Modification of DNA with Octadiynyl Side Chains: Synthesis, Base Pairing, and Formation of Fluorescent Coumarin Dye Conjugates of Four Nucleobases by the Alkyne−Azide “Click” Reaction

Abstract

Oligonucleotides incorporating 5-(octa-1,7-diynyl)-2'-deoxycytidine 1a, 5-(octa-1,7-diynyl)-2'-deoxyuridine 2a and 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyguanosine 3a, 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyadenosine 4a were prepared. For this, the phosphoramidites 7, 10, and 13 were synthesized and employed in solid-phase oligonucleotide synthesis. The octa-1,7-diynyl nucleosides 1a- 4a were obtained from their corresponding iodo derivatives using the palladium-assisted Sonogashira cross-coupling reaction. The Tm values demonstrated that DNA duplexes containing octa-1,7-diynyl nucleosides show a positive influence on the DNA duplex stability when they are introduced at the 5-position of pyrimidines or at the 7-position of 7-deazapurines. The terminal alkyne residue of oligonucleotides were selectively conjugated to the azide residue of the nonfluorescent 3-azido-7-hydroxycoumarin ( 38) using the protocol of copper(I)-catalyzed [3 + 2] Huisgen--Sharpless--Meldal cycloaddition "click chemistry" resulting in the formation of strongly fluorescent 1,2,3-triazole conjugates. The fluorescence properties of oligonucleotides with covalently linked coumarin--nucleobase assemblies were investigated. Among the four modified bases, the 7-deazapurines show stronger fluorescence quenching than that of pyrimidines.

Click chemistry with DNA

“…A pyrrolo dC analogue of deoxycytidine with a terminal alkyne was also prepared and incorporated into oligonucleotides. 26 The terminal alkyne residues of oligonucleotides bearing the above alkyne and octadiynyl groups 27 were selectively conjugated by the CuAAC reaction to the non-fluorescent 3-azido-7-hydroxycoumarin 4 to give strongly fluorescent 1,2,3-triazole conjugates (Scheme 1). The fluorescence properties of oligonucleotides with these covalently linked coumarin-nucleobases displayed the expected pH-dependence of fluorescence intensity.…”

“…The fluorescence properties of oligonucleotides with these covalently linked coumarin-nucleobases displayed the expected pH-dependence of fluorescence intensity. 27 The generation of fluorescence by the ''click'' reaction can be used for the visualization of DNA in free solution or embedded in DNA-protein complexes, and can also be used for the labelling and visualization of biomolecules in vivo.…”

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

“…If a copper catalyst were used then it is highly likely that only 1,4-substituted triazoles would be produced and that the reaction would proceed much faster. Seela et al took DNA labeling one step further and synthesized nucleosides that each contained a single terminal alkyne on their aromatic nucleobase (125). Modified deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and deoxythymidine (dT) were all included.…”

DNA with Branched Internal Side Chains: Synthesis of 5‐Tripropargylamine‐dU and Conjugation by an Azide‐Alkyne Double Click Reaction

“…The 5-substituted coumarin conjugate 24 has already been reported from our laboratory. [19] The formation of click products was confirmed by MALDI-TOF mass spectrometry as well as by enzymatic hydrolysis, which revealed the complete consumption of the starting oligonucleotides.…”

“…Fluorescent dye conjugates of all four DNA building blocks were prepared. [19,20] The side chains were located in the 7-positions of 7-deazapurines or the 5-positions of pyrimidines. This guarantees that bulky dye residues protrude into the major groove of B-DNA.…”