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Preparation of Metal-Containing Peptide Nucleic Acid Bioconjugates on the Solid Phase

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Peptide Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1050))

Abstract

Peptide nucleic acids (PNAs) are a class of artificial DNA/RNA analogues that have unique physicochemical properties, which include a high chemical stability, resistance to nucleases and proteases, and higher mismatch sensitivity than DNA. PNAs were initially anticipated to be useful for application in antisense and antigene therapies; however, their poor cellular uptake has limited their use for such purposes in the “real world.” Recently, it has been shown that the addition of metal complexes to these oligonucleotide analogues could open up new avenues for their utilization in various research fields. Such metallo-constructs have shown great promise, for a diverse range of applications, most notably in the biosensing area. In this book chapter, we report on the recent synthetic advances towards the preparation of these “(multi-)metallic PNAs” on the solid phase.

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Abbreviations

A:

Adenine

Bhoc:

Benzhydryloxycarbonyl

Boc:

Tert-butyloxycarbonyl

Bpy:

2,2′-bipyridine

C:

Cytosine

Cpp-L-PNA-OH:

[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine

DIC:

N,N′-diisopropyl carbodiimide

DhBTOH:

3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

DIPEA:

Diisopropylethylamine

DMF:

Dimethylformamide

Fc:

Ferrocene

Fmoc:

Fluorenylmethoxycarbonyl

G:

Guanine

Gly:

Glycine

HATU:

2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate

HPLC:

High-performance liquid chromatography

Lys:

Lysine

MALDI-TOF:

Matrix-assisted laser/desorption ionization-time of flight

Mtt:

4-methyltrityl

PNA:

Peptide nucleic acid

T:

Thymine

TBTU:

O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

TFA:

Trifluoroacetic acid

TIS:

Triisopropylsilane

References

  1. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500

    Article  PubMed  CAS  Google Scholar 

  2. Jensen KK, Orum H, Nielsen PE, Norden B (1997) Kinetics for hybridisaton of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry 36:5072–5077

    Article  PubMed  CAS  Google Scholar 

  3. Wittung P, Nielsen PE, Buchardt O, Egholm M, Norden B (1994) DNA-like double helix formed by peptide nucleic acid. Nature 368:561–563

    Article  PubMed  CAS  Google Scholar 

  4. Gasser G, Sosniak AM, Metzler-Nolte N (2011) Metal-containing peptide nucleic acid conjugates. Dalton Trans 40:7061–7076

    Article  PubMed  CAS  Google Scholar 

  5. Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365:566–568

    Article  PubMed  CAS  Google Scholar 

  6. Nielsen PE (1999) Peptide nucleic acids as therapeutic agents. Curr Opin Struct Biol 9:353–357

    Article  PubMed  CAS  Google Scholar 

  7. Nielsen PE (2010) Targeted gene repair facilitated by peptide nucleic acids (PNA). ChemBioChem 11:2073–2076

    Article  PubMed  CAS  Google Scholar 

  8. Bendifallah N, Winther Rasmussen F, 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 17:750–758

    Article  CAS  Google Scholar 

  9. Koppelhus U, Nielsen PE (2003) Cellular delivery of peptide nucleic acid (PNA). Adv Drug Deliv Rev 55:267–280

    Article  PubMed  CAS  Google Scholar 

  10. Whitney A, Gavory G, Balasubramanian S (2003) Site-specific cleavage of human telomerase RNA using PNA-neocuproine.Zn(II) derivatives. Chem Commun 36–37

    Google Scholar 

  11. Watson RM, Skorik YA, Patra GK, Achim C (2005) Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes. J Am Chem Soc 127:14628–14639

    Article  PubMed  CAS  Google Scholar 

  12. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ (1997) In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA). J Nucl Med 38:907–913

    PubMed  CAS  Google Scholar 

  13. Lewis MR, Jia F, Gallazzi F, Wang Y, Zhang J, Shenoy N, Lever SZ, Hannink M (2002) Radiometal-labeled peptide-PNA conjugates for targeting bcl-2 expression: preparation, characterization, and in vitro mRNA binding. Bioconjugate Chem 13:1176–1180

