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Immobilized acyl-transfer molecular reactors enable the solid-phase synthesis of sterically hindered peptides

Nature Chemistry volume 17, pages 1596–1606 (2025)Cite this article

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Abstract

Incorporating unnatural amino acids, such as hindered N-methylated or α,α-disubstituted amino acid(s), into peptides can improve their properties for application in the pharmaceutical and biomedical fields. However, the current solid-phase peptide synthesis (SPPS) faces sluggish reaction rates and low yields when incorporating sterically hindered amino acids, owing to the poor kinetics of the two-phase acyl-transfer process from solution to solid. Here we introduce an immobilized ribosome-mimicking molecular reactor to facilitate on-resin proximity-induced intra(inter)-reactor acyl transfers. This strategy bypasses the two-phase acyl-transfer mechanism in conventional SPPS and boosts coupling efficiency in the solid-phase synthesis of N-methylated and/or α,α-disubstituted amino acid(s)-containing sterically hindered peptides, including cyclosporin A and alamethicin F analogues. The ribosome-mimicking molecular reactor SPPS can be integrated into existing SPPS platforms using commercially available resins and reagents, and displays high compatibility with standard synthesizers, enabling the automated synthesis of pharmaceutically important, sterically hindered difficult peptides.

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Fig. 1: The RMMR-based protocol in comparison with traditional SPPS.
Fig. 2: Possible acyl-transfer pathways in the RMMR-based protocol.
Fig. 3: The assembly of RMMRs and capability validation to peptide synthesis.
Fig. 4: Syntheses of sterically hindered peptides containing N-methylated amino acid(s).
Fig. 5: Synthesis of NMeLeu-CsA (CsA with NMeLeu7 replacing NMeBmt7).
Fig. 6: Syntheses of sterically hindered peptides containing α,α-disubstituted amino acid(s).

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All data are available in the main text and the Supplementary Information. Source data are provided with this paper.

References

  1. Jensen, K. J., Shelton, P. T. & Pedersen, S. L. (eds.) Peptide Synthesis and Applications 2nd edn (Humana, 2013).

  2. Vinogradov, A. A., Yin, Y. & Suga, H. Macrocyclic peptides as drug candidates: recent progress and remaining challenges. J. Am. Chem. Soc. 141, 4167–4181 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Muttenthaler, M., King, G. F., Adams, D. J. & Alewood, P. F. Trends in peptide drug discovery. Nat. Rev. Drug Discov. 20, 309–325 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Henninot, A., Collins, J. C. & Nuss, J. M. The current state of peptide drug discovery: back to the future? J. Med. Chem. 61, 1382–1414 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Chatterjee, J., Gilon, C., Hoffman, A. & Kessler, H. N-Methylation of peptides: a new perspective in medicinal chemistry. Acc. Chem. Res. 41, 1331–1342 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Hamman, J. H., Enslin, G. M. & Kotzé, A. F. Oral delivery of peptide drugs: barriers and developments. BioDrugs 19, 165–177 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Chatterjee, J., Rechenmacher, F. & Kessler, H. N-Methylation of peptides and proteins: an important element for modulating biological functions. Angew. Chem. Int. Ed. 52, 254–269 (2013).

    Article  CAS  Google Scholar 

  8. Biron, E. et al. Improving oral bioavailability of peptides by multiple N-methylation: somatostatin analogues. Angew. Chem. Int. Ed. 47, 2595–2599 (2008).

    Article  CAS  Google Scholar 

  9. Driggers, E. M., Hale, S. P., Lee, J. & Terrett, N. K. The exploration of macrocycles for drug discovery—an underexploited structural class. Nat. Rev. Drug Discov. 7, 608–624 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Toniolo, C., Formaggio, F., Kaptein, B. & Broxterman, Q. B. You are sitting on a gold mine! Synlett 9, 1295–1310 (2006).

    Article  Google Scholar 

  11. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).

    Article  CAS  Google Scholar 

  12. Merrifield, R. B. Automated synthesis of peptides. Science 150, 178 (1965).

    Article  CAS  PubMed  Google Scholar 

  13. Naoum, J. N., Alshanski, I., Mayer, G., Strauss, P. & Hurevich, M. Stirring peptide synthesis to a new level of efficiency. Org. Process Res. Dev. 26, 129–136 (2022).

    Article  CAS  Google Scholar 

  14. Alshanski, I. et al. Enhancing the efficiency of the solid phase peptide synthesis (SPPS) process by high shear mixing. Org.Process Res. Dev. 22, 1318–1322 (2018).

    Article  CAS  Google Scholar 

  15. Naoum, J. N. et al. Diffusion-enhanced amide bond formation on a solid support. Org. Process Res. Dev. 23, 2733–2739 (2019).

    Article  CAS  Google Scholar 

  16. Falb, E., Yechezkel, T., Salitra, Y. & Gilon, C. In situ generation of Fmoc-amino acid chlorides using bis-(trichloromethyl) carbonate and its utilization for difficult couplings in solid-phase peptide synthesis. J. Pept. Res. 53, 507–517 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Frérot, E., Coste, J., Pantaloni, A., Dufour, M.-N. & Jouin, P. PyBOP and PyBroP: Two reagents for the difficult coupling of the α,α-dialkyl amino acid, Aib. Tetrahedron 47, 259–270 (1991).

