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Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units

Abstract

Hydroxyhydrocarbon (Hhc) moieties in the backbone of peptidic natural products can exert a substantial influence on the bioactivities of the products, making Hhc units an attractive class of building blocks for de novo peptides. However, despite advances in in vitro genetic code reprogramming, the ribosomal incorporation of Hhc units remains challenging. Here we report a method for in vitro ribosomal synthesis of natural-product-like peptides bearing Hhc units. A series of azide/hydroxy acids were designed as chemical precursors of Hhc units and incorporated into the nascent peptide chain by means of genetic code reprogramming. Post-translational reduction of the azide induced an O-to-N acyl shift to rearrange the peptide backbone. This method allows for one-pot ribosomal synthesis of designer macrocycles bearing various β-, γ- and δ-type Hhc units. We also report the synthesis of a statine-containing peptidomimetic inhibitor of β-secretase 1 as a showcase example.

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Fig. 1: Schematic illustration of the post-translational backbone-acyl shift reaction for in vitro ribosomal synthesis of peptides containing Hhc units.
Fig. 2: Post-translational backbone-acyl shift reaction yielding a γ-peptide linkage on a model peptide (cyPep1-γN3).
Fig. 3: Optimization of the post-translational backbone-acyl shift conditions.
Fig. 4: Scope and limitation of the post-translational backbone-acyl shift to yield various Hhc units.
Fig. 5: In vitro expression of peptides containing statine analogues.
Fig. 6: Application of the post-translational backbone-acyl shift for the in vitro synthesis of the β-secretase 1 inhibitor P10–P4′statV.

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Data availability

Methods and data, including for the synthesis of acyl-donor substrates, characterization of acyl-donor substrates, primer sequences, DNA template assembly schemes, additional LC-MS data for the backbone-acyl shift reactions, MS/MS identification of P10–P4′statV, optimization of flexizyme-mediated acylation conditions and summary of all LC-MS chromatograms discussed in this paper are available in the Supplementary Information. Alternatively, data are available from the corresponding authors upon reasonable request.

References

  1. Umezawa, H., Aoyagi, T., Morishima, H., Matsuzaki, M. & Hamada, M. Pepstatin, a new pepsin inhibitor produced by Actinomycetes. J. Antibiot. (Tokyo) 23, 259–262 (1970).

    Article  CAS  Google Scholar 

  2. Nyfeler, R. & Keller-Schierlein, W. [Metabolites of microorganisms. 143. Echinocandin B, a novel polypeptide-antibiotic from Aspergillus nidulans var. echinulatus: isolation and structural components]. Helv. Chim. Acta 57, 2459–2477 (1974).

    Article  CAS  Google Scholar 

  3. Trevisi, L. et al. Callipeltin A, a cyclic depsipeptide inhibitor of the cardiac sodium-calcium exchanger and positive inotropic agent. Biochem. Biophys. Res. Commun. 279, 219–222 (2000).

    Article  CAS  Google Scholar 

  4. Nagano, Y. et al. Pyloricidins, novel anti-Helicobacter pylori antibiotics produced by Bacillus sp. II. Isolation and structure elucidation. J. Antibiot. (Tokyo) 54, 934–947 (2001).

    Article  CAS  Google Scholar 

  5. Plaza, A. et al. Celebesides A-C and theopapuamides B-D, depsipeptides from an Indonesian sponge that inhibit HIV-1 entry. J. Org. Chem. 74, 504–512 (2009).

    Article  CAS  Google Scholar 

  6. Choi, H. et al. The hoiamides, structurally intriguing neurotoxic lipopeptides from Papua New Guinea marine cyanobacteria. J. Nat. Prod. 73, 1411–1421 (2010).

    Article  CAS  Google Scholar 

  7. Festa, C. et al. Solomonamides A and B, new anti-inflammatory peptides from Theonella swinhoei. Org. Lett. 13, 1532–1535 (2011).

    Article  CAS  Google Scholar 

  8. Tajima, H., Wakimoto, T., Takada, K., Ise, Y. & Abe, I. Revised structure of cyclolithistide A, a cyclic depsipeptide from the marine sponge Discodermia japonica. J. Nat. Prod. 77, 154–158 (2014).

    Article  CAS  Google Scholar 

  9. Wakimoto, T., Egami, Y. & Abe, I. Calyculin: Nature’s way of making the sponge-derived cytotoxin. Nat. Prod. Rep. 33, 751–760 (2016).

    Article  CAS  Google Scholar 

  10. Bott, R., Subramanian, E. & Davies, D. R. Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5 Å resolution. Biochemistry 21, 6956–6962 (1982).

    Article  CAS  Google Scholar 

  11. Rich, D. H. et al. Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition. Biochemistry 24, 3165–3173 (1985).

    Article  CAS  Google Scholar 

  12. Tumminello, F. M., Bernacki, R. J., Gebbia, N. & Leto, G. Pepstatins: aspartic proteinase inhibitors having potential therapeutic applications. Med. Res. Rev. 13, 199–208 (1993).

    Article  CAS  Google Scholar 

  13. Wada, S., Matsunaga, S., Fusetani, N. & Watabe, S. Theonellamide F, a bicyclic peptide marine toxin, induces formation of vacuoles in 3Y1 rat embryonic fibroblast. Mar. Biotechnol. (N. Y.) 1, 337–341 (1999).

    Article  CAS  Google Scholar 

  14. Okada, Y., Matsunaga, S., van Soest, R. W. & Fusetani, N. Nagahamide A, an antibacterial depsipeptide from the marine sponge Theonella swinhoei. Org. Lett. 4, 3039–3042 (2002).

    Article  CAS  Google Scholar 

  15. Kuranaga, T. et al. Total synthesis and structural revision of kasumigamide, and identification of a new analogue. ChemBioChem 21, 3329–3332 (2020).

    Article  CAS  Google Scholar 

  16. Sinha, S. et al. Purification and cloning of amyloid precursor protein β-secretase from human brain. Nature 402, 537–540 (1999).

    Article  CAS  Google Scholar 

  17. Turner, R. T. 3rd et al. Subsite specificity of memapsin 2 (β-secretase): implications for inhibitor design. Biochemistry 40, 10001–10006 (2001).

    Article  CAS  Google Scholar 

  18. Hong, L. et al. Crystal structure of memapsin 2 (β-secretase) in complex with an inhibitor OM00-3. Biochemistry 41, 10963–10967 (2002).

    Article  CAS  Google Scholar 

  19. Radics, G., Koksch, B., El-Kousy, S. M., Spengler, J. & Burger, K. l-α-methylhomoisoserine: a new versatile building block for peptide and depsipeptide modification. Synlett 2003, 1826–1829 (2003).

    Google Scholar 

  20. Brenner, M. & Seebach, D. Synthesis and CD spectra in MeCN, MeOH and H2O of γ-oligopeptides, with hydroxy groups on the backbone—preliminary communication. Helv. Chim. Acta 84, 1181–1189 (2001).

    Article  CAS  Google Scholar 

  21. Gessier, F., Noti, C., Rueping, M. & Seebach, D. Synthesis and CD spectra of fluoro- and hydroxy-substituted β-peptides. Helv. Chim. Acta 86, 1862–1870 (2003).

    Article  CAS  Google Scholar 

  22. Rodriguez, F. et al. Conformational preferences of chiral acyclic homooligomeric β2,2-peptides. Curr. Top. Med. Chem. 14, 1225–1234 (2014).

    Article  CAS  Google Scholar 

  23. Gademann, K., Hane, A., Rueping, M., Jaun, B. & Seebach, D. The fourth helical secondary structure of β-peptides: the (P)-28-helix of a β-hexapeptide consisting of (2R,3S)-3-amino-2-hydroxy acid residues. Angew. Chem. Int. Ed. 42, 1534–1537 (2003).

    Article  CAS  Google Scholar 

  24. Dobrowolski, J. C. et al. IR low-temperature matrix, X-ray and ab initio study on l-isoserine conformations. Phys. Chem. Chem. Phys. 12, 10818–10830 (2010).

    Article  CAS  Google Scholar 

  25. Zhang, W., Bando, T. & Sugiyama, H. Discrimination of hairpin polyamides with an α-substituted-γ-aminobutyric acid as a 5′-TG-3′ reader in DNA minor groove. J. Am. Chem. Soc. 128, 8766–8776 (2006).

    Article  CAS  Google Scholar 

  26. Bandyopadhyay, A., Malik, A., Kumar, M. G. & Gopi, H. N. Exploring β-hydroxy γ-amino acids (statines) in the design of hybrid peptide foldamers. Org. Lett. 16, 294–297 (2014).

    Article  CAS  Google Scholar 

  27. Malik, A., Kumar, M. G., Bandyopadhyay, A. & Gopi, H. N. Helices with additional H-bonds: crystallographic conformations of α,γ-hybrid peptides helices composed of β-hydroxy γ-amino acids (statines). Pept. Sci. 108, e22978 (2017).

    Article  Google Scholar 

  28. Maini, R. et al. Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center. Bioorg. Med. Chem. 21, 1088–1096 (2013).

