Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Site-selective protein editing by backbone extension acyl rearrangements

Nature Chemical Biology volume 21, pages 1621–1630 (2025)Cite this article

Subjects

Abstract

Protein and polypeptide heteropolymers containing non-α-backbone monomers are highly desirable as potential materials and therapeutics but many remain difficult or impossible to biosynthesize in cells using traditional genetic code expansion. Here we describe a next-generation approach to such materials that relies instead on proximity-guided intramolecular rearrangements that edit the protein backbone post-translationally. This approach relies on orthogonal aminoacyl-tRNA synthetase enzymes that accept α-hydroxy acid monomers whose side chains contain masked nucleophiles. Introduction of such an α-hydroxy acid into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored intramolecular backbone extension acyl rearrangement (BEAR) reaction that edits the protein to install an extended-backbone monomer. In the examples described here, BEAR reactions are used to generate protein heteropolymers containing a β-backbone, γ-backbone or δ-backbone. This report represents a general strategy to install extended backbones into genetically encoded proteins and peptides expressed in cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: BEAR reactions create extended backbones in full-length proteins expressed in vivo.
Fig. 2: Computational analysis provides evidence that BEAR reactions to establish β-linkages, γ-linkages and δ-linkages are thermodynamically favorable.
Fig. 3: BEAR reactions generate a β2-linkage in NanoLuc G159-TAG-V160 in vivo.
Fig. 4: BEAR reactions generate a γ-linkage in SUMO–GFP in vivo.
Fig. 5: BEAR reactions generate one or two δ-linkages in SUMO–GFP in vivo.

Similar content being viewed by others

Data availability

Data that support the main findings of this study are available within the article and Supplementary Information. All primary data for figures included as Supplementary Information are provided in Supplementary Data 1. Alternatively, data included in this study are also available from the corresponding authors upon request. Source data are provided with this paper.

References

  1. Kwon, D. How scientists are hacking the genetic code to give proteins new powers. Nature 618, 874–876 (2023).

    Article  CAS  PubMed  Google Scholar 

  2. Kritzer, J. A., Lear, J. D., Hodsdon, M. E. & Schepartz, A. Helical β-peptide inhibitors of the p53–hDM2 interaction. J. Am. Chem. Soc. 126, 9468–9469 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Sadowsky, J. D. et al. Chimeric (α/β+α)-peptide ligands for the BH3-recognition cleft of Bcl-xL: critical role of the molecular scaffold in protein surface recognition. J. Am. Chem. Soc. 127, 11966–11968 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Hamuro, Y., Schneider, J. P. & DeGrado, W. F. De novo design of antibacterial β-peptides. J. Am. Chem. Soc. 121, 12200–12201 (1999).

    Article  CAS  Google Scholar 

  5. Cheloha, R. W., Maeda, A., Dean, T., Gardella, T. J. & Gellman, S. H. Backbone modification of a polypeptide drug alters duration of action in vivo. Nat. Biotechnol. 32, 653–655 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wang, P. S. P., Nguyen, J. B. & Schepartz, A. Design and high-resolution structure of a β3-peptide bundle catalyst. J. Am. Chem. Soc. 136, 6810–6813 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Girvin, Z. C., Andrews, M. K., Liu, X. & Gellman, S. H. Foldamer-templated catalysis of macrocycle formation. Science 366, 1528–1531 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Daniels, D. S., Petersson, E. J., Qiu, J. X. & Schepartz, A. High-resolution structure of a β-peptide bundle. J. Am. Chem. Soc. 129, 1532–1533 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Seebach, D. et al. Biological and pharmacokinetic studies with β-peptides. Chimia 52, 734–734 (1998).

    Article  CAS  Google Scholar 

  10. Adaligil, E., Song, A., Hallenbeck, K. K., Cunningham, C. N. & Fairbrother, W. J. Ribosomal synthesis of macrocyclic peptides with β2- and β2,3-homo-amino acids for the development of natural product-like combinatorial libraries. ACS Chem. Biol. 16, 1011–1018 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Salveson, P. J. et al. Expansive discovery of chemically diverse structured macrocyclic oligoamides. Science 384, 420–428 (2024).

    Article  CAS  PubMed  Google Scholar 

  12. Cheloha, R. W. et al. Consequences of periodic α-to-β3 residue replacement for immunological recognition of peptide epitopes. ACS Chem. Biol. 10, 844–854 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cheloha, R. W. et al. Recognition of class II MHC peptide ligands that contain β-amino acids. J. Immunol. 203, 1619–1628 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Melo Czekster, C., Robertson, W. E., Walker, A. S., Söll, D. & Schepartz, A. In vivo biosynthesis of a β-amino acid-containing protein. J. Am. Chem. Soc. 138, 5194–5197 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Scott, T. A. et al. Widespread microbial utilization of ribosomal β-amino acid-containing peptides and proteins. Chem 8, 2659–2677 (2022).