    Article  Google Scholar 

  18. Otake, Y. et al. N-Methylated peptide synthesis via generation of an acyl N-methylimidazolium cation accelerated by a Broensted acid. Angew. Chem. Int. Ed. 59, 12925–12930 (2020).

    Article  CAS  Google Scholar 

  19. Wang, X., Li, J. & Hayashi, Y. Highly sterically hindered peptide bond formation between α,α-disubstituted α-amino acids and N-alkyl cysteines using α,α-disubstituted α-amidonitrile. J. Am. Chem. Soc. 144, 10145–10150 (2022).

    Article  CAS  PubMed  Google Scholar 

  20. Crick, F. Central dogma of molecular biology. Nature 227, 561–563 (1970).

    Article  CAS  PubMed  Google Scholar 

  21. Steitz, T. A. A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Biol. 9, 242–253 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Mäde, V., Els-Heindl, S. & Beck-Sickinger, A. G. Automated solid-phase peptide synthesis to obtain therapeutic peptides. Beilstein J. Org. Chem. 10, 1197–1212 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  23. König, W. & Geiger, R. Eine neue methode zur synthese von peptiden: aktivierung der carboxylgruppe mit dicyclohexylcarbodiimid unter zusatz von 1-hydroxy-benzotriazolen. Chem. Ber. 103, 788–798 (1970).

    Article  PubMed  Google Scholar 

  24. Subirós-Funosas, R., Prohens, R., Barbas, R., El-Faham, A. & Albericio, F. Oxyma: an efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion[1]. Chem. Eur. J. 15, 9394–9403 (2009).

    Article  PubMed  Google Scholar 

  25. Bhattacharya, S. & Kumar, V. P. Ester cleavage properties of synthetic hydroxybenzotriazoles in cationic monovalent and gemini surfactant micelles. Langmuir 21, 71–78 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. He, X. & Jabbari, E. Solid-phase synthesis of reactive peptide crosslinker by selective deprotection. Protein Pept. Lett. 13, 715–718 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Hines, D. A. & Kamat, P. V. Quantum dot surface chemistry: ligand effects and electron transfer reactions. J. Phys. Chem. C 117, 14418–14426 (2013).

    Article  CAS  Google Scholar 

  28. Greenwald, R. B. PEG drugs: an overview. J. Control. Release 74, 159–171 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Suzuki, Y. et al. Inflammation and angiotensin II. Int. J. Biochem. Cell Biol. 35, 881–900 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Kumar, P., Paterson, N. G., Clayden, J. & Woolfson, D. N. De novo design of discrete, stable 310-helix peptide assemblies. Nature 607, 387–392 (2022).

    Article  CAS  PubMed  Google Scholar 

  31. Yang, Y. & Hansen, L. Optimized Fmoc-removal strategy to suppress the traceless and conventional diketopiperazine formation in solid-phase peptide synthesis. ACS Omega 7, 12015–12020 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Subirós-Funosas, R., El-Faham, A. & Albericio, F. PyOxP and PyOxB: the Oxyma-based novel family of phosphonium salts. Org. Biomol. Chem. 8, 3665–3673 (2010).

    Article  PubMed  Google Scholar 

  33. Nzama, H., Manne, S. R., Akintayo, D. C., de la Torre, B. G. & Albericio, F. Ethylthio-1H-tetrazole (ETT) as coupling additive for the solid-phase synthesis (SPS) of hindered amino acid-containing peptides. Tetrahedron Lett. 142, 155108 (2024).

    Article  CAS  Google Scholar 

  34. Ahlbach, C. L. et al. Beyond cyclosporine A: conformation-dependent passive membrane permeabilities of cyclic peptide natural products. Future Med. Chem. 7, 2121–2130 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Ruegger, A. et al. Cyclosporin A, ein immunsuppressiv wirksamer peptidm-etabolit aus trichoderma polysporum (link ex pers.) rifai. Helv. Chim. Acta 59, 1075–1092 (1976).

    Article  CAS  PubMed  Google Scholar 

  36. Attur, M. G. et al. Differential anti-inflammatory effects of immunosuppressive drugs: cyclosporin, rapamycin and FK-506 on inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and PGE 2 production. Inflammation Res. 49, 20–26 (2000).

    Article  CAS  Google Scholar 

  37. Corbett, K. M., Ford, L., Warren, D. B., Pouton, C. W. & Chalmers, D. K. Cyclosporin structure and permeability: from A to Z and beyond. J. Med. Chem. 64, 13131–13151 (2021).

    Article  CAS  PubMed  Google Scholar 

  38. Li, P. & Xu, J. C. Total synthesis of cyclosporin O both in solution and in the solid phase using novel thiazolium-, immonium-, and pyridinium-type coupling reagents: BEMT, BDMP, and BEP1. J. Org. Chem. 65, 2951–2958 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Angell, Y. M., Thomas, T. L., Flentke, G. R. & Rich, D. H. Solid-phase synthesis of cyclosporin peptides. J. Am. Chem. Soc. 117, 7279–7280 (1995).

