5-Bromo-4′,5′-bis(dimethylamino)fluorescein: Synthesis and Photophysical Studies

In this study, three new fluorescein derivatives—5-bromo-4′,5′-dinitrofluorescein (BDNF), 5-bromo-4′,5′-diaminofluorescein (BDAF), and 5-bromo-4′,5′-bis(dimethylamino)fluorescein (BBDMAF)—were synthesized and their pH-dependent protolytic equilibria were investigated. In particular, BBDMAF exhibited pH-dependent fluorescence, showing strong emission only at pH 3–6. BBDMAF bears a bromine moiety and thus, can be used in various cross-coupling reactions to prepare derivatives and take advantage of its unique emission properties. To confirm this, the Suzuki and Sonogashira reactions of BBDMAF with phenylboronic acid and phenylacetylene, respectively, were performed, and the desired products were successfully obtained.


Introduction
Fluorescein is one of the most widely used fluorophores in the chemical, biochemical, and medicinal fields due to its ease of synthesis and brilliant green fluorescence [1][2][3][4][5][6]. Fluorescein shows complex protolytic equilibria in aqueous solution. The dianionic and monoanionic forms, which are present at pH > 6, exhibit bright fluorescence; however, the fluorescence of the neutral and monocationic forms, which are present at pH < 6, is drastically reduced. Therefore, great care must be taken when using fluorescein as a fluorescent probe under acidic or pH-ambiguous conditions. To overcome this disadvantage, studies have been carried out to lower the pK a by attaching an electron-attracting group such as a halogen to the xanthene ring of fluorescein [7,8]. Nevertheless, these fluorescein derivatives, such as fluorescein, present the same disadvantage in that their emission decreases upon decreasing the pH.
We recently reported 4 ,5 -bis(dimethylamino)fluorescein (BDMAF)-bearing dimethylamino groups at the 4 and 5 positions of the xanthene ring ( Figure 1) [9]. The most prominent feature of this fluorescein derivative is that, between pH 3 and 6, the photoinduced electron transfer (PET) process is inhibited by the protonation of the unpaired electrons of the nitrogen atoms resulting in an increased fluorescence in this pH region. In order to extend the applicability of BDMAF, we designed 5-bromo-4 ,5 -bis(dimethylamino)fluorescein (BBDMAF) containing a bromine moiety at the 5 position of the benzoic acid. Since fluorescein derivatives bearing a bromine moiety can be used in various cross-coupling reactions [10][11][12], BDMAF is expected to be a key compound with applications in sensors and biomarkers. Here, we report the synthesis of BBDMAF, its protolytic equilibria, and its pH-dependent fluorescence.

pKa Determination of BDNF, BDAF, and BBDMAF
The ultraviolet-visible (UV-Vis) absorption spectra of BDNF (12.5 μM), BDAF (2.5 μM), and BBDMAF (2.5 μM) were recorded at various pH levels in mixtures of EtOH and 10 mM phosphate buffer (1:1, v/v) or in 10 mM phosphate buffer ( Figure 2 and Figure S1). Relatively high amounts of BDNF compared to BDAF and BBDMAF were dissolved in 50% EtOH to dissociate the absorption bands of the monoanionic and dianionic forms, as previously reported for DNF [14]. The UV-Vis absorption spectra and pH-dependent mole fractions of each protolytic species of BDNF, BDAF, and

pKa Determination of BDNF, BDAF, and BBDMAF
The ultraviolet-visible (UV-Vis) absorption spectra of BDNF (12.5 μM), BDAF (2.5 μM), and BBDMAF (2.5 μM) were recorded at various pH levels in mixtures of EtOH and 10 mM phosphate buffer (1:1, v/v) or in 10 mM phosphate buffer ( Figure 2 and Figure S1). Relatively high amounts of BDNF compared to BDAF and BBDMAF were dissolved in 50% EtOH to dissociate the absorption bands of the monoanionic and dianionic forms, as previously reported for DNF [14]. The UV-Vis absorption spectra and pH-dependent mole fractions of each protolytic species of BDNF, BDAF, and Scheme 1. Synthesis of BDNF, BDAF, and BBDMAF.

pK a Determination of BDNF, BDAF, and BBDMAF
The ultraviolet-visible (UV-Vis) absorption spectra of BDNF (12.5 µM), BDAF (2.5 µM), and BBDMAF (2.5 µM) were recorded at various pH levels in mixtures of EtOH and 10 mM phosphate buffer (1:1, v/v) or in 10 mM phosphate buffer ( Figure 2 and Figure S1). Relatively high amounts of BDNF compared to BDAF and BBDMAF were dissolved in 50% EtOH to dissociate the absorption bands of the monoanionic and dianionic forms, as previously reported for DNF [14]. The UV-Vis absorption spectra and pH-dependent mole fractions of each protolytic species of BDNF, BDAF, and BBDMAF were predicted and their pK a values were determined through the DATAN program analysis ( Figure 3 and Table 1) [15][16][17].
Similar to DNF, which does not contain a bromine moiety, BDNF displayed very low absorbance in the neutral form, and the absorption bands of the monoanionic and dianionic forms were very close to each other (Table S1). There was no significant difference in the pK a1 and pK a2 values of BDNF, as observed with DNF [14]. The values of pK a1 and pK a2 , which relate to the protonation of the benzoate and phenolate forms of BDNF, were 3.19 and 3.22, respectively. This result indicated that the introduction of a bromine moiety into BDNF causes an increase in the acidity over that of DNF. BDAF and BBDMAF also displayed absorption bands similar to those of DAF and BDMAF, respectively [9]. Comparing the pK a values of BDAF and BBDMAF with those of DAF and BDMAF, it was found that the introduction of a bromine moiety slightly reduced the pK a1 and pK a2 values, which relate to the protonation of the amino group and the benzoate, respectively. On the other hand, the value of pK a3 , which represents the protonation of the phenolate, slightly increased. Based on these data, we suggest the protolytic equilibria of BDAF and BBDMAF, as shown in Scheme 2. BBDMAF were predicted and their pKa values were determined through the DATAN program analysis ( Figure 3 and Table 1) [15][16][17]. Similar to DNF, which does not contain a bromine moiety, BDNF displayed very low absorbance in the neutral form, and the absorption bands of the monoanionic and dianionic forms were very close to each other (Table S1). There was no significant difference in the pKa1 and pKa2 values of BDNF, as observed with DNF [14]. The values of pKa1 and pKa2, which relate to the protonation of the benzoate and phenolate forms of BDNF, were 3.19 and 3.22, respectively. This result indicated that the introduction of a bromine moiety into BDNF causes an increase in the acidity over that of DNF. BDAF and BBDMAF also displayed absorption bands similar to those of DAF and BDMAF, respectively [9]. Comparing the pKa values of BDAF and BBDMAF with those of DAF and BDMAF, it was found that the introduction of a bromine moiety slightly reduced the pKa1 and pKa2 values, which relate to the protonation of the amino group and the benzoate, respectively. On the other hand, the value of pKa3, which represents the protonation of the phenolate, slightly increased. Based on these data, we suggest the protolytic equilibria of BDAF and BBDMAF, as shown in Scheme 2.   BBDMAF were predicted and their pKa values were determined through the DATAN program analysis ( Figure 3 and Table 1) [15][16][17]. Similar to DNF, which does not contain a bromine moiety, BDNF displayed very low absorbance in the neutral form, and the absorption bands of the monoanionic and dianionic forms were very close to each other (Table S1). There was no significant difference in the pKa1 and pKa2 values of BDNF, as observed with DNF [14]. The values of pKa1 and pKa2, which relate to the protonation of the benzoate and phenolate forms of BDNF, were 3.19 and 3.22, respectively. This result indicated that the introduction of a bromine moiety into BDNF causes an increase in the acidity over that of DNF. BDAF and BBDMAF also displayed absorption bands similar to those of DAF and BDMAF, respectively [9]. Comparing the pKa values of BDAF and BBDMAF with those of DAF and BDMAF, it was found that the introduction of a bromine moiety slightly reduced the pKa1 and pKa2 values, which relate to the protonation of the amino group and the benzoate, respectively. On the other hand, the value of pKa3, which represents the protonation of the phenolate, slightly increased. Based on these data, we suggest the protolytic equilibria of BDAF and BBDMAF, as shown in Scheme 2.

Fluorescence of BDNF, BDAF, and BBDMAF
The pH-dependent emission behaviors of BDNF, BDAF, and BBDMAF were next examined. Excitation was performed at the maximum absorption wavelength of the protolytic species of each dye at pH 0.00, 1.23, 1.97, 3.00, 3.97, 4.97, 5.94, 6.92, 7.98, 8.99, 9.58, 9.99, and 10.97 ( Figure S2). The fluorescence of BDNF and BDAF was almost quenched, regardless of the pH and excitation wavelength. However, BBDMAF was found to show a similar pH-dependent fluorescence behavior as BDMAF. Excitation at the maximum absorption wavelength of the cationic BBDMAF form (440 nm) gave the maximum fluorescence intensity at pH 3.00. Excitation at the maximum absorption wavelengths of the neutral, monoanionic, and dianionic forms (495, 503, and 510 nm, respectively) led to the highest fluorescence intensity at both pH 3.97 and 4.97 ( Figure 4).   The pK a values were calculated using the DATAN program. 2 Reported pK a values [3,6]. 3 Not detectable.

Fluorescence of BDNF, BDAF, and BBDMAF
The pH-dependent emission behaviors of BDNF, BDAF, and BBDMAF were next examined. Excitation was performed at the maximum absorption wavelength of the protolytic species of each dye at pH 0.00, 1.23, 1.97, 3.00, 3.97, 4.97, 5.94, 6.92, 7.98, 8.99, 9.58, 9.99, and 10.97 ( Figure S2). The fluorescence of BDNF and BDAF was almost quenched, regardless of the pH and excitation wavelength. However, BBDMAF was found to show a similar pH-dependent fluorescence behavior as BDMAF. Excitation at the maximum absorption wavelength of the cationic BBDMAF form (440 nm) gave the maximum fluorescence intensity at pH 3.00. Excitation at the maximum absorption wavelengths of the neutral, monoanionic, and dianionic forms (495, 503, and 510 nm, respectively) led to the highest fluorescence intensity at both pH 3.97 and 4.97 ( Figure 4).
The quantum yield at pH 4.55, where the neutral form of BBDMAF is predominant, was 0.78, which is slightly lower than that for the neutral form of BDMAF (0.85) [9]. In contrast, the quantum yields of the cationic, monoanionic, and dianionic forms were 0.11, 0.013 and 0.00080, respectively (Table S1). We then compared the radiative and nonradiative decay rate constants of neutral and monoanionic BBDMAF. The radiative decay rate was 41-fold higher for the neutral form than for the monocationic form, but the nonradiative decay rate was 6.5-fold lower. This indicated that the dramatic increase in the quantum yield of the neutral form of BBDMAF results from the enhancement of radiative decay and suppression of nonradiative decay ( Figure S3). Similar to BDMAF, the analysis of the pH-dependent emission behavior of BBDMAF revealed a significant reduction in the emission intensities at pH > 6 due to PET caused by the lone pair of electrons on the amino groups [9,18]. Instead, between pH 3 and 6, where protonation of the amino groups of BBDMAF occurs to some extent, PET was suppressed and very strong fluorescence was observed. The fluorescence of BBDMAF, which was expected to increase at pH < 3 due to further suppression of PET, decreased due to the formation of the ammonium xanthene form, which shows very low absorption. In addition, the absorbance of BBDMAF increased in the pH region where the cationic xanthene form (dicationic form) prevailed because its absorption is known to be greater than that of the ammonium xanthene form [9,19,20]. Accordingly, we measured the absorption and emission spectra of BDMAF and BBDMAF at pH −0.30, −0.56, and −0.94 ( Figure S4). As expected, the absorbance increased upon decreasing the pH. However, the fluorescence intensities of the dicationic forms were quenched for BDMAF or decreased for BBDMAF when the pH was lowered, suggesting that the ammonium xanthene and cationic xanthene forms of BBDMAF, present at pH < 3, displayed very low emission. Notably, this pH-dependent fluorescence behavior of BBDMAF under illumination with a transilluminator is readily observable with the naked eye, as shown in Figure 4b.

Fluorescence of BDNF, BDAF, and BBDMAF
The pH-dependent emission behaviors of BDNF, BDAF, and BBDMAF were next examined. Excitation was performed at the maximum absorption wavelength of the protolytic species of each dye at pH 0.00, 1.23, 1.97, 3.00, 3.97, 4.97, 5.94, 6.92, 7.98, 8.99, 9.58, 9.99, and 10.97 ( Figure S2). The fluorescence of BDNF and BDAF was almost quenched, regardless of the pH and excitation wavelength. However, BBDMAF was found to show a similar pH-dependent fluorescence behavior as BDMAF. Excitation at the maximum absorption wavelength of the cationic BBDMAF form (440 nm) gave the maximum fluorescence intensity at pH 3.00. Excitation at the maximum absorption wavelengths of the neutral, monoanionic, and dianionic forms (495, 503, and 510 nm, respectively) led to the highest fluorescence intensity at both pH 3.97 and 4.97 (Figure 4).

Suzuki and Sonogashira Reactions of BBDMAF
The suitability of BBDMAF as a starting material for cross-coupling reactions was evaluated against the Suzuki and Sonogashira couplings, and the desired products 2 and 3 were obtained in 50% and 66% yield, respectively (Scheme 3) [21,22]. Therefore, it is assumed that BBDMAF can be employed in various cross-coupling reactions if the appropriate reaction conditions are used. The quantum yield at pH 4.55, where the neutral form of BBDMAF is predominant, was 0.78, which is slightly lower than that for the neutral form of BDMAF (0.85) [9]. In contrast, the quantum yields of the cationic, monoanionic, and dianionic forms were 0.11, 0.013 and 0.00080, respectively (Table S1). We then compared the radiative and nonradiative decay rate constants of neutral and monoanionic BBDMAF. The radiative decay rate was 41-fold higher for the neutral form than for the monocationic form, but the nonradiative decay rate was 6.5-fold lower. This indicated that the dramatic increase in the quantum yield of the neutral form of BBDMAF results from the enhancement of radiative decay and suppression of nonradiative decay ( Figure S3).
Similar to BDMAF, the analysis of the pH-dependent emission behavior of BBDMAF revealed a significant reduction in the emission intensities at pH > 6 due to PET caused by the lone pair of electrons on the amino groups [9,18]. Instead, between pH 3 and 6, where protonation of the amino groups of BBDMAF occurs to some extent, PET was suppressed and very strong fluorescence was observed. The fluorescence of BBDMAF, which was expected to increase at pH < 3 due to further suppression of PET, decreased due to the formation of the ammonium xanthene form, which shows very low absorption. In addition, the absorbance of BBDMAF increased in the pH region where the cationic xanthene form (dicationic form) prevailed because its absorption is known to be greater than that of the ammonium xanthene form [9,19,20]. Accordingly, we measured the absorption and emission spectra of BDMAF and BBDMAF at pH −0.30, −0.56, and −0.94 ( Figure S4). As expected, the absorbance increased upon decreasing the pH. However, the fluorescence intensities of the dicationic forms were quenched for BDMAF or decreased for BBDMAF when the pH was lowered, suggesting that the ammonium xanthene and cationic xanthene forms of BBDMAF, present at pH < 3, displayed very low emission. Notably, this pH-dependent fluorescence behavior of BBDMAF under illumination with a transilluminator is readily observable with the naked eye, as shown in Figure 4b.

Suzuki and Sonogashira Reactions of BBDMAF
The suitability of BBDMAF as a starting material for cross-coupling reactions was evaluated against the Suzuki and Sonogashira couplings, and the desired products 2 and 3 were obtained in 50% and 66% yield, respectively (Scheme 3) [21,22]. Therefore, it is assumed that BBDMAF can be employed in various cross-coupling reactions if the appropriate reaction conditions are used. In order to assess whether the photophysical properties of BBDMAF were maintained in compounds 2 and 3, their pH-dependent absorbance and protolytic equilibria were investigated (Figure 5a, Figures S5 and S6). As shown in Table 1, the absorption behavior and pKa values of compounds 2 and 3 resemble those of BBDMAF (Table 1). In addition, compounds 2 and 3 exhibited maximum emissions at 512 and 514 nm, respectively, regardless of the excitation wavelength ( Figures  S7 and S8), and exhibited strong fluorescence only at pH 3-6, as observed for BDMAF and BBDMAF (Figures 5b,c). These results indicate that the products of the cross-coupling reaction of BBDMAF retain the characteristic pH-dependent photophysical properties of BDMAF and BBDMAF. In order to assess whether the photophysical properties of BBDMAF were maintained in compounds 2 and 3, their pH-dependent absorbance and protolytic equilibria were investigated (Figure 5a, Figures S5 and S6). As shown in Table 1, the absorption behavior and pK a values of compounds 2 and 3 resemble those of BBDMAF (Table 1). In addition, compounds 2 and 3 exhibited maximum emissions at 512 and 514 nm, respectively, regardless of the excitation wavelength  Figures S7 and S8), and exhibited strong fluorescence only at pH 3-6, as observed for BDMAF and BBDMAF (Figure 5b,c). These results indicate that the products of the cross-coupling reaction of BBDMAF retain the characteristic pH-dependent photophysical properties of BDMAF and BBDMAF.

Synthetic Procedures
Sulfuric acid (32 mL) was added to 5-bromofluorescein diacetate 1 (1.0 g, 2.0 mmol), and the resulting mixture was stirred at −5 • C until 1 dissolved completely. After addition of nitric acid (2.3 mL), the mixture was stirred for a further 20 min at −5 • C. The mixture was then poured into cold water and extracted with EtOAc. The extracts were combined and dried over Na 2 SO 4 . After evaporation of the solvent under vacuum, the residue was purified by column chromatography on silica gel (CH 2 Cl 2 /MeOH = 4:1) to afford BDNF as an orange solid, which was recrystallized from EtOAc/hexane ( Tin chloride (4.5 g, 20 mmol) was added to a solution of BDNF (1.0 g, 2.0 mmol) in ethanol (70 mL), and the mixture was then refluxed for 6 h. After evaporation of the solvent under vacuum, 1 M NaOH (20 mL) was added, and the mixture was filtered to remove tin chloride. The filtrate was washed with water. The solution was acidified with 1 M HCl to pH 4-5 and extracted with EtOAc. The combined extracts were dried (Na 2 SO 4 ), and the solvent was removed under vacuum to afford BDAF as a purple solid, which was recrystallized from hot acetone (0.