A Novel Approach to the Preparation of Peptide-Oligonucleotide Conjugates

Abstract

A novel approach to the synthesis of peptide-oligonucleotide conjugates (POC) based on the oxathiaphospholane chemistry has been developed. Peptide and oligonucleotide fragments, which were separately prepared, were linked postsynthetically by the functionalization of the peptide either by attachment of the oxathiaphospholane residue directly at the N-terminus or at the hydroxy group of the linker connected to N-terminus. The conjugation reaction, based on DBU-assisted nucleophilic attack of hydroxy group of oligonucleotide on phosphorus atom in the oxathiaphospholane derivatives of peptide, furnished the desired POC.

Key words

conjugation - peptides - oligonucleotides - phosphorylations - solid-phase synthesis

References and Notes

• 1 Zamecnik PC. Stephenson ML. Proc. Natl. Acad. Sci. U.S.A.  1978,  75:  280 
• 2 Stein CA. Narayan R. Perspect. Drug Discovery Des.  1996,  4:  41 
• 3 Lebedeva I. Benimetskaya L. Stein CA. Vilenchik M. Eur. J. Pharmacol. Biopharm.  2000,  50:  101 
• 4a Gait MJ. Cell. Mol. Life Sci.  2003,  60:  844 
• 4b Dokka S. Toledo-Velasquez D. Shi X. Wang L. Rojanasukul Y. Pharm. Res.  1997,  14:  1759 
• 5a Seksek O. Bolard J. Methods Mol. Biol.  2004,  252:  545 
• 5b Fischer PM. Krausz E. Lane DP. Bioconjugate Chem.  2001,  12:  825 
• 6 Tung ChH. Stein S. Bioconjugate Chem.  2000,  11:  605 
• 7 Venkatesan N. Kim BH. Chem. Rev.  2006,  106:  3712 
• 8a Eritja R. In Solid-Phase Synthesis   Kates S. A., Albericio F., Marcel Dekker; New York: 2000.  p.529-548  
• 8b Virta P. Katajisto J. Niittymaki T. Lonnberg H. Tetrahedron  2003,  59:  5137 
• 8c Stetsenko DA. Gait MJ. Methods Mol. Biol.  2005,  288:  205 
• 9 Grandas A. Robles J. Pedroso E. Nucleosides Nucleotides  1995,  14:  825 
• 10 Mironova NL. Pyshnyi DV. Ivanova EM. Nucleic Acids Mol. Biol.  2004,  13:  151 
• 11a Stec WJ. Grajkowski A. Karwowski B. Kobylańska A. Koziożkiewicz M. Misiura K. Okruszek A. Wilk A. Guga P. Boczkowska M. J. Am. Chem. Soc.  1995,  117:  12019 
• 11b Guga P. Stec WJ. In Current Protocols in Nucleic Acid Chemistry   Vol. 4:  Beaucage SL. Bergstrom DE. Glick GD. Jones RA. Wiley and Sons; New York: 2003.  p.1-28  
• 12 Baraniak J. Wasilewska E. Korczyński D. Stec WJ. Tetrahedron Lett.  1999,  40:  8603 
• 13 Baraniak J. Kaczmarek R. Korczyński D. Wasilewska E. J. Org. Chem.  2002,  67:  7267 
• 14a Baraniak J. Kaczmarek R. Stec WJ. Tetrahedron Lett.  2000,  41:  9139 
• 14b Baraniak J. Kaczmarek R. Wasilewska E. Korczyński D. Stec WJ. Tetrahedron Lett.  2004,  45:  4269 
• 15 Misiura K. Szymanowicz D. Olesiak M. Stec WJ. Tetrahedron Lett.  2004,  45:  4301 
• 19 Haralambidis J. Duncan L. Tregear GW. Tetrahedron Lett.  1987,  28:  5199 

16

Reaction Conditions: The synthesis of resin bound tetrathymidine phosphorothioates (4) was carried out manually by the syringe technique on 1 µmol scale and after oligonucleotide-chain assembly the coupling reaction was performed using 20-fold molar excess of 3 and 50-fold molar excess of DBU, coupling time 15 min.

17

Compound 3 underwent cyclization immediately into 5 in the presence of 1.2 equiv of DBU in CH₂Cl₂ solution. Data for 5: ^³¹P NMR (81 MHz, CD₃CN): δ = 60.70, 53.30 ppm. MS-FAB: m/z = 322 [M - 1]. Yield 79%.

18

HPLC analysis were performed using Econosil C-18 column (4.6 × 250 mm), 1.0 mL/min flow rate; buffer A, 0.1 M TEAB (pH 7.5); buffer B, 40% MeCN in 0.1 M TEAB; gradient from 0% → 60% B over 30 min, 60% over 5 min, 60% → 0% over 5 min. Yield was calculated based on A₂₆₀ units of starting crude oligonucleotide and conjugate.