    Article  CAS  Google Scholar 

  40. Sharma, A. et al. N-methylation in amino acids and peptides: scope and limitations. Biopolymers 109, 23110–23124 (2018).

    Article  Google Scholar 

  41. Urban, J. A. N., Vaisar, T. O. M., Shen, R. & Lee, M. S. Lability of N-alkylated peptides towards TFA cleavage. Int. J. Pept. Protein Res. 47, 182–189 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Sheppard, R. C. & Williams, B. J. Acid-labile resin linkage agents for use in solid phase peptide synthesis. Int. J. Pept. Protein Res. 20, 451–454 (1982).

    Article  CAS  PubMed  Google Scholar 

  43. Zieleniewski, F., Woolfson, D. N. & Clayden, J. Automated solid-phase concatenation of Aib residues to form long, water-soluble, helical peptides. Chem. Commun. 56, 12049–12052 (2020).

    Article  CAS  Google Scholar 

  44. Pike, S. J., Raftery, J., Webb, S. J. & Clayden, J. Conformational analysis of helical aminoisobutyric acid (Aib) oligomers bearing C-terminal ester Schellman motifs. Org. Biomol. Chem. 12, 4124–4131 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Zhang, Y., Vanderghinste, J., Wang, J. & Das, S. Challenges and recent advancements in the synthesis of α,α-disubstituted α-amino acids. Nat. Commun. 15, 1474 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Whitmore, L. & Wallace, B. A. Analysis of peptaibol sequence composition: implications for in vivo synthesis and channel formation. Eur. Biophys. J. 33, 233–237 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Chugh, J. K. & Wallace, B. A. Peptaibols: models for ion channels. Biochem. Soc. Trans. 29, 565–570 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Casagrande, N. et al. Analogs of a natural peptaibol exert anticancer activity in both cisplatin-and doxorubicin-resistant cells and in multicellular tumor spheroids. Int. J. Mol. Sci. 22, 8362 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Shi, M. et al. Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells. Mol. Cancer 9, 1–16 (2010).

    Article  Google Scholar 

  50. Whitmore, L. & Wallace, B. A. The Peptaibol Database: a database for sequences and structures of naturally occurring peptaibols. Nucleic Acids Res. 32, D593–D594 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Nagaraj, R. & Balaram, P. Alamethicin, a transmembrane channel. Acc. Chem. Res. 14, 356–362 (1981).

    Article  CAS  Google Scholar 

  52. Siow, A., Hung, K. Y., Harris, P. W. R. & Brimble, M. A. Solid-phase synthesis of the peptaibol alamethicin U-22324 by using a double-linker strategy. Eur. J. Org. Chem. 2017, 350–354 (2017).

    Article  CAS  Google Scholar 

  53. Hjørringgaard, C. U., Pedersen, J. M., Vosegaard, T., Nielsen, N. C. & Skrydstrup, T. An automatic solid-phase synthesis of peptaibols. J. Org. Chem. 74, 1329–1332 (2009).

    Article  PubMed  Google Scholar 

  54. Ben Haj Salah, K. & Inguimbert, N. Efficient microwave-assisted one shot synthesis of peptaibols using inexpensive coupling reagents. Org. Lett. 16, 1783–1785 (2014).

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank the National Natural Science Foundation of China (grant no. 22450003) and Shenzhen Science and Technology Program (grant no. KQTD20190929174023858) for financial support, and Y. Du for assistance in mass spectroscopy analysis.

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Authors and Affiliations

  1. State Key Laboratory of Coordination Chemistry, and Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Siyuan Wei, Xuchun Zhang, Xiaoliang Yang, Fa Liu & Zhu-Jun Yao

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  1. Siyuan Wei
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  2. Xuchun Zhang
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  3. Xiaoliang Yang
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Contributions

S.W. developed and optimized the RMMRs, completed most of the experiments and data collection. X.Z. and X.Y. participated in the partial synthetic experiments, NMR experiments and spectra analyses. S.W., F.L. and Z.-J.Y. wrote the manuscript, with inputs from all authors. F.L. and Z.-J.Y. conceptualized the project and participated in the design and coordination of the experiments.

Corresponding authors

Correspondence to Fa Liu or Zhu-Jun Yao.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–19, experimental procedures, compound characterizations, LC-MS analysis reports and copies of 1H and 13C NMR spectra.

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Packed raw NMR data

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Source Data Fig. 3

Source data for HPLC analysis in Fig. 3c,d,e.

Source Data Fig. 4

Source data for HPLC analysis in Fig. 4b.

Source Data Fig. 5

Source data for HPLC analysis in Fig. 5.

Source Data Fig. 6

Source data for HPLC analysis in Fig. 6ab.

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Wei, S., Zhang, X., Yang, X. et al. Immobilized acyl-transfer molecular reactors enable the solid-phase synthesis of sterically hindered peptides. Nat. Chem. 17, 1596–1606 (2025). https://doi.org/10.1038/s41557-025-01896-8

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