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

Abstract

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') (tc(ee)-T and tc(hd)-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 ΔT(m)/mod of +0.4 to +2.0 °C. The only destabilizing residue was tc(hd)-T, most likely due to self-aggregation of the lipophilic side chains in the single stranded oligonucleotide. A decamer containing five tc(hd)-T residues was readily taken up by HeLa and HEK 293T cells without the use of a transfection agent.

Synthesis and Properties of 6′-Fluoro-tricyclo-DNA

“…-selective nucleosidation, 24,25 compound 5 was converted to enolether 6 with TMSOTf, which was then reacted with in situ persilylated thymine and NIS, yielding iodo-nucleoside 7 in 85% yield in a stereospecific manner. Removal of the iodine via radical reduction with Bu3SnH finally gave the expected O-protected 6'-fluoro tricyclothymidine 8 in excellent yields.…”

“…21 At the same time it is in agreement with the properties of other 6'-modified tc-DNA derivatives for which it was shown before that this position can be chemically modified without compromising RNA affinity. 24 In the C-series, the 5-methyl group of cytosine brings about 0.2-1.2 °C/mod of additional thermal stability also in the context of the tricyclic nucleoside structure. As for the case of 5-methyldeoxycytidine in DNA duplexes, this is most likely the consequence of improved stacking interactions and/or improved hydrogen bonding induced by the molecular polarizability of the size extended base.…”

Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

“…The main bottleneck of their use, as for many other modified ONs, is their poor cellular uptake. Therefore, to address this issue, Leumann et al synthesized “pro-tricyclo-ONs” bearing two different metabolically labile ethyl and hexadecyl esters at position C6’ that were expected to promote cell penetration ( Scheme 12 ) [ 56 ]. It was shown that the cellular uptake of a decamer containing five tc hd -T units with a C 16 side chain was increased in two different cell lines (HeLa and HEK 293T) without using a transfection agent.…”

“… Prodrugs of tricyclo-ONs functionalized with A) ethyl (tc ee -T) and B) hexadecyl (tc hd -T) ester functions at C6 obtained from corresponding thymidine phosphoramidites [ 56 ]. …”

Nuclease resistant oligonucleotides with cell penetrating properties

“…18,19 In that a number of positive charges are included in the structure, one could consider AECM oligonucleotides to somewhat (crudely) resemble an oligonucleotide-cell penetrating peptide (CPP) complex. 20 Since CPPs can enhance oligonucleotide uptake 21 it seemed that it would be interesting to see how a fully AECM modified oligonucleotide behaves with respect to cellular uptake.…”