Synthesis and photophysical properties of 2′-deoxyguanosine derivatives labeled with fluorene and fluorenone units: toward excimer probes

Abstract

In this study we prepared two fluorescent nucleosides, G^FL and G^FO, comprising 2′-deoxyguanosine units covalently bound to 2-ethynylfluorene and 2-ethynyl-9-fluorenone moieties, respectively. The photophysical properties (fluorescence emission shifts and emission intensities) of these fluorescent nucleosides are solvent-dependent. Most notably, G^FO, which bears a guanine nucleobase as its electron-donating group, displays an excimer emission in nonpolar solvents; accordingly, we used excimer emission titration to determine the binding constants for the interactions between G^FO and nucleobases.

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Introduction

Fluorescent nucleosides that are sensitive to their physical environment, including the presence of other molecular species in solution, resulting in specific changes in their fluorescence properties, are powerful tools for investigating nucleic acid structures, recognizing single nucleotide polymorphisms, and studying enzymatic processes involving DNA.¹

Among the fluorophores that have been used to design fluorescent nucleosides, fluorene (FL) and 9-fluorenone (FO) have moderate quantum yields and relatively low bulk (cf. pyrene, perylene or fluorescein). Although FL and FO are structural analogs, they have dramatically different photophysical properties.² Previously, we prepared FL- and FO-labeled deoxyuridines (U^FL and U^FO, respectively) to examine the effect of electronic modification of the fluorophore scaffold on their potential use as microenvironment-sensitive probes and quencher-free molecular beacon probes (Fig. 1).³ Solutions of such fluorescent deoxyuridine derivatives exhibit solvent-dependent photophysical properties, with dramatic changes in their emission intensities and emission wavelengths.

Fig. 1 FL- and FO-labeled fluorescent nucleosides.

In continuing studies toward the development of practically useful fluorescent nucleosides and examinations of the effects of different nucleoside scaffolds (other than deoxyuridine) on the behavior of microenvironment-sensitive probes, we became interested in fluorescent deoxyguanosine derivatives; these attractive targets can act not only as environment-sensitive probes⁴ and DNA probes⁵ but also as essential elements for G-quadruplex formation.⁶ Here, we report the synthesis and photophysical properties of the FL- and FO-conjugated 2′-deoxyguanosine analogs G^FL and G^FO (Fig. 1).

Results and discussion

Synthesis of G^FL and G^FO

We synthesized G^FL and G^FO through palladium-catalyzed Sonogashira coupling⁷ of the 2-N-protected 8-bromo-2′-deoxyguanosine 1^4a,c with 2-ethynylfluorene⁸ and 2-ethynyl-9H-fluoren-9-one,⁹ respectively, and subsequent conversion of species 2 into the corresponding free nucleosides (Scheme 1).

Scheme 1 Reagents and conditions: (a) 2-ethynylfluorene (for 2a), 2-ethynyl-9H-fluoren-9-one (for 2b), Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 50 °C, 5 h, 78% (2a), 86% (2b); (b) NH₄OH, MeOH, 55 °C, 25 h, 64% (3a), 60% (3b).

Photophysical properties of G^FL and G^FO

We first measured the absorption and emission spectra of the nucleosides G^FL and G^FO in 14 solvents of various polarities. The solvent polarity affected the absorption marginally (Fig. S1†), but had a significant influence on both the emission maximum and intensity (Fig. 2a and b and S2†); thus, both G^FL and G^FO are environmentally sensitive. In particular, the fluorescence change of G^FO under illumination with a transilluminator at 365 nm was readily observable by the naked eye (Fig. 2c).

Fig. 2 Emission spectra of (a) G^FL and (b) G^FO in various solvents at 25 °C (all at 3 μM concentration). Excitation wavelength: 366 nm. All samples contained 0.5% DMSO to ensure solubility. (c) Fluorescence image of G^FO in various solvents, under illumination with a transilluminator (365 nm).

