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Asymmetric and Reduced Xanthene Fluorophores: Synthesis, Photochemical Properties, and Application to Activatable Fluorescent Probes for Detection of Nitroreductase

Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. “Turn on” activatable fluorescent probes, that can be turned on in response to enzymatic reactions, have been developed and prepared to reduce the high background signal of “always-on” fluorescent probes. However, the development of activity-based fluorescent probes for biological applications, using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol, and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.


Introduction
The xanthene scaffold-based fluorophores, including fluorescein, rhodamine, and their hybrid structure (rhodol), are among the most commonly used fluorophores, which have been widely applied as fluorescent probes to detect various biological and cellular processes [1,2]. Rhodol and rhodamine fluorophores in the form of activity-based fluorescent probes have attracted much interest, due to their high quantum yields in aqueous solutions, over a broad pH range and better photostability, as compared to fluorescein in detecting various cellular phenomena [3]. Previously, fluorescent probes carrying macromolecules or small-molecule ligands for targeted delivery have been developed and used in biomedical imaging. These fluorescent probes are disadvantageous, due to their "always-on" nature for imaging which lead to poor target-to-background ratios, that result from non-specific binding. Activatable "turn-on" fluorescent probes were assisted in overcoming this drawback, as they emit fluorescence only under specific conditions, such as binding to the target protein [1,4,5]. Our research group has attempted the development of nitroreductase-responsive fluorescent probes for hypoxia imaging, based on xanthene fluorophores, such as fluorescein and rhodol fluorophores, which bear a Asymmetric and reduced xanthene fluorophores based on fluorescein can be synthesized from commercially available fluorescein via the reduction and subsequent oxidation of the carboxylate group of fluorescein (Figure 2A), whereas reduced rhodol and rhodamine fluorophores can be prepared by a convergent synthesis of the xanthene moiety via the reaction of 2-(4-(dialkylamino)-2-hydroxybenzoyl)benzoic acid with resorcinol or 4-aminophenol, followed by reduction and subsequent oxidation ( Figure 2B,C) [9][10][11]14,23]. However, the current synthetic methods have limitations in effectively generating diverse asymmetric and reduced xanthene fluorophores. Not only different types of fluorophores, such as fluorescein, rhodol, and rhodamine, but also the same type of fluorophores with different substituents should be individually synthesized. Thus, we have designed a diversity-oriented strategy, that is based on continuous Pd-catalyzed cross-coupling reactions, using fluorescein as the starting material for the efficient synthesis of a series of asymmetric and reduced xanthene fluorophores ( Figure 2D).

Molecules 2019, 24, x FOR PEER REVIEW 3 of 17
Pd-catalyzed cross-coupling reactions, using fluorescein as the starting material for the efficient synthesis of a series of asymmetric and reduced xanthene fluorophores ( Figure 2D). Herein, we describe the synthesis and photochemical properties of a series of asymmetric and reduced xanthene fluorophores with fluorescein, rhodol, and rhodamine scaffolds, that enable efficient asymmetric N-functionalization or O-functionalization. Nitroreductase (NTR)-responsive fluorescent probes were developed as activatable fluorescent probes using the various reduced xanthene fluorophores, which can be applied to the design of new pro-drugs for therapeutics and imaging agents targeting hypoxia [6,23].

