Diels-Alder bioconjugation of diene-modified oligonucleotides

In an effort to offer complementary technology for covalent biomolecule mod ification (bioconjugation), we have developed a method that exploits the aq ueous acceleration of Diels-Alder reactions for this purpose. Three differe nt diene phosphoramidite reagents have been synthesized that enable diene m odification of synthetic oligonucleotides prepared by the phosphoramidite m ethod. Clean and efficient Diels-Alder cycloaddition of these diene oligonu cleotides with maleimide dieneophiles was carried out, and the labeled olig onucleotide bioconjugates were characterized by HPLC and electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parame ters that were shown to influence the efficiency of the process.