Table 1 summarizes the photophysical properties of G^FL and G^FO in the 14 different solvents. We determined the fluorescence quantum yields (Φ_F) of the nucleosides using an EtOH solution of 9,10-diphenylanthracene (λ_ex = 366 nm) as the standard.¹⁰ The quantum yields of G^FL are higher than those of G^FO. The quantum yields of G^FL were highest in 1,4-dioxane and CH₂Cl₂, while those of G^FO were highest in toluene and benzene. The emission maximum of G^FO was substantially red-shifted in relatively nonpolar solvents (toluene, benzene, 1,4-dioxane, THF, EtOAc, CHCl₃), while that of G^FL was substantially red-shifted in DMSO. Thus, G^FL and G^FO exhibit highly distinct solvent-dependent photophysical properties, despite their structural similarities.

Table 1 Photophysical characteristics of nucleosides in various solvents at 25 °C

Compound	Solvent^a	λmax^b (nm)	ε^c (M^−1 cm^−1)	λem^d^,^e (nm)	ΦF^c^,^f
a All samples contained 0.5% DMSO to ensure solubility.b Only the largest absorption maxima are listed.c Presented data are means from three independent experiments.d Wavelength of emission maximum when excited at the absorption maximum.e Maximum values of excimer emission band listed in parentheses.f Quantum efficiencies determined using an EtOH solution of 9,10-diphenylanthracene as the standard; λex = 366 nm.
G^FL	Toluene	368	15 000	407	0.32
	Benzene	368	18 500	405	0.50
	1,4-Dioxane	344	37 100	406	0.72
	THF	366	30 400	419	0.51
	EtOAc	363	26 100	412	0.53
	CHCl3	366	19 100	404	0.61
	CH2Cl2	364	20 100	408	0.63
	DMSO	349	40 800	456	0.19
	MeCN	340	35 500	425	0.17
	^iPrOH	364	30 400	406	0.37
	EtOH	363	30 000	409	0.17
	MeOH	362	28 200	408	0.071
	Ethylene glycol	368	29 100	415	0.28
	H2O	340	15 100	419	0.085
G^FO	Toluene	365	14 800	412 (544)	0.15
	Benzene	365	12 600	412 (543)	0.12
	1,4-Dioxane	359	21 300	409 (551)	0.088
	THF	361	19 100	410 (561)	0.027
	EtOAc	358	17 800	411 (564)	0.031
	CHCl3	365	13 500	411 (565)	0.053
	CH2Cl2	358	15 800	412	0.037
	DMSO	360	19 700	410	0.017
	MeCN	355	18 700	410	0.027
	^iPrOH	356	20 100	409	0.021
	EtOH	355	18 600	410	0.016
	MeOH	355	17 500	409	0.017
	Ethylene glycol	350	19 000	410	0.044
	H2O	345	15 100	417	0.044

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Interestingly, a highly red-shifted emission peak appeared near 550 nm for G^FO in nonpolar solvents (Fig. 2b and Table 1). The emission spectra of FO in aprotic solvents typically feature a new red-shifted band as a result of excimer formation.¹¹ In addition, FO derivatives presenting an electron donor moiety [e.g., 1-hydroxyfluorenone, 3-methylaminofluorenone, 2,7-bis(4-diethylaminophenyl)fluorenone] display large Stokes shifts of their monomer and excimer emission bands relative to those of FO itself.¹² The spectra of these electron-rich FO derivatives could feature emission bands from locally excited (LE) and twisted intramolecular charge transfer (TICT) of an isomer in which the electron donor moiety donates charge to the skeleton of FO. When these FO derivatives form an excimer in typical solvents, the ratio of TICT emission decreases and excimer emission appears. This switching between TICT and excimer emissions is readily facilitated simply through changes in solvent.¹² The guanine unit in our G^FO system acts as a good electron donor because the oxidation potential of guanine is the lowest among the nucleobases.¹³ Thus, we could not measure distinct TICT bands in most solvents, instead observing broad emissions in the region 370–450 nm arising from overlapping of the LE and TICT bands. Nevertheless, we could observe excimer emissions at longer wavelength in toluene, benzene, 1,4-dioxane, THF, EtOAc, and CHCl₃—the relatively nonpolar solvents tested in this study. It is possible that LE and TICT processes are more stable in polar solvents, resulting in the absence of excimer emissions.