Chemistry
We started the synthesis of a series of asymmetric and reduced xanthene fluorophores from commercially available fluorescein, in order to produce fluorophores with synthetic advantage in Oand N-functionalization of the xanthene ring, and can be applied as an activatable fluorescent probes. The asymmetric and reduced fluorescein derivative (3) for O-functionalization was first prepared from fluorescein via dimethylation, LiAlH4 reduction, and subsequent oxidation with p-chloranil (Scheme 1) [10]. We attempted to extend our synthetic strategy to various reduced rhodafluors, including rhodol and rhodamine fluorophores, starting from 3. Asymmetric and reduced rhodol derivatives with a monoalkylamino (n-propylamino, 7), dialkylamino Herein, we describe the synthesis and photochemical properties of a series of asymmetric and reduced xanthene fluorophores with fluorescein, rhodol, and rhodamine scaffolds, that enable efficient asymmetric N-functionalization or O-functionalization. Nitroreductase (NTR)-responsive fluorescent probes were developed as activatable fluorescent probes using the various reduced xanthene fluorophores, which can be applied to the design of new pro-drugs for therapeutics and imaging agents targeting hypoxia [6,23].
The cross-coupling reaction, that has been used to prepare the reduced rhodol fluorophores was optimized by the reaction of triflate 5 with diethylamine, in the presence of conventional palladium catalysts (Pd(PPh 3 ) 4 , Pd(OAc) 2 , and Pd 2 (dba) 3 ·CHCl 3 ), ligands (BINAP, Johnphos, and Xantphos), and base (Cs 2 CO 3 and t-BuONa, data not shown). The use of Pd(OAc) 2 (10 mol%), BINAP (16 mol%), and Cs 2 CO 3 (3 eq.) in toluene at 105 • C led to the formation of reduced rhodol derivative 8 bearing a diethylamino group in moderate yield (41%). Under the same condition for the synthesis of 8, cross-coupling reactions of 5 with n-propylamine and benzophenone imine produced reduced rhodol 7, with an n-propylamino group in 20% yield and fluorophore 9, which was further hydrolyzed, using 1 N HCl in THF to give reduced rhodol derivative 12 with an -NH 2 group (55% yield over 2 steps). The reactions for reduced rhodol 7 and 8 required an excess of n-propylamine (20 eq.) and diethylamine (10 eq.), due to their low boiling points, which resulted in relatively low yields.
Next, we attempted to synthesize reduced rhodol 13 and 14 with a -OH group, which are key intermediates for the reduced rhodamine 17 and 18 with an -NH 2 group. We designed a synthetic route, using intermediate 4, bearing a methoxymethyl (MOM)-protected hydroxyl group, instead of the methoxy group (Scheme 1). Triflate 6 was synthesized in the same manner as 5, except that the first methylation was replaced by MOM protection using MOMCl. Based on the cross-coupling reaction of 5, Pd(OAc) 2 and BINAP were used to synthesize the O-MOM protected rhodol fluorophores, containing an ethylamino (10) and diethylamino (11) group, but the reactions were unsuccessful. Thus, the Pd-catalyzed cross-coupling reaction of 6 for reduced rhodols (10 and 11) was optimized using a variety of Pd catalysts (Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd(dppf)Cl 2 , and Pd 2 (dba) 3 ·CHCl 3 ) and ligands (BINAP, Xantphos, and Johnphos) (data not shown). Thus, 10 was synthesized in good yield (quant.) under the reaction conditions with Pd 2 (dba) 3 ·CHCl 3 (10 mol%), Xantphos (15 mol%), and Cs 2 CO 3 (3 eq.) in toluene at 105 • C, while 11 was prepared in a moderate yield (47%) by using Pd(PPh 3 ) 4 (10 mol%), BINAP (15 mol%) and Cs 2 CO 3 (3 eq.). O-MOM protected rhodol derivatives (10 and 11) were treated with trifluoroacetic acid to give rhodol fluorophores 13 and 14 bearing a phenolic -OH group for O-functionalization. Subsequently, we tried to synthesize reduced rhodamine fluorophores for N-functionalization. Triflation of rhodol derivative 13, using triflic anhydride, gave the desired triflate 15 in very low yield. However, triflation by phenyl triflimide [N-phenyl-bis(trifluoromethanesulfonimide)] and K 2 CO 3 in acetonitrile afforded the desired rhodol triflates 15 and 16 in moderate yields (69%, and 57%, respectively). We then performed Pd-catalyzed cross-coupling reactions of 15 and 16 to prepare reduced rhodamine fluorophores possessing an -NH 2 group for N-functionalization. The cross-coupling reaction of 15 and 16 with benzophenone imine, using Pd(OAc) 2 , BINAP, and Cs 2 CO 3 in toluene, followed by acidic hydrolysis produced the desired rhodamine fluorophores 17 and 18 in 44%, and 30% yields, respectively. Previously, various attempts were made for the synthesis of reduced xanthene fluorophores, which resulted in the synthesis of various fluorescein, rhodol, and rhodamine fluorophores from more than one synthetic scheme [9][10][11]14,23]. The continuous Pd-catalyzed cross-coupling reactions enabled the rapid synthesis of novel series of reduced fluorescein, rhodol, and rhodamine fluorophores in highly efficient and concise synthetic scheme. The library of chemically-stable reduced fluorescein, rhodols, and rhodamines could be constructed with this synthetic strategy in moderate-to high-yields. It can be applied to synthesize various reduced xanthene-based fluorophores with N-functionalization or O-functionalization, which are useful to the design and discovery of novel activity-based fluorescent probes.
Next, we investigated the reactivity of the reduced xanthene fluorophores in comparison with typical xanthene fluorophores for the O-alkylation and amide coupling reactions. We performed O-alkylation of reduced fluorescein 3 and typical fluorescein 22, using methyl iodide and benzyl bromide, under basic conditions (Scheme 2). Both methylation and benzylation of 3 produced the desired O-alkylated product in high yields (86%, and 98%, respectively), whereas 22 only gave undesired ester products, which could not be used as activatable fluorescent probes. But the amide coupling reactions of two types of rhodol fluorophores (reduced rhodol 12 and typical rhodol 23) were different from the O-alkylation reactions of 3 and 22. In the amide coupling reactions, both 12 and 23 afforded the desired amide products (21, and 26, respectively). However, under the reaction conditions, using HOBt and iPrNEt 2 in DMF, EDC-coupling of 12 exhibited a six-fold higher yield (50%) than 23 (8%). From the results of alkylation and amide coupling of the two types of xanthene fluorophores, we concluded that reduced xanthene fluorophores are more beneficial for phenolic O-alkylation and terminal N-amide coupling, compared to typical xanthene fluorophores. Scheme 2. Reactivity of reduced fluorescein and typical fluorescein fluorophores on alkylation and amide coupling reactions . Reagents and conditions: i) CH 3 I, K 2 CO 3 , DMF, rt, 86% for 19, 87% for 24; ii) benzyl bromide, DBU, acetone, rt, 98% for 20, 71% for 25; iii) EDC, HOBt, iPrNEt 2 , DMF, rt, 50% for 21, 8% for 26.
We further attempted to synthesize activatable fluorescent probes based on the reduced xanthene fluorophores for sensing nitroreductase, an enzyme that catalyzes the reduction of a nitro group to an amine via a hydroxyl amine in the presence of NADH and a representative biomarker of hypoxic cells including solid tumors [28,29] (Scheme 3). We envisioned that the reduced fluorescein (3), rhodol (12 and 14), and rhodamine (18) fluorophores will be potential candidates for activatable fluorescent probes, because they have a free -OH or NH 2 group and show strong fluorescence at physiological pH (pH~7). The only exception is 12, which shows weak fluorescence emission at pH~7, but we prepared an activatable fluorescent probe, based on 12 because the concentration of fluorophores released from activatable fluorescent probes can be calculated by the concentration-dependent calibration curve of the corresponding fluorophore. O-Alkylated NTR-responsive fluorescent probes (27 and 28), based on reduced fluorescein 2 and rhodol 14 containing an -OH group were synthesized using Ag 2 O in toluene, according to a previously reported method [6]. Reduced rhodol and rhodamine-based NTR-responsive fluorescent probes (29 and 30), bearing a carbamate linker, were prepared from reduced xanthene fluorophores 12 and 18 containing an -NH 2 group, respectively, by using 4-nitrobenzyl chloroformate and iPrNEt 2 .

