A Cell Trappable Methyl Rhodol-Based Fluorescent Probe for Hydrogen Sulfide Detection

Abstract

Hydrogen sulfide is a biologically important molecule and developing chemical tools that enable further investigations into the functions of H₂S is essential. Fluorescent turn-on H₂S probes have been developed for use in cellulo and in vivo, but the membrane permeability of these probes can lead to probe leakage and signal attenuation over time. Here we report a cell trappable fluorescent probe for H₂S, CT-MeRhoAz, which is based on a methylrhodolazide scaffold derivatized with an acetoxymethyl ester group. Prior to ester cleavage, the CT-MeRhoAz probe generates a 2500-fold turn-on response to H₂S, which is enhanced to a 3000-fold response for the carboxylic acid form of the probe. Additionally, the probe is highly selective for H₂S over other biologically relevant sulfur, oxygen, and nitrogen-based analytes. Live cell imaging experiments confirmed the biocompatibility of CT-MeRhoAz and also that it is cell trappable, unlike the parent MeRhoAz scaffold.

Graphical Abstract

We report a bright cell-trappable fluorescent probe for H₂S that is based on a methylrhodol azide scaffold. Reaction with H₂S generates a >2500-fold fluorescence turn-on and the probe shows excellent selectivity for H₂S over other biological thiols.

Introduction

Hydrogen sulfide (H₂S) and related reactive sulfur species (RSS) are highly involved in the health and homeostasis of humans, animals, bacteria, and other organisms.^[1] H₂S is one of three commonly recognized gasotransmitters and is produced endogenously by numerous processes, including the enzymatic breakdown of cysteine by cystathionine γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST).^[2] Decreased H₂S levels have been observed in various diseases, such as Huntington’s disease, asthma, and Crohn’s disease.^[3] Whether the downregulation of H₂S production is a contributing cause or resulting symptom of different diseases remains an ongoing question. For example, the mutation that causes Huntington’s disease also causes a decrease in brain CSE, which leads to decreased cysteine and H₂S production and ultimately increases oxidative stress and damage.^[4] Disentangling the separate impacts of genetic mutation, oxidative damage, and Hcy/H₂S levels remains a challenge.

Aligned with the rapid expansion of the roles of H₂S and other RSS in biology, the last decade has seen a significant increase in the available tools for H₂S detection. These approaches can broadly be divided into two classes: those that allow for bulk H₂S measurement and quantification (e.g. colorimetric assays, H₂S-selective electrodes, electrophilic trapping, etc.) and those that allow for in cellulo or in vivo imaging (e.g. fluorescent probes).^[5] Fluorescent probes for H₂S have been developed based on a number of strategies including electrophilic reactions with H₂S, azide reduction, metal precipitation, and other methods.^[6] Of these approaches, azide reduction has emerged as the most commonly used approach, in part due to the ease of modifying chromophores with fluorogenic amines into azides and the established mechanism of H₂S-mediated azide reduction.^[7] Such approaches have generated a broad array of fluorescent probes with different properties, including different emission wavelengths, sensitivities, and targets.^[8]

One challenge for many fluorescent probes, including those developed for H₂S, is that most small molecule probes are freely membrane permeable and can diffuse both into and out of cells, making signals less reliable over longer time course experiments. A common approach to address this limitation is to append ester motifs to small molecule probes, which can be cleaved by intracellular esterase enzymes and converted into the corresponding carboxylate. The resulting negative charge of the carboxylate at physiological pH makes the probe cell impermeable, thus trapping the probe within the cell. Despite the high fidelity of this approach to impart trappability in other activity-based probes, relatively few examples of cell trappable H₂S fluorescent probes have been reported. These include BODIPY-, rhodamine-, and fluorescein-based fluorescent probes that incorporate acetoxymethyl ester (AM) groups^[9] to impart cell trappability.^[10] Key examples of azide-based cell trappable probes based on rhodamine fluorophores include SF5-AM and SF7-AM, developed by the Chang group in 2013,^[10a] and Rho630-AM-H₂S, developed by the Kong group^[10b] in 2019 (Figure 1). These platforms generated cell-trappable azide-based probes for H₂S detection and provide moderate fluorescence turn-on responses (4–20 fold) upon probe activation.

Figure 1.

(a) Prior rhodamine-based cell-trappable H₂S probes based on azide reduction and associated fluorescent responses for H₂S (F/F₀). (b) This work reports a cell-trappable methyl rhodol azide-based H₂S probe, CT-MeRhoAz.

Inspired by this approach, we viewed the addition of an enzyme-cleavable ester group onto a bright, otherwise non-trappable H₂S probe could increase the usefulness of this platform. In prior work, we reported the azide-based H₂S fluorescent probe azidