Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions

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Article Cite This: ACS Omega 2019, 4, 5479−5485

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Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions Based on 9‑Iminopyronin Derivatives Peter Horvat́ h,†,‡ Peter Š ebej,*,‡ David Kovaŕ ,̌ ‡,§,∥ Jirí̌ Damborský,‡,§,∥ Zbyneǩ Prokop,*,‡,§,∥ and Petr Klań *,†,‡ Department of Chemistry, Faculty of Science, ‡RECETOX, Faculty of Science, and §Loschmidt Laboratories and Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic ∥ International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic Downloaded via 37.9.40.95 on March 19, 2019 at 16:38:41 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: Monitoring enzymatic activities at pH ranges compatible with their physiological optimum using fluorescent assays is important for high-throughput screening and engineering of novel biocatalysts as well as applications in biosensing and diagnostics. However, the number of fluorescent dyes exhibiting necessary optical properties at alkaline pHs is limited. Here, we report the design, synthesis, and biochemical application of fluorescent 9-iminopyronin derivatives possessing unique and distinct emission properties to provide optimal sensitivity at neutral to near-alkaline conditions. They exist in two acid−base forms, allowing for a dual-emission ratiometric sensing with a linear response of pH between 6.0 and 8.5 and detection sensitivity of a 0.06 pH unit. Selected fluorophores were used as the sensors for monitoring the enzymatic hydrolytic activity of a model enzyme haloalkane dehalogenase.

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INTRODUCTION Small-molecule organic fluorescent dyes represent a common detection platform in molecular biology, biochemistry, and medicine.1,2 State-of-the-art designs3 based on the structure− activity relationship and modern synthetic approaches allow for precise tuning of their optical properties.4 Fluorescence and colorimetric probes responding to pH have been extensively explored for a wide range of applications in science and technology. In particular, fluorescence sensors have become widespread because they are easy to implement and offer high sensitivity and specificity.5 A large number of fluorescence probes for pH measurements have been developed for in vivo studies of biological and pathological processes. These indicators typically operate at acidic to neutral pHs, useful for monitoring of intracellular pH in the range between 6.8 and 7.4 in the cytosol and between 4.5 and 6.0 in the cell’s acidic organelles.6−8 Fluorescence pH sensors are frequently used in other research fields, for example, for monitoring enzyme activity in conventional or microfluidic screening for novel industrially attractive biocatalysts either from the diverse pool of genomic databases9 or libraries constructed during directed evolution.2 The key prerequisite for sensitivity of such pH-based assays is the compatibility of the dye, buffer pKa and pH optimum of enzyme activity.10,11 However, among various fluorescence pH sensors developed in the past, there is only a limited availability of sensitive and stable probes, which are easy to operate at a neutral to alkaline pH range or applicable for wide pH-range monitoring.5,12 Such probes are particularly attractive for © 2019 American Chemical Society

applications in pH-based activity assays for numerous enzyme families that are important for industrial or medical applications, such as carboxylesterases, triacylglycerol lipases, acetylcholinesterases, ureases, and haloalkane dehalogenases (HLDs). It is worth to note that more than half of the enzymes listed in the database BRENDA are reported to operate optimally at pH 7−9.13 Therefore, the design and synthesis of fluorophores sensitive for neutral to near-alkaline conditions have significant merit. Pyronin derivatives are chemically stable fluorophores that possess high molar absorption coefficients and high fluorescence quantum yields.14−17 In the past two decades, novel pyronin/rhodamine analogues (Figure 1) with different heteroatoms (e.g., the groups 14−16 elements18−22) and functionalities in the 10 position have been developed, exhibiting considerable bathochromic shifts of the absorption and emission maxima up to the near-infrared (NIR) region.3,23

Figure 1. Pyronin (left) and rhodamine (right) chromophores. Received: February 8, 2019 Accepted: March 7, 2019 Published: March 19, 2019 5479

DOI: 10.1021/acsomega.9b00362 ACS Omega 2019, 4, 5479−5485

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There are only a few known pyronin-based fluorescent dyes with a substituted amine functionality in the 9 position, such as 9-N-arylpyronins used as sensors for peroxynitrites24 and NO,25,26 or 9-N-alkylpyronins used as cell-permanent dyes.27 Several criteria, such as favorable photophysical properties (high molar absorption coefficients, strong fluorescence, and large Stokes shifts to avoid self-quenching),6,28 good solubility in aqueous solutions, thermal and photochemical stabilities, and biocompatibility, have to be fulfilled when novel fluorescent pH indicators are designed for applications in biological systems.2 Recently introduced 9-iminopyronins have been shown to fulfill many of those criteria, as they are both thermally and photochemically stable in aqueous solutions and exhibit substantially different photophysical properties at low and high pHs, and possess pKas in the neutral to alkaline region.29 In this work, we designed and synthesized a series of (9acylimino)pyronins with the O, C(CH3)2, Si(CH3)2, and Ge(CH3)2 motifs in the 10 position with the aim of offering novel pH probes for neutral to alkaline conditions applicable in biology. We evaluated their photophysical properties and compared their sensitivity with the commercially available fluorescence pH-sensor 8-hydroxy-pyrene-1,3,6-trisulfonic acid (HPTS), previously applied in monitoring the enzymatic activity at near-alkaline conditions.30,31 We demonstrate the applicability of the selected derivatives to monitor the activity of a model enzyme, HLDs. The application of the new pH sensors compatible with the enzymes operating at alkaline conditions will enable the development of advanced sensitive biosensors,30,31 high throughput screening for novel biocatalysts,9 and sensitive microfluidic devices for advanced enzymology research and engineering.32

Figure 2. Fluorescence emission spectra of 3f (c = 5 × 10−6 mol dm−3, λexc = 500 nm) in HEPES buffer (see the Supporting Information for details) in the pH in range from 7.80 (black thick line, λem(max) = 539 nm; form B) to 5.10 (red thick line, λem(max) = 599 nm; form A) measured under constant settings at a 90° angle. Inset: pH dependence of the emission intensity ratio for 3f (c = 5 × 10−6 mol dm−3) at λem = 540 and 600 nm (λexc = 490 nm) in an aqueous solution for a series of HEPES buffers with pH in the range of 5.0− 10.0 (see Supporting Information for details), acquired using a microplate reader in a front-face mode (black squares); the Boltzmann sigmoidal function was fitted to the data (black solid line).

ionic strength I = 0.1 mol dm−3; KCl) were obtained by a global analysis of the spectra and fitting of a Boltzmann titration function to the data (e.g., Figures 2, S16, and S40; Tables 2 and S6). Enzyme Preparation. Expression of HLD LinB was achieved using Escherichia coli BL21(DE3) containing the expression vector pET21b carrying the linB gene. Bacterial cells were cultivated in a Luria-Bertani (LB) medium with ampicillin. Expression of the dehalogenase was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) (c = 0.5 mmol dm−3). The His-tagged LinB was purified using a single-step nickel affinity chromatography and transferred to the HEPES (c = 1 mmol dm−3; pH = 8.0) buffer using a HiTrap desalting column (GE Healthcare).33 The concentration of the enzyme was determined by the Bradford method.34 pH Sensor-Based Enzyme Activity Assay. The specific activity of the LinB enzyme for conversion of 1,2-dibromoethane was determined using 3a and 3f as fluorescence acid− base indicators monitoring the change in the concentrations of HBr formed upon enzymatic dehalohydration of the model substrate in the weak HEPES buffer. The enzymatic dehalogenase reaction was initiated by addition of the LinB enzyme (c(stock) = 2.163 mg mL−1; to give a reaction mixture with a final concentration of LinB: c = 10.6 μmol dm−3 and the final volume V = 4.5 mL) to the HEPES buffer (c = 1 × 10−3 mol dm−3, pH = 8.0, see the Supporting Information for further details) containing a fluorescent pH sensor (3, c = 5 × 10−6 mol dm−3) and 1,2-dibromoethane (substrate; c = 2 × 10−3 mol dm−3). The reaction mixture was kept in a 1 × 1 × 5 cm quartz fluorescence cuvette equipped with a new poly(tetrafluoroethylene) septum preventing leakage of the substrate from the cuvette. The temperate was controlled by a thermostat at T = 37.0 ± 0.1 °C. Fluorescence emission spectra (λexc = 490 nm with a 5 nm slit width) were recorded in the range of λem = 495−750 nm (measured in 2 nm steps with a 2 nm slit width) in the corresponding time intervals (the overall reaction time was 30 min) using a fluorimeter. The pH of the solution was calculated from the ratios of the emission intensities I540/I600 (Figures 3b and S18) using a titration

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EXPERIMENTAL SECTION Photophysical Properties of 3 in Methanol and Aqueous Solutions. Absorption and fluorescence emission spectra of 3 were measured in unbuffered solutions in commercial methanol (Figures S12, S22, S28, and S34), and the two major absorption bands in the visible region were attributed to the acid−base forms A and B (Figure 2). Photophysical properties of pure forms A and B of 3 were further determined in methanol solutions containing NaOH (c = 1 mmol dm−3) to convert the compounds to the imino form B (Table S3; Figures S13, S23, S29, and S35) and in the methanol solutions containing HCl (c = 1 mmol dm−3) to convert the compounds to the form A (Table S4; Figures S14, S24, S30, and S36). Absorption and fluorescence emission spectra of 3 were also measured in unbuffered aqueous solutions containing 1% dimethyl sulfoxide (DMSO) (v/v) as a co-solvent with KCl to keep the ionic strength, I, equal to 0.1 mol dm−3. HCl (c = 1 mmol dm−3) or NaOH (c = 1 mmol dm−3) was used to convert 3 to its respective acid−base forms; form A or form B (Scheme 2). The photophysical properties of both pure forms are summarized in Table 2. Determination of pKa (3a−f) from Fluorescence Emission Spectra. A series of the fluorescence emission spectra of 3a, 3b, and 3f (c = 5 × 10−6 mol dm−3) in 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffers (see the Supporting Information for details) within the pH ranges relevant for each derivative were measured either by luminescence spectrometer or using a plate reader. The acid dissociation constants (pKa; ionization quotients at the used 5480


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Figure 3. (a) Fluorescence emission intensity ratios (3f, c = 5 × 10−6 mol dm−3; black squares) obtained in an aqueous solution upon addition of HLD LinB (c = 2.163 mg mL−1) in HEPES buffer c = 1 mmol dm−3 HEPES (V = 20 μL) in one portion to a solution of 1,2-dibromoethane (c = 5 mmol dm−3, V = 0.1 mL) in the same HEPES buffer at 37 °C; (b) c(HBr) calculated from the change of emission intensity ratios (Figure 3a); (c) Concentrations of 1,2-dibromoethane (black squares) and 2-bromoethanol (red triangles) during the reaction with the LinB enzyme determined by GC under the same conditions. The solid lines represent the best fit to the data.

5% diphenylsiloxane; 30 m × 0.25 mm i.d. × 0.25 μm) was used for separations under the following temperature conditions: start at 40 °C, held for 1 min, then increased by 25 °C min−1 to 150 °C. The total chromatographic run-time was 5.40 min. Helium was used as a carrier gas (constant flow of 1.2 mL min−1). The mass spectrometer was operated in an electron ionization mode (70 eV). The ion source temperature was maintained at 250 °C, and solvent delay of 2.35 min was used. A set of eight calibration solutions of 1,2-dibromoethane and 2-bromoethanol in methanol (c = 0−2 mmol dm3) were used as standards with 1,2-dichloroethane as an internal standard (c = 1 × 10−3 mol dm−3). Analytical Sensitivity. The linear section of the sigmoidal calibration curves was determined by using OriginPro 2018b and the Quick Fit Linear regression gadget, applying the method of least squares. The slope determines the analytical sensitivity of a fluorescent probe and shows the response of the probe toward the pH change. The HPTS was chosen as a benchmark operating close to the target pH and possessing double excitation behavior. The emission of HPTS at λem = 510 nm was monitored upon excitation with two different light sources at λexc = 450 and 405 nm and the fitting of a titration function to the intensity ratio I450/I405 plot versus pH. The smallest changes in the emission ratio of I540/I600 allowing to unambiguously quantify pH change were interpreted as the limit of detection (LOD) (see the Supporting Information for details).

equation obtained from the pKa determination. The obtained pH values were then recalculated to c(HBr) formed in enzymatic dehalogenation reaction using the equation for the HEPES buffer response to HBr addition (Supporting Information). The reaction mixture without substrate was used in control experiments under the same conditions. To provide a comparison (Table 1), aliquots (V = 50 μL) were simultaneously taken by a syringe form the reaction Table 1. Specific Activity of the LinB Enzyme for Monodehalogenation of 1,2-Dibromoethane Determined Using Different Analytical Methods method 35

colorimetry GC; c(BrCH2CH2OH) 3a as a pH sensor 3f as a pH sensor

specific activity/μmol s−1 mg−1 0.133 0.063 0.374 0.124

± ± ± ±

0.008 0.003 0.005 0.007

mixture in the corresponding time intervals and immediately injected into methanol (V = 50 μL) containing 1,2-dichloroethane (internal standard; c = 1 × 10−3 mol dm−3) in crimped gas chromatography (GC) vials. These mixtures were immediately analyzed using GC/mass spectrometry (MS) to determine the concentrations of 1,2-dibromoethane and 2bromoethanol using the independently constructed calibration curves. Determination of the Concentrations of 1,2-Dibromethane and 2-Bromoethanol. A GC system coupled with a mass-selective detector equipped with a split/splitless injector and an autosampler was used. The inlet was operated at 250 °C, septum purge flow was set to 3 mL min−1, and the split ratio was set to 20:1 with the total flow of 24 mL min−1. The injection volume was 1 μL. A narrow-bore chromatographic column (stationary phase: 95% dimethylsiloxane with

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RESULTS AND DISCUSSION Pyronin derivatives are chemically stable fluorophores that possess high molar absorption coefficients and high fluorescence quantum yields.15,36−38 Many pH sensors exhibiting a high sensitivity in near-alkaline pH suffer from some drawbacks, such as low fluorescence quantum yields, high

Scheme 1. Synthesis of the Derivatives 3

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3 (form B; Table S3) obeyed a single-exponential rate law with lifetimes similar to those of other 9-iminopyronins.29 Upon addition of HCl in methanol (up to c(HCl) = 0.001 mol dm−3), the form B (spectra in Figures S13, S23, S29, and S35) was reversibly29 converted to its conjugate acid (form A; Table S4, Scheme 2; Figures S14, S24, S30, and S36). This form A retained high fluorescence quantum yields, but exhibited considerably smaller Stokes shifts (Δν̃ < 1 × 103 cm−1) as a result of a significant bathochromic shift of its λmax abs values. The acid−base properties of 3 were also studied in aqueous media containing 1% DMSO (v/v) as a co-solvent. The absorption and emission band maxima were bathochromically shifted (Figures S16, S19, S25, and S31) compared to those in methanol. Their pKas were determined from a series of absorption spectra obtained during titration at the constant ionic strength upon addition of either HCl or NaOH solutions.29 The global analyses provided species spectra identical to those determined at high and low pHs. The pKa values were also independently determined from a series of the fluorescence emission spectra (Supporting Information). The compounds 3 possess pKas in the interval of ∼6.8 for 3d to ∼8.0 for 3b (Table 2). The small difference between the values obtained from absorption and emission spectra (up to 0.5 pH unit, see Table S6 for details) can be related to different experimental concentrations, media composition, and/or instrumental limitations. Fluorescence emission spectra of the tested probes 3 (c ≈ 5 × 10−6 mol dm−3, aqueous HEPES) were recorded (Figures 2, S16, and S41) in the pH range at least 2 pH units above and below their pKas (Table 2). The plot of the emission intensity ratio at 540 and 600 nm, near the emission maxima of the forms B and A, respectively, against pH was used to construct the calibration, and the Boltzmann sigmoidal function was fitted to the data (Figures 2, S16, and S41). The pKa values obtained from emission spectra (e.g., pKa(3f)em ≈ 7.2) were usually higher by ∼0.5 pH unit than those obtained by absorption spectroscopy (e.g., pKa(3f) abs = 6.8 ± 0.1) (Table S6). The linear part of the calibration curve was analyzed by linear regression (Table S7). The sensitivities (the slopes of the linear section of the calibration curve close to the pKa) of 3a and 3f were compared to that of HPTS. It was found to decrease in the order of 3f > HPTS > 3a (Supporting Information). Although the pKa of 3a is slightly higher than that of 3f, the sensitivity and the linear range are preferably better in the case of the 3f derivative, which provides improved characteristics in comparison with HPTS. In addition, we tested the influence of temperature on the sensitivity of 3f. The

lipophilicity, or emit only in the blue region of visible light (see the Supporting Information for details, Table S10). Here, we designed and synthesized a library of novel fluorescent 9iminopyronin derivatives possessing unique and distinct emission properties with the aim of providing optimal sensitivity at neutral to near-alkaline conditions (Supporting Information). Xanthenone derivatives 139,40 were treated with trifluoromethanesulfonic anhydride to give 9-triflyloxypyronins as synthetic intermediates, which were in situ purged29 with gaseous ammonia (Scheme 1) to give 9-aminopyronin analogues 2 in good yields (82−99%). Subsequently, compounds 2 were converted to 9-acyliminopyronin analogues 3 in 74−94% yields using the corresponding carboxylic acid halides (Scheme 1). The synthesized derivatives 3 are soluble in methanol (∼3 × 10−5 mol dm−3) and relatively stable in the dark (Table S2) and upon irradiation with light-emitting diodes (λexc = 370 nm; Table S5; Figure S42). Compound 3a is reasonably soluble in aqueous solutions at pH = 6 (c(3a) ∼1.5 × 10−5 mol dm−3), but the solution becomes turbid because of precipitation when strongly basified to pH = 9 with aqueous NaOH. The derivative 3f, specifically modified by an oligoethylene glycol chain to enhance its aqueous solubility, forms aqueous solutions at pH = 6 with c < 4.0 × 10−5 mol dm−3. Physicochemical and optical properties of 3 are strongly influenced by the electronic character of the 10-position substituents. We have already demonstrated that compounds 3b, d have relatively high pKas (6.5−8) in water.29 Their absorption spectra in pure methanol possess two absorption bands, one in the region below 400 nm, attributed to the form B (conjugate base), and the other one in the region of 550− 650 nm attributed to the form A (conjugate acid) (Scheme 2, Tables S3−S4, Figures S12, S22, S28, and S34). Scheme 2. Acid−Base Equilibrium of 3 in Protic Solvents

The forms B of 3 were found to be fluorescent with λmax em between 513 and 599 nm and large Stokes shifts (Δν̃ ≈ 7−9 × 103 cm−1; Table S3). The fluorescence quantum yields were moderate to high (Φf = 0.17−0.56). The fluorescence decay of

Table 2. Absorption and Emission Properties of Acid−Base Forms of 3a−f in Aqueous Solutionsa form Ab

form Bc

compound

λmax abs /nm

λmax em /nm

Δν̃/cm−1e

λmax abs /nm

λmax em /nm

Δν̃/cm−1e

3a 3b 3c 3d 3e 3f

572 560 623 663 653 577

601 583 648 683 670 606

844 704 619 442 389 829

495 491 536 400 397 490

539 526 641 605 597 540

1649 1355 3056 8471 8438 1890

pKad 7.76 7.61 7.79 6.77 7.14 7.16

± ± ± ± ± ±

0.20 0.08 0.06f 0.06g 0.06f 0.28

a Measured in aqueous solutions containing 1% DMSO (v/v) as a co-solvent, I = 0.1 mol dm−3 (KCl). bHCl (c = 1 mmol dm−3). cNaOH (c = 1 mmol dm−3). dCalculated by global analyses of fluorescence emission spectra. eStokes shifts. fCalculated by global analyses of absorption spectra. g From the literature.29

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calibration curves obtained for 3f at 25 and 37 °C were identical; thus, it is confirmed that there is no effect of temperature within the conditions relevant for biological applications. Using the formalism of the LOD, we calculated the LOD for 3f to be 6 × 10−5 mol dm−3 Δ[H+], which is lower than that found for HPTS or 3a by a factor of ≈5 (Table S7). However, this value does not correspond to the probe detection limit of proton concentration changes; therefore, a different procedure to determine the LOD of fluorescent dualemission pH sensors was used. The LOD calculated from the smallest measurable change of the emission ratio I540/I600 (using both the signal and noise of the signal to provide the emission ratio and its uncertainty) depends on solution pH and is typically smallest near the pKa of the sensor (a sensor is the most sensitive here); for example, 3f was found to detect changes as low as ∼0.057 pH unit (at pH ≈ 7.2) and increases to ∼0.1 pH unit at both pH = 6 and pH = 9 (Table S8). In comparison, 3a was found to be less sensitive with an LOD of ∼0.072 pH unit (at pH ≈ 7.8; Table S9). To compare our results with those of sensors based on other physical characteristics, such as HPTS (dual-excitation sensor), we used the emission ratio I450/I405 as an input in a similar way and determined its LOD to be ∼0.062 pH unit (at pH ≈ 7.3; Table S9). However, the system became significantly less sensitive in regions far from the pKa value of the sensors; thus, the range of its effective use is restricted to pH 7−9. To complement these results, we calculated the pH values of calibration solutions from the Boltzmann titration function fit to the data (the emission intensities ratio dependence on the measured pH) and subtracted the pH values measured by a glass electrode. For both 3f and HPTS, the obtained residuals were ≤0.1 pH unit in the pH range of 6−9 (they exceeded 0.1 pH unit at pH ≥ 9.5; Figure S44). In the case of 3a, the residuals exceeded 0.1 pH unit at pH ≤ 7. Next, we tested the novel probes as pH sensors to monitor the activity of a model biotechnologically attractive enzyme, HLDs. HLDs catalyze substitution of alkyl halides (chlorides, bromides, and iodides) to the corresponding alkanols along with the release of a hydrohalic acid.41 The reaction progress was monitored by aliquoting samples from the reaction mixture and determination of the concentration of remaining alkyl halide and/or produced alkanol by GC/MS.42 The concomitantly released hydrohalic acid decreases the pH of a solution, which was recently used for the development of biosensors and detection stripes for analysis of warfare chemicals.30,43 The sensitivity and analytical performance of these applications are strongly limited by the properties of the available pH probes (e.g., the discrepancy in the pKa values of the probe and pH optimum of an enzyme narrow the useful analytical range). The optimal pH for the majority of reported HLD variants is 8−9.9,44 Therefore, for sensitive monitoring of the enzymatic reaction progress of representative HLD LinB from Sphingobium japonicum UT26 in this work, we selected 3a as a probe with the highest pKa, and 3f as a probe which provided the highest solubility in aqueous buffer and the lowest LOD. The enzymatic reaction was initiated by the addition of a LinB solution into a weakly buffered aqueous solution of 1,2dibromoethane. The reaction mixture aliquots were sampled for the determination of 1,2-dibromoethane and 2-bromoethanol (as a product; Supporting Information) concentrations, and the fluorescence emission spectra were measured

simultaneously in the given time intervals. An exponential function fit to the 2-bromoethanol and HBr concentration values plotted against time (Figure 3c) gave the parameters allowing the determination of the initial rate and specific activity of LinB (Table 1; see the Supporting Information for details). The specific activity of the LinB enzyme for dehalogenation of 1,2-dibromoethane was determined from the emission intensity ratios of 3a and 3f at 540 and 600 nm, respectively (Figures 3, S18, and S39, Table 1), and the results are in accordance with the previously published data.35 The parallel GC analyses of the same reaction mixture provided relatively comparable quantitative measures of enzymatic activity (Table 1). We found that a smaller value in the case of GC analyses is most probably related to an unavoidable manipulation with sample solutions of volatile compounds prior to GC measurements. Therefore, this observation further advocates the utilization of experimentally less-demanding applications of pH molecular sensors for monitoring of volatile compounds. In comparison with the conventional GC, our technique allows simple and fast pH readout and real-time monitoring of related enzymatic transformations with no need for elaborate sample manipulation and treatment.

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CONCLUSIONS We designed, synthesized, and fully characterized the library of thermally and photochemically stable fluorescent 9-iminopyronin pH sensors. They exhibit high fluorophore brightness in protic solvents and exist in two, spectroscopically very distinct acid−base forms with pKas at neutral to the near alkaline pH range (6.8−8.0). These sensors show dual emission ratiometric response at neutral and near-alkaline conditions and are emitting in the visible-light region. Such properties provide two significant advantages: (i) a highly sensitive H+ analysis with ability to detect pH changes as small as 0.06 pH unit (3f) in the pH range 6−9 (see the Supporting Information for more details) and (ii) lower requirements on the optical setup. In comparison to dual-excitation ratiometric dyes (e.g., HPTS), employment of a single light source is advantageous particularly for the development of portable biosensors and miniaturized microfluidic lab-on-a-chip devices. The excitation wavelength of the 9-iminopyronin derivatives is well compatible with the most common laser sources used in fluorescence-activated cell sorting and fluorescence microscopy. Selected compounds, successfully used as sensors to closely monitor activity of model enzyme, HLD LinB in conversion of 1,2-dibromoethane in this work, demonstrate that 9-iminopyronin dyes are promising pH sensors which can be further structurally modified in six different positions, thus allowing further fine-tuning of the sensor properties. These probes should particularly be attractive as biosensors, highthroughput systems for screening of novel biocatalysts, as well as for applications in droplet-based microfluidics and cell biology research. A broader array of xanthene-based pH probes is currently under investigation in our laboratories.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00362. 5483


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Details of materials and methods; details of synthesis; photophysical properties of fluorophores; NMR, UV− vis, fluorescence spectra; acid−base titration curves and their detailed analysis; stability determination experiments; details on analytical properties’ determination and on enzymatic dehalogenation (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.S.). *E-mail: [email protected] (Z.P.). *E-mail: [email protected] (P.K.). ORCID

Peter Š ebej: 0000-0003-0317-0630 David Kovár:̌ 0000-0002-5550-6143 Jiří Damborský: 0000-0002-7848-8216 Zbyněk Prokop: 0000-0001-9358-4081 Petr Klán: 0000-0001-6287-2742 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this work was provided by the Czech Science Foundation (P. K.: GA18-12477S; Z. P.: GA16-07965S) and the RECETOX Research Infrastructure (LM2015051 and CZ.02.1.01/0.0/0.0/16_013/0001761). P. H. was supported by the AXA Fund. In addition, we express our thanks to Lukás ̌ Maier, Miroslava Bittová, Lubomı ́r Prokeš, Aleš Stýskalı ́k, and David Š koda (all Masaryk University) for their help with the NMR and mass spectrometry measurements. Hana Moskalı ́ková is acknowledged for her assistance with enzymatic experiments, Luboš Jı ́lek for his assistance with light sources, and Ján Krausko (all from Masaryk University) for his help with the fluorescence measurements. Jiřı ́ Kalina (Masaryk University) is acknowledged for discussions on some statistical analyses.

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Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions

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