Synthesis of a New Amino-Furopyridine-Based Compound as a Novel Fluorescent pH Sensor in Aqueous Solution.

Development of new fluorescent molecules, especially pH-sensitive fluorescent dyes, is always in high demand due to their wide applications in various fields and the limited number of common chromophores. In this work, a family of 3-amino-N-phenylfuro[2,3-b]pyridine-2-carboxamides (AFP) was synthesized as novel fluorescent compounds. Besides fluorescence in an organic solvent, AFP 1 and AFP 2 exhibit good fluorescence properties in both acidic and basic aqueous solution, which could be explained by protonation or different conformations formed in solution. Density functional theory (DFT) calculations on the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) of various conformations were performed for further support.

Introduction

Development of small fluorescent molecules,[1−5] especially pH-sensitive fluorescent dyes, has gained substantial attention owing to their promising applications in various fields, including analytical chemistry,[6,7] biomedicinal research,[8−12] clinical diagnostics,[13−15] biology,[16−19] and environmental science.[20] They are used as strainers,[21] biomolecular labels,[22,23] environmental indicators,[24] etc. Although different methods for pH measurement have been well documented in literature,[25−29] optical sensors could serve as an effective alternative in specific cases when traditional methods are not applicable, such as in cellular process or when high throughput screening is needed.

Despite the wide use and huge number of small fluorescent probes, they come from a relatively small set of “core” scaffolds. Common pH-sensitive dyes include BODIPY,[30] n>an class="Chemical">rhodamine,[31] some cyanine dyes,[32−36] etc. Derivatization is usually necessary for binding with proton. A novel fluorophore with multiple binding sites and at different wavelengths is still in demand. Here, we have synthesized four analogs with 3-amino-furo[2,3-b]pyridine-2-carboxamide (AFP) as a novel fluorophore (Figure a). It not only emits at a different wavelength from common fluorescent molecules but also contains multiple functional groups as binding sites for potential sensing and easy derivatization. Among the four synthesized molecules, AFP 1 and AFP 2 displayed good fluorescence properties in aqueous solutions under both acidic and basic conditions (Figure b).

Figure 1

(a) Chemical structures of 3-amino-furo[2,3-b]pyridine-2-carboxamides (AFPs) and (b) the proposed fluorescent mechanism under different conditions.

(a) Chemical structures of pan class="Chemical">3-amino-furo[2,3-b]pyridine-2-carboxamides (n>an class="Chemical">AFPs) and (b) the proposed fluorescent mechanism under different conditions.

Results and Discussion

Synthesis of 3-Amino-furo[2,3-b]pyridine-2-carboxamide (AFP)

AFPs (1–4) were synthesized via Barker’s method.[37] The mixture of n>an class="Chemical">glycolic acid 1 and substituted aniline 2a-b was heated together with no solvent to provide 2-hydroxyacetamides 3a-b, which were used directly in the next step with no purification. The following SNAr substitution of 3a-b with chloropyridines 4c-d gave four cyanopyridines 5a-d. The intramolecular cyclization of cyanopyridines 5a-d was carried out with KOBu to eventually provide 3-amino-furo[2,3-b]pyridine-2-carboxamide (AFP 1 to AFP 4) in 81–98% yield (Figure ). The chemical structures of AFP 1 to AFP 4 were clearly identified by 1H NMR and 13C NMR as well as high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (see the Supporting Information).

Figure 2

Synthesis of 3-amino-furo[2,3-b]pyridine-2-carboxamides (AFPs). Reagents and conditions: (a) 1 equiv substituted aniline, 130 °C, 5 h; (b) 0.95 equiv 4c-d, Na2CO3, EtOH, reflux, 40 h, 5a-d, 33–48%; (c)1.2 equiv KOBu, THF, 80 °C, 3 h, AFP 1 to AFP 4, 81–98%.

Synthesis of 3-amino-furo[2,3-b]pyridine-2-carboxamides (n>an class="Chemical">AFPs). Reagents and conditions: (a) 1 equiv substituted aniline, 130 °C, 5 h; (b) 0.95 equiv 4c-d, Na2CO3, EtOH, reflux, 40 h, 5a-d, 33–48%; (c)1.2 equiv KOBu, THF, 80 °C, 3 h, AFP 1 to AFP 4, 81–98%.

UV–Vis Absorption of AFP 1 to AFP 4 in Organic and Aqueous Solutions

The absorption spectra of AFP 1 to n>an class="Chemical">AFP 4 were monitored in acetonitrile and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) solution (1 × 10–5 M, pH = 7), respectively. As shown in Figure a, the UV–vis absorption of AFP 1 is very much like AFP 2 with three absorbance bonds centered at 262 (266 nm for AFP 2), 301 (302 nm for AFP 2), and 339 nm (the same for AFP 2), while AFP 3 and AFP 4 share almost the same absorption patterns at 270, 317, and 350 nm (348 nm for AFP 4). The red shift from AFP 1 to AFP 3 is due to the slightly electro-withdrawing −Br atom on the furo[2,3-b]pyridine fluorophore, but no major effect on the absorbance wavelength was observed by introducing −Cl on the far right benzene ring.

Figure 3

UV–vis absorption of AFP 1 to AFP 4 in (a) acetonitrile and (b) HEPES solution (pH = 7).

UV–vis absorption of AFP 1 to n>an class="Chemical">AFP 4 in (a) acetonitrile and (b) HEPES solution (pH = 7).

When switching from acetonitrile to aqueous solution (Figure b), the absorpn>tion of n>an class="Chemical">AFP 4 becomes much weaker, very likely due to its poor solubility in water. A slight blue shift occurs on AFP 3 to 254, 316, and 343 nm. No obvious shift of AFP 1 and AFP 2 was observed (λmax = 257, 301, and 336 nm for AFP 1; 257, 301, and 338 nm for AFP 2), but an absorption at 301 nm became more apparent. Besides diverse transitions between different energy levels, these multiple absorption peaks could also be explained by various conformations of AFP in solution such as the examples shown in Figure . In acetonitrile, the two conformations may stay in an equilibrium favoring AFP 2a, while in aqueous solution, the intramolecular hydrogen bond is very possibly broken owing to the surrounding water molecule, causing different distribution of absorption peaks.

Figure 4

Two possible conformations of AFP 2 in solution.

Two possible conformations of pan class="Chemical">AFP 2 in solution.

Fluorescence Emission of AFP 1 to AFP 4 in Organic and Aqueous Solutions

Fluorescence spectra of AFP 1 to n>an class="Chemical">AFP 4 in acetonitrile (Figure a) show a consistent pattern with their absorption spectra, that is, fluorescence emission spectra of AFP 1 and AFP 2 are almost the same (λem = 443 nm for AFP 1; λem = 442 nm for AFP 2) while those of AFP 3 and AFP 4 are about identical. Substitution on furo[2,3-b]pyridine with −Br displayed a red-shifted λem for AFP 3 and AFP 4 to 469 and 467 nm, respectively. These further demonstrate that the chromophore mainly locates on the furo[2,3-b]pyridine moiety. Fluorescence of AFP 1 to AFP 4 in acetone is almost identical to that in acetonitrile (Figure b). Not surprisingly, the fluorescence of all four compounds decreased when switched from acetonitrile to aqueous HEPES solution at pH = 7 (Figure c). The large diminution of the fluorescence of AFP 3 and AFP 4 is probably due to the heavy atom effect introduced by −Br. AFP 1 and AFP 2 still show fluorescent activity in aqueous solution with a blue-shifted emission wavelength λem around 380 nm and another weak emission wavelength around 486 nm for AFP 2. As mentioned earlier, the merging peak around 380 nm is very likely from some newly formed conformation in aqueous solution. AFP 1 and AFP 2 were then further tested against different pH values.

Figure 5

Fluorescence emission spectra of AFP 1 to AFP 4 in (a) acetonitrile, (b) acetone, and (c) HEPES solution (pH = 7).

Fluorescence emission spectra of AFP 1 to n>an class="Chemical">AFP 4 in (a) acetonitrile, (b) acetone, and (c) HEPES solution (pH = 7).

UV–Vis Absorption and Fluorescence Emission of AFP 1 and AFP 2 in HEPES Solution at Different pH Values

As shown in Figure a, UV–vis absorption spectra of AFP 1 in aqueous n>an class="Chemical">HEPES solution at different pH values display three absorbance bands around 257, 301, and 336 nm. Upon excitation at 335 nm, AFP 1 emits strong fluorescence centered at 378 nm under strong acidic conditions, which decreases as the pH value increases until it reaches 7 (Figure b). Very interestingly, when the solution becomes basic, its fluorescence starts to increase instead as the pH value continuously goes up and exhibits a slight red shift.

Figure 6

(a) UV–vis absorption and (b) fluorescence emission of AFP 1 and (c) UV–vis absorption and (d) fluorescence emission of AFP 2 in HEPES solution at different pH values.

(a) UV–vis absorption and (b) fluorescence emission of AFP 1 and (c) UV–vis absorpn>tion and (d) fluorescence emission of n>an class="Chemical">AFP 2 in HEPES solution at different pH values.

UV–vis absorption spectra of AFP 2 in aqueous n>an class="Chemical">HEPES solution at different pH values show three absorbance bands around 257, 301, and 335 nm as well (Figure c). Likewise, AFP 2 exhibits a similar luminescence pattern (Figure d) compared to AFP 1. Its fluorescence around 381 nm diminishes gradually, while the pH value increases until pH = 5. Under alkaline conditions, the fluorescence gradually enhances and red-shifts slightly when the pH value goes from 7 to 13. In addition, a weak fluorescent peak centered at 486 nm exists in AFP 2’s spectra among most pH ranges.

The strong fluorescence around 380 nm for AFP 1 and n>an class="Chemical">AFP 2 under acidic conditions is supposed to be from the protonation of the chromophore, namely, protonation of the pyridine ring in the furo[2,3-b]pyridine fluorophores. When the solution becomes more acidic, more pyridine N atoms are protonated, resulting in stronger fluorescence. While under basic conditions, according to the p−π conjugation of the amide, the other resonance form AFP 2e (Figure b) may be responsible for the increase of the fluorescent peak, even though it is not sure why the fluorescent peak is slightly red-shifted. Density functional theory (DFT) calculation was performed, as shown in the next part for further explanation.

DFT Calculation

To have more insight into the fluorescent mechanism in aqueous solution, calculations by density functional theory at the level of the B3LYP and 6-31+G(d,p) standard basis set were carried out. First of all, calculations on the minimized energy of protonated n>an class="Chemical">AFP 2b-d were carried out to confirm the protonation site in the molecule. According to Figure a, pyridine nitrogen is most likely deprotonated under acidic conditions since AFP 2b is the most stable species among the three. Then, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of AFP 2, its protonated form AFP 2b, and its resonance form AFP 2e were all calculated. As shown in Figure b, both the HOMO and LUMO orbitals of AFP 2 before protonation are well distributed through the molecule in aqueous solution with a relatively big HOMO-LUMO gap. However, when pyridine is protonated, the HOMO is largely shifted to the chlorophenylacetamide moiety, while the LUMO mainly stays on the aminofuropyridinium end. The intramolecular charge transfer (ICT) is very likely more efficient when pyridine N is protonated, which may cause the large enhancement of the fluorescence around 380 nm in acidic aqueous solution. As in AFP 2e, the possible major resonance form in basic solution, the HOMO-LUMO gap is also reduced compared to AFP 2, making it more close to the excitation energy. It might be responsible for the enhancement of fluorescence in basic solutions, demonstrating wide applications.[38−46]

Figure 7

(a) Minimized energies of various protonated AFP 2; (b) HOMO and LUMO orbitals of AFP 2, AFP 2b, and AFP 2e in aqueous solution.

(a) Minimized energies of various protonated AFP 2; (b) HOMO and n>an class="Chemical">LUMO orbitals of AFP 2, AFP 2b, and AFP 2e in aqueous solution.

Conclusions

In summary, we have synthesized a series of 3-amino-furo[2,3-b]pyridine-2-carboxamides in good yields. All four analogs with/without n>an class="Chemical">halogen on the furo[2,3-b]pyridine chromophore show good fluorescent activity in acetonitrile, while in aqueous solution, only the ones without −Br (AFP 1 and AFP 2) exhibit good fluorescence response. Generally, they show strong fluorescence under acidic conditions, which becomes weaker as the pH value increases. A very interesting phenomenon is that the downward trend turned into an upward trend when the pH keeps increasing into the basic range. The fluorescence properties could be explained by different conformations formed in solution under different conditions, which was further supported by DFT calculation. Therefore, we envision 3-amino-furo[2,3-b]pyridine (AFP) as a novel fluorophore and pH sensor in aqueous conditions, which could be easily attached due to its multiple functional groups.

Experimental Section

General Note

All commercial reagents were used directly without further purification. All of the reactions were carried out using standard techniques. Column chromatography was performed using silica gel (200–300 mesh) with n>an class="Chemical">ethyl acetate/petroleum ether as an eluent mixture. Solvent removal was carried out by using a RE-52AA rotary evaporator under reduced pressure. The 1H NMR and 13C NMR spectra were recorded on 400 MHz and a Bruker Avance III using d6-DMSO solutions with tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported in parts per million (ppm), and coupling constants J were indicated in hertz. HRMS was recorded on a Thermo Scientific LTQ Orbitrap Discovery spectrometer (ESI). Absorption spectra data were recorded on a UV-2550 UV–visible spectrophotometer in a quartz cuvette with a path length of 1 cm. Steady-state emission data were collected at room temperature using a Hitachi F-7000 spectrophotometer with slit widths set at 5 nm.