Fluorescent nucleosides which are structurally noninvasive, forming stable Watson-Crick base pairs, and sensitive to their physical conditions and molecular species in solution, exhibiting environmental-specific changes in their fluorescent properties, have become powerful tools for the investigation of nucleic acid structure, recognition of single nucleotide polymorphisms (SNPs), and studies on enzymatic processes involving DNA [1,2,3,4,5,6,7,8].

In order to design fluorescent nucleosides, we utilized an ethynyl linker at the 5 position of uracil to maintain the hybridization properties of the parent nucleoside. This substitution is expected to have very little influence on the stability of the resulting duplex DNA [9,10,11,12,13,14,15,16,17,18,19,20]. Among fluorophores, fluorene derivatives have moderate quantum yields and are less bulky than other commonly used fluorophores, e.g., pyrene, fluorescein, rhodamine, and cyanine dyes [21]. Previously, we reported fluorene (FL)- and 9-fluorenone (FO)-labeled deoxyuridine (U^FL and U^FO), which we incorporated at the central positions of oligodeoxynucleotides in an attempt to examine the effect of electronic modification of the fluorophore scaffold on the potential of the molecular beacon (MB) for SNP typing (Figure 1) [9,10,11]. When such a quencher-free MB hybridizes with its perfectly matched target DNA, it exhibits strong fluorescence. In contrast, when it forms duplexes with single-base-mismatched target DNAs, the U^FL and U^FO units display quenched fluorescence as a result of photoinduced charge transfer originating from interactions with neighboring nucleobases. These changes in fluorescence are extremely dependent on the electronic and conformational microenvironments of the flanking bases. Therefore, we sought to synthesize other fluorescent uridines labeled with new FL derivatives, dibenzofuran (DBF) and dibenzothiophene (DBT), in order to examine changes in their photophysical properties through modifications of the fluorene unit and to develop these nucleosides as microenvironment-sensitive fluorescent nucleosides [17,22,23]. Although FL, FO, DBF, and DBT are structural analogs that differ only in the type of atoms bridging the two aromatic rings, they have dramatically different photophysical properties [24,25,26]. Here, we report the synthesis and photophysical properties of fluorescent FL derivative-conjugated 2′-deoxyuridine analogs.

The synthetic route of the DBF- and DBT-labeled 2′-deoxyuridine derivatives U^DBF and U^DBT is outlined in Scheme 1. 3-Ethynyldibenzofuran (3a) was prepared by Pd/Cu-catalyzed Sonogashira coupling [27,28] of 3-bromodibenzofuran (1) with trimethylsilylacetylene followed by desilylation. 3-Ethynyldibenzothiophene (3b) was also synthesized according to the reported protocol [29]. We synthesized U^DBF and U^DBT from the corresponding 2′-deoxy-5-iodouridine (4) through a palladium catalyzed cross-coupling reaction with 3-ethynyldibenzofuran (3a) or 3-ethynyldibenzothiophene (3b). The syntheses of U^FL and U^FO were conducted as reported [9,10,11].

Generally, solvent polarity is of primary interest when considering environmental effects [30]. Therefore, we first measured the absorption and emission spectra of nucleosides in thirteen solvents of different polarities. Solvent marginally affected the absorption, probably due to the weak interaction between the nucleosides and solvent in the ground state (Figure 2). However, solvent polarity had a significant influence on both the emission maximum and intensity (Figure 3). All nucleosides exhibited different emission intensities and maxima depending on the solvent they were in, indicating that they are all environmentally sensitive.

Table 1 summarizes the photophysical properties of nucleosides in thirteen different solvents. The fluorescence quantum yields (Φ_F) of the nucleosides were determined using a 0.1 N aqueous H₂SO₄ solution of quinine sulfate (λ_ex = 350 nm) as a standard [31]. There are some noteworthy features: (a) generally, the presence of a heteroatom in the fluorene unit of nucleoside U^FO, U^DBF, and U^DBT diminishes its fluorescence yield and fluorescence brightness (i.e., the product of its molar extinction coefficient and quantum yield) drastically when compared with U^FL. (2) U^DBF and U^DBT showed very similar photophysical properties in various solvents. (3) The quantum yield and fluorescence brightness of nucleosides is highest in ⁱPrOH for U^FL, ethyl acetate for U^FO, and ethylene glycol for U^DBF and U^DBT. The lowest fluorescence brightness, however, was observed in ethylene glycol for U^FO and water for U^FL, U^DBF, and U^DBT. These results indicate that the nucleosides exhibit highly solvent-dependent photophysical properties despite their structural similarities. U^FO, interestingly, exhibited a strong solvent dependency–namely, higher fluorescence brightness in aprotic solvents relative to those in protic solvents such as ⁱPrOH, EtOH, MeOH, ethylene glycol, and water which was attributable to the hydrogen bonding between the carbonyl group of U^FO and solvent.

In polar solvents such as ⁱPrOH, EtOH, MeOH, acetonitrile, ethylene glycol, DMSO, and water substantially larger red-shifts in emission maxima of nucleosides were observed. Because it is instructive to calculate the magnitude of the expected spectral shifts due to solvent polarity effects, we plotted the fluorescence emission maxima and Stokes shifts (ν_abs–ν_em) of nucleosides in thirteen different solvents against Reichardt’s microscopic solvent parameter, E_T(30) (Figure 4) [32]. It is interesting to note that there is a linear correlation between emission maxima and E_T(30) regardless of the aproticity of the solvent. The red-shift of the fluorescence could be due to the significant difference between the excited‐state charge distribution in the solute and the ground‐state charge distribution, resulting in stronger interactions with polar solvents in the excited state.

Emission maxima of U^FO were red-shifted relative to those of other nucleosides. This higher Stokes shift of U^FO is probably because the carbonyl group allows for hydrogen bonding and charge separation better than do the other nucleosides [30]. Interestingly, the Stokes shifts of U^DBF and U^DBT exhibited a more gradual shift to longer wavelengths with increasing solvent polarity compared to the slopes of other nucleosides, as shown in Figure 4b. In order to compare the sensitivity of our molecules of interest to environmental polarity with that of reported polarity-sensitive nucleosides [12,33], we examined the photophysical properties of fluorescent nucleosides in binary water/1,4-dioxane mixtures (Table S1, Figure 5), which is an established method for estimating the microenvironment polarity of fluorophores [34]. The Stokes shifts plotted against the E_T(30) values of the samples is shown in Figure 5. The slopes obtained from the linear plots indicated that U^FO, U^DBF, and U^DBT are highly sensitive to environmental polarity and are comparable to the slopes of reported nucleosides such as pyridine- and furan-labeled uridines. U^DBF and U^DBT revealed a seemingly exponential trend, leading us to conclude that a more appropriate expression for the interactions between these nucleosides and solvents should be explored.

We designed structurally similar fluorescent 2′-deoxyuridine derivatives that exhibit solvent-dependent photophysical properties via drastic changes in emission intensity as well as emission wavelength. These microenvironment-sensitive nucleosides may be used as probes for investigating nucleic acid dynamics and the recognition process. A deeper understanding of how the photophysical properties relate to chemical structures may allow for the design of ideal environmentally sensitive fluorescent nucleosides towards the development of DNA probes. Efforts in these directions are currently in progress.

Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/10/12061/s1.