    Article  CAS  Google Scholar 

  16. Hamlish, N. X., Abramyan, A. M., Shah, B., Zhang, Z. & Schepartz, A. Incorporation of multiple β2-hydroxy acids into a protein in vivo using an orthogonal aminoacyl-tRNA synthetase. ACS Cent. Sci. 10, 1044–1053 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dunkelmann, D. L. et al. Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism. Nature 625, 603–610 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Katoh, T. & Suga, H. Ribosomal incorporation of consecutive β-amino acids. J. Am. Chem. Soc. 140, 12159–12167 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Fricke, R. et al. Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo. Nat. Chem. 15, 960–971 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Watson, Z. L. et al. Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates. Nat. Chem. 15, 913–921 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Cruz-Navarrete, F. A. et al. β-Amino acids reduce ternary complex stability and alter the translation elongation mechanism. ACS Cent. Sci. 10, 1262–1275 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kobayashi, T., Yanagisawa, T., Sakamoto, K. & Yokoyama, S. Recognition of non-α-amino substrates by pyrrolysyl-tRNA synthetase. J. Mol. Biol. 385, 1352–1360 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Li, Y.-M. et al. Ligation of expressed protein α-hydrazides via genetic incorporation of an α-hydroxy acid. ACS Chem. Biol. 7, 1015–1022 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Spinck, M. et al. Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles. Nat. Chem. 15, 61–69 (2023).

    Article  CAS  PubMed  Google Scholar 

  26. Wan, W., Tharp, J. M. & Liu, W. R. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta 1844, 1059–1070 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huguenin-Dezot, N. et al. Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid. Nature 565, 112–117 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Koch, N. G., Goettig, P., Rappsilber, J. & Budisa, N. Engineering pyrrolysyl-tRNA synthetase for the incorporation of non-canonical amino acids with smaller side chains. Int. J. Mol. Sci. 22, 11194 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Sohma, Y., Sasaki, M., Hayashi, Y., Kimura, T. & Kiso, Y. Novel and efficient synthesis of difficult sequence-containing peptides through O–N intramolecular acyl migration reaction of O-acyl isopeptides. Chem. Commun. 124–125 (2004).

  31. Dos Santos, S. et al. Switch-peptides: controlling self-assembly of amyloid β-derived peptides in vitro by consecutive triggering of acyl migrations. J. Am. Chem. Soc. 127, 11888–11889 (2005).

    Article  PubMed  Google Scholar 

  32. Schissel, C. K. et al. Ribosomal synthesis of ketone-containing peptide backbone via O to C acyl shift. J. Am. Chem. Soc. 147, 6503–6513 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Dose, C. & Seitz, O. New isocysteine building blocks and chemoselective peptide ligation. Org. Biomol. Chem. 2, 59–65 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Kuroda, T., Huang, Y., Nishio, S., Goto, Y. & Suga, H. Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units. Nat. Chem. 14, 1413–1420 (2022).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, C., Guo, Q.-X. & Fu, Y. Theoretical analysis of the detailed mechanism of native chemical ligation reactions. Chem. Asian J. 6, 1241–1251 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Grimme, S., Hansen, A., Ehlert, S., & Mewes, J.-M. r2SCAN-3c: a “Swiss army knife” composite electronic-structure method. J. Chem. Phys. 154, 064103 (2021).

  37. Mardirossian, N. & Head-Gordon, M. ωB97M-V: a combinatorially optimized, range-separated hybrid, meta-GGA density functional with VV10 nonlocal correlation. J. Chem. Phys. 144, 214110 (2016).

    Article  PubMed  Google Scholar 

  38. Mardirossian, N. & Head-Gordon, M. Thirty years of density functional theory in computational chemistry: an overviewand extensive assessment of 200 density functionals. Mol. Phys. 115, 2315–2372 (2017).

    Article  CAS  Google Scholar 

  39. Marenich, A. V., Cramer, C. J. & Truhlar, D. G. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113, 6378–6396 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Volkwein, W., Maier, C., Krafczyk, R., Jung, K. & Lassak, J. A versatile toolbox for the control of protein levels using Nε-acetyl-l-lysine dependent amber suppression. ACS Synth. Biol. 6, 1892–1902 (2017).

    Article  PubMed  Google Scholar 

  42. Leisle, L. et al. Cellular encoding of Cy dyes for single-molecule imaging. eLife 5, e19088 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Lee, J., Schwarz, K. J., Kim, D. S., Moore, J. S. & Jewett, M. C. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nat. Commun. 11, 4304 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Agouridas, V. et al. Native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations. Chem. Rev. 119, 7328–7443 (2019).

