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Preparation of Semisynthetic Peptide Macrocycles Using Split Inteins

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Split Inteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1495))

Abstract

Cyclic peptides are highly desired molecules not only for basic research but also for many biomedical and pharmacological applications. Due to their potentially superior physicochemical properties as compared to their linear counterparts, they are considered as ideal candidates for studying protein–protein interactions, among others. Most of the methods developed in recent years to prepare cyclic peptides focus either on a synthetic or a recombinant route. While the former provides access to diversified, noncanonical peptides, including unnatural and d-amino acid, for example, the latter can harness the power of genetic randomization to generate and select from large peptide libraries. Only few approaches have been reported to prepare semisynthetic macrocycles that would benefit from both the advantages associated with synthetic and genetically encoded parts. We describe in this chapter a chemo-enzymatic method to make semisynthetic cyclic peptides in vitro from two fragments using protein trans-splicing and bioorthogonal oxime ligation.

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References

  1. Clardy J, Walsh C (2004) Lessons from natural molecules. Nature 432(7019):829–837

    Article  CAS  PubMed  Google Scholar 

  2. Driggers EM et al (2008) The exploration of macrocycles for drug discovery--an underexploited structural class. Nat Rev Drug Discov 7(7):608–624

    Article  CAS  PubMed  Google Scholar 

  3. Arnison PG et al (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30(1):108–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Trauger JW et al (2000) Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407(6801):215–218

    Article  CAS  PubMed  Google Scholar 

  5. Tseng CC et al (2002) Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase. Biochemistry 41(45):13350–13359

    Article  CAS  PubMed  Google Scholar 

  6. Grunewald J, Sieber SA, Marahiel MA (2004) Chemo- and regioselective peptide cyclization triggered by the N-terminal fatty acid chain length: the recombinant cyclase of the calcium-dependent antibiotic from Streptomyces coelicolor. Biochemistry 43(10):2915–2925

    Article  CAS  PubMed  Google Scholar 

  7. Mootz HD, Schwarzer D, Marahiel MA (2000) Construction of hybrid peptide synthetases by module and domain fusions. Proc Natl Acad Sci U S A 97(11):5848–5853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Passioura T et al (2014) Selection-based discovery of druglike macrocyclic peptides. Annu Rev Biochem 83:727–752

    Article  CAS  PubMed  Google Scholar 

  9. Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97(2):391–410

    Article  CAS  PubMed  Google Scholar 

  10. Antos JM et al (2009) A straight path to circular proteins. J Biol Chem 284(23):16028–16036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nguyen GK et al (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 10(9):732–738

    Article  CAS  PubMed  Google Scholar 

  12. Scott CP et al (1999) Production of cyclic peptides and proteins in vivo. Proc Natl Acad Sci U S A 96(24):13638–13643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Morimoto J, Hayashi Y, Suga H (2012) Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew Chem Int Ed Engl 51(14):3423–3427

    Article  CAS  PubMed  Google Scholar 

  14. Kawakami T et al (2009) Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol 5(12):888–890

    Article  CAS  PubMed  Google Scholar 

  15. Schlippe YVG et al (2012) In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors. J Am Chem Soc 134(25):10469–10477

    Article  CAS  PubMed  Google Scholar 

  16. Timmerman P et al (2005) Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces. Chembiochem 6(5):821–824

    Article  CAS  PubMed  Google Scholar 

  17. Heinis C et al (2009) Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat Chem Biol 5(7):502–507

    Article  CAS  PubMed  Google Scholar 

  18. Day JW et al (2013) Identification of metal ion binding peptides containing unnatural amino acids by phage display. Bioorg Med Chem Lett 23(9):2598–2600

    Article  CAS  PubMed  Google Scholar 

  19. Ng S, Jafari MR, Derda R (2012) Bacteriophages and viruses as a support for organic synthesis and combinatorial chemistry. ACS Chem Biol 7(1):123–138

    Article  CAS  PubMed  Google Scholar 

  20. Smith JM et al (2011) Modular assembly of macrocyclic organo-peptide hybrids using synthetic and genetically encoded precursors. Angew Chem Int Ed Engl 50(22):5075–5080

    Article  CAS  PubMed  Google Scholar 

  21. Satyanarayana M et al (2012) Diverse organo-peptide macrocycles via a fast and catalyst-free oxime/intein-mediated dual ligation. Chem Commun (Camb) 48(10):1461–1463

    Article  CAS  Google Scholar 

  22. Palei S, Mootz HD (2016) Cyclic peptides made by linking synthetic and genetically encoded fragments. Chembiochem 17(5):378–382

    Article  CAS  PubMed  Google Scholar 

  23. Appleby-Tagoe JH et al (2011) Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. J Biol Chem 286(39):34440–34447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Volkmann G, Mootz HD (2013) Recent progress in intein research: from mechanism to directed evolution and applications. Cell Mol Life Sci 70(7):1185–1206

    Article  CAS  PubMed  Google Scholar 

  25. Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5:446–461

    Article  CAS  PubMed  Google Scholar 

  26. Mootz HD (2009) Split inteins as versatile tools for protein semisynthesis. Chembiochem 10(16):2579–2589

    Article  CAS  PubMed  Google Scholar 

  27. Ludwig C et al (2006) Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew Chem Int Ed Engl 45(31):5218–5221

    Article  CAS  PubMed  Google Scholar 

  28. Ludwig C, Schwarzer D, Mootz HD (2008) Interaction studies and alanine scanning analysis of a semi-synthetic split intein reveal thiazoline ring formation from an intermediate of the protein splicing reaction. J Biol Chem 283(37):25264–25272

    Article  CAS  PubMed  Google Scholar 

  29. Wang L et al (2003) Addition of the keto functional group to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A 100(1):56–61

    Article  CAS  PubMed  Google Scholar 

  30. Dirksen A, Hackeng TM, Dawson PE (2006) Nucleophilic catalysis of oxime ligation. Angew Chem Int Ed Engl 45(45):7581–7584

    Article  CAS  PubMed  Google Scholar 

  31. Wasmuth A, Ludwig C, Mootz HD (2013) Structure-activity studies on the upstream splice junction of a semisynthetic intein. Bioorg Med Chem 21(12):3495–3503

    Article  CAS  PubMed  Google Scholar 

  32. Böcker JK et al (2015) Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides. Angew Chem Int Ed Engl 54(7):2116–2120

    Article  CAS  PubMed  Google Scholar 

  33. Binschik J, Zettler J, Mootz HD (2011) Photocontrol of protein activity mediated by the cleavage reaction of a split intein. Angew Chem Int Ed Engl 50(14):3249–3252

    Article  CAS  PubMed  Google Scholar 

  34. Thiel IV et al (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angew Chem Int Ed Engl 53(5):1306–1310

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Peter G. Schultz (Scripps Research Institute, La Jolla) for providing the plasmid for AcF incorporation. We acknowledge financial support from the DFG (MO1073/3-2, SPP1623 and Cells in Motion cluster EXC1003) and the International Graduate School of Chemistry in Münster (GSC-MS; Ph.D. stipend to S. P.).

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Authors and Affiliations

  1. Institute of Biochemistry, Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149, Münster, Germany

    Shubhendu Palei

  2. Institute of Biochemistry, Department of Chemistry and Pharmacy, University of Muenster, Wilhelm-Klemm-Str. 2, 48149, Münster, Germany

    Henning D. Mootz

Authors
  1. Shubhendu Palei
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  2. Henning D. Mootz
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Correspondence to Henning D. Mootz .

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Editors and Affiliations

  1. Institute of Biochemistry Department of Chemistry and Pharmacy, University of Muenster, Münster, Germany

    Henning D. Mootz

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Palei, S., Mootz, H.D. (2017). Preparation of Semisynthetic Peptide Macrocycles Using Split Inteins. In: Mootz, H. (eds) Split Inteins. Methods in Molecular Biology, vol 1495. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6451-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6451-2_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6449-9

  • Online ISBN: 978-1-4939-6451-2

  • eBook Packages: Springer Protocols

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