Expanded Fluorescent Nucleoside Analog as Hybridization Probe

The design and synthesis of fluorescent nucleosides has been the subject of intensive research because these nucleoside derivatives can be used as tools in molecular biology and diagnostics. Fluorescent nucleoside analogs that are sensitive to the local environment in DNA duplexes are attractive candidate probes for DNA hybridization and for investigating nucleic acid structure. They display a strong signal change upon hybridization with a target DNA, and structural changes in DNA such as the formation of Gquadruplexes and i-motifs. Although a broad range of substituted fluorescent dyes are suitable for labelling nucleic acids, rapid growth in this area requires new and efficient fluorescent nucleoside analogs. Our strategy to synthesize an efficient fluorescent analog is based on the use of expanded nucleobase analogs, which have good fluorescent properties such as high quantum efficiency and high sensitivity to the microenvironment. This system does not change the conformation of stable BDNA and also yields higher duplex stability owing to the bulky hydrophobic planar structure. In this context, we synthesized a new fluorescent nucleoside analog, 1-(2Deoxy-β-D-erythro-pentofuranosyl) benzothieno[3,2,-d]pyrimidine 2,4(3H)-dione( U). Scheme 1 shows our synthetic strategy to prepare the expanded fluorescent nucleoside U. Compound 2 was prepared from 1 in three steps, as shown in Scheme 1. The initial step is nucleophilic displacement of o-halobenzonitrile (2-fluorobenzonitrile) 1 with thioglycolate anion, followed by spontaneous base-induced aldol cyclization with ethyl carboxyisocyanate and base treatment to produce 2 as a white solid in an overall yield of 40% starting from compound 1. Compound 2 was coupled to a sugar moiety to afford an unresolved anomeric mixture of compound 3 in 72%. After detoluoylation, attempts to protect the 5'position of the α/β anomeric mixture with DMT-Cl failed. Thus, instead of DMT-Cl, 4,4'-dimethoxytrityl tetrafluoroborate (DMTBF4) in pyridine was employed for selective protection of the 5'-hydroxyl group of the nucleoside to obtain the protected product. The 5'-ODMT anomeric mixture was separated on a flash silica gel column. A small amount of each anomer was deprotected using aqueous acetic acid to afford the free nucleosides, which were subjected to extensive spectroscopic analysis to determine the anomeric configuration unambiguously and to measure the quantum yield. ROESY showed a correlation spot for H-1' and H-4', as expected for the β configuration of 6. The β anomer of 4 was treated with standard phosphoramidite reagent to yield the nucleoside 3'-O-phosphoramidite derivative, 5. To evaluate the sensitivity of U to its microenvironment, fluorescence spectra of U were measured in different solvents (Figure 1A). The emission intensity of U was markedly affected by solvent polarity. U showed higher quantum efficiency in polar than in non-polar solvents (Table 1). We also measured the emission change for U at different pH values to evaluate its sensitivity. U exhibited different signals, depending on the pH (acidic, neutral, basic) (Figure 1B). This difference may arise from O-4 protonation in