Oligonucleotides incorporating 7-(octa-1,7-diynyl) derivatives of 8-aza-7-deaza-2-deoxyguanosine (2d) were prepared by solid-phase synthesis. The side chain of 2d was introduced by the Sonogashira cross coupling reaction and phosphoramidites (3a, 3b) were synthesized. Duplexes containing 2d are more stabilized compared to those incorporating the non-functionalized 8-aza-7-deaza-2-deoxyguanosine (2a) demonstrating that these side chains have steric freedom in duplex DNA. Nucleoside 2d as well as 2d-containing oligonucleotides were conjugated to the non-fluorescent 3-azido-7-hydroxycoumarin 15 by the Huisgen-Meldal-Sharpless click reaction. Pyrazolo[3,4-d]pyrimidine nucleoside conjugate 16 shows a much higher fluorescence intensity than that of the corresponding pyrrolo[2,3-d]pyrimidine derivative 17. The quenching in the dye conjugate 17 was found to be stronger on the stage of monomeric conjugates than in single-stranded or duplex DNA. Nucleobase-dye contact complexes are suggested which are more favourable in the monomeric state than in the DNA chain when the nucleobase is part of the stack. The side chains with the bulky dye conjugates are well accommodated in DNA duplexes thereby forming hybrids which are slightly more stable than canonical DNA.

Clickable oligonucleotides with ethynyl residues in the 5-position of pyrimidines ((eth)dC and (eth)dU) or the 7-position of 7-deazaguanine ((eth)c(7)G(d)) are hydrated during solid-phase oligonucleotide synthesis and workup conditions. The side products were identified as acetyl derivatives by MALDI-TOF mass spectra of oligonucleotides and by detection of modified nucleosides after enzymatic phosphodiester hydrolysis. Ethynyl → acetyl group conversion was also studied on ethynylated nucleosides under acidic and basic conditions. It could be shown that side chain conversion depends on the nucleobase structure. Triisopropylsilyl residues were introduced to protect ethynyl residues from hydration. Pure, acetyl group free oligonucleotides were isolated after desilylation in all cases.

Duplex DNA with terminal and internal sugar cross-links were synthesized by the CuAAC reaction from oligonucleotides containing 2'-O-propargyl-2-aminoadenosine as a clickable site and a bifunctional azide (4). Stepwise click chemistry was employed to introduce cross-links at internal and terminal positions. Copper turnings were used as catalyst, reducing the copper load of the reaction mixture and avoiding complexing agents. For oligonucleotide building block synthesis, a protecting group strategy was developed for 2'-O-propargyl-2-aminoadenosine owing to the rather different reactivities of the two amino groups. Phosphoramidites were synthesized bearing clickable 2'-O-propargyl residues (14 and 18) as well as a 2'-deoxyribofuranosyl residue (10). Hybridization experiments of non-cross-linked oligonucleotides with 2,6-diaminopurine as nucleobase showed no significant thermal stability changes over those containing adenine. Surprisingly, an isobutyryl group protecting the 2-amino function has no negative impact on the stability of DNA-DNA and DNA-RNA duplexes. Oligonucleotide duplexes with cross-linked 2'-O-propargylated 2-aminoadenosine (1) and 2'-O-propargylated adenosine (3) at terminal positions are significantly stabilized (ΔT(m) = +29 °C). The stability results from a molecularity change from duplex to hairpin melting and is influenced by the ligation position. Terminal ligation led to higher melting duplexes than corresponding hairpins, while duplexes with central ligation sites were less stable.