Fluorescent pH probes applicable in staining lysosomes in live human cells have been synthesized and characterized. These aminobenzocoumarins display large Stokes shifts, emission in the visible region, and up to 300-fold emission intensity increase upon activation in acidic conditions.

Abstract

Regulation of pH plays an essential role in orchestrating the delicate cellular machinery responsible for life as we know it. Its abnormal values are indicative of aberrant cellular behavior and associated with pathologies including cancer progression or solid tumors. Here, we report a series of bent and linear aminobenzocoumarins decorated with different substituents. We investigate their photophysical properties and demonstrate that the probes display strong pH-responsive fluorescence “turn on” behavior in highly acidic environments, with enhancement up to 300-fold. In combination with their low cytotoxicity, this behavior enabled their application in bioimaging of acidic lysosomes in live human cells. We believe that these molecules serve as attractive lead structures for future rational design of novel biocompatible fluorescent pH probes.

Introduction

Maintenance of cellular homeostasis is crucial for ensuring the viability of living cells. pH regulation plays a pivotal role in preserving the delicate balance essential for cellular functionality through the control of diverse biological processes such as cellular metabolism, tissue regeneration, and ion transport.¹ Cellular compartments exhibit varying pH levels due to differing influx and efflux of protons and differing metabolic activity. The unique functionalities of organelles hinge on the establishing and maintaining distinct pH values, influencing critical processes such as cell proliferation, cell cycle regulation,² apoptosis,³ and endocytosis.⁴ The internal pH spectrum spans from basic to highly acidic values, manifesting in both prokaryotic species and distinct subcellular compartments of eukaryotic cells.^{1a, 1b, 2, 3} In eukaryotic cells, pH typically ranges from 7.2–7.4 in cytosol and extracellular space, to 4.5–5.5 in lysosomes, whereas the mitochondrial matrix exhibits basic environment (7.9–8.1). However, deviations from these norms are indicative of various diseases. Anomalous intracellular pH serves as a hallmark in cancer progression, with acidic tumor extracellular fluid (pH 6.2–6.9) being a consistent feature in solid tumors, irrespective of genotypic or phenotypic variations.⁵ Beyond extracellular fluids, lysosomal environment in cancer cells exhibits increased acidity (pH ~3.8–4.7) compared to normal cells.^{5c, 6} Consequently, the sensing and monitoring of intracellular pH fluctuations are of paramount importance to comprehend physiological and pathological processes, explore cellular metabolisms, diagnose relevant diseases, and facilitate targeted drug delivery.⁷

Fluorescence imaging is a well-established non-invasive molecular imaging technology with important parameters being high sensitivity, selectivity, cost-effectiveness, excellent spatiotemporal resolution, and real-time, in-situ detection capabilities. Notably, lysosomal probes hold particular significance, as lysosomes are intricately linked to pH regulation.^{7i, 8} pH probes exhibit a highly sensitive fluorescence response within a narrow pH range proximal to their respective pK_a values (pK_a±1). Two distinct approaches in pH sensing are generally recognized: (1) off-on response fluorescence probes, manifesting increased emission upon alterations in the solution‘s pH.⁹ (2) Ratiometric fluorescent probes, distinguished by their dual emission spectra that undergo a transition from one state to another in response to a stimulus, provide the inherent benefit of self-calibration.¹⁰ However, the expansive emission profile of ratiometric probes presents a significant drawback, constraining their simultaneous application with other probes within a single experimental configuration in for instance fluorescent microscopy.

To date, a multitude of small molecule fluorescent pH probes, employing organic fluorophores as signaling units, have been developed.¹¹ Coumarins, a classical fluorescent dyes, have been widely used as a fluorophore moieties because of the excellent photophysical and chemical properties emphasizing a high fluorescence quantum yield, robust and enduring fluorescence emission, structural flexibility,¹² efficient cell permeability without inducing cell death,^{11e, 12a} straightforward de novo preparation,¹³ and low toxicity.¹⁴ Furthermore, these structures demonstrate large Stokes shifts substantial to prevent significant overlap between excitation and emission spectra.^12b

The past few years have witnessed progress in the development of pH probes based on two organic fluorophores, one of which was coumarin.^{11d, 11e, 12a} However, only few examples of pH probes for acidic environment based on coumarin as a sole fluorophore (Figure 1, Table S1) have been reported.¹⁵ Recently, we have reported that electron-donating substituent in the C7 position of 3-aminocoumarins (Figure 1) facilitated their application as fluorescent probes for extremely acidic environment (pH<4).^{9a, 9b} These compounds were utilized for rapid yeast vacuolar lumen staining, with working concentrations 10–100-fold lower than for commercial 7-amino-4-chloromethylcoumarin derivates (CellTracker™ Blue CMAC and CMAC-Ala-Pro).¹⁶ The short absorption and emission wavelengths typical for these intramolecular charge transfer (ICT) dyes, are the primary bottlenecks in advancing their applications. The efforts to overcome these limitations led to the extension of the C7,C3-push-pull system^{7a, 17} as well as to the development of π-expanded coumarins.¹⁸ Harnessing these observations and our previous experience,^{9a, 9b} we synthesized six new pH-responsive benzocoumarin fluorescent probes 4 a–f (Scheme 1).

Details are in the caption following the image — **Figure 1**
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Previously reported coumarin low pH probes and their comparison with the molecules reported here.

Five of them (4 a–d, 4 f) exhibit strong off-on fluorescent response at low pH compared to neutral environment (Figure 2). We showcase their applicability in staining lysosomes in live human cells, establishing them as attractive lead structures for biocompatible pH probes.

Results and Discussion

The probes 4 a–f were synthesized from o-hydroxy naphthaldehydes 3 a–e as key intermediates in Perkin type condensation that introduces the dimethylamino substituent at the position 3 with a concomitant formation of the lactone ring according to Hrnčiar and colleagues. (Scheme 1).^{19a, 19b} Commercially unavailable 1-naphthaldehydes 3 b and 3 c were prepared from corresponding naphthols 1 b or 1 c by direct formylation using Vilsmeier-Haack or Reimer-Tiemann reactions, respectively, affording the corresponding 1-formylated compound exclusively. In contrast, 2-naphthaldehydes 3 d and 3 e were synthesized by multistep protocol. Protection of the hydroxyl groups of 2-naphthol 1 a or 2,7-dihydroxynaphthalene 1 b with tetrahydropyranyl (THP) ether or methoxymethyl (MOM) ether, respectively, allowed for the Snieckus-type lithiation of 2 a, b. Treating these lithioorganic intermediates with N,N-dimethylformamide (DMF) as a formylation agent, followed by acidic hydrolysis, yields the naphthaldehydes 3 d, e with a moderate regioselectivity (e.g. 2 : 1 ratio of 2-formyl/1-formyl isomers for MOM derivatives). Perkin type reaction of aldehydes 3 with anhydrous betaine (trimethylglycine) in Ac₂O results in formation of dimethylamino derivatives 4 a–e in moderate yields. The first step of the reaction is the formation of a trimethylammonium ylide from betaine and acetic anhydride followed by Perkin type condensation with salicylaldehyde. The reaction is catalyzed by acetate anion formed in situ. We assume that the presence of acetate anion in reaction mixture can also promote the demethylation of such trimethylammonium salt as reported before.^{9a, 19c, 19d} On the other hand, use of dimethylglycine instead of betaine, neither the Mashraqui's method^19e did not led to the desired coumarins. The observed moderate yields are consistent with deactivation of the aldehyde functional group by the introduction of electron donating substituents. The use of Ac₂O as the solvent resulted in acetylation of the hydroxyl groups in 4 b-Ac and 4 e-Ac, and was efficiently removed using HCl/EtOH or SOCl₂/MeOH deacetylation protocol,²⁰ followed by treatment with methyl iodide which afforded the derivative 4 f.

Given the motivation to apply the probes in biological settings, we investigated their spectroscopic properties in detail by UV-vis absorption and fluorescence emission spectroscopies in buffered aqueous media – phosphate buffered saline (PBS, pH 7.4, 10 mM) or Briton-Robinson buffer (BRB, pH range 1.81–7.0). However, all derivatives 4 a–f exhibit decreased solubility in water, presumably due to aggregation induced by π–π stacking.²¹ To compensate for this, DMSO was used as co-solvent (5 % or 10 %). The compounds show absorption spectra typical for the coumarin chromophore with absorption maxima in 348–367 nm range, and moderate molar absorption coefficients (ϵ) of ~14–30×10³ mol⁻¹ dm³ cm⁻¹. All derivatives except for 4 e were only very weakly fluorescent at pH 7.4 due to deactivation of the excited state via intramolecular PET (photoinduced electron transfer) from electron rich amine substituent (vide supra).