    Article  CAS  Google Scholar 

  14. Metzler-Nolte N (2010) Biomedical applications of organometal-peptide conjugates. In: Jaouen G, Metzler-Nolte N (eds) Medicinal organometallic chemistry. Springer, Heidelberg, pp 195–217

    Chapter  Google Scholar 

  15. Kirin SI, Noor F, Metzler-Nolte N, Mier W (2007) Manual solid phase peptide synthesis of metallocene-peptide bioconjugates. J Chem Educ 84:108–111

    Article  CAS  Google Scholar 

  16. Metzler-Nolte N (2006) Conjugates of peptides and PNA with organometallic complexes: syntheses and applications. In: Jaouen G (ed) Bioorganometallics: biomolecules, labeling, medicine. Wiley-VCH, Weinheim, pp 125–179

    Chapter  Google Scholar 

  17. Dirscherl G, König B (2008) The use of solid-phase synthesis techniques for the preparation of peptide-metal complex conjugates. Eur J Org Chem 597–634

    Google Scholar 

  18. Heinze K, Beckmann M, Hempel K (2008) Solid-phase synthesis of transition metal complexes. Chem Eur J 14:9468–9480

    Article  PubMed  CAS  Google Scholar 

  19. Dueholm KL, Egholm M, Behrens C, Christensen L, Hansen HF, Vulpius T, Pettersen EF, Berg RH, Nielsen PE, Buchardt O (1994) Synthesis of peptide nucleic acid monomers containing the four natural nucleobases: thymine, cytosine, adenine, and guanine and their oligomerization. J Org Chem 59:5767–5773

    Article  CAS  Google Scholar 

  20. Thomson SA, Josey JA, Cadilla R, Gaul MD, Hassman CF, Luzzio MJ, Pipe AJ, Reed KL, Ricca DJ, Wiethe RW, Noble SA (1995) Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron 51:6179–6194

    Article  CAS  Google Scholar 

  21. Amant AHS, Hudson RHE (2012) Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine. Org Biomol Chem. doi:10:876-881

    Google Scholar 

  22. Hyrup B, Nielsen PE (1996) Peptide nucleic acids (PNA): synthesis, properties and potential applications. Bioorg Med Chem 4:5–23

    Article  PubMed  CAS  Google Scholar 

  23. Amblard M, Fehrentz J-A, Martinez J, Subra G (2005) Peptide synthesis and applications. In: Howl J (ed) Methods in molecular biology. Humana Press Inc., Totowa, NJ, pp 3–24

    Google Scholar 

  24. Verheijen JC, van der Marel GA, van Boom JH, Metzler-Nolte N (2000) Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding. Bioconjuguate Chem 11:741–743

    Article  CAS  Google Scholar 

  25. Kersebohm T, Kirin SI, Metzler-Nolte N (2006) Insertion of an internal dipeptide into PNA oligomers: thermal melting studies and further functionalization. Bioorg Med Chem Lett 16:2964–2968

    Article  PubMed  CAS  Google Scholar 

  26. Sun X, Fang H, Li X, Rossin R, Welch MJ, Taylor J-S (2005) MicroPET imaging of MCF-7 tumors in mice via unr mRNA-targeted peptide nucleic acids. Bioconjugate Chem 16:294–305

    Article  CAS  Google Scholar 

  27. Gasser G, Hüsken N, Köster SD, Metzler-Nolte N (2008) Synthesis of organometallic PNA oligomers by click chemistry. Chem Commun 3675–3677

    Google Scholar 

  28. Gasser G, Jäger K, Zenker M, Bergmann R, Steinbach J, Stephan H, Metzler-Nolte N (2010) Preparation, 99mTc-Labeling and Biodistribution Studies of a PNA Oligomer Containing a New Ligand Derivative of 2,2′-Dipicolylamine. J Inorg Biochem 104:1133–1140

