Synthesis, Pairing, and Cellular Uptake Properties of C(6′)-Functionalized Tricyclo-DNA

Tricyclo-DNA (tc-DNA) is a promising candidate for oligonucleotide-based
therapeutic applications exhibiting increased affinity to RNA and
increased resistance to nucleases. However, as many other oligonucleotide
analogs, tc-DNA does not readily cross cell membranes. We wished to
address this issue by preparing a prodrug of tc-DNA containing a metabolically
labile group at C(6′) that promotes cellular uptake. Two monomeric
nucleoside building blocks bearing an ester function at C(6′)
(tcee-T and tchd-T) were synthesized starting
from a known C(6′) functionalized bicyclic sugar unit to which
the cyclopropane ring was introduced via carbene addition. NIS-mediated
nucleosidation of the corresponding glycal with in situ persilylated
thymine afforded the β-iodonucleoside exclusively that was dehalogenated
via radical reduction. Diversity in the ester function was obtained
by hydrolysis and reesterification. The two nucleosides were subsequently
incorporated into DNA or tc-DNA by standard phosphoramidite chemistry.
The reactivity of the ester function during oligonucleotide deprotection
was explored and the corresponding C(6′) amide, carboxylic
acid, or unchanged ester functions were obtained, depending on the
deprotection conditions. Compared to unmodified DNA, these tc-DNA
derivatives increased the stability of duplexes investigated with
ΔTm/mod of +0.4 to +2.0 °C.
The only destabilizing residue was tchd-T, most likely
due to self-aggregation of the lipophilic side chains in the single
stranded oligonucleotide. A decamer containing five tchd-T residues was readily taken up by HeLa and HEK 293T cells without
the use of a transfection agent.