Introduction of methoxy group at the C8 and C9 positions of benzo[g]coumarin 4 f and benzo[f]coumarin 4 c, respectively, causes a red-shift of both the absorption and emission maxima compared to the unsubstituted parent molecules (Table 1). The electron-donating substituent effect on absorption/emission maxima can be explained by their resonance contribution with respect to the electron-accepting 2-pyranone moiety. These changes can be further rationalized by different lengths of the conjugated system in the chromophores. In contrast to benzo[g]coumarins, benzo[f]coumarins are bent-shaped molecules with a shorter conjugated system, resulting in a decrease in the transition dipole moment. Hence these benzocoumarins 4 a, 4 c possess the emission maxima in the shorter wavelength region compared to the linear analogues 4 d, and 4 f. (Table 1). The methoxy and hydroxy substituents introduce additional equilibria in the system. Highly acidic environment can protonate these electron rich substituents in derivatives 4 b, 4 c, 4 e, 4 f, which is most likely the reason why a single isosbestic point in the titration experiments was not observed (see Figures S57–S59).

Table 1. Photophysical properties and pK_a values of probes 4.

	λ_abs. [nm]^[a]	λ_em. [nm]^[b]	ϵ_max^[a,c]	Φ_F ^[d]	ϵΦ_F^[e]	enhancement^[f]	pK_a^[g]
4 a	356	449	16 200	0.37±0.007	6 000	292	2.54±0.03
4 b	360	443	15 600	<0.02	n.d.	25	1.70±0.09
4 c	358	539	16 700	0.14±0.005	2 300	64	2.54±0.01
4 d	348	541	14 200	<0.02	n.d.	15	2.48±0.02
4 e	349	422	30 200	<0.02	n.d.	–^[i]	2.14±0.07; 3.72±0.03
4 f	367	549	16 000	0.05±0.0004	800	15	2.65±0.01

^[a] Measured in PBS (pH ~7.4). ^[b] Determined in Britton-Robinson buffer (BRB) at pH 1.81. ^[c] The molar absorption coefficient, ϵ_max/L ⋅ mol⁻¹ ⋅ cm⁻¹. ^[d] Determined for the protonated form (at pH ~1.8) by absolute method using an integrating sphere in BRB. ^[e] Brightness, ϵΦ_F/L ⋅ mol⁻¹ ⋅ cm^{−1 [f]} Ratio of emission maxima I(pH 1.8)/I(pH 7.4). ^[g] Determined in BRB at pH 1.81. ^[h] 4 e exhibits on-off behavior under measurement conditions.

We subsequently probed the effects of pH on the photophysical properties by titrating their solutions and following it by UV-vis and fluorescence emission spectroscopies. Except for 4 e, all derivatives showed a strong increase of the fluorescence with decreasing pH. The spectral change of the probes (λ_ex=350–361 nm) in various pH solutions are illustrated in Figures 3 and S51–S59. The probes were silent up to pH 4–5 and then showed a rapid increase of fluorescence with change of pH up to 1.5. Fitting these data with a sigmoidal curve provided the pK_a values of all probes 4 a–f which were in the range of 1.7–2.7 (Table 1). This behavior is consistent with blocking of the deactivating PET pathway by protonation of the electron-donating amine functionality. It should be noted that ideal values for lysosomal imaging would be higher by ~1.5, and their optimization offers room for future investigations. The only exception was the probe 4 e which behaved as on-off probe (Figure S55); i.e. displays diminishing fluorescence intensity in low pH. We attribute this difference to protonation of the carbonyl moiety in 4 e under highly acidic conditions (pH<2.1) resulting in PET quenching due to acceptor-acceptor instead of donor-acceptor character of the compound. Similar behavior was observed for structurally related hydroxy derivative in our previous study.^9b Changes with decreasing pH values could be observed also in the absorption spectra. Absorption maxima of benzo[f]coumarins 4 a–c gradually decreased and experienced a minor red shift, whereas the maxima of benzo[g]coumarin derivatives 4 d–f displayed a minor blue shift.

