Modifed
oligonucleotides are routinely employed as analytical probes for use in diagnostics, e.g. in the examination of specific RNA sequences for
infectious diseases, however, a major limiting factor in
oligonucleotide-based diagnostics is poor cellular uptake of naked
oligonucleotides. This problem can be overcome by covalent attachment of a so-called '
cell-penetrating peptide' to form an
oligonucleotide peptide conjugate. Stepwise solid phase synthesis of such a conjugate is difficult and expensive due to the conflicting chemistries of
oligonucleotides and
peptides. A simple approach to overcome this is post-synthetic conjugation. Diels-Alder cycloaddition is an attractive methodology for
oligonucleotide peptide conjugation; the reaction is fast, chemoselective and the reaction rate is greatly enhanced in aqueous media - ideal conditions for
biological moieties. An
oligodeoxyribonucleotide sequence has been derivatised with a series of dienes at the 5'-terminus, using a series of unique dienyl-modified phosphoramidites, and investigation into the effect of diene type on the efficiency of conjugation, using Diels-Alder cycloaddition with a maleimido-derivatised cell-penetrating (TAT)
peptide, has been performed. This led to the observation that the optimal diene for conjugation was
cyclohexadiene, allowing conjugation of
oligodeoxyribonucleotides to a
cell-penetrating peptide by Diels-Alder cycloaddition for the first time.