Uridine cannot be utilized as fluorescent probe due to its extremely low quantum yield. For improving the uracil fluorescence characteristics we extended the natural chromophore at the C5 position by coupling substituted aromatic rings directly or via an alkenyl or alkynyl linker to create fluorophores. Extension of the uracil base was achieved by treating 5-I-uridine with the appropriate boronic acid under the Suzuki coupling conditions. Analogues containing an alkynyl linker were obtained from 5-I-uridine and the suitable boronic acid in a Sonogashira coupling reaction. The uracil fluorescent analogues proposed here were designed to satisfy the following requirements: a minimal chemical modification at a position not involved in base-pairing, resulting in relatively long absorption and emission wavelengths and high quantum yield. 5-((4-Methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6b, was found to be a promising fluorescent probe. Probe 6b exhibits a quantum yield that is 3000-fold larger than that of the natural chromophore (Φ 0.12), maximum emission (478 nm) which is 170 nm red shifted as compared to uridine, and a Stokes shift of 143 nm. In addition, since probe 6b adopts the anti conformation and Ssugar puckering favored by B-DNA, it makes a promising nucleoside analogue to be incorporated in an oligonucleotide probe for detection of genetic material.

Note: In lieu of an abstract, this is the article's first page.

Natural nucleotides are not useful as fluorescent probes because of their low quantum yields. Therefore, a common methodology for the detection of RNA and DNA is the application of extrinsic fluorescent dyes coupled to bases in oligonucleotides. To overcome the many limitations from which fluorescent nucleotide–dye conjugates suffer, we have developed novel purine nucleosides with intrinsic fluorescence to be incorporated into oligonucleotide probes. For this purpose we synthesized adenosine and guanosine fluorescent analogues 7–25, conjugated at the C8 position with aryl/heteroaryl moieties either directly, or via alkenyl/alkynyl linkers. Directly conjugated analogues 7–14, exhibited high quantum yields, ϕ >0.1, and short λ_em (<385 nm). Alkynyl conjugated analogues 22–25, exhibited low quantum yields, ϕ <0.075, and λ_em<385 nm. The alkenyl conjugated analogues 15–21, exhibited λ_em 408–459 nm. While analogues 15,16, and 20 bearing an EDG on the aryl moiety, exhibited ϕ <0.02, analogues 17, and 21 with EWG on the aryl moiety, exhibited extremely high quantum yields, ϕ ≈ 0.8, suggesting better intramolecular charge transfer. We determined the conformation of selected adenosine analogues. Directly conjugated analogue 8 and alkynyl conjugated analogue 22, adapted the syn conformation, whereas alkenyl conjugated analogue 15 adapted the anti conformation. Based on the long emission wavelengths, high quantum yields, anti conformation and base-paring compatibility, we suggest analogues 17 and 21 for further development as fluorescent probes for the sensitive detection of genetic material.