We then set forth to determine the absolute quantum yields of fluorescence (Φ_F) using an integrating sphere. The quantum yields of all the neutral forms of 4 a–d, 4 f were below the detection limit, corroborating their off-state at neutral pH. At low pH values, derivatives 4 a, 4 c and 4 f showed Φ_F of 37 %, 14 % and 5 %, respectively, translating to fluorescence enhancements of 292, 64 and 15-fold, respectively. The values of Φ_F for derivatives for 4 b, 4 d and 4 e at low pH remained below the detection limit of our setup. Nevertheless, we could still determine the enhancement of fluorescence even for 4 b and 4 d by direct comparison of the emission spectra intensities (integrals), which increased upon protonation 15–25-fold. Our observation that 4 f exhibits Φ_F of only 5 % was rather surprising considering that alkoxycoumarins are intensely fluorescent in water, which is why they have been used extensively as fluorescent polarity probes and biological markers.²² This phenomenon has been attributed to an inversion of the nature of the lowest excited singlet state, which is responsible for fluorescence.²³ We expect that in our case, the methoxy substituents are protonated in the highly acidic environment, which completely changes the nature of the chromophore. This notion is corroborated by the accompanying drastic changes in UV-vis spectra of 4 f compared to 4 a (Figures S57, S59), indicative of additional process. Alternatively, the low Φ_F of 4 f could be explained by aggregation-induced quenching due to decreased aqueous solubility of the extended aromatic system. Despite the largest Stokes shifts and red shifted emission maxima in benzocoumarins 4 c and 4 f, we chose not to pursue them in subsequent cellular studies due to their lower quantum yields of fluorescence. Nevertheless, the studied probes exhibit good (4 b), up to excellent (4 a) fluorescence enhancements compared to other recently published coumarin pH probes (Table S1). The scaffolds designed and synthesized by us is also advantageous because, despite the presence of electron donor atoms (N, O) in the molecule, their fluorescence at low pH is likely insignificantly affected by the presence of different metal ions and biological relevant species (Phe, Gly) as we have shown earlier on 3-aminocoumarin derivatives.^9a

Using the best performing pH probe 4 a (pK_a=2.54), we carried out a cell viability study using HeLa cells. After 24 h treatment, no sign of toxicity for concentrations up to 5 μM were observed. Higher concentrations cause non-specific toxicity with an estimated GI₅₀ >50 μM, accompanied by precipitation of the probe from the solution above c ~20 μM. Our observation validates the applicability of benzocoumarins in mammalian cell studies at these concentrations.²⁴ Encouraged by these results, we reasoned that the pH-specific “turn-on” behavior of the probe 4 a might be applicable to track acidic cellular compartments in live human cells, i. e. lysosomes. To corroborate this hypothesis, we performed a colocalization experiment using confocal fluorescence microscopy on HeLa cells in the presence of 4 a and a commercial label Lysotracker DeepRed with absorption/emission shifted in the far-red region (647 nm/688 nm). The results shown in Figure 4 indicate that probe 4 a exhibits emission signal in the locations of cells identical to Lysotracker (Figure 4D). The observed mediocre intensity results from the mediocre pK_a of probe 4 a, and consequently incomplete fluorescence turn on at the pH levels expected in lysosomes (4.5–5.5 in normal cells,^{1a, 1b, 2, 3} 3.8–4.7 in cancer cells).⁵ This precluded calculation of Pearson's correlation coefficient and thereby precise quantification of the colocalization

Conclusions

Our results show that aminobenzocoumarins are promising biosafe lysosomal pH probes with low working concentrations and rapid staining. Their simple structures are easily synthesized from simple naphthols by a multi-step synthesis. We believe that further rational engineering of this scaffold, especially in the context of pK_a values – by a combination of more basic amines (e. g. by harnessing β-silicon effect) and modifying the substituents on the aromatic rings – will make these molecules strong contenders in the field of pH-responsive fluorescent probes.

Experimental Section

Synthesis

General experimental details and synthesis of naphthaldehydes – please see Supporting Information.

General procedure. Mixture of betaine (2 equiv.) and acetic anhydride (3 mL/1 mmol of aldehyde) was heated in a sealed tube to 150 °C until the change of color to dark brown (1–2 h) was observed. Corresponding naphtaldehyde (1 equiv.) was added, and the mixture was stirred at 150 °C for 6 h, cooled down, and poured over ice. The precipitate was filtered, washed with cold water, and dried. The crude product was purified by column chromatography.

