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Chiral PNAs with Constrained Open-Chain Backbones

  • Roberto Corradini
  • Tullia Tedeschi
  • Stefano Sforza
  • Rosangela Marchelli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1050)

Abstract

Chiral open-chain PNAs have been shown to have improved properties in terms of control of helical handedness, DNA affinity, sequence selectivity, and cellular uptake. They can be synthesized either using preformed chiral monomers or by means of a submonomeric strategy. The former is preferred when only a stereogenic center is present at C-5, whereas for PNA-bearing substituents at C-2, the submonomeric approach is preferred, since racemization, generally occurring during the solid-phase synthesis, can be minimized by this procedure. Here we describe the protocols for the synthesis of PNA oligomers containing C-2- or C-5- (or both) modified monomers and a GC method for checking the optical purity of C-2-modified PNAs.

Key words

Stereochemistry Chiral PNA Submonomeric strategy Optical purity Solid-phase synthesis 

Acronym List

BTSA

N,O-Bis(trimethylsilyl)acetamide

CMB

Carboxymethyl nucleobase

DCC

N,N-dicyclohexylcarbodiimide

DCM

Dichloromethane

DIC

N,N-diisopropylcarbodiimide

DIPEA

N,N-diisopropylethylamine

DhBTOH

3-hydroxy-1,2,3-benzotriazin-4(3H)-one

DMF

N,N-dimethylformamide

EDC

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

HBTU

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HOBt

1-hydroxy-1,2,3-benzotriazole

MBHA

4-methyl benzhydrylamine

NMP

N-methylpyrrolidone

TFA

Trifluoroacetic acid

TFMSA

Trifluoromethanesulfonic acid

Notes

Acknowledgments

This work was supported by the Italian Ministry of University and Research (MIUR) with the national projects PRIN 2007 and PRIN 2009.

References

  1. 1.
    Corradini R, Sforza S, Tedeschi T et al (2011) Peptide nucleic acids with a structurally biased backbone. Updated review and emerging challenges. Curr Top Med Chem 11:1535–1554PubMedCrossRefGoogle Scholar
  2. 2.
    Kl D, Petersen KH, Jensen DK et al (1994) Peptide nucleic-acid (PNA) with a chiral backbone based on alanine. Bioorg Med Chem Lett 4:1077–1080CrossRefGoogle Scholar
  3. 3.
    Haaima G, Lohse A, Buchardt O et al (1996) Peptide nucleic acids (PNAs) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of D-lysine PNA. Angew Chem Int Ed Engl 35:1939–1941CrossRefGoogle Scholar
  4. 4.
    Puschl A, Sforza S, Haaima G et al (1998) Peptide nucleic acids (PNAs) with a functional backbone. Tetrahedron Lett 39:711–714CrossRefGoogle Scholar
  5. 5.
    Sforza S, Haaima G, Marchelli R et al (1999) Chiral peptide nucleic acids (PNAs): helix handedness and DNA recognition. Eur J Org Chem 197–204Google Scholar
  6. 6.
    Sforza S, Corradini R, Ghirardi S et al (2000) DNA binding of a D-lysine-based chiral PNA: direction control and mismatch recognition. Eur J Org Chem 2905–2913Google Scholar
  7. 7.
    Sforza S, Tedeschi T, Corradini R et al (2003) Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry. Chem Commun 1102–1103Google Scholar
  8. 8.
    Kosynkina L, Wang W, Liang TC (1994) A convenient synthesis of chiral peptide nucleic acid (PNA) monomer. Tetrahedron Lett 35:5173–5176CrossRefGoogle Scholar
  9. 9.
    Dragulescu-Andrasi A, Rapireddy S, Frezza BM et al (2006) A simple γ-backbone modification preorganizes peptide nucleic acid into a helical structure. J Am Chem Soc 128:10258–10267PubMedCrossRefGoogle Scholar
  10. 10.
    Rapireddy S, He G, Roy S et al (2007) Strand invasion of mixed-sequence B-DNA by acridine-linked, γ-peptide nucleic acid (g-PNA). J Am Chem Soc 129:15596–15600PubMedCrossRefGoogle Scholar
  11. 11.
    Englund EA, Appella DH (2005) Synthesis of γ-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding. Org Lett 7:3465–3467PubMedCrossRefGoogle Scholar
  12. 12.
    Englund EA, Appella DH (2007) γ-substituted peptide nucleic acids constructed from L-lysine are a versatile scaffold for multifunctional display. Angew Chem Int Ed 46:1414–1418CrossRefGoogle Scholar
  13. 13.
    Tedeschi T, Sforza S, Corradini R et al (2005) Synthesis of new chiral PNAs bearing a dipeptide-mimic monomer with two lysine-derived stereogenic centres. Tetrahedron Lett 46:8395–8399CrossRefGoogle Scholar
  14. 14.
    Sforza S, Tedeschi T, Corradini R, et al (2007) Induction of helical handedness and dna binding properties of peptide nucleic acids (pnas) with two stereogenic centres. Eur J Org Chem 5879–5885Google Scholar
  15. 15.
    Bentin T, Nielsen PE (2003) Superior duplex DNA strand invasion by acridine conjugated peptide nucleic acids. J Am Chem Soc 125:6378–6379PubMedCrossRefGoogle Scholar
  16. 16.
    Ishizuka T, Yoshida J, Yamamoto Y et al (2008) Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences. Nucleic Acid Res 36:1464–1471PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Ishizuka T, Tedeschi T, Corradini R et al (2009) SSB-assisted duplex invasion of preorganized PNA into double-stranded DNA. ChemBioChem 10:2607–2612PubMedCrossRefGoogle Scholar
  18. 18.
    Aguado GP, Rua F, Branchadell V et al (2006) Cyclobutyl-carbonyl substituted PNA: synthesis and study of a novel PNA derivative. Tetrahedron Asymm 17:2499–2503CrossRefGoogle Scholar
  19. 19.
    Hamzavi R, Meyera C, Metzler-Nolte N (2005) Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer. Org Biomol Chem 4:3648–3651CrossRefGoogle Scholar
  20. 20.
    Corradini R, Feriotto G, Sforza S et al (2004) Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acids probes by a surface Plasmon resonance biosensor. J Mol Rec 17:76–84CrossRefGoogle Scholar
  21. 21.
    Tedeschi T, Chiari M, Galaverna G et al (2005) Detection of the R553X DNA single point mutation related to cystic fibrosis by a “chiral box” D-lysine-peptide nucleic acid probe by capillary Electrophoresis. Electrophoresis 26:4310–4316PubMedCrossRefGoogle Scholar
  22. 22.
    Manicardi A, Calabretta A, Bencivenni M et al (2010) Affinity and selectivity of C2- and C5-substituted “chiral-box” PNA in solution and on microarrays. Chirality 22:E161–E17223PubMedCrossRefGoogle Scholar
  23. 23.
    Tedeschi T, Calabretta A, Bencivenni M et al (2011) A PNA microarray for tomato genotyping. Mol BioSyst 7:1902–1907PubMedCrossRefGoogle Scholar
  24. 24.
