Synthesis and Photophysical Study of 2′-Deoxyuridines Labeled with Fluorene Derivatives

We examined microenvironment-sensitive fluorescent 2′-deoxyuridines labeled with fluorene derivatives that exhibited solvent-dependent photophysical properties. The high sensitivity of the fluorescence shift and the nucleoside intensity dependence on solvent polarity provided information useful for estimating the polarity of the environment surrounding the fluorescent nucleoside.


Introduction
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 microenvironmentsensitive 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.
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 H 2 SO 4 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 i PrOH 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 i PrOH, 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 i PrOH, 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.

General
All reactions were performed in dry glassware under Ar atmospheres.Analytical thin layer chromatography (TLC) was performed using Merck 60 F 254 silica gel plates; column chromatography was performed using Merck 60 silica gel (230-400 mesh).Melting points were determined using an Electrothermal IA 9000 series melting point apparatus and are uncorrected.Infrared (IR) spectra were recorded using a JASCO FT/IR-4100 spectrometer. 1 H-and 13 C-NMR spectra were recorded using a Bruker NMR spectrometer (AVANCE digital 400 MHz).High-resolution electron impact (EI) mass spectra were recorded using a JEOL JMS-700 mass spectrometer at the Daegu center of KBSI, Korea.

Figure 1 .
Figure 1.Fluorescent nucleosides used in this study.

Scheme 1 .
Scheme 1. Route for the synthesis of U DBF and U DBT .

Figure 3 .
Figure 3. Emission spectra of (a) U FL , (b) U FO , (c) U DBF , and (d) U DBT in different solvent at 25 C (all at 3 M concentration).The excitation wavelengths were 370 nm for U FL and 340 nm for the others.All samples contain 0.5% THF/MeOH (1:1 v/v) to ensure solubility.

Figure 4 .
Figure 4. Effect of E T (30) on (a) the fluorescence emission maxima and (b) the Stokes shifts of nucleosides.

Figure 5 .
Figure 5. Dependence of the Stokes shift of nucleosides in water/1,4-dioxane binary solvent mixture on the empirical solvent polarity parameter, E T (30).

Table 1 .
Photophysical characteristics of nucleosides in different solvents at 25 C.