Pd-catalysed synthesis of functionalised adenosine analogues and their characterisation as base-discriminating fluorescent probes

A novel class of 7-modified 7-deazaadenosine nucleosides has been synthesised containing diarylacetylene groups at C7. Diarylacetylene boronate esters were synthesised by chemoselective Sonogashira cross-coupling. The boronate esters were then coupled to 7-iodo-7-deazaadenine nucleosides using aqueous Suzuki-Miyaura cross-coupling in good to excellent yields (68%->99%). The modified nucleosides showed very promising UV-vis absorption and fluorescence emission properties in DMSO, with high quantum yields and UV absorbance maxima bathochromically shifted with respect to intrinsic biomolecular absorption bands. In water, the parent compound 20a had a decreased quantum yield (0.15) and the absorbance maximum occurred at a lower wavelength (302 nm). The protected phosphoramidite of 20a was synthesised, and the fluorescent nucleoside was incorporated at a central position in a series of 13-base deoxyoligonucleotides, covering a range of neighbouring DNA sequence contexts. The oligonucleotides had absorbance maxima similar to the nucleoside in DMSO (ca. 322 nm), and quantum yields comparable to the nucleoside in water (0.024-0.237). Melting curve analysis of the oligonucleotide duplexes showed some destabilisation due to the fluorescent substituent, but less than expected for a completely mismatched base pair. Circular dichroism spectra highlighted the range of ordered, helix-like structures present in the single-strand oligonucleotides, and confirmed that the duplexes formed a B-type DNA helix. An investigation of base-pair mismatch discrimination showed a clear fluorescence quenching effect for c7A∙C mismatch pairs. Other mismatches did not show discriminatory fluorescence quenching or enhancement. Investigation of the pH dependence of the oligonucleotide fluorescence emission showed high sensitivity to changes in pH, with acidic conditions causing significant quenching. The evidence strongly suggests that mismatched base-pair discrimination is due to formation of a protonated wobble pairing between the 7-deazaadenosine and cytidine, as previously proposed by Seela and co-workers. This base-pair mismatch causes an increase in the pKa of the 7-deazaadenosine heterocycle, so the mismatch acts as a template for protonation. Potential applications for the 7-modified 7-deazaadenosine nucleoside analogues are discussed.