Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

Abstract

The azides 1 and 2 bearing a phenoxazinium and a coumarin fluorophore, respectively, were applied in postsynthetic "click"-type bioconjugation and coupled to oligonucleotides modified with alkyne groups using two alternative approaches: (i) as a nucleotide modification at the 2′-position of uridine and (ii) as a nucleotide substitution using (S)-(-)-3-amino-1,2-propanediol as an acyclic linker between the phosphodiester bridges. The corresponding alkynylated phosporamidites 3 and 6 were used as DNA building blocks for the preparation of alkyne-bearing DNA duplexes. The base pairs adjacent to the site of modification and the base opposite to it were varied in the DNA sequences. The modified duplexes were investigated by UV/vis absorption spectroscopy (including melting temperatures) and fluorescence spectroscopy in order to study the different optical properties of the two chromophores and to evaluate their potential for bioanalytical applications. The sequence-selective fluorescence quenching of phenoxazinium 1 differs only slightly and does not depend on the type of modification, meaning whether it has been attached to the 2′-position of uridine or as DNA base surrogate using the acyclic glycol linker. The 2′-chromophore-modified uridine still recognizes adenine as the counterbase, and the duplexes exhibit a sufficient thermal stability that is comparable to that of unmodified duplexes. Thus, the application of the 2′-modification site of uridine is preferred in comparison to glycol-assisted DNA base surrogates. Accordingly, the coumarin dye azide 2 was attached only to the 2′-position of uridine. The significant Stokes shift of ∼100 nm and the good quantum yields make the coumarin chromophore a powerful fluorescent label for nucleic acids.

Click chemistry with DNA

“…A suitable position for label attachment to DNA is the 2-position of the ribose sugar as in compound 19 (Scheme 3), as substitution here does not affect duplex stability greatly. Wagenknecht et al have used this strategy 31 to incorporate alkyne functions into oligonucleotides and label them with dye azides to give oligonucleotides 20 (blue) and 21 (red).…”

Synthesis and Optical Properties of Cyanine Dyes as Fluorescent DNA Base Substitutions for Live Cell Imaging

“…As previously, (S)-1-aminopropane-2,3-diol serves as an acyclic linker between the phosphodiesters. Similar propanediol linkers have been used by others in order to prepare glycol nucleic acids (GNA), [35] twisted intercalating nucleic acids (TINA), [36] alkyne-modified oligonucleotides for the click-type postsynthetic modification, [37][38][39] and by our group for fluorescent DNA base substitutions by ethidium, [40] indole, [41] perylene bisimide, [20,21] phenothiazine [42] as DNA base surrogates. Avoiding the acid/base labile glycosidic bond of natural nucleosides, [43] the application of a flexible acyclic linker provides a high chemical stability during the preparation of chromophore-DNA conjugates via automated phosphoramidite chemistry, and allows the chromophore to intercalate.…”

Significantly Enhanced DNA Thermal Stability Resulting from Porphyrin H‐Aggregate Formation in the Minor Groove of the Duplex

“…[21] This work uses a click chemistry approach to combine DNAs containing commercially available 2'-O-propargyl uridine or 2'-O-propargyl adenosine [22] with methylazidostyryl-substituted porphyrins; this allows us to place functional entities in the minor groove of the DNA duplex. [23] In addition, contrary to the common functionalization of the porphyrin through the meso positions [6-8, 10, 19] that results in a system orthogonal to the porphyrin core, we used a porphyrin modified through the b-pyrrolic position that provides a planar system between the porphyrin core and the adjacent benzene ring (Scheme 1).…”