    Article  PubMed  CAS  Google Scholar 

  29. Gasser G, Neumann S, Ott I, Seitz M, Heumann R, Metzler-Nolte N (2011) Preparation and biological evaluation of di-hetero organometallic-containing pna bioconjugates. Eur J Inorg Chem 5471–5478

    Google Scholar 

  30. Gasser G, Pinto A, Neumann S, Sosniak AM, Seitz M, Merz K, Heumann R, Metzler-Nolte N (2012) Synthesis, characterisation and bioimaging of a fluorescent rhenium-containing PNA bioconjugate. Dalton Trans 41:2304–2313

    Article  PubMed  CAS  Google Scholar 

  31. Gasser G, Sosniak AM, Leonidova A, Braband H, Metzler-Nolte N (2011) Towards the preparation of novel Re/99mTc tricarbonyl-containing peptide nucleic acid bioconjugates. Aust J Chem 64:265–272

    Article  CAS  Google Scholar 

  32. Hüsken N, Gasser G, Köster SD, Metzler-Nolte N (2009) “Four-potential” ferrocene labelling of PNA oligomers via click chemistry. Bioconjugate Chem 20:1578–1586

    Article  CAS  Google Scholar 

  33. Patra M, Gasser G, Bobukhov D, Merz K, Shtemenko AV, Metzler-Nolte N (2010) Sequencial insertion of three different organometallics into a versatile building block containing a PNA backbone. Dalton Trans 39:5617–5619

    Article  PubMed  CAS  Google Scholar 

  34. Sosniak A, Gasser G, Metzler-Nolte N (2009) Thermal melting studies of modified PNA oligomers. Org Biomol Chem 7:4992–5000

    Article  PubMed  CAS  Google Scholar 

  35. Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed 40:2004–2021

    Article  CAS  Google Scholar 

  36. Tørnoe CW, Christensen C, Meldal L (2002) J Org Chem 67:3057–3064

    Article  PubMed  CAS  Google Scholar 

  37. Gasser G, Neukamm MA, Ewers A, Brosch O, Weyhermüller T, Metzler-Nolte N (2009) Synthesis and characterisation of dicobalthexacarbonyl-alkyne amino acid, peptide and PNA monomer derivatives. Inorg Chem 48:3157–3166

    Article  PubMed  CAS  Google Scholar 

  38. Brunner J, Barton JK (2006) Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide. Biochemistry 45:12295–12302

    Article  PubMed  CAS  Google Scholar 

  39. Joshi T, Barbante GJ, Francis PS, Hogan CF, Bond AM, Gasser G, Spiccia L (2012) Electrochemiluminescent monomers for solid support syntheses of Ru(II) PNA bioconjugates - multimodal biosensing tools with enhanced duplex stability. Inorg Chem 51:3302–3315

    Article  PubMed  CAS  Google Scholar 

  40. Joshi T, Barbante GJ, Francis PS, Hogan CF, Bond AM, Spiccia L (2011) Electrochemiluminescent PNA like monomers containing Ru(II)-dipyridoquinoxaline and Ru(II)-dipyridophenazine complexes. Inorg Chem 50:12172–12183

    Article  PubMed  CAS  Google Scholar 

  41. Joshi T, Gasser G, Martin LL, Spiccia L (2012) Specific uptake and interactions of peptide nucleic acid derivatives with biomimetic membranes. RSC Adv 2:4703–4712

    Article  CAS  Google Scholar 

  42. Baldoli C, Cerea P, Giannini C, Licandro E, Rigamonti C, Maiorana S (2005) The metal-conjugated peptide nucleic acid challenge. Synlett 13:1984–1994

    Google Scholar 

  43. Hudson RHE, Li G, Tse J (2002) The use of Sonogashira coupling for the synthesis of modified uracil peptide nucleic acid. Tetrahedron Lett 43:1381–1386