2-(Dimethylamino)-3H-benzo[f]chromen-3-one (4 a). Prepared from 2-hydroxy-1-napthaldehyde (3 a) (1.72 g; 10 mmol). The crude product was purified by column chromatography (SiO₂, hexanes : EtOAc, gradient 50 : 1 to 4 : 1) to afford product 4 a. Yellow solid, 50 % yield (1.21 g). M.p. (hexanes/EtOAc): 102.5–104.0 °C. R_f: 0.42 (hexanes:EtOAc, 4 : 1). ¹H NMR (500 MHz, DMSO-d₆) δ 8.58–8.53 (m, 1H), 8.01–7.97 (m, 1H), 7.89 (d, J=8.9 Hz, 1H), 7.67 (ddd, J=8.4, 6.9, 1.4 Hz, 1H), 7.63 (s, 1H), 7.57 (ddd, J=8.1, 6.9, 1.1 Hz, 1H), 7.50 (d, J=8.9 Hz, 1H), 3.00 (s, 6H) ppm. ¹³C NMR (126 MHz, DMSO-d₆) δ 158.1, 147.9, 137.9, 130.7, 129.1, 128.6, 128.4, 127.6, 126.1, 123.2, 116.5, 115.2, 113.6, 41.8 ppm. HRMS (ESI⁺) Calc. for C₁₅H₁₄NO₂ [M+H]⁺ 240.1025, found 240.1022.

2-(Dimethylamino)-3-oxo-3H-benzo[f]chromen-9-yl acetate (4 b–Ac). Prepared from 2,7-dihydroxy-1-naphthaldehyde (3 b) (0.40 g; 4.25 mmol). The crude product was purified by column chromatography (SiO₂, hexanes:EtOAc, gradient 100 : 0 to 2 : 1) and crystallized (methanol) to afford product 4 b–Ac. Yellow solid, 60 % yield (0.38 g). M.p. (MeOH): 146–149 °C. R_f: 0.19 (hexanes:EtOAc, 4 : 1). ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (d, J=2.2 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.91 (d, J=8.9 Hz, 1H), 7.54 (s, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.37 (dd, J=8.8 Hz, 2.2 Hz, 1H), 2.99 (s, 6H), 2.36 (s, 1H) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 169.9, 158.0, 150.1, 148.3, 137.9, 130.6, 129.4, 128.7, 128.1, 121.7, 116.3, 115.1, 115.1, 113.4, 41.8, 21.4 ppm. HRMS (ESI⁺) Calc. for C₁₇H₁₆NO₄ [M+H]⁺ 298.1079, found 298.10751.

Synthesis of 2-(dimethylamino)-9-hydroxy-3H-benzo[f]chromen-3-one (4 b). 2-(Dimethylamino)-3-oxo-3H-benzo[f]chromen-9-yl acetate (4 b-Ac) (220 mg; 0.74 mmol) was refluxed in the mixture of HCl (aq.) (36 %, 5 mL) and ethanol (2.5 mL) for 2 h. The cooled reaction mixture was poured over ice and the precipitate was filtered and dissolved in ethyl acetate. pH of the filtrate was adjusted to ~7 using NaHCO₃ (s) and extracted with ethyl acetate (3×30 mL). The combined organic phase was washed with brine and dried over MgSO₄. The volatiles were evaporated and the crude product was purified by column chromatography (SiO₂, hexanes:EtOAC, 1 : 1) to afford product 4 b. Yellow solid, yield 47 % (90 mg,). M.p. (hexanes/EtOAc): 220 °C (decomposition). R_f: 0.49 (hexanes:EtOAC, 1 : 1). ¹H NMR (500 MHz, DMSO-d₆) δ 9.99 (s, 1H), 7.84 (d, J=8.8 Hz, 1H), 7.77 (d, J=8.9 Hz, 1H), 7.68 (d, J=2.2 Hz, 1H), 7.42 (s, 1H), 7.25 (d, J=8.9 Hz, 1H), 7.14 (dd, J=8.8, 2.3 Hz, 1H), 2.98 (s, 6H) ppm. ¹³C NMR (126 MHz, DMSO-d₆) δ 158.2, 157.4, 148.6, 137.2, 130.8, 130.5, 128.6, 125.0, 118.0, 114.2, 113.5, 113.0, 105.3, 41.7 ppm. HRMS (ESI⁺) Calc. for C₁₅H₁₄NO₃ [M+H]⁺ 256.0974, found 256.09693.

2-(Dimethylamino)-9-methoxy-3H-benzo[f]chromen-3-one (4 c). Prepared from 2-hydroxy-7-methoxy-1-naphthaldehyde (3 c) (0.5 g, 2.47 mmol). The crude product was purified by column chromatography (SiO₂, hexanes:EtOAc, gradient 4 : 1 to 1 : 1) to afford product 4 c. Yellow solid, 51 % yield (330 mg). M.p. (hexanes/EtOAc): 118–119 °C. R_f: 0.25 (hexanes:EtOAc, 4 : 1). ¹H NMR (500 MHz, DMSO-d₆) δ 7.91 (d, J=8.9 Hz, 1H), 7.82 (d, J=8.9 Hz, 1H), 7.79 (d, J=2.5 Hz, 1H), 7.58 (s, 1H), 7.32 (d, J=8.8 Hz, 1H), 7.23 (dd, J=8.9, 2.4 Hz, 1H), 3.99 (s, 3H), 3.00 (s, 6H) ppm. ¹³C NMR (126 MHz, DMSO-d₆) δ 159.1, 158.2, 148.7, 137.5, 130.8, 130.2, 128.4, 125.8, 117.6, 114.3, 114.2, 113.8, 103.2, 56.2, 41.9 ppm. HRMS (ESI⁺) Calc. for C₁₆H₁₆NO₃ [M+H]⁺ 270.1130, found 270.11287.

3-(Dimethylamino)-2H-benzo[g]chromen-2-one (4 d). Prepared from 3-hydroxy-2-naphthaldehyde (3 d) (500 mg, 2.90 mmol). The crude product was purified by column chromatography (SiO₂, hexanes:EtOAc, gradient 8 : 1 to 4 : 1) to afford product 4 d. Yellow solid, 16 % yield (0.694 mg). M.p. (hexanes/EtOAc): 151.7–152.5 °C. ¹H NMR (400 MHz, DMSO) δ 8.07 (s, 1H), 7.93 (d, J=7.7 Hz, 2H), 7.79 (s, 1H), 7.49 (dd, J=7.7, 5.8 Hz, 2H), 7.11 (s, 1H), 2.93 (d, J=1.5 Hz, 6H) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 158.1, 147.9, 138.0, 132.1, 130.7, 127.9, 127.8, 126.9, 125.9, 124.9, 121.6, 117.0, 111.4, 41.8 ppm. HRMS (ESI⁺) Calc. for C₁₅H₁₄NO₂ [M+H]⁺ 240.1025, found 240.10200.

3-(Dimethylamino)-2-oxo-2H-benzo[g]chromen-8-yl acetate (4 e-Ac). Prepared from 3,6-dihydroxy-2-naphthaldehyde (3 e) (140 mg, 0.74 mmol). The crude product was purified by column chromatography (SiO₂, hexanes:EtOAc, gradient 15 : 1 to 4 : 1) to afford product 4 e-Ac. Yellow solid, 12 % yield (26 mg). M.p. (hexanes/EtOAc): 181.7–183.0 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (s, 1H), 7.98 (d, J=8.9 Hz, 1H), 7.80 (s, 1H), 7.68 (d, J=2.3 Hz, 1H), 7.28 (dd, J=8.9, 2.3 Hz, 1H), 7.10 (s, 1H), 2.93 (s, 6H), 2.33 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 169.8, 149.0, 138.0, 132.5, 129.6, 128.7, 124.9, 121.8, 121.5, 118.5, 116.8, 111.2, 41.8, 21.4 ppm. HRMS (ESI⁺) Calc. for C₁₇H₁₆NO₄ [M+H]⁺ 298.1079, found 298.10749.

Synthesis of 3-(dimethylamino)-8-hydroxy-2H-benzo[g]chromen-2-one (4 e). SOCl₂ (0,1 mL; 1.34 mmol) was added to a suspension of (3-(dimethylamino)-2-oxo-2H-benzo[g]chromen-8-yl acetate (4 e-Ac) (100 mg; 0.34 mmol) in anhydrous methanol (5 mL). The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the residue was triturated with Et₂O (2 mL) and dried to give product 4 e. Yellow solid, yield 93 % (80 mg). M.p. (Et₂O): 237 °C (decomposition). R_f: 0.50 (hexanes:EtOAc, 1 : 1). ¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (s, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.52 (s, 1H), 7.14–6.99 (m, 3H), 2.84 (s, 6H) ppm. ¹³C NMR (101 MHz, DMSO) δ 158.3, 156.6, 148.6, 136.8, 134.2, 129.9, 125.6, 125.3, 119.2, 118.8, 118.4, 109.5, 108.4, 41.9 ppm. HRMS (ESI⁺) Calc. for C₁₅H₁₄NO₃ [M+H]⁺ 256.0974, found 256.09693.

