Conjugates of Uridine and GFP-like Chromophore for Detection of Genetic Material

We recently showed that dU^TO, 1, in an oligonucleotide probe targeting cyclin D1 mRNA (a breast cancer marker), in total RNA extract from cancerous cells, allowed the sensitive detection of the target based on a 7-fold enhanced fluorescence of the probe.¹ Yet, a useful fluorescent probe for living cell imaging has to have an excellent signal/background ratio. Therefore, here, we aimed to prepare single stranded 2’-OMe-RNA probes which covalently bind, via a suitable spacer, a duplex-intercalating moiety which has an extremely low Φ in water, and which upon probe hybridization with target mRNA will exhibit significant Φ and brightness. Specifically, we developed novel uridine analogues bearing a GFP-like chromophore as an intercalator, 2, for incorporation into an oligonucleotide probe, to be used for the detection of breast cancer markers, HER-2. We successfully synthesized analogues of the GFP-chromophore, 2 and then conjugated them to uridine via a 4-atom nitrogen-containing spacer to get compound 3. Nucleotide conjugate 3 was prepared from 5-(3″-Aminoallyl)-2′-deoxyuridine, which in turn was obtained by Heck reaction between 5-iodo-2`-deoxyuridine and N-allyltrifluoroacetamide in 57% yield. We then removed the trifluoroacetamide protecting group by hydrolysis in ammonium hydroxide in 99% yield. 5-(3″-Aminoallyl)-2′-deoxyuridine was coupled with 2, to give 3 in 47% yield. Finally, the corresponding monomer for oligonucleotide synthesis was prepared by 5’-OH DMT protection and activation of 3’-OH with phosphoroamidite, 55% yield. Chromophore 2 both in methanol and glycerol exhibited λ_abs 381 (nm) and ϵ (M^-1 cm^-1) 20600. Addition of two ortho flourine atoms in the phenol provided compound 4, improving the absorption values: λ_abs (nm) and ϵ (M^-1 cm^-1) to 386 and 25600, respectively. Compound 3 did not fluoresce in methanol; however, in glycerol, which is 3000 times more viscous than methanol, emission was observed at 460 nm (Φ= 0.31, Brightness= 11.7). This finding supports our hypothesis that a rigid medium (e.g. base-pairs of an RNA duplex) limits the free-rotation of the intercalator, resulting in a fluorescent signal.