the Cambridge Crystallographic Data Centre at www.ccdc.cam.ac.uk/data_request/cif. The source data for Supplementary Figs. 1, 2, 5, 6a,b, 10–14, 17 and 18 can be obtained free of charge from Figshare at https://doi.org/10.6084/m9.figshare.23302427 (ref. 60). Source data are provided with this paper.

Code availability

The codes used in the calculations of data from vesicle-based halide transport HPTS assays can be obtained free of charge from Figshare at https://doi.org/10.6084/m9.figshare.23302427 (ref. 60).

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Acknowledgements

We acknowledge support from the US National Science Foundation (CHE-2304878 to B.G. for the conceptualization, design, data collection, analysis, decision to publish or preparation of the manuscript; CHE-1905094 and 2108538 to B.G. for preparing some of the synthetic building blocks; and CHE-2108597 to D.P.M. for the computational studies) for financial support; the Cystic Fibrosis Foundation (BUTTON19G0) and the NIH (R01HL125280) to B.B. for assessing the effect of c5a on restoring the ASL of cultured CF cells; the National Key R&D Program of China (2020YFA0908100), the NSFC (81627801) and the K. C. Wong Education Foundation (Hong Kong) to Z.S. for the design and performance of experiments on anion transport, for the determination of transport selectivity and for the preparation of the manuscript; and the Rabinowitz Honors College Research Assistant Program and a Lister Endowed Fellowship from Hofstra University (to D.P.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Computations were carried out at the Center for Computational Research at the University at Buffalo (http://hdl.handle.net/10477/79221).

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  1. These authors contributed equally: Ruikai Cao, Robert B. Rossdeutcher, Yulong Zhong.

Authors and Affiliations

  1. Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, USA

    Ruikai Cao, Robert B. Rossdeutcher, Yulong Zhong, Thomas A. Sobiech, Eva Zurek, Thomas Szyperski & Bing Gong

  2. State Key Laboratory of Systems Medicine for Cancer, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

    Yi Shen & Zhifeng Shao

  3. Department of Chemistry, Hofstra University, Hempstead, NY, USA

    Daniel P. Miller, Laura Sánchez Buitrago, Karishma Ramcharan & Pia Luthra

  4. Academy of Chinese Medical Science, Henan University of Chinese Medicine, Zhengzhou, Henan, China

    Xiangxiang Wu

  5. Marsico Lung Institute, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Mark I. Gutay, Miriam Frankenthal Figueira & Brian Button

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Contributions

R.C. and R.B.R. designed and conducted the syntheses and also performed the binding studies. T.A.S. performed ITC experiments. Y.S., Y.Z., R.B.R. and Z.S. performed the vesicle-based assays on anion transport. X.W. processed the X-ray data. D.P.M., L.S.B., K.R. and E.Z. performed the computational studies. B.B designed and, along with M.I.G. and M.F.F., performed experiments on cystic fibrosis cell cultures. T.S. helped with the NMR experiments. B.G. conceived and supervised the project. Z.S. and B.G. co-wrote the paper. All authors participated in discussion and editing of the manuscript.

Corresponding authors

Correspondence to Zhifeng Shao or Bing Gong.

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Competing interests

A provisional US patent application (PCT/US2021/050041) regarding the synthesis, anion binding, anion transport and potential therapeutic uses involving macrocycles c5 has been filed61. The remaining authors declare no competing interests.

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Nature Chemistry thanks Maija Nissinen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–19, Table 1, synthetic procedures, additional NMR and mass spectrometry spectra, descriptions of anion transport, cell culture and imaging and computational studies.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound c5c; CCDC reference 2124841.

Supplementary Data 2

Crystallographic data for compound c5a, including cif file, checkcif file, structural factors and justification for A- and B-level alerts.

Supplementary Data 3

The xyz coordinates of computed structures.

Source data

Source Data Fig. 2

UV and NMR titration data of c5a with Cl− for Fig. 2a,b.

Source Data Fig. 3

Values of binding constants of c5a with different anions versus the diameter of the corresponding anions for Fig. 3c.

Source Data Fig. 4

The numerical values of data for halide ion transport by vesicle assay with and without c5a for Fig. 4b–d.

Source Data Fig. 5

The numerical values of the thickness of the ASL layers of control and treated (with c5a) CF cells for Fig. 5c.

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Cao, R., Rossdeutcher, R.B., Zhong, Y. et al. Aromatic pentaamide macrocycles bind anions with high affinity for transport across biomembranes. Nat. Chem. 15, 1559–1568 (2023). https://doi.org/10.1038/s41557-023-01315-w

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