Synthesis of 3-(dimethylamino)-8-methoxy-2H-benzo[g]chromen-2-one (4 f). NaH (7 mg; 0.29 mmol) was added to a solution of 3-(dimethylamino)-8-hydroxy-2H-benzo[g]chromen-2-one (4 e) (50 mg; 0.19 mmol) in anhydrous DMF (1 mL), followed by iodomethane (28 mg; 0.19 mmol). The reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure and the residue was crystallized (heptane) to give product 4 f. Green solid, yield 30 % (16 mg). M.p. (heptane): 174.3-175.9 °C. R_f: 0.30 (hexanes : EtOAc, 4 : 1). ¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (s, 1H), 7.84 (d, J=9.0 Hz, 1H), 7.67 (s, 1H), 7.33 (d, J=2.5 Hz, 1H), 7.13 (dd, J=9.1, 2.5 Hz, 1H), 7.08 (s, 1H), 3.88 (s, 3H), 2.89 (s, 6H) ppm. ¹³C NMR (101 MHz, DMSO) δ 158.3, 158.2, 148.6, 137.3, 133.9, 129.7, 126.3, 125.1, 119.2, 119.1, 118.0, 110.4, 105.7, 55.7, 41.8 ppm. HRMS (ESI⁺) Calc. for C₁₆H₁₆NO₃ [M+H]⁺ 270.1130, found 270.11263.

Photophysical and Photochemical Measurements Methodology

UV-Vis Absorption Measurements. Absorption spectra and molar absorption coefficients were obtained on a UV-vis spectrometer with matched 1.0-cm quartz cells. The solvent used was PBS:DMSO mixture (95 : 5 (v/v) for 4 a–c or 90 : 10 (v/v) for 4 d–f).

Fluorescence Measurements and Quantum Yields. Fluorescence emission spectra were measured in BRB buffer : DMSO mixture (95 : 5 (v/v) for 4 a–c or 90 : 10 (v/v) for 4 d–f) using a fluorescence spectrometer in a 1.0 cm quartz fluorescence cuvette at 20 °C. The sample concentrations were adjusted to keep the absorbance <0.15 at the corresponding excitation wavelength. Each sample was measured five times, and the spectra were averaged. Fluorescence emission spectra were normalized and corrected by the photomultiplier sensitivity function using correction files supplied by the manufacturer and corrected for Raman scattering. The fluorescence quantum yields (Φ_F) were determined using integration sphere, each sample was measured five times using independent solutions keeping A<0.15, and the values were averaged. Three independent samples were measured and averages with standard deviation of the mean are given.

Determination of pK_a. Solution of benzocoumarins 4 a–c (c ~20 μM) in Britton-Robinson buffer (pH=1.81, 5 % DMSO, I=100 mM adjusted using KI) and 4 d–f in Britton-Robinson buffer (pH=1.81, 10 % DMSO, I=100 mM adjusted using KI) was titrated by 10 M NaOH solution and pH values were continuously monitored with a pH meter. To reach pH values below pH=1.81, concentrated H₃PO₄ (25 μL, 85 %) was added to the respective samples. Absorption and fluorescence emission spectra were recorded gradually in indicated pH range and were corrected for the dilution resulting from the addition of NaOH aliquots. The pK_a value has been calculated using the Henderson-Hasselbach type mass action equation: log(I_max–I)/(I–I_min)=pH–pK_a, where I_max, I_min, and I represent the maximum, minimum, and observed fluorescence intensity at a given pH value, respectively.