    Article  CAS  Google Scholar 

  29. Fujino, T., Goto, Y., Suga, H. & Murakami, H. Ribosomal synthesis of peptides with multiple β-amino acids. J. Am. Chem. Soc. 138, 1962–1969 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. 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  Google Scholar 

  33. Dedkova, L. M., Fahmi, N. E., Golovine, S. Y. & Hecht, S. M. Enhanced d-amino acid incorporation into protein by modified ribosomes. J. Am. Chem. Soc. 125, 6616–6617 (2003).

    Article  CAS  Google Scholar 

  34. Goto, Y., Murakami, H. & Suga, H. Initiating translation with d-amino acids. RNA 14, 1390–1398 (2008).

    Article  CAS  Google Scholar 

  35. Fujino, T., Goto, Y., Suga, H. & Murakami, H. Reevaluation of the d-amino acid compatibility with the elongation event in translation. J. Am. Chem. Soc. 135, 1830–1837 (2013).

    Article  CAS  Google Scholar 

  36. Katoh, T., Tajima, K. & Suga, H. Consecutive elongation of d-amino acids in translation. Cell Chem. Biol. 24, 46–54 (2017).

    Article  CAS  Google Scholar 

  37. Merryman, C. & Green, R. Transformation of aminoacyl tRNAs for the in vitro selection of ‘drug-like’ molecules. Chem. Biol. 11, 575–582 (2004).

    Article  CAS  Google Scholar 

  38. Kawakami, T., Murakami, H. & Suga, H. Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem. Biol. 15, 32–42 (2008).

    Article  CAS  Google Scholar 

  39. Subtelny, A. O., Hartman, M. C. & Szostak, J. W. Ribosomal synthesis of N-methyl peptides. J. Am. Chem. Soc. 130, 6131–6136 (2008).

    Article  CAS  Google Scholar 

  40. Maini, R. et al. Ribosomal formation of thioamide bonds in polypeptide synthesis. J. Am. Chem. Soc. 141, 20004–20008 (2019).

    Article  CAS  Google Scholar 

  41. Sando, S. et al. Unexpected preference of the E. coli translation system for the ester bond during incorporation of backbone-elongated substrates. J. Am. Chem. Soc. 129, 6180–6186 (2007).

    Article  CAS  Google Scholar 

  42. Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem. Biol. 14, 1315–1322 (2007).

    Article  CAS  Google Scholar 

  43. Goto, Y. & Suga, H. Translation initiation with initiator tRNA charged with exotic peptides. J. Am. Chem. Soc. 131, 5040–5041 (2009).

    Article  CAS  Google Scholar 

  44. Ad, O. et al. Translation of diverse aramid- and 1,3-dicarbonyl-peptides by wild type ribosomes in vitro. ACS Cent. Sci. 5, 1289–1294 (2019).

    Article  CAS  Google Scholar 

  45. Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat. Commun. 10, 5097 (2019).

    Article  Google Scholar 

  46. Takatsuji, R. et al. Ribosomal synthesis of backbone-cyclic peptides compatible with in vitro display. J. Am. Chem. Soc. 141, 2279–2287 (2019).

    Article  CAS  Google Scholar 

  47. Nakajima, E., Goto, Y., Sako, Y., Murakami, H. & Suga, H. Ribosomal synthesis of peptides with C-terminal lactams, thiolactones, and alkylamides. ChemBioChem 10, 1186–1192 (2009).

    Article  CAS  Google Scholar 

  48. 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  Google Scholar 

  49. Trobro, S. & Aqvist, J. Mechanism of peptide bond synthesis on the ribosome. Proc. Natl Acad. Sci. USA 102, 12395–12400 (2005).

    Article  CAS  Google Scholar 

  50. Gindulyte, A. et al. The transition state for formation of the peptide bond in the ribosome. Proc. Natl Acad. Sci. USA 103, 13327–13332 (2006).

    Article  CAS  Google Scholar 

  51. Voorhees, R. M., Weixlbaumer, A., Loakes, D., Kelley, A. C. & Ramakrishnan, V. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat. Struct. Mol. Biol. 16, 528–533 (2009).

    Article  CAS  Google Scholar 

  52. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).

    Article  CAS  Google Scholar 

  53. Adaligil, E., Song, A., Cunningham, C. N. & Fairbrother, W. J. Ribosomal synthesis of macrocyclic peptides with linear γ4- and β-hydroxy-γ4-amino acids. ACS Chem. Biol. 16, 1325–1331 (2021).

    Article  CAS  Google Scholar 

  54. Sohma, Y. & Kiso, Y. Synthesis of O-acyl isopeptides. Chem. Rec. 13, 218–223 (2013).

    Article  CAS  Google Scholar 

  55. Goto, Y. et al. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 3, 120–129 (2008).

    Article  CAS  Google Scholar 

  56. Iwasaki, K., Goto, Y., Katoh, T. & Suga, H. Selective thioether macrocyclization of peptides having the N-terminal 2-chloroacetyl group and competing two or three cysteine residues in translation. Org. Biomol. Chem. 10, 5783–5786 (2012).

    Article  CAS  Google Scholar 

  57. Back, J. W. et al. Mild and chemoselective peptide-bond cleavage of peptides and proteins at azido homoalanine. Angew. Chem. Int. Ed. 44, 7946–7950 (2005).

    Article  CAS  Google Scholar 

  58. Deslongchamps, P. & Taillefer, R. J. The mechanism of hydrolysis of imidate salts. The importance of stereoelectronic control and pH of the reaction medium on the cleavage of tetrahedral intermediates. Can. J. Chem. 53, 3029–3037 (2011).

    Article  Google Scholar 

  59. Kwan, J. C., Eksioglu, E. A., Liu, C., Paul, V. J. & Luesch, H. Grassystatins A-C from marine cyanobacteria, potent cathepsin E inhibitors that reduce antigen presentation. J. Med. Chem. 52, 5732–5747 (2009).

    Article  CAS  Google Scholar 

  60. Al-Awadhi, F. H., Law, B. K., Paul, V. J. & Luesch, H. Grassystatins D-F, potent aspartic protease inhibitors from marine cyanobacteria as potential antimetastatic agents targeting invasive breast cancer. J. Nat. Prod. 80, 2969–2986 (2017).

    Article  CAS  Google Scholar 

  61. Kanamori, Y. et al. Izenamides A and B, statine-containing depsipeptides, and an analogue from a marine cyanobacterium. J. Nat. Prod. 81, 1673–1681 (2018).

    Article  CAS  Google Scholar 

  62. Walsh, C. T., O’Brien, R. V. & Khosla, C. Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew. Chem. Int. Ed. 52, 7098–7124 (2013).

    Article  CAS  Google Scholar 

  63. Guibejampel, E. & Wakselman, M. Selective cleavage of para-nitrobenzyl esters with sodium dithionite. Synthetic Commun. 12, 219–223 (1982).

    Article  CAS  Google Scholar 

  64. Istvan, E. S. et al. Esterase mutation is a mechanism of resistance to antimalarial compounds. Nat. Commun. 8, 14240 (2017).

    Article  CAS  Google Scholar 

  65. Goto, Y. & Suga, H. The RaPID platform for the discovery of pseudo-natural macrocyclic peptides. Acc. Chem. Res. 54, 3604–3617 (2021).

    Article  CAS  Google Scholar 

  66. Seebeck, F. P. & Szostak, J. W. Ribosomal synthesis of dehydroalanine-containing peptides. J. Am. Chem. Soc. 128, 7150–7151 (2006).

    Article  CAS  Google Scholar 

  67. Goto, Y., Iwasaki, K., Torikai, K., Murakami, H. & Suga, H. Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides. Chem. Commun. (Camb.) 21, 3419–3421 (2009).

    Article  Google Scholar 

  68. Seebeck, F. P., Ricardo, A. & Szostak, J. W. Artificial lantipeptides from in vitro translations. Chem. Commun. (Camb.) 47, 6141–6143 (2011).

    Article  CAS  Google Scholar 

  69. Kato, Y. et al. Chemoenzymatic posttranslational modification reactions for the synthesis of Ψ[CH2NH]-containing peptides. Angew. Chem. Int. Ed. 59, 684–688 (2020).

    Article  CAS  Google Scholar 

  70. Tsutsumi, H., Kuroda, T., Kimura, H., Goto, Y. & Suga, H. Posttranslational chemical installation of azoles into translated peptides. Nat. Commun. 12, 696 (2021).

    Article  CAS  Google Scholar 

  71. Yamagishi, Y. et al. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem. Biol. 18, 1562–1570 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Murakami for invaluable discussions and helpful suggestions. This work was supported by KAKENHI grants (JP16H06444 to H.S. and Y.G.; JP20H05618 to H.S.; JP17H04762, JP19H01014, JP19K22243 and JP20H02866 to Y.G.; JP19J14230 to T.K.) from the Japan Society for the Promotion of Science and Human Frontier Science Program (to H.S.).

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Y.G. and H.S. conceived and supervised the study. All authors designed experiments. T.K., Y.H. and Y.G. synthesized acyl-donor substrates. T.K. and S.N. performed expression and modification of AzHyA-containing peptides. All authors analysed the experimental results. T.K., Y.G. and H.S. wrote the manuscript with input from all authors. Y.G. prepared manuscript figures.

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Correspondence to Yuki Goto or Hiroaki Suga.

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Kuroda, T., Huang, Y., Nishio, S. et al. Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units. Nat. Chem. 14, 1413–1420 (2022). https://doi.org/10.1038/s41557-022-01065-1

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