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.