Cell Viability and Fluorescence Microscopy Experiments

Cell Viability Assays. HeLa cells were seeded in 96 well plates at a density of 4×10³ cells per well and grown to 30 % confluency for 24 hours 37 °C at 5 % CO₂ atmosphere. Half of the media (50 μL) was removed and substituted with media containing different concentrations of 4 a (DMSO stock solution with c ~ 1×10⁻² M of the parent compound was diluted with DMEM (4.5 g/L glucose, L-glutamine, 1 % pyruvate, 10 % FBS, 1 % P/S) to obtain stock solution with 1 % DMSO. This solution was further diluted with DMEM (4.5 g glucose, L-glutamine, 1 % pyruvate, 10 % FBS, 1 % P/S) to reach the final concentration of compound). The content of DMSO in wells was kept stable at 0.1 %. The cells were incubated for 24 h at 37 °C at 5 % CO₂ atmosphere after the addition of compound. The cells were then treated with resazurin (10 uL of 860 μM solution in PBS) and incubated at 37 °C at 5 % CO₂ atmosphere for 90 minutes. The emission was detected using a plate reader in fluorescence kinetic mode (ex. 560 nm, em. 590 nm, 10 points, average value taken for each well) and a plot of cell viability was obtained from three replicates. All experiments were repeated three times using cells from different passages. The cell viability was calculated using cells without the tested compounds as a reference.

Confocal Microscopy Imaging. HeLa cells were seeded in 8 well microscopy slides (ibidi) at density of 1.2×10⁴ cells per well and grown to 80 % confluency for 24 hours at 37 °C at 5 % CO₂ atmosphere. Half of the media (150 μL) was removed and substituted with DMEM (4.5 g/L glucose, L-glutamine, 1 % pyruvate, 10 % FBS, 1 % P/S, w/o phenol red) containing 4 a (c ~10 μM, prepared as for the cell viability studies) and Lysotracker DeepRed (purchased from ThermoFischer) (c ~50 nM, used according to supplier recommendations), or no compound. After 1 h of incubation at 37 °C at 5 % CO₂ atmosphere, the media was removed and PBS (300 μL) was added. The cells were placed in the confocal microscope and kept at 37 °C at 5 % CO₂ atmosphere throughout the duration of experiments. The cells were visualized using Leica DMi8 CS with AFC, Model SP8 fully motorized, inverted microscope with at 63× magnification, using coumarin fluorescence channel (exc. 405 nm with diode laser, 1.5 % intensity, 408–539 nm detection), and red channel (exc. 640 nm, 4 % intensity, 774–779 nm detection) at scanning speed 400 Hz, accumulation of 4 lines. The images were processed using ImageJ. Background noise was reduced using smoothing and sharping function and by applying removal of outliers (2 px, threshold 20).

Supporting Information

The authors have cited additional references within the Supporting Information (Ref. [25–41]). The Supporting Information includes general experimental details, synthesis of naphtaldehydes, MS and NMR spectra of compounds, absorption and fluorescence emission spectra as well as comparison of synthesized probes with known acidic pH probes with coumarin scaffold.

Acknowledgments

This study was supported by the Operation Program of Integrated Infrastructure for the project, UpScale of Comenius University Capacities and Competence in Research, Development and Innovation, ITMS2014+: 313021BUZ3, co-financed by the European Regional Development Fund. Henrieta and Martin would also like to thank the Slovak Grant Agency (VEGA 1/0804/21) for funding the study. Karin acknowledges National Scholarship Programme of the Slovak Republic for founding her stay at UZH Zurich under contract No. 41172. Peter and Hana acknowledge Swiss National Science Foundation (P.Š/PZ00P2_193425) and the Department of Chemistry, University of Zurich (H.J./UZH Candoc), for funding this research project. Peter and Hana would like to thank Prof. Cristina Nevado and Prof. Karl Gademann and (University of Zurich) for the generous support of their research. Open Access funding provided by Universität Zürich.

Conflict of interests

The authors declare no conflict of interest.

Open Research

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Supporting Information

References

Volume30, Issue28

May 17, 2024

e202400111

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pH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity

Graphical Abstract

Abstract

Introduction

Results and Discussion

Conclusions

Experimental Section

Synthesis

Photophysical and Photochemical Measurements Methodology

Cell Viability and Fluorescence Microscopy Experiments

Supporting Information

Acknowledgments

Conflict of interests

Open Research

Data Availability Statement

Supporting Information

References

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References

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pH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity

Graphical Abstract

Abstract

Introduction

Results and Discussion

Conclusions

Experimental Section

Synthesis

Photophysical and Photochemical Measurements Methodology

Cell Viability and Fluorescence Microscopy Experiments

Supporting Information

Acknowledgments

Conflict of interests

Open Research

Data Availability Statement

Supporting Information

References

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