Photochemical Properties and NTR Reaction
The photochemical properties of the asymmetric and reduced xanthene fluorophores, including quantum yield, concentration-dependent fluorescence emission, stability, and solvent effect, were evaluated. The quantum yields of newly synthesized fluorophores were calculated in comparison with the reference standard, fluorescein (0.1 N NaOH, Φ r = 0.85; Table 1). Most of reduced xanthene fluorophores exhibited significant quantum yields proving their promising fluorogenic nature. Reduced fluorophores with a -OH showed high quantum yield compared to fluorophore with an -NH 2 . Among all fluorophores, rhodol (13) and rhodamine (17) bearing monoethylamine showed highest quantum yields of 0.824, and 0.399 respectively, showing their strong fluorogenic nature. Next, the fluorescent emission at the maximal absorption wavelength (λ max ) for the asymmetric and reduced xanthene fluorophores in PBS (phosphate buffered saline; 10 mM, pH 7.4) was evaluated in a concentration-dependent manner ( Figure 3). The reduced fluorescein (3), rhodol (13 and 14), and rhodamine (17 and 18), containing a -OH or -NH 2 group, showed very strong fluorescence emission at physiological pH~7.4, whereas reduced rhodol 7 and 8, without the -OH or -NH 2 group, showed relatively weak fluorescence emission at pH~7.4. Reduced rhodol 8 with a -OCH 3 and -NEt 2 groups exhibited extremely low fluorescence and quantum yield, implying that an acidic hydrogen on the terminal oxygen or nitrogen atom on the xanthene ring is essential for sufficiently intense fluorescence emission in this series of fluorophores. The rhodol 12 showed exceptionally low fluorescent emission (about 2000 RFU at 5 µM) in the series, even though it contains an -NH 2 group. Of all the reduced xanthene fluorophoresm containing the novel rhodol (7, 8 and 12) and rhodamine (17) derivatives, the rhodamine 17 containing -NHEt and -NH 2 groups showed the strongest fluorescence emission (about 80,000 RFU at 2 µM; Figure 3G). However, the pH-dependent fluorescence spectra showed a different trend from physiological pH (Figure 4). Reduced rhodol fluorophores (13 and 14) showed strong fluorescence in the pH range of 5-11, indicating that it is feasible to apply them to activatable fluorescent probes for use under physiological conditions, as demonstrated previously [11,12]. On the other hand, the reduced fluorescein (3) and rhodamine (17 and 18) fluorophores exhibited, not only strong fluorescence at physiological pH, but also significantly enhanced fluorescence emission upon lowering the pH. Interestingly, reduced rhodol 7 and 12, containing a -OCH 3 at the end and mono-substituted amine (-NHPr), or free amine (-NH 2 ), at the other end exhibited very weak fluorescence at physiological pH 7.4, but showed a significant increase in fluorescence below pH 6. We then investigated the solvent effect on the fluorescence emission of the asymmetric and reduced xanthene fluorophores ( Figure 5). It is well known that the fluorescence of xanthene dyes is complicated by the presence of a solvent-dependent equilibrium, between the colored open form bearing a zwitterion, and the colorless closed lactone form in protic solvents [30][31][32]. Urano's group reported a series of reduced rhodol and rhodamine fluorophores, and concluded that the lifetime of the open form of reduced xanthene dyes is very important in determining fluorescence emission [11]. In addition to their results, we envisioned that the nucleophilicity of the benzylic alkoxide will shift the equilibrium towards non-fluorescent closed form, as the spirocyclization of the open form is induced by the nucleophilic addition of benzyl alkoxide ( Figure 1B). As observed in many S N 2 and nucleophilic addition reactions, polar protic solvents, such as water and methanol, can stabilize the nucleophile via solvation by hydrogen bonding and decrease its reactivity, whereas polar aprotic solvents, such as DMSO (dimethyl sulfoxide), which has a strong dipole moment, but cannot form H-bonds, enhance the reactivity of the nucleophile. Thus, we investigated the solvent effect on the fluorescence emission of reduced xanthene dyes, in order to evaluate the nucleophilicity of benzyl alkoxide in equilibrium between the closed and open forms (  Finally, we applied our asymmetric and reduced xanthene fluorophores as activatable fluorescent probes targeting nitroreductase. We chose four representative fluorophores, including fluorescein 3, rhodols 12 and 14, and rhodamine 18, to develop NTR-responsive fluorescent probes. Reduced rhodol 12 showed weaker fluorescence as compared to the other fluorophores, employed as NTR-responsive probes. Nevertheless, we chose 12 as a fluorophore for activatable fluorescent probe, as the concentration-dependent fluorescence calibration curve could be established, so that the concentration of the fluorophore, released from the NTR-responsive fluorescent probe, could be determined. As reported in our previous work on NTR-responsive fluorescent probes [6], xanthene fluorophores are linked to the p-nitrobenzyl group via an ether (27 and 28) or carbamate (29 and 30) moiety, and reduction of the nitro group in the probe triggers the release of the fluorophore via 1,6-rearrangement elimination of the p-aminobenzyl group, leading to a turn-on fluorescent response.
The stability of the linkage in the NTR-responsive fluorescent probes (27)(28)(29)(30) was assessed by estimating the fluorescence emission under varying temperature and pH conditions ( Figure 6). All the probes showed weak, but stable, fluorescence emissions in the temperature range 25 to 45 • C and pH range 5-13, implying that they can be applied as activatable fluorescent probes for sensing a specific protein, NTR, under physiological conditions. Exceptionally, the strong fluorescence emission of reduced rhodamine-based probe 30, containing a carbamate linkage, present in acidic pH range between 2 to 4, is the result of the fluorophore via hydrolysis under acidic conditions, which is highly correlated with the pH-dependent fluorescence spectrum of fluorophore 18 ( Figure 4H). We performed kinetic studies (1.0 µM of probes) of probes during the NTR reaction, using 10 µg/mL of this protein as a function of time, in order to investigate the activatable response of probes to NTR (Figure 7). Probe 27 and 28, which are fluorescein-and rhodol-based activatable fluorescent probes, containing an ether linkage, showed strong fluorescence responses over time in the presence of NTR ( Figure 7A). On the other hand, probes 29 and 30, bearing a carbamate linkage, which were prepared via N-functionalization of the reduced rhodol and rhodamine fluorophores containing an -NH 2 group, showed relatively weak fluorescence emission over time during the NTR reaction. We determined the concentration of the released fluorophores in the NTR reaction using the concentration-dependent calibration curves obtained for the fluorophores (Figure 7B-F). Distinctly different results were obtained from the calibration curves: Probes 27 and 29 showed the completion of the NTR reaction with almost 1.0 µM of the released fluorophore (3 and 12), whereas the concentration of the fluorophore released from probes 28 and 30, reached only 0.167 µM (14) and 0.065 µM (18) ( Figure 7B). Although, probe 29 gave 100% yield for the NTR reaction, the corresponding fluorophore 12 could not be applied as an activatable imaging probe at physiological pH due to its photochemical nature, i.e., weak fluorescence emission at physiological pH. Rhodamine-based fluorophore 18 showed enough fluorescence at physiological pH, but the corresponding NTR-responsive fluorescent probe 30 exhibited a poor release of the fluorophore, during the NTR reaction, demonstrating that 18 is not a good candidate for an activatable fluorescent probe. The reduced fluorescein 3 and rhodol 14 fluorophores, bearing an -OH group showed strong fluorescence at physiological pH, and the corresponding NTR-responsive fluorescent probes (27 and 28), also emitted strong fluorescence during the NTR reaction. However, probe 28, based on the reduced rhodol gave a relatively low yield (~17%) in the NTR reaction, as compared to probe 27. Taken together, the NTR reaction revealed that 3 and 14 are promising candidates for activatable fluorescent probes, and among all the asymmetric and reduced fluorophores studied, fluorescein 3 is the best choice for an activatable fluorescent probe.

Materials and Instrumentation
All reagents and solvents were purchased from Sigma-Aldrich Chemical Co. were used for thin-layer chromatography. 1 H and 13 C-NMR spectra were measured on a JEOL JNM-ECZ400s/L1 (400 MHz) spectrometer (Jeol, Tokyo, Japan), with CDCl 3 or DMSO-d 6 as the NMR solvent (Cambridge Isotope Laboratories, Tewksbury, MA, USA). Chemical shifts are expressed in parts per million (ppm), and the coupling constant J is reported in hertz (Hz). Chemical shifts (in ppm) in 1 H-NMR are based on the chemical shift of tetramethylsilane (δ = 0 ppm) in CDCl 3 as an internal standard. The chemical shifts in 13 C-NMR are reported in ppm relative to the centerline of the triplet at 77.0 ppm observed for CDCl 3 or 39.5 ppm for DMSO-d 6 . All in vitro enzyme assays were performed by recording the absorbance and emission using a Synergy™ H1 microplate reader from BioTek Instruments (Winooski, VT, USA). Nitroreductase from Escherichia coli and NADH were purchased from Sigma-Aldrich Chemical Co. The lyophilized nitroreductase powder was dissolved in deionized water, fractionated, and immediately stored at −80 • C.

General Synthetic Procedures
3.2.1. General Procedure A: Alkylation K 2 CO 3 (2.5 eq.) was added to a solution of fluorescein (1.0 eq.) in DMF, and the reaction mixture stirred at rt for 1 h under N 2 atmosphere. Methyl iodide or chloromethyl methyl ether (3.0 eq.) was added dropwise to the reaction mixture, using a syringe pump with the rate of 5 mL/1hr, and the reaction mixture stirred at rt for 3-12 h. After completion of the reaction, ice water was added to the reaction mixture and stirred at 0 • C for 30 min. The resulting yellow solid was filtered (in the case of the MOM protection reaction, extraction was performed using ethyl acetate) and washed with water to completely remove K 2 CO 3 . The resulting solid was dried or purified by column chromatography to afford the desired compound.

General Procedure B: LiAlH 4 Reduction and p-Chloranil Oxidation
To a solution of compound 1 or 2 (1.0 eq.) in anhydrous THF was added LiAlH 4 (2.0 eq.) at 0 • C. The reaction mixture was stirred at 0 • C for 4 h. After completion of the reaction, sodium sulfate decahydrate (5.6 eq.) was added to the reaction mixture at 0 • C and then stirred at rt for 30 min. The reaction mixture was filtered through a short pad of Celite, which was washed with CH 2 Cl 2 . The filtrate was concentrated in vacuo, and the crude product used in the next reaction without further purification. The crude compound, obtained from the LiAlH 4 reduction, was dissolved in MeOH, followed by the addition of p-chloranil (3.0 eq.), and stirred at rt for 2 h. The reaction mixture was filtered, and the filtrate concentrated in vacuo. The residue was purified by flash column chromatography on silica gel to give the desired product.

General Procedure C: Triflation
To a solution of the phenol derivative (1.0 eq.) in anhydrous CH 2 Cl 2 or CH 3 CN was added pyridine, or K 2 CO 3 (4.0 eq.), respectively, and the reaction mixture stirred at 0 • C for 20 min. Triflic anhydride or N-phenyl-bis(trifluoromethanesulfonimide) (2.0 eq.) was slowly added to the reaction mixture over 30 min, and then, the mixture was allowed to warm to rt and stirred for 3 h. The reaction was quenched with water and extracted with CH 2 Cl 2. The organic layer was washed with aqueous 1 N HCl solution or saturated NH 4 Cl aqueous solution, followed by water and brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel to give the desired product.

General Procedure D: Pd-Catalyzed Cross Coupling Reaction
All glassware was dried in an oven before use. To a solution of the corresponding triflate (1.0 eq.) and amine (10 or 20 eq.) or benzophenone imine (1.2 eq.) in anhydrous toluene were added Pd(OAc) 2 , Pd(PPh 3 ) 4 , or Pd 2 (dba) 3 .CHCl 3 (0.1 eq.), BINAP or Xantphos (0.16 eq.), and Cs 2 CO 3 (3.0 eq.), and the reaction mixture was heated at 105 • C for 4-12 h under N 2 atmosphere. After completion of the reaction, the mixture was filtered through a short pad of Celite and washed with CH 2 Cl 2 . The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel to give the desired product.

General Procedure E: MOM-Deprotection
To a solution of the corresponding MOM protected compound (100 mg) in anhydrous CH 2 Cl 2 (1 mL) at 0 • C, a solution of trifluoroacetic acid in CH 2 Cl 2 [1 mL, TFA:CH 2 Cl 2 = 1:1 (v/v)] was slowly added dropwise at 0 • C. After the addition of TFA was complete, the reaction mixture was allowed to warm to rt and stirred at rt for 1 h. The reaction was quenched with 1 N NaOH solution and extracted with CH 2 Cl 2 . The organic layer was dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel to give the desired product.

Determination of Fluorescence Quantum Yield
The quantum yield (Φ s ) of the newly synthesized reduced xanthene fluorophores was determined by comparing the integrated area under the curve of the sample, excited at 490 nm, with the reference fluorophore. Fluorescein (Φ r = 0.85, 0.1 N NaOH) is used as the reference fluorophore for determining the quantum yield of reduced xanthene fluorophore. Absorption spectra and fluorescence spectra of fluorophores were recorded on Synergy H1 TM microplate reader (BioTek Instruments, Winooski, VT, USA) and FluoroMate FS-2 fluorescence spectrometer (Scinco, Seoul, Korea), respectively. The absorbance values are kept below 0.1 in order to minimize re-absorption effects. Fluorescence quantum yield (Φ s ) of reduced xanthene fluorophores was calculated by using following Equation (1):

Concentration-Dependent Fluorescence Study of Fluorophores
A concentration-dependent study was performed by incubating different concentration, such as 0.5, 1, 2, and 5 µM of the fluorophore in PBS (pH 7.4) at 25 • C, and recording the fluorescence spectra at each concentration in 96-well microplate using Synergy H1 reader.

Effect of pH on Fluorescence Intensity of Fluorophores
A pH-dependent fluorescence change of fluorophore was performed by incubating 1 µM of the probe in a range of pH buffer solutions (pH 2 to 13) at 25 • C, and recording the fluorescence at each pH in 96-well microplate, using Synergy TM H1 (BioTek Instruments).

Solvent Effect on the Fluorescence Emission of Fluorophores
The effect of solvent polarity on the fluorescent intensity was measured by incubating 1 µM of the fluorophore in different solvents, including water, methanol, ethanol, isopropanol, and DMSO. The fluorescence was measured at the respective excitation wavelength of the fluorophore, in a 96-well microplate, using Synergy TM H1 (BioTek Instruments).

In vitro Nitroreductase Assay
All spectroscopic readings were recorded on Synergy TM H1 (BioTek Instruments) using a 96-well microplate. The NTR reaction was performed in a total volume of 200 µL with the addition of 100 µL of PBS (10 mM, pH 7.4), 10 µL of probe stock solution (20 µM in DMSO), 20 µL of NADH solution (5 mM in 0.01 M aq. NaOH), and an appropriate volume of NTR solution (10 µg/100 µL in distilled water). The final volume was adjusted to 200 µL using PBS. The plate was incubated at 37 • C for an appropriate length of time with continuous shaking, and the emission spectra were recorded at the respective wavelengths with respect to time to prepare kinetic graph.

pH and Thermal Stability of Fluorescent Probes
A temperature-dependent assay was performed by incubating 1 µM of the probe in PBS (pH 7.4) at different temperatures for 20 min, and recording the fluorescence at each temperature. A pH-dependent study was performed by incubating 1 µM of the probe in a range of pH buffer solutions (pH 2 to 13) at 25 • C and recording the fluorescence at each pH in 96-well microplate, using Synergy TM H1 (BioTek Instruments).
All synthetic procedures, 1H-NMR, 13C-NMR and HRMS of all compounds can be seen in the Supplementary Materials.

Conclusions
In conclusion, we developed a highly efficient and versatile synthetic route to a series of asymmetric and reduced xanthene fluorophores, including fluorescein, rhodol, and rhodamine derivatives, which are representative xanthene scaffold-based dyes, and employed them as activatable fluorescent probes for sensing nitroreductase. A variety of asymmetric and reduced xanthene fluorophores, bearing an -OH or -NH 2 group, capable of Oor N-functionalization to prepare activatable fluorescent probes, were synthesized from commercially available fluorescein by continuous Pd-catalyzed cross-coupling reactions. Their photochemical properties, including quantum yields, fluorescence emission under various conditions (variation in concentrations, pH, and solvents) were characterized. Two fluorophores, including fluorescein (3) and rhodol (14) bearing an -OH group for O-functionalization, and two other fluorophores, including rhodol (12) and rhodamine (18), containing an -NH 2 group for N-functionalization were subjected to develop NTR-responsive fluorescent probes. These probes exhibited turn-on fluorescence by releasing the fluorophore in the NTR reaction. This work demonstrates that the asymmetric and reduced xanthene fluorophores synthesized using our strategy are useful in developing activatable fluorescent probes for diverse biological applications under physiological conditions.