The 7-tripropargylamine-7-deaza-2'-deoxyguanosine (2) containing two terminal triple bonds in the side chain was synthesized by the Sonogashira cross-coupling reaction from the corresponding 7-iodo nucleoside 1b. This was protected at the 2-amino group with an iso-butyryl residue, affording the protected intermediate 5. Then, compound 5 was converted to the 5'-O-DMT derivative 6, which on phosphitylation afforded the phosphoramidite 7. This was employed in solid-phase synthesis of a series of oligonucleotides. T(m) measurements demonstrate that a covalently attached tripropargylamine side chain increases duplex stability. Both terminal triple bonds of nucleoside 2 and corresponding oligonucleotides were functionalized by the Cu(I)-mediated 1,3-dipolar cycloaddition "double click reaction" with 1-azidomethyl pyrene 3, decorating the side chain with two proximal pyrenes. While the monomeric tripropargylamine nucleoside with two proximal pyrenes (4) shows strong excimer fluorescence, the ss-oligonucleotide containing 4 does not. This was also observed for ds-oligonucleotides when the complementary strand was unmodified. However, duplex DNA bearing pyrene residues in both strands exhibits strong excimer fluorescence when each strand contains two pyrene residues linked to the tripropargylamine moiety. This pyrene-pyrene interstrand interaction occurs when the pyrene modification sites of the duplex are separated by two base pairs which bring the fluorescent dyes in a proximal position. Molecular modeling indicates that only two out of four pyrene residues are interacting forming the exciplex while the other two do not communicate.

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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.

A new, easy-to-prepare and highly selective pyrene-linked tris-triazole amine fluorescent chemosensor has been designed from tripropargylamine and pyrene azide using Cu(I)-catalyzed click chemistry. The fluorescence on-off sensor 1 is highly selective for Zn(2+) displaying a ratiometric change in emission. The relative intensity ratio of monomer to excimer fluorescence (M(376)/E(465)) of the sensor increases 80-fold upon the addition of 10 equiv of Zn(2+) ions (with a detection limit of 0.2 μM).

5-Aza-7-deaza-2'-deoxyguanosine (dZ) forms a silver-mediated base pair with dC. The metal ion pair represents a mimic of the H-bonded Watson-Crick dG-dC pair. The modified nucleoside displays a similar shape as the parent 2'-deoxyguanosine from which it can be constructed by transposition of nitrogen-7 to the bridgehead position-5. It lacks the major groove binding site as the positional change moves the dG- acceptor position from nitrogen-7 to nitrogen-1. As a shape mimic of dG, it fits nicely in the DNA double helix. The purine-pyrimidine dZ-dC hetero pair shows a relationship to the pyrimidine-pyrimidine dC-dC homo base pair. The dZ-dC pair forms a mismatch in the absence of silver ions and matches after addition of metal ions. Base-pair formation was verified on self-complementary 6-mer duplexes and 12-mer DNA with random composition by UV-dependent T measurements. Modified silver-mediated and hydrogen-bonded canonical base pairs can coexist. The dZ-Ag -dC base pair is slightly less stable than the dG-dC pair, shows sequence dependence, and consumes one or two silver ions. These properties make the dZ-Ag -dC pair suitable for programmable incorporation of silver ions in DNA which cannot be achieved by canonical base pairs. If the silver ion content is higher than the total number of base pairs the duplexes turn into very stable structures in which all base pairs are considered to be in the silver-mediated pairing mode.

8-Phenylimidazolo-dC ((ph) ImidC, 2) forms metal-mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent "purine" 2'-deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6. Owing to the ease of nucleobase deprotonation, the new Ag(+) -mediated base pair containing a "purine" skeleton is much stronger than that derived from the pyrrolo- [3,4-d]pyrimidine system ((ph) PyrdC, 1). The silver-mediated (ph) ImidC-(ph) ImidC base pair fits well into the DNA double helix and has the stability of a covalent cross-link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.