    Article  CAS  PubMed  Google Scholar 

  45. Baldwin, J. E. Rules for ring closure. J. Chem. Soc. Chem. Commun. 18, 734–736 (1976).

    Article  Google Scholar 

  46. Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Young, T. S. & Schultz, P. G. Beyond the canonical 20 amino acids: expanding the genetic lexicon. J. Biol. Chem. 285, 11039–11044 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, S., Ji, X., Gao, M., Dedkova, L. M. & Hecht, S. M. In cellulo synthesis of proteins containing a fluorescent oxazole amino acid. J. Am. Chem. Soc. 141, 5597–5601 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to members of the A.S., S.J.M. and A.C. labs for comments and suggestions, and to J. Chin (Medical Research Council) and N. Budisa (University of Manitoba) for sharing materials. We thank H. Celik for assistance with nuclear magnetic resonance spectroscopy, National Institutes of Health grant S10OD024998 for spectrometer support and the Chemical and Biophysical Instrumentation Center at Yale University (RRID:SCR_021738). This work was supported by the National Science Foundation (NSF) Center for Genetically Encoded Materials (CHE 2002182 to A.S.). L.T.R., T.L.D. and D.A.D. were supported by the NSF Graduate Research Fellowship Program (DGE-1752814, DGE-2139841 and DGE-1122492, respectively). C.K.S. was supported by the Miller Institute for Basic Research in Science (University of California, Berkeley). A.S. is a Chan-Zuckerberg Biohub San Francisco Investigator and an Arc Innovation Investigator.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Leah T. Roe, Isabel M. Piper, Carly K. Schissel, Arjun M. Garapaty, Shuai Zheng, Nicole Wong, Matthew B. Francis & Alanna Schepartz

  2. Department of Chemistry, Yale University, New Haven, CT, USA

    Taylor L. Dover, Diondra A. Dilworth & Scott J. Miller

  3. Process Development, Attribute Sciences, Amgen, Inc., Thousand Oaks, CA, USA

    Bhavana Shah & Zhongqi Zhang

  4. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Noah X. Hamlish & Alanna Schepartz

  5. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    Abhishek Chatterjee

  6. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, USA

    Alanna Schepartz

  7. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Alanna Schepartz

  8. ARC Institute, Palo Alto, CA, USA

    Alanna Schepartz

Authors
  1. Leah T. Roe
    View author publications

    Search author on:PubMed Google Scholar

  2. Isabel M. Piper
    View author publications

    Search author on:PubMed Google Scholar

  3. Carly K. Schissel
    View author publications

    Search author on:PubMed Google Scholar

  4. Taylor L. Dover
    View author publications

    Search author on:PubMed Google Scholar

  5. Bhavana Shah
    View author publications

    Search author on:PubMed Google Scholar

  6. Noah X. Hamlish
    View author publications

    Search author on:PubMed Google Scholar

  7. Arjun M. Garapaty
    View author publications

    Search author on:PubMed Google Scholar

  8. Shuai Zheng
    View author publications

    Search author on:PubMed Google Scholar

  9. Diondra A. Dilworth
    View author publications

    Search author on:PubMed Google Scholar

  10. Nicole Wong
    View author publications

    Search author on:PubMed Google Scholar

  11. Zhongqi Zhang
    View author publications

    Search author on:PubMed Google Scholar

  12. Abhishek Chatterjee
    View author publications

    Search author on:PubMed Google Scholar

  13. Matthew B. Francis
    View author publications

    Search author on:PubMed Google Scholar

  14. Scott J. Miller
    View author publications

    Search author on:PubMed Google Scholar

  15. Alanna Schepartz
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Study conceptualization and design, L.T.R., A.C., M.B.F., S.J.M. and A.S. Preparation of materials, L.T.R., I.M.P., C.K.S., T.L.D., N.X.H., A.M.G., S.Z., D.A.D. and N.W. Data collection, L.T.R., I.M.P., C.K.S., T.L.D., B.S., N.X.H., A.M.G., S.Z., N.W. and M.B.F. Analysis and interpretation of results, L.T.R., I.M.P., C.K.S., T.L.D., B.S., Z.Z., M.B.F., S.J.M. and A.S. Paper preparation, L.T.R., C.K.S., T.L.D., M.B.F., S.J.M. and A.S.

Corresponding authors

Correspondence to Matthew B. Francis, Scott J. Miller or Alanna Schepartz.

Ethics declarations

Competing interests

L.T.R. and A.S. have submitted a patent application related to this work through The Regents of the University of California (provisional application 63/587,179 filed October 2, 2023; PCT Application PCT/US2024/048032 filed 9/23/2024; aspect covered: methods for synthesis of protein heteropolymers). The other authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information (download PDF )

Supplementary Methods, Figs. 1–21, Tables 1–4, References and processed NMR spectra.

Reporting Summary (download PDF )

Supplementary Data 1 (download ZIP )

This file contains all source data for the supplementary figures included in Supplementary Information.

Source data

Source Data Fig. 2 (download XLSX )

Free energy plot coordinates.

Source Data Fig. 3 (download XLSX )

Statistical source data.

Source Data Fig. 4 (download XLSX )

Statistical source data.

Source Data Fig. 5 (download XLSX )

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Roe, L.T., Piper, I.M., Schissel, C.K. et al. Site-selective protein editing by backbone extension acyl rearrangements. Nat Chem Biol 21, 1621–1630 (2025). https://doi.org/10.1038/s41589-025-01999-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41589-025-01999-w

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing