Skip to main content
SpringerLink
Log in

Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications

  • Protocol
  • First Online:
Non-Ribosomal Peptide Biosynthesis and Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2670))

Abstract

Noncanonical peptide backbone structures, such as heterocycles and non-α-amino acids, are characteristic building blocks present in peptidic natural products. To achieve ribosomal synthesis of designer peptides bearing such noncanonical backbone structures, we have devised translation-compatible precursor residues and their chemical posttranslational modification processes. In this chapter, we describe the detailed procedures for the in vitro translation of peptides containing the precursor residues by means of genetic code reprogramming technology and posttranslational generation of objective noncanonical backbone structures.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Walsh CT, O’Brien RV, Khosla C (2013) Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed Engl 52:7098–7124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Arnison PG, Bibb MJ, Bierbaum G et al (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Montalban-Lopez M, Scott TA, Ramesh S et al (2020) New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep 38:130–239

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Kofman C, Lee J, Jewett MC (2021) Engineering molecular translation systems. Cell Syst 12:593–607

    Article  CAS  PubMed  Google Scholar 

  6. Hartman MCT (2022) Non-canonical amino acid substrates of E. coli aminoacyl-tRNA synthetases. ChemBioChem 23:e202100299

    CAS  PubMed  Google Scholar 

  7. Hecht SM (2022) Expansion of the genetic code through the use of modified bacterial ribosomes. J Mol Biol 434:167211

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Goto Y, Iwasaki K, Torikai K et al (2009) Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides. Chem Commun (Camb) 23:3419–3421

    Article  Google Scholar 

  10. Nakajima E, Goto Y, Sako Y et al (2009) Ribosomal synthesis of peptides with C-terminal lactams, thiolactones, and alkylamides. ChemBioChem 10:1186–1192

    Article  CAS  PubMed  Google Scholar 

  11. Kato Y, Kuroda T, Huang Y et al (2020) Chemoenzymatic posttranslational modification reactions for the synthesis of Psi[CH2 NH]-containing peptides. Angew Chem Int Ed Engl 59:684–688

    Article  CAS  PubMed  Google Scholar 

  12. Tsutsumi H, Kuroda T, Kimura H et al (2021) Posttranslational chemical installation of azoles into translated peptides. Nat Commun 12:696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kuroda T, Huang Y, Nishio S et al (2022) Posttranslational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units. Nat Chem:in press

    Google Scholar 

  14. Murakami H, Ohta A, Ashigai H et al (2006) A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3:357–359

    Article  CAS  PubMed  Google Scholar 

  15. Goto Y, Katoh T, Suga H (2011) Flexizymes for genetic code reprogramming. Nat Protoc 6:779

    Article  CAS  PubMed  Google Scholar 

  16. Wipf P, Fritch PC, Geib SJ et al (1998) Conformational studies and structure-activity analysis of lissoclinamide 7 and related cyclopeptide alkaloids. J Am Chem Soc 120:4105–4112

    Article  CAS  Google Scholar 

  17. Siodlak D, Stas M, Broda MA et al (2014) Conformational properties of oxazole-amino acids: effect of the intramolecular N-H...N hydrogen bond. J Phys Chem B 118:2340–2350

    Article  CAS  PubMed  Google Scholar 

  18. Ahlbach CL, Lexa KW, Bockus AT et al (2015) Beyond cyclosporine A: conformation-dependent passive membrane permeabilities of cyclic peptide natural products. Future Med Chem 7:2121–2130

    Article  CAS  PubMed  Google Scholar 

  19. Marshall CG, Burkart MD, Keating TA et al (2001) Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate. Biochemistry 40:10655–10663

    Article  CAS  PubMed  Google Scholar 

  20. Schneider TL, Shen B, Walsh CT (2003) Oxidase domains in epothilone and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation. Biochemistry 42:9722–9730

    Article  CAS  PubMed  Google Scholar 

  21. Dunbar KL, Melby JO, Mitchell DA (2012) YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations. Nat Chem Biol 8:569–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Koehnke J, Bent AF, Zollman D et al (2013) The cyanobactin heterocyclase enzyme: a processive adenylase that operates with a defined order of reaction. Angew Chem Int Ed Engl 52:13991–13996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Melby JO, Li X, Mitchell DA (2014) Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases. Biochemistry 53:413–422

    Article  CAS  PubMed  Google Scholar 

  24. Chowdhury SR, Maini R, Dedkova LM et al (2015) Synthesis of fluorescent dipeptidomimetics and their ribosomal incorporation into green fluorescent protein. Bioorg Med Chem Lett 25:4715–4718