    Menchise V, De Simone G, Tedeschi T et al (2003) Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine based chiral PNA-DNA duplex. Proc Natl Acad Sci U S A 100:12021–12026PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Rasmussen H, Sandholm J (1997) Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 angstrom resolution. Nat Struct Biol 4:98–101PubMedCrossRefGoogle Scholar
  26. 26.
    Govindaraju T, Madhuri V, Kumar VA et al (2006) Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding. Tuning of dihedral angle β in PNAs for DNA/RNA discrimination. J Org Chem 71:14–21PubMedCrossRefGoogle Scholar
  27. 27.
    Topham CM, Smithyz JC (2007) Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form. Biophys J 92:769–786PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Calabretta A, Tedeschi T, Corradini R et al (2011) DNA and RNA binding properties of an arginine-based “Extended Chiral Box” peptide nucleic acid. Tetrahedron Lett 52:300–304CrossRefGoogle Scholar
  29. 29.
    Calabretta A, Tedeschi T, Di Cola G et al (2009) Arginine-based PNA microarrays for APOE genotyping. Mol BioSyst 5:1323–1330PubMedCrossRefGoogle Scholar
  30. 30.
    Koppelhus U, Awasthi SK, Zachar V et al (2002) Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic Acid Drug Dev 12:51–63PubMedCrossRefGoogle Scholar
  31. 31.
    Zhou P, Dragulescu-Andrasi A, Bhattacharya B et al (2006) Synthesis of cell-permeable peptide nucleic acids and characterization of their hybridization and uptake properties. Bioorg Med Chem Lett 16:4931–4935PubMedCrossRefGoogle Scholar
  32. 32.
    Zhou P, Wang MM, Du L et al (2003) Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA). J Am Chem Soc 125:6878–6879PubMedCrossRefGoogle Scholar
  33. 33.
    Dragulescu-Andrasi A, Zhou P, He GF et al (2005). Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA. Chem Comm 244–246Google Scholar
  34. 34.
    Dragulescu-Andrasi A, Rapireddy S, He G et al (2006) Cell-permeable peptide nucleic acid designed to bind to the 5′-untraslated region of E-cadherin transcript induces potent and sequence-specific antisense effects. J Am Chem Soc 128:16104–16112PubMedCrossRefGoogle Scholar
  35. 35.
    Pianowski Z, Gorska K, Oswald L et al (2009) Imaging of mRNA in live cells using nucleic acid-templated reduction of azidothodamine probes. J Am Chem Soc 131:6492–6497PubMedCrossRefGoogle Scholar
  36. 36.
    Sahu B, Chenna V, Lathrop KL et al (2009) Synthesis of conformational preorganized and cell-permeable guanidine-based γ-peptide nucleic acid(γGPNAs). J Org Chem 74:1509–1516PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Sforza S, Tedeschi T, Calabretta A et al (2010) A peptide nucleic acid embedding a pseudo peptide nuclear localization sequence in the backbone behave as a peptide mimic. Eur J Org Chem 2441–2444Google Scholar
  38. 38.
    Falkiewicz B, Kolodziejczyk A, Wisniewsky K (2001) Synthesis of achiral and chiral peptide nucleic acid (PNA) monomers using Mitsunobu reaction. Tetrahedron 57:7909–7917Google Scholar
  39. 39.
    Sforza S, Tedeschi T, Corradini R et al (2003) Fast, solid-phase synthesis of chiral peptide nucleic acids with a high optical purity by a submonomeric strategy. Eur J Org Chem 1056–1063Google Scholar
  40. 40.
    Tedeschi T, Corradini R, Marchelli R et al (2002) Racemization of chiral PNAs during solid-phase synthesis: effect of the coupling conditions on enantiomeric purity. Tetrahedron Asymm 13:1629–1636CrossRefGoogle Scholar
  41. 41.
    Tedeschi T, Sforza S, Maffei F et al (2008) A Fmoc-based submonomeric strategy for the solid phase synthesis of optically pure chiral PNAs. Tetrahedron Lett 49:4958–4961CrossRefGoogle Scholar
  42. 42.
    Corradini R, Di Silvestro G, Sforza S et al (1999) Direct enantiomeric separation of N-aminoethyl amino acids: determination of the optical purity of chiral peptide nucleic acids (PNAs) by GC. Tetrahedron Asymm 10:2063–2066CrossRefGoogle Scholar
  43. 43.
    Thomson SA, Josey JA, Cadilla R et al (1995) Fmoc mediated synthesis of peptide nucleic-acids. Tetrahedron 51:6179–6194CrossRefGoogle Scholar
  44. 44.
    Nielsen PE (2004) Peptide nucleic acids: methods and protocols, vol 208, Method in molecular biology. Humana Press Inc., Towota, NJGoogle Scholar
  45. 45.
    Christensen L, Fitzpatrick R, Gildea B et al (1995) Solid-phase synthesis of peptide nucleic acids. J Pept Sci 1:175–183PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Roberto Corradini
    • 1
  • Tullia Tedeschi
    • 2
  • Stefano Sforza
    • 2
  • Rosangela Marchelli
    • 1
  1. 1.Department of ChemistryUniversity of ParmaParmaItaly
  2. 2.Department of Food ScienceUniversity of ParmaParmaItaly

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