To confirm its excimer formation, we examined the concentration-dependence of the fluorescence of G^FO in 1,4-dioxane (Fig. 3). Upon increasing the concentration of G^FO from 0.1 to 1.0 μM, we observed slight increases in the intensities of fluorescence at both shorter (monomer band) and longer (excimer band) wavelengths. The emission intensity of the peak at longer wavelength increased dramatically upon increasing the concentration to greater than 2.0 μM, indicating that this additional fluorescence band was due to excimer formation. Interestingly, the excimer emission of G^FO underwent a slight red-shift as we increased its concentration. On the other hand, we did not observe the excimer emission for G^FL; we suspect that its absence arose from the FL moiety being a relatively poor electron-acceptor.

Fig. 3 Fluorescence spectra of G^FO at various concentrations in 1,4-dioxane; excitation: 350 nm.

Next, we plotted the emission maxima of G^FL and G^FO in various solvents against Reichardt's microscopic solvent polarity parameter, E_T(30),¹⁴ to examine the effect of solvent polarity. Generally, fluorophores exhibit strong fluorescence emissions in nonpolar solvents, but very weak red-shifted emissions in polar solvents.¹⁵ We observed, however, no linear correlations between the monomer emission maxima of these nucleosides and E_T(30), regardless of the aproticity of the solvent (Fig. S3a and S3b†), presumably because of specific solvent–fluorophore interactions (e.g., preferential solvation, charge-transfer interactions or hydrogen bonding).¹⁵ In contrast, the excimer emission maxima of G^FO could be plotted linearly against the values of E_T(30) (Fig. S3c†), but the solvent polarity had little effect on the intensity of this excimer band (Fig. 2b).

G^FO as an excimer probe

FO derivatives presenting electron donor groups would be useful excimer-based probes with high sensitivity if these host systems could bind selectively to guest molecules in typical solvents. Although the guanine unit of G^FO can be considered as an electron donor group, it also functions as a host recognition site. To emphasize the utility of the excimer emission band of G^FO having in nonpolar solvents, we performed fluorescence titrations of G^FO with nucleobases in 1,4-dioxane at a concentration of 3 μM (Fig. 4).

Fig. 4 Changes in the fluorescence spectra of G^FO (3 μM) in 1,4-dioxane in the presence (0–30 μM) of (a) cytosine and (b) guanine (λ_ex = 345 nm). Inset: mole ratio plots of the emissions of the excimer and monomer.

The addition of up to approximately 1 equiv. of cytosine or guanine to the solution of G^FO increased the intensity of the monomer band, but quenched the excimer band (Fig. 4 inset). Job's plots revealed the formation of 1 : 1 complexes between G^FO and both nucleobases (Fig. S4†). The excimer emission was presumably quenched as a result of disrupted excimer formation upon binding of G^FO to cytosine or guanine, thereby enhancing the monomer emission. The fluorescence emission intensity was not significantly affected, however, upon the addition of thymine or adenine, due to the low binding affinity of G^FO toward these nucleobases (Fig. S5†).

We used the emission intensity data and the steady state fluorometric method to determine the binding constants (K_a) for the interactions of G^FO with cytosine and guanine.¹⁶ When considering the excimer bands, the binding mode of G^FO with cytosine, determined from the linear regression curve with a good linear correlation coefficient (R = 0.9950 for cytosine; 0.96099 for guanine), was 1 : 1, providing values of K_a of 394 M⁻¹ for cytosine and 505 M⁻¹ for guanine (error limits: <10%) (Fig. S6†). In contrast, use of the monomer band provided poor linear correlation coefficients (R = 0.93346 for cytosine; 0.93519 for guanine). Accordingly, the excimer band of G^FO is more useful for fluorescence titration.

Recently, Temps and coworkers examined the formation of hydrogen-bonded homo- and heterodimers of 2′,3′,5′-TBDMS-protected guanosine (G) and 3′,5′-TBDMS-protected 2′-deoxycytidine (dC) in CHCl₃, using FTIR spectroscopy; they reported values of K_a of (3.4 ± 0.8) × 10⁴ M⁻¹ for the Watson–Crick base pair G:dC and 1010 ± 20.0 M⁻¹ for the G:G pair, which features two intermolecular hydrogen bonds in a reverse-Hoogsteen structure.¹⁷ In comparison, our data, determined through excimer emission titration, indicates that the value of K_a for the Watson–Crick base pair formed between G^FO and cytosine is 86-fold lower than that for G:dC pairing. This discrepancy presumably originated from hydrogen bonding of the cytosine units with the free OH groups of G^FO as well as the oxygen atoms of 1,4-dioxane,¹⁸ thereby disrupting the Watson–Crick base pairing.

The FO–donor pairs would presumably provide an opportunity to detect guest species, such as ions and small molecules, through monitoring of the excimer band if their electron-donating units would also function as host species. Excimer formation from a single FO–donor molecule would, however, be limited in terms of solvent selection and the demand for a somewhat high concentration. Nevertheless, we suspect that the FO–donor systems could be employed as spatially sensitive fluorophores if they could be brought into close proximity with other moiety, such as pyrene, which can form long-wavelength excimer states with neighboring pyrene units.¹⁹ There may be significant advantages in utilizing FO–donor systems over pyrene for excimer formation because FO is smaller than pyrene and more sensitive to its local environment. Furthermore, there is a wide range of possible modifications of the donor moiety of FO–donor probes. Therefore, more judicious selection of the donor group should enable these FO–donor systems to become useful biophysical tools (e.g., to evaluate distances between biomolecules; to detect nucleic acids or proteins).

Conclusions

We have designed structurally similar fluorescent 2′-deoxyguanosine derivatives labeled with FL and FO units that display solvent-dependent photophysical properties. In particular, we employed G^FO as a host molecule for the detection of nucleobases through monitoring of the excimer emission band in nonpolar solvents; thus, this electron-rich system can be considered as an excimer probe. Modification of such FO–donor systems might enable such systems to be used as excimer probes for investigations of biomolecule dynamics and recognition processes. Efforts in these directions are currently in progress.

Experimental

General

Analytical thin layer chromatography (TLC) was performed using Merck 60 F₂₅₄ 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. ¹H and ¹³C NMR spectra were recorded using a Bruker NMR spectrometer (AVANCE digital 400 MHz and AVANCE III 500 MHz). Multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). High-resolution fast atom bombardment (FAB) mass spectra were recorded using a JEOL JMS-700 mass spectrometer at the Daegu center of KBSI, Korea. All commercially available chemicals were used without further purification; solvents were carefully dried and distilled prior to use. Compound 1 was synthesized according to the reported protocol.^4a,c

2′-Deoxy-8-N-[(dimethylamino)methylene]-(2-ethynylfluorenyl)guanosine (2a)

The 2-N–protected 8-bromo-2′-deoxyguanosine 1 (307 mg, 0.842 mmol) and 2-ethynylfluorene (331 mg, 1.74 mmol) were added to a mixture of (Ph₃P)₂PdCl₂ (49.1 mg, 76.5 μmol) and CuI (13.3 mg, 76.5 μmol) in DMF (3.5 mL) under a Ar atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Ten pump/purge cycles were applied with the addition of Ar gas Et₃N (1.2 mL) was added and then the mixture was heated at 45–50 °C for 5 h. After evaporation of the solvent under reduced pressure, the product was purified through column chromatography [SiO₂; MeOH–CH₂Cl₂, 1 : 50 (v/v)] to yield 2a (305 mg, 78%). Mp > 174 °C dec.; IR (film): ν 3752, 3643, 3207, 3107, 2926, 2670, 2329, 2191, 1663, 1621, 1514, 1409, 1340, 1275, 1098, 1048, 968, 864, 731 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 11.57 (s, 1H; NH), 8.55 (s, 1H; N=CH), 8.03 (d, J = 7.9 Hz, 1H; FL–H), 7.99 (d, J = 7.2 Hz, 1H; FL–H), 7.87 (s, 1H; FL–1H), 7.68 (d, J = 8.0 Hz, 1H; FL–H), 7.64 (d, J = 8.0 Hz, 1H; FL–H), 7.45–7.36 (m, 2H; FL–H), 6.49 (t, J = 8.0 Hz, 1H; H-3′), 5.38 (d, J = 4.0 Hz, 1H; OH-3′), 4.87 (t, J = 6.0 Hz, 1H; OH-5′), 4.52–4.49 (m, 1H; H-3′), 4.01 (s, 2H; FL–CH₂), 3.88–3.84 (m, 1H; H-4′), 3.70–3.64 (m, 1H; H-5′), 3.59–3.53 (m, 1H; H-5′), 3.17 (s, 3H; NCH₃), 3.16–3.08 (m, 1H; H-2′), 3.06 (s, 3H; NCH₃), 2.29–2.23 (m, 1H; H-2′); ¹³C NMR (125 MHz, DMSO-d₆): δ 158.4, 157.8, 157.0, 149.6, 143.7, 143.6, 142.8, 140.1, 130.7, 130.5, 128.3, 127.8, 127.0, 125.3, 120.8, 120.6, 118.3, 94.2, 87.7, 84.0, 79.4, 70.9, 62.0, 40.9, 37.7, 36.4, 34.7; HRMS-FAB (m/z): [M + Na]⁺ calcd for C₂₈H₂₆N₆O₄Na, 533.1916; found, 533.1916.