    Article  CAS  Google Scholar 

  44. Gasser G, Belousoff MJ, Bond AM, Spiccia L (2006) Facile synthesis and detailed characterization of a new ferrocenyl uracil peptide nucleic acid monomer. J Org Chem 71:7565–7573

    Article  PubMed  CAS  Google Scholar 

  45. Gasser G, Spiccia L (2008) Synthesis of a ferrocenyl uracil PNA monomer for insertion into PNA sequences. J Organomet Chem 693:2478–2482

    Article  CAS  Google Scholar 

  46. Baldoli C, Maiorana S, Licandro E, Zinzalla G, Perdicchia D (2002) Synthesis of chiral chromium tricarbonyl labeled thymine PNA monomers via the Ugi reaction. Org Lett 4:4341–4344

    Article  PubMed  CAS  Google Scholar 

  47. Baldoli C, Falciola L, Licandro E, Maiorana S, Mussini P, Ramani P, Rigamonti C, Zinzalla G (2004) A new ferrocene conjugate of a tyrosine PNA monomer: synthesis and electrochemical properties. J Organomet Chem 689:4791–4802

    Article  CAS  Google Scholar 

  48. Baldoli C, Licandro E, Maiorana S, Resemini D, Rigamonti C, Falciola L, Longhi M, Mussini PR (2005) Electrochemical activity of new ferrocene-labelled PNA monomers to be applied for DNA detection: effects of the molecular structure and of the solvent. J Electroanal Chem 585:197–205

    Article  CAS  Google Scholar 

  49. Baldoli C, Rigamonti C, Maiorana S, Licandro E, Falciola L, Mussini PR (2006) A new triferrocenyl-tris(hydroxymethyl)aminomethane derivative as a highly sensitive electrochemical marker of biomolecules: application to the labelling of PNA monomers and their electrochemical characterization. Chem Eur J 12:4091–4100

    Article  PubMed  CAS  Google Scholar 

  50. Kaiser E, Colescot RL, Bossinge CD, Cook PI (1970) Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal Biochem 34:595–598

    Article  PubMed  CAS  Google Scholar 

  51. Hüsken N, Gębala M, La Mantia F, Schuhmann W, Metzler-Nolte N (2011) Mechanistic studies of Fc-PNA(⋅DNA) surface dynamics based on the kinetics of electron-transfer processes. Chem Eur J 17:9678–9690

    Article  PubMed  CAS  Google Scholar 

  52. Hüsken N, Gebala M, Schuhmann W, Metzler-Nolte N (2010) A single-electrode, dual-potential ferrocene-PNA biosensor for the detection of DNA. ChemBioChem 11:1754–1761

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The Swiss National Science Foundation and the University of Zurich are gratefully acknowledged for their generous financial support (Professorship No. PP00P2_133568 and Research Grant No. 200021_129910). I would also like to thank my previous supervisors and advisors Prof. Leone Spiccia and Prof. Nils Metzler-Nolte, who have initiated me into the wonderful world of (metal-containing) PNAs. Anna Leonidova, Cristina Mari, Anna M. Sosniak, and Dr. Tanmaya Joshi are acknowledged for carefully proofreading this manuscript.

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Authors and Affiliations

  1. Institute of Inorganic Chemistry, University of Zurich, Zurich, Switzerland

    Gilles Gasser

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  1. Gilles Gasser
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Editors and Affiliations

  1. Dept. Cellular & Molecular Medicine, University of Copenhagen Faculty of Health Sciences, Copenhagen N, Denmark

    Peter E Nielsen

  2. NIH NIDDK, Bethesda, USA

    Daniel H. Appella

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Gasser, G. (2014). Preparation of Metal-Containing Peptide Nucleic Acid Bioconjugates on the Solid Phase. In: Nielsen, P., Appella, D. (eds) Peptide Nucleic Acids. Methods in Molecular Biology, vol 1050. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-553-8_5

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  • DOI: https://doi.org/10.1007/978-1-62703-553-8_5

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-552-1

  • Online ISBN: 978-1-62703-553-8

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