Advertisement

Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimide-Thioether Linkage

  • Anna Mette Hansen
  • Ashif Yasin Shaikh
  • Henrik FranzykEmail author
Protocol
  • 119 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2105)

Abstract

Conjugation of a delivery peptide containing a thiol functionality (e.g., a cysteine residue) with a PNA oligomer displaying a single unprotected aliphatic primary amine (e.g., the N-terminus or a C-terminal lysine residue) can be achieved via a one-pot modification with a bisfunctional maleimide linker also displaying a reactive N-hydroxysuccinimidyl ester group (e.g., Mal-PEG2-OSu). Here, an optimized protocol with respect to ratios between the reactants as well as recommended reaction times is presented. Formation and conversion of the maleimide-PNA intermediate was followed by analytical HPLC as exemplified by its conjugation to (KFF)3K-Cys-NH2. In addition, the reaction time required for direct conversion of a preformed Mal-(CH2)2-(C=O)-PNA oligomer in the presence of a slight excess of thiol-modified peptide (with a varying degree of sterical hindrance: HS-(CH2)2-CONH-(KFF)3K-NH2, (KFF)3K-hCys-NH2 and (KFF)3K-Cys-NH2) is provided.

Key words

Peptide nucleic acid Peptide Conjugation Thiol Maleimide linker 

Notes

Acknowledgments

This research was supported by the Department of Drug Design and Pharmacology and Center for Peptide-Based Antibiotics (Cepan).

References

  1. 1.
    Ray A, Nordén B (2000) Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB J 14:1041–1060CrossRefGoogle Scholar
  2. 2.
    Koppelhus U, Nielsen PE (2003) Cellular delivery of peptide nucleic acid (PNA). Adv Drug Deliv Rev 55:267–280CrossRefGoogle Scholar
  3. 3.
    Gait MJ (2003) Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues. Cell Mol Life Sci 60:844–853CrossRefGoogle Scholar
  4. 4.
    Guidotti G, Brambilla L, Rossi D (2017) Cell-penetrating peptides; from basic research to clinics. Trends Pharmacol Sci 38:406–424CrossRefGoogle Scholar
  5. 5.
    Ramsey JD, Flynn NH (2015) Cell-penetrating peptides transport therapeutics into cells. Pharmacol Ther 154:78–86CrossRefGoogle Scholar
  6. 6.
    Kurrikoff K, Gestin M, Langel U (2016) Recent in vivo advances in cell-penetrating peptide-assisted drug delivery. Expert Opin Drug Deliv 13:373–387CrossRefGoogle Scholar
  7. 7.
    Lebleu B, Moulton HM, Abes R, Ivanova GD, Abes S, Stein DA, Iversen PL, Arzumanov AA, Gait MJ (2008) Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev 60:517–529CrossRefGoogle Scholar
  8. 8.
    Bendifallah N, Rasmussen FW, Zachar V, Ebbesen P, Nielsen PE, Koppelhus U (2006) Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA). Bioconjugate Chem 17:750–758CrossRefGoogle Scholar
  9. 9.
    Basu S, Wickstrom E (1997) Synthesis and characterization of a peptide nucleic acid conjugated to a d-peptide analog of insulin-like growth factor 1 for increased cellular uptake. Bioconjugate Chem 8:481–488CrossRefGoogle Scholar
  10. 10.
    de Koning MC, van der Marel GA, Overhand M (2003) Synthetic developments towards PNA–peptide conjugates. Curr Opin Chem Biol 7:734–740CrossRefGoogle Scholar
  11. 11.
    Fabani MM, Ivanova GD, Gait MJ (2008) Chapter 4: Peptide-peptide nucleic acid conjugates for modulation of gene expression. In: Kurreck J (ed) RSC biomolecular sciences: therapeutic oligonucleotides. Royal Chemical Society, Cambridge, UK, pp 80–102CrossRefGoogle Scholar
  12. 12.
    Svensen N, Díaz-Mochón JJ, Bradley M (2008) Microwave-assisted orthogonal synthesis of PNA-peptide conjugates. Tetrahedron Lett 49:6498–6500CrossRefGoogle Scholar
  13. 13.
    Fabani MM, Abreu-Goodger C, Williams D, Lyons PA, Torres AG, Smith KG, Enright AJ, Gait MJ, Vigorito E (2010) Efficient inhibition of miR-155 function in vivo by peptide nucleic acids. Nucleic Acids Res 38:4466–4475CrossRefGoogle Scholar
  14. 14.
    Hansen AM, Bonke G, Hogendorph WFJ, Björkling F, Nielsen J, Nielsen PE, Kongstad KT, Zabicka D, Franzyk H (2019) Microwave-assisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae. Eur J Med Chem 168:134–145CrossRefGoogle Scholar
  15. 15.
    Abes S, Ivanova GD, Abes R, Arzumanov AA, Williams D, Owen D, Lebleu B, Gait MJ (2009) Peptide-based delivery of steric-block PNA oligonucleotides. Methods Mol Biol 480:85–99CrossRefGoogle Scholar
  16. 16.
    Patil NA, Karas JA, Turner BJ, Shabanpoor F (2019) Thiol-cyanobenzothiazole ligation for the efficient preparation of peptide−PNA conjugates. Bioconjugate Chem 30:793–799CrossRefGoogle Scholar
  17. 17.
    Awasthi SK, Nielsen PE (2002) Synthesis of PNA-peptide conjugates. Methods Mol Biol 208:43–52PubMedGoogle Scholar
  18. 18.
    Neuner P, Gallo P, Orsatti L, Fontana L, Monaci P (2003) An efficient and versatile synthesis of bisPNA-peptide conjugates based on chemoselective oxime formation. Bioconjugate Chem 14:276–281CrossRefGoogle Scholar
  19. 19.
    de Koning MC, Filippov DV, Meeuwenoord N, Overhand M, van der Marel GA, van Boom JH (2001) Synthesis of a PNA-peptide conjugate by chemical ligation. Synlett 10:1516–1518CrossRefGoogle Scholar
  20. 20.
    Równicki M, Wojciechowska M, Wierzba AJ, Czarnecki J, Bartosik D, Gryko D, Trylska J (2017) Vitamin B12 as a carrier of peptide nucleic acid (PNA) into bacterial cells. Sci Rep 7:7644CrossRefGoogle Scholar
  21. 21.
    Deuss PJ, Arzumanov AA, Williams DL, Gait MJ (2013) Parallel synthesis and splicing redirection activity of cell-penetrating peptide conjugate libraries of a PNA Cargo. Org Biomol Chem 11:7621–7630CrossRefGoogle Scholar
  22. 22.
    Good L, Awasthi SK, Dryselius R, Larsson O, Nielsen PE (2001) Bactericidal antisense effects of peptide-PNA conjugates. Nat Biotechnol 19:360–364CrossRefGoogle Scholar
  23. 23.
    Harrison JG, Frier C, Laurant R, Dennis R, Raney KD, Balasubramanian S (1999) Inhibition of the human telomerase by PNA-cationic peptide conjugates. Bioorg Med Chem Lett 9:1273–1278CrossRefGoogle Scholar
  24. 24.
    Nanaware-Kharade N, Gonzalez GA III, Lay JO Jr, Hendrickson HP, Peterson EC (2012) Therapeutic anti-methamphetamine antibody fragment-nanoparticle conjugates: synthesis and in vitro characterization. Bioconjugate Chem 23:1864–1872CrossRefGoogle Scholar
  25. 25.
    Taylor SK, Pei R, Moon BC, Damera S, Shen A, Stojanovic MN (2009) Triggered release of an active peptide conjugate from a DNA device by an orally administrable small molecule. Angew Chem Int Ed 48:4394–4397CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Anna Mette Hansen
    • 1
  • Ashif Yasin Shaikh
    • 1
  • Henrik Franzyk
    • 1
    Email author
  1. 1.Department of Drug Design and Pharmacology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations