Color-Tunable Indolizine-Based Fluorophores and Fluorescent pH Sensor

A new fluorescent indolizine-based scaffold was developed using a straightforward synthetic scheme starting from a pyrrole ring. In this fluorescent system, an N,N-dimethylamino group in the aryl ring at the C-3 position of indolizine acted as an electron donor and played a crucial role in inducing a red shift in the emission wavelength based on the ICT process. Moreover, various electron-withdrawing groups, such as acetyl and aldehyde, were introduced at the C-7 position of indolizine, to tune and promote the red shift of the emission wavelength, resulting in a color range from blue to orange (462–580 nm). Furthermore, the ICT effect in indolizine fluorophores allowed the design and development of new fluorescent pH sensors of great potential in the field of fluorescence bioimaging and sensors.


Introduction
Optical fluorescence imaging for the non-invasive study of living systems has contributed to the organic fluorophore and fluorescent probe exploitation in the fields of life sciences, medicine, chemical biology, and biotechnology [1][2][3][4][5][6][7][8]. Due to a high demand for the development of novel fluorescent core skeletons, considerable progress has been made over the past few decades, which has resulted in the synthesis of various new fluorophores characterized by specific photophysical properties allowing the development of novel fluorescent probes [9][10][11][12][13]. As an example, indolizine-based organic molecules were successfully exploited in new fluorophores and fluorescent probes [14][15][16][17][18][19][20]. Synthetic methods for various types of indolizine scaffolds have led to the development of welldefined fluorophore scaffolds with an in-depth studied structure-fluorescence relationship. Figure 1a reports an emission-tunable and predictable indolizine-based fluorescent core skeleton, 9-aryl-1,2-dihydropyrrolo [3,4-b]indolizin-3-one, named Seoul-Fluor, and various fluorescent probes developed by Park et al. Perturbation of the electron density of the substituents at the C-7 and C-9 positions in Seoul-Fluor dramatically changed the emission wavelength, allowing a systematic tuning of the emission in the range 420-613 nm [21][22][23]. Moreover, computational studies provided guidelines for tuning the emission wavelength of Seoul-Fluor and developing Seoul-Fluor analogs with improved molar absorptivity [24]. More recently, Kim et al. prepared a new colorful fluorescent scaffold, named Kaleidolizine (KIz), with unique aggregation-induced emission (AIE) properties, on which the resulting fluorogenic probes were based [25,26]. However, previous studies typically focused on the synthesis of indolizine via the cycloaddition reaction of pyridinium ylide with alkenes or alkynes, with results critically influenced by the substrates [27][28][29]. Thus, most of these reactions are limited by incorporation of various functional groups at specific indolizine positions [30]. To address this, substantial efforts have been devoted to the development of straightforward synthetic methods for the incorporation of functional groups into indolizine. An indolizine-based full-color-tunable (405-616 nm) fluorophore, named C3-Indo-Fluoro, was developed by Lan et al. through the metal catalytic C-H activation of indolizine [31]. Despite the notable advances in indolizine-based fluorophores, the development of a straightforward synthetic strategy for the versatile functionalization of the indolizine core, providing convenient access to new indolizine fluorophores with unique photophysical properties, is challenging. To this end, we conceived a new synthetic route to prepare 3,7-disubstitued indolizine from the pyrrole ring (Figure 1b). The methodology reported in this study revealed a systematic tunability of the emission wavelength (462-580 nm) through the perturbation of the substituent electron density based on the intramolecular charge transfer (ICT) process [32]. Finally, the introduction of a pH-sensitive functional group into this new indolizine-based fluorescent scaffold allowed the successful development of a fluorescent pH sensor.

Results and Discussion
Based on the retrosynthetic analysis, the synthetic pathway employed to provide 3,7-disubstitued indolizine compounds is illustrated in Scheme 1. First, the SN2 reaction of 1H-pyrrole-2-carboxaldehyde (1) with ethyl 4-bromobutyrate afforded compound 2, followed by aldol condensation using sodium hydride (NaH), resulting in the cyclic compound 3. Regioselective bromination of compound 3 with N-bromosuccinimide (NBS) generated compound 4. Afterwards, the dehydrogenative oxidation with manganese dioxide (MnO 2 ) generated the indolizine compound 5 with bromo and ester functional groups at the C-3 and C-7 positions, respectively, which allow facile conversion into other functional groups. Finally, to introduce an aryl moiety into the C-3 position for the new π-conjugated indolizine, we adopted the Suzuki cross-coupling reaction. The optimized cross-coupling conditions were conventional heating in water in the presence of palladium(II) acetate (Pd(OAc) 2 ) and tetrabutylammonium bromide (TBAB) without any organic co-solvents [33]. Under the optimized reaction conditions, we successfully prepared a series of 7-aryl substituted indolizine-based fluorescent compounds with various R 1 substituents to change the electronic characteristics of the phenyl ring. Our systematic evaluation of the structure-photophysical property relationship included the maximum absorption and emission wavelengths, Stokes shifts, and quantum yields measurements (Table 1). Normalized absorption and emission spectra of each compound are shown in Figure S1 in Supplementary Materials. Interestingly, for all the functional groups introduced at the R 1 position (from an electron-donating group, EDG, such as the methoxy group, to an electron-withdrawing group, EWG, such as the cyano group), the maximum absorption and emission wavelength change was negligible. However, compound 9 bearing an N,N-dimethylamino group, which is the most electron-donating group of the series, exhibited a red shift in the emission spectra (533 nm), compared to other compounds (462-492 nm). We hypothesized that this feature originated from the ICT process between the N,N-dimethylamino group at the R 1 position and the ester group at the C-7 position of indolizine. Contrarily to other donating groups, with the ester group acting as an electron acceptor, in this electron-push-pull π-conjugated indolizine-based system, the N,N-dimethylamino group acted as a powerful electron donor, triggering an ICT process. In the electronic push-pull system in fluorescent scaffolds, the effects of the substituent electronic characteristics on the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) resulted in a red or blue shift in emission [34]. In the case of compound 9, the N,N-dimethylamino group on the phenyl ring might increase the HOMO energy level, with a minimal effect on the LUMO energy level, causing a red shift of the emission wavelength. Moreover, ICT causes a large change in the excited state dipole moment, promoting a solvent-dependent emission shift [35]. To determine the effect of ICT, we further studied the solvatochromic fluorescence emission change of compound 9. As shown in Figure 2, the solvent polarity had limited effects on the absorption wavelength. Conversely, an increase in solvent polarity induced a bathochromic shift of the emission wavelength from 485 nm in non-polar diethyl ether to 533 nm in polar methanol. This positive emission solvatochromism suggested that the ICT process was the dominant mechanism for the red-shift emission of compound 9.  Based on this observation, we designed and synthesized indolizine-based fluorescent compounds to tune the emission wavelength, which can cover a wide range of colors. As the N,N-dimethylamino group at the R 1 position could successfully reduce the HOMO-LUMO energy gap by increasing the HOMO energy level, we hypothesized that the EWG group at the C-7 position influences the stabilization of the LUMO state, allowing a red-shift emission. As shown in Scheme 2, to introduce a stronger electron-withdrawing group than the ester group at the C-7 position, compound 9 was subjected to a hydrolysis reaction in the presence of sodium hydroxide (NaOH), to afford compound 14. Compound 15 was generated through an amide coupling reaction with N,O-dimethylhydroxylamine after the activation of carboxylic acids using carbonyldiimidazole (CDI). The N-methoxy-N-methyl amide group (Weinreb amide) was converted into aldehyde or ketone after treatment with lithium aluminum hydride (LiAlH 4 ) or methylmagnesium bromide (CH 3 MgBr), to produce compounds 16 and 17, respectively. Compounds 16 and 17 bearing aldehyde and acetyl groups, respectively, which are stronger electron-withdrawing groups than ester, showed a 47 nm and 45 nm red shift of emission wavelength, respectively, compared to compound 9 (Table 1). Nevertheless, the carboxylic acid group at the C-7 position induced a 28 nm blue shift in emission (Table 1). This was reasonably due to the reduction of the electron-withdrawing strength upon the introduction of the carboxylic acid group at the C-7 position. Figure 3 clearly shows a positive solvatochromism (from 499 to 574 nm) and a reduction in fluorescence intensity under polar conditions in compound 17. These more considerable bathochromic shifts and changes in fluorescence intensity compared to compound 9 can be ascribed to the strengthening the ICT process through the introduction of more electron-withdrawing acetyl groups than the ester group at the C-7 position of the indolizine fluorophores. Overall, the synthesized new indolizine fluorophore compounds covered blue to orange-red color emission ranges (462-580 nm). The normalized emission spectra of representative compounds 12 (blue), 14 (green), 9 (yellow), and 16 (red-orange) are shown in Figure 4a. The colors, brought about irradiation at 365 nm, were visible to the naked eye, as shown in the fluorescence image ( Figure 4b).   Because ICT processes are extensively used in the design of fluorescent probe [36][37][38][39], we hypothesized that the unique features of this indolizine-based fluorescent scaffold could lead to new applications for environmental changes as a fluorescent pH sensor. Therefore, compound 14 was selected as a potential pH sensor probe, owing to its pHresponsive functional group, namely the N,N-dimethylamino group, at the R 1 position and carboxylic acid group at the C-7 position as well as considering high solubility in water solution. The protonation and deprotonation processes of these pH-responsive groups can induce changes in their electronic characteristics according to their pKa values, generating subsequent dramatic shifts in the emission wavelength. As an example, under acidic conditions, protonation of the dialkylamino group at the R 1 position can promote the blue shift of the emission wavelength through the reduction of electron-donating characteristics. Nevertheless, under neutral conditions, the deprotonation of carboxylic acid can reduce the electron-donating character, causing a red-shifted emission. To demonstrate this, the emission response of compound 14 at different pH values (3.0-7.0) was examined. We observed a blue shift of the emission wavelength from 529 nm to 473 nm and an improvement of fluorescence intensity with the pH decreasing from 7.0 to 3.0 (Figure 5a,b). This result suggested that the protonation and deprotonation of the functional groups on indolizine fluorophores perturbed the electronic states, which could control the ICT process. In neutral conditions, the dimethylamino group could strengthen the ICT process compared to protonated dimethylamino group in acid conditions, leading to a red shift of the emission wavelength and reduction of emission intensity. As shown in Figure 5c, the emission intensity at 473 nm gradually decreased at higher pH values, revealing the potential of compound 14 as a fluorescent pH sensor. The inflection point of the sigmoid curve for compound 14 was pH 4.07. Besides compound 14, the emission response of compounds 9, 12, 16, and 17 at different pH values (3.0, 5.0, and 7.0) was examined. As expected, compound 9, containing a pH-responsive element such as a dimethylamino group, clearly shows a blue shift of the emission wavelength from 522 nm to 476 nm and improvement of fluorescence intensity with the pH decreasing from 7.0 to 3.0, similar to compound 14. In addition, compounds 16 and 17 exhibit a more dramatic reduction of emission intensity with the pH increasing from 3.0 to 7.0 compared to compound 9, which is caused by strengthening the ICT process through the introduction of a more electronwithdrawing group (aldehyde and acetyl group) at the C-7 position, consistent with the findings in a previous solvatochromic study (Figure 6c,d). On the other hand, in the case of compound 12 containing cyano group at the R 1 position, the emission wavelength and intensity cannot be obviously changed depending on the pH value, which can be ascribed to less intense ICT through the introduction of an electron-withdrawing group into the R 1 position (Figure 6b). Thus, we clearly demonstrated that the ICT phenomenon in the indolizine fluorophore allowed the successful development of fluorescent pH sensors by introducing pH-responsive functional groups such as N,N-dimethylamino, revealing the potential of this new indolizine fluorophore for versatile fluorescent probes to explore biological systems and environmental changes.

Materials and Methods
All commercially available reagents were used without further purification unless noted otherwise. Dried solvents were passed through a solvent purification system equipped with activated alumina columns (glass contours). NMR analyses were carried out using a JEOL ECZ500/S1 spectrometer (500 MHz, Jeol, Tokyo, Japan). The chemical shifts of the proton ( 1 H) and carbon ( 13 C) NMR spectra were reported in parts per million (δ), with respect to the internal standard tetramethylsilane (TMS) or to the residual solvent peak (CDCl 3 , 1 H: 7.26, 13 C: 77.00; DMSO-d 6 , 1 H: 2.49, 13 C: 39.52; CD 2 Cl 2 , 1 H: 5.32, 13 C: 53.8). The multiplicities of the 1 H NMR peaks are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), quintet, m (multiplet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets), and br s (broad singlet). Signals marked with an asterisk (*) correspond to the peaks assigned to the minor rotamer conformation. The coupling constants (J) were reported in Hz. High-resolution mass spectrometry (HRMS) spectra were obtained on a compact QToF spectrometer (Bruker, Billerica, MA, USA) using the electrospray ionization (ESI) method. Low-resolution mass spectrometry (LRMS) spectra were obtained on a compact mass spectrometer (Advion, Ithaca, NY, USA) using the atmospheric pressure chemical ionization (APCI) method. To monitor the progress of the reactions, analytical thin-layer chromatography (TLC) analysis was performed using glass plates pre-coated with silica gel (60 F254, Merck, Darmstadt, Germany), and the components were observed under UV light (254 and 365 nm). Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany). Absorption spectra were recorded using a UV-3101PC spectrophotometer (Shimadzu, Kyoto, Japan) or V-770 spectrophotometer (Jasco, Easton, MD, USA). Fluorescence emission spectra were obtained with a RF-5301PC Fluorescence spectrophotometer (Shimadzu, Kyoto, Japan), and the absolute quantum yield was measured by QE-2000 (Otsuka Electronics, Osaka, Japan).

Conclusions
In summary, a novel 3,7-disubstituted indolizine-based fluorophore, whose emission wavelengths were tunable to cover a wide color range from blue to red-orange (462-580 nm), was designed and synthesized through a straightforward synthetic scheme starting from the pyrrole ring. Most of the functional groups introduced into the aryl ring at the C-3 position of indolizine did not induce changes in the photophysical properties, such as the absorption and emission wavelength. However, compound 9 bearing an N,N-dimethylamino group at the aryl ring exhibited a pronounced red-shift emission spectra (533 nm), compared to the other compounds (462-492 nm). This result was caused by the ICT process between the N,N-dimethylamino group and the ester group at the C-7 position in the indolizine scaffold. Furthermore, indolizine-based fluorophores showed more evident red-shift emission spectra (580 nm) through strengthening the ICT process by the introduction of more electron-withdrawing groups than the ester group, finally successfully leading to the potential fluorescent pH probe. Unfortunately, these newly developed indolizine fluorophores show broad emission spectra, which can cause fluorescent cross-talk problems [40][41][42]. In addition, they have absorption wavelengths in the UV and blue light range (~400 nm). UV and blue light range possess higher energy than visible light (> 400 nm), can be more damaging to the live cell, and usually cause high scattering more than other color light, which occasionally leads to a lower bioavailability [43]. Despite these drawbacks, many other advantages, such as easy access for absorption and emission tunability and predictability guided by computational studies, have rendered indolizine-based fluorophores versatile tools to generate fluorescent and fluorogenic probes. The past decade has witnessed remarkable advances in the development of new indolizine-based fluorophores. Thus, we are sure that our indolizine-based fluorophores could act as the new molecular template for the development of fluorophores with better photophysical properties and fluorescent probes, which leads to providing convenience for live-cell imaging. The following study will be reported in due course.
Supplementary Materials: The following are available online, Figure S1. Absorption and Emission Spectra of Compounds, Figure S2. 1 H and 13 C NMR Spectra.
Author Contributions: T.K. and J.K. conceived and designed the project. T.K. performed all experiments. T.K. and J.K. analyzed the data. T.K. and J.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.