Reactivity of PNA Thioesters in Chemical Ligation Reactions

Abstract

The ligation rate and efficiency of PNA thioesters in the reaction with the pentapeptide CRYNK has been determined and proved to be substantially lower in comparison with the corresponding C-terminal glycine thioester.

Key words

native chemical ligation - thioesters - peptide nucleic ­acid - reactivity - convergent synthesis

References

• 1 Dawson PE. Muir TW. Clark-Lewis I. Kent SBH. Science  1994,  266:  776 
• For leading references see:
• 2a Albericio F. Curr. Opin. Chem. Biol.  2004,  8:  211 
• 2b Muir TW. Annu. Rev. Biochem.  2003,  72:  249 
• 2c Casi G. Hilvert D. Curr. Opin. Struct. Biol.  2003,  13:  589 
• 2d Dawson PE. Kent SBH. Annu. Rev. Biochem.  2000,  69:  923 
• 3a McPherson M. Wright MC. Lohse PA. Synlett  1999,  978 
• 3b Stetsenko DA. Gait MJ. J. Org. Chem.  2000,  65:  4900 
• 3c Macmillan D. Bertozzi CR. Tetrahedron  2000,  56:  9515 
• 4a de Koning MC. Filippov DV. Meeuwenoord N. Overhand M. van der Marel GA. van Boom JH. Synlett  2001,  1516 
• 4b de Koning MC. Filippov DV. Meeuwenoord N. Overhand M. van der Marel GA. van Boom JH. Tetrahedron Lett.  2002,  43:  8173 
• 4c de Koning MC. Filippov DV. Overhand M. van der Marel GA. van Boom JH. Eur. J. Org. Chem.  2004,  850 
• 5a Nielsen PE. Egholm M. Berg RM. Buchardt O. Science  1991,  254:  1497 
• 5b Dueholm KL. Nielsen PE. New J. Chem.  1997,  21:  19 
• 5c Hyrup B. Nielsen PE. Bioorg. Med. Chem.  1996,  4:  5 
• 6a Cutrona G. Carpaneto EM. Ulivi M. Roncella S. Ladt M. Ferrarini M. Boffa LC. Nat. Biotech.  2000,  18:  300 
• 6b Good L. Awasthi SK. Dryselius R. Larsson O. Nielsen PE. Nat. Biotech.  2001,  19:  360 
• 6c de Koning MC. van der Marel GA. Overhand M. Curr. Opin. Chem. Biol.  2003,  7:  734 ; and references cited herein
• 7a Rapozzi V. Burm BEA. Cogoi S. van der Marel GA. van Boom JH. Quadrifoglio F. Xodo LE. Nucleic Acids Res.  2002,  30:  3712 
• 7b Cutrona G. Carpaneto EM. Ulivi M. Roncella S. Landt O. Ferrarini M. Boffa LC. Nat. Biotech.  2000,  18:  300 
• 7c Brandén LJ. Christensson B. Edvard Smith CI. Gene Ther.  2001,  8:  84 
• 8 Petersen L. de Koning MC. van Kuik-Romeijn P. Weterings J. Pol CJ. Platenburg G. Overhand M. van der Marel GA. van Boom JH. Bioconjugate Chem.  2004,  15:  576 
• 9 Hackeng TM. Griffin JH. Dawson PE. Proc. Natl. Acad. Sci. U. S. A.  1999,  96:  10068 
• 10 Apart from this, side-reactions were observed with the ligation of Lys-thioesters (see ref. 4c) and at Asp-Cys and Glu-Cys sites. See: Villain M. Gaertner H. Botti P. Eur. J. Org. Chem.  2003,  17:  3267 
• 11 Aimoto S. Biopolymers  1999,  51:  247 
• 12a Ellman GL. Arch. Biochem. Biophys.  1959,  82:  70 
• 12b Ellman GL. Biochem. Pharmacol.  1961,  7:  68 

13

MS: m/z found (calculated) for:
Compound 6: [M + H⁺]: 1340.9 (1340.5), [M + 2 H⁺]: 670.9 (670.8).
Compound 7: [M + H⁺]:1283.6 (1283.5), [M + 2 H⁺]: 642.6 (642.3).
Compound 8: [M + H⁺]: 1308.5 (1308.3), [M + 2 H⁺]: 655.2 (654.7).
Compound 10: [M + H⁺]: 1847.1 (1846.2), [M + 2 H⁺]: 923.9 (923.6), [M + 3 H⁺]: 616.2 (616.1).
Compound 11: [M + H⁺]: 1788.9 (1788.8), [M + 2 H⁺]: 895.2 (894.9).
Compound 12: [M + H⁺]: 1815.2 (1814.8), [M + 2 H⁺]: 907.8 (907.9), [M + 3 H⁺]: 605.7 (605.6).

14

On the basis of similar studies to assess the ligation efficiency of amino acid thioesters (see ref. 9) the pentapeptide CRANK was initially used. However, it proved to be impossible to resolve the PNA-conjugate of this peptide and the starting PNA thioester on RP-HPLC column.