2′-Deoxy-8-N-[(dimethylamino)methylene]-(2-ethynyl-9H-fluoren-9-onyl)guanosine (2b)

1 (200 mg, 0.548 mmol) and 2-ethynyl-9H-fluoren-9-one (232 mg, 1.14 mmol) were added to a mixture of (Ph₃P)₂PdCl₂ (35.0 mg, 49.8 μmol) and CuI (9.48 mg, 49.8 μmol) in DMF (2.33 mL) under a N₂ atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Ten pump/purge processes were applied with the addition of Ar gas. Et₃N (0.78 mL) was added and then the mixture heated at 45–50 °C for 5 h. After evaporation of the solvent under reduced pressure, the product was purified through column chromatography [SiO₂; MeOH–CH₂Cl₂, 1 : 20 (v/v)] as eluent (161 mg, 56%). Mp > 176 °C dec.; IR (film): ν 3832, 3748, 3676, 3377, 3205, 3131, 2928, 2367, 1680, 1633, 1511, 1417, 1345, 1291, 1240, 1185, 1109, 1050, 985, 868, 822, 739, 652 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 11.58 (s, 1H; NH), 8.56 (s, 1H; N=CH), 7.96–7.90 (m, 3H; FO–H), 7.83 (s, 1H; FO–H), 7.70–7.66 (m, 2H; FO–H), 7.45 (t, J = 8.0 Hz, 1H; FO–H), 6.48 (t, J = 6.0 Hz, 1H; H-3′), 5.38 (d, J = 4.0 Hz, 1H; OH-3′), 4.85 (t, J = 6.0 Hz, 1H; OH-5′), 4.53–4.48 (m, 1H; H-3′), 3.86–3.73 (m, 1H; H-4′), 3.69–3.63 (m, 1H; H-5′), 3.57–3.51 (m, 1H; H-5′), 3.18 (s, 3H; NCH₃), 3.11–3.08 (m, 1H; H-2′), 3.06 (s, 3H; NCH₃), 2.30–2.24 (m, 1H; H-2′); ¹³C NMR (125 MHz, DMSO-d₆): δ 192.0, 158.4, 157.9, 157.0, 149.6, 144.6, 143.2, 138.5, 135.7, 133.7, 133.5, 130.2, 130.0, 130.0, 126.5, 124.2, 121.9, 121.8, 121.2, 120.8, 92.5, 87.7, 84.0, 80.7, 70.8, 61.9, 40.9, 37.8, 34.7; HRMS-FAB (m/z): [M + Na]⁺ calcd for C₂₈H₂₄N₆O₅Na, 547.1708; found, 547.1704.

2′-Deoxy-8-(2-ethynylfluorenyl)guanosine (G^FL)

NH₄OH (20.4 mL) was added to a solution of 2a (130 mg, 0.255 mmol) in MeOH (20.4 mL) and then the mixture was heated at 55 °C for 20 h. After evaporation of the solvent under reduced pressure, the residue was crystallized with EtOH (74.3 mg, 64%). Mp > 239 °C dec.; IR (film): ν 3847, 3653, 3030, 2916, 2589, 2369, 2193, 1672, 1573, 1354, 1183, 1040, 819, 712 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 10.95 (br s, 1H; NH), 8.03–7.97 (m, 2H; FL–H), 7.84 (s, 1H; FL–H), 7.65 (t, J = 8.8 Hz, 2H; FL–H), 7.45–7.36 (m, 2H; FL–H), 6.65 (s, 2H; NH₂), 6.38 (t, J = 7.2 Hz, 1H; H-1′), 5.31 (s, 1H; OH-3′), 4.91 (s, 1H; OH-5′), 4.45 (s, 1H; H-3′), 4.00 (s, 2H; FL–CH₂), 3.83 (d, J = 3.1 Hz, 1H; H-4′), 3.66–3.63 (m, 1H; H-5′), 3.54–3.52 (m, 1H; H-5′), 3.12 (q, J = 6.8 Hz, 1H; H-2′), 2.22–2.18 (m, 1H; H-2′); ¹³C NMR (100 MHz, DMSO-d₆): δ 156.6, 154.4, 151.3, 144.0, 143.9, 143.0, 140.5, 130.9, 129.6, 128.6, 128.1, 127.4, 125.7, 121.1, 120.9, 118.8, 118.0, 94.0, 88.2, 84.1, 80.0, 71.5, 62.5, 37.6, 36.7; HRMS-FAB (m/z): [M + H]⁺ calcd for C₂₅H₂₂N₅O₄, 456.1674; found, 456.1675.

2′-Deoxy-8-(2-ethynyl-9H-fluoren-9-onyl)guanosine (G^FO)

NH₄OH (48.0 mL) was added to a solution of 2a (480 mg, 0.915 mmol) in MeOH (73.1 mL) and then the mixture was heated at 55 °C for 20 h. After evaporation of the solvent under reduced pressure, the residue was crystallized with MeOH–CHCl₃ (1 : 4, v/v) to yield the title compound (129 mg, 30%). Mp > 240 °C dec.; IR (film): ν 3844, 3742, 3612, 3043, 2919, 2359, 1671, 1522, 1366, 1173, 1085, 1035, 952, 867, 716 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 11.80 (s, 1H; NH), 7.96–7.84 (m, 3H; FO–H), 7.80 (s, 2H; FO–H), 7.68 (s, 1H; FO–H), 7.58 (d, J = 8.0 Hz, 1H; FO–H), 7.45 (t, J = 7.2 Hz, 1H; FO–H), 6.74 (s, 2H; NH₂), 6.38 (t, J = 6.0 Hz, 1H; H-1′), 5.31 (s, 1H; OH-3′), 5.08 (br s, 1H; OH-5′), 4.44 (br s, 1H; H-3′), 3.83 (br s, 1H; H-4′), 3.65–3.62 (m, 1H; H-5′), 3.53 (s, 1H; H-5′), 3.14–3.05 (m, 1H; H-2′), 2.22–2.21 (m, 1H; H-2′); ¹³C NMR (125 MHz, DMSO-d₆): δ 192.0, 156.2, 154.2, 151.0, 144.5, 143.2, 138.4, 135.7, 133.7, 133.5, 130.2, 128.7, 126.5, 124.2, 121.9, 121.8, 121.4, 117.8, 92.0, 87.8, 83.6, 81.1, 71.0, 62.1, 37.4; HRMS-FAB (m/z): [M + H]⁺ calcd for C₂₅H₂₀N₅O₅, 470.1466; found, 470.1464.

UV and fluorescence measurements

UV spectra were recorded using a Cary 100 UV-Vis spectrophotometer and a 10 mm-path quartz cell, with respect to a pure-solvent reference. Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer. All samples contained 0.5% DMSO to ensure solubility. The excitation and emission bandwidth was 1 nm. The fluorescence quantum yields (Φ_F) were determined using an EtOH solution of 9,10-diphenylanthracene (λ_ex = 366 nm) as the ref. 10.

Binding constants

The binding constants (K_a) for the interactions of G^FO with nucleobases were determined from the excimer emission intensity data, following the steady state fluorometric method.¹⁶ I₀ refers to the intensity of the excimer emission of the solution containing the free nucleobase. After fluorescence titration, I₀/(I − I₀) was plotted with respect to the reciprocal of the nucleobase concentration (1/[M]). The value of K_a was obtained from the ratio of the intercept to the slope.

Acknowledgements

This study was supported by a grant (no. 2012M2B2A4029626) from the National Research Foundation of Korea (NRF), funded by the Korean government (MEST).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Absorption spectra; effect of E_T(30) on emission maxima; fluorescence Job's plots; titration curves with thymine and adenine; linear regression curves; ¹H and ¹³C NMR spectra. See DOI: 10.1039/c3ra47383j

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