    Article  PubMed Central  Google Scholar 

  25. Chowdhury SR, Chauhan PS, Dedkova LM et al (2016) Synthesis and evaluation of a library of fluorescent dipeptidomimetic analogues as substrates for modified bacterial ribosomes. Biochemistry 55:2427–2440

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  27. Bott R, Subramanian E, Davies DR (1982) Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5-A resolution. Biochemistry 21:6956–6962

    Article  CAS  PubMed  Google Scholar 

  28. Rich DH, Bernatowicz MS, Agarwal NS et al (1985) Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition. Biochemistry 24:3165–3173

    Article  CAS  PubMed  Google Scholar 

  29. Umezawa H, Aoyagi T, Morishima H et al (1970) Pepstatin, a new pepsin inhibitor produced by actinomycetes. J Antibiot (Tokyo) 23:259–262

    Article  CAS  PubMed  Google Scholar 

  30. Tumminello FM, Bernacki RJ, Gebbia N et al (1993) Pepstatins: aspartic proteinase inhibitors having potential therapeutic applications. Med Res Rev 13:199–208

    Article  CAS  PubMed  Google Scholar 

  31. Kuranaga T, Matsuda K, Takaoka M et al (2020) Total synthesis and structural revision of kasumigamide, and identification of a new analogue. ChemBioChem 21:3329–3332

    Article  CAS  PubMed  Google Scholar 

  32. Lee J, Schwarz KJ, Kim DS et al (2020) Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nat Commun 11:4304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Trobro S, Aqvist J (2005) Mechanism of peptide bond synthesis on the ribosome. Proc Natl Acad Sci U S A 102:12395–12400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Voorhees RM, Weixlbaumer A, Loakes D et al (2009) Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat Struct Mol Biol 16:528–533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shimizu Y, Inoue A, Tomari Y et al (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755

    Article  CAS  PubMed  Google Scholar 

  36. Charlton A, Zachariou M (2008) Immobilized metal ion affinity chromatography of histidine-tagged fusion proteins. Methods Mol Biol 421:137–149

    CAS  PubMed  Google Scholar 

  37. Block H, Maertens B, Spriestersbach A et al (2009) Immobilized-metal affinity chromatography (IMAC): a review. Methods Enzymol 463:439–473

    Article  CAS  PubMed  Google Scholar 

  38. Martinis SA, Schimmel P (1992) Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine. Proc Natl Acad Sci U S A 89:65–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Putz J, Wientges J, Sissler M et al (1997) Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids. Nucleic Acids Res 25:1862–1863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ohta A, Murakami H, Higashimura E et al (2007) Synthesis of polyester by means of genetic code reprogramming. Chem Biol 14:1315–1322

    Article  CAS  PubMed  Google Scholar 

  41. Terasaka N, Hayashi G, Katoh T et al (2014) An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat Chem Biol 10:555–557

    Article  CAS  PubMed  Google Scholar 

  42. Katoh T, Iwane Y, Suga H (2017) Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation. Nucleic Acids Res 45:12601–12610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Goto Y, Iseki M, Hitomi A et al (2013) Nonstandard peptide expression under the genetic code consisting of reprogrammed dual sense codons. ACS Chem Biol 8:2630–2634

    Article  CAS  PubMed  Google Scholar 

  44. Olins PO, Devine CS, Rangwala SH et al (1988) The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Gene 73:227–235

    Article  CAS  PubMed  Google Scholar 

  45. Sako Y, Goto Y, Murakami H et al (2008) Ribosomal synthesis of peptidase-resistant peptides closed by a nonreducible inter-side-chain bond. ACS Chem Biol 3:241–249

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank H. Tsutsumi and T. Kuroda for their major contributions to the development of the methods presented in this chapter. We also appreciate the financial supports from the Japan Society for the Promotion of Science, KAKENHI (JP16H06444 to H.S. and Y.G.; JP20H05618 to H.S.; JP17H04762, JP18H04382, JP19K22243, JP20H02866 to Y.G.).

Author information

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Yuki Goto & Hiroaki Suga

Authors
  1. Yuki Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroaki Suga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yuki Goto or Hiroaki Suga .

Editor information

Editors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

    Michael Burkart

  2. Faculty of Pharmacy, Kindai University, Higashi-Osaka, Osaka, Japan

    Fumihiro Ishikawa

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Goto, Y., Suga, H. (2023). Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications. In: Burkart, M., Ishikawa, F. (eds) Non-Ribosomal Peptide Biosynthesis and Engineering. Methods in Molecular Biology, vol 2670. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3214-7_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3214-7_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3213-0

  • Online ISBN: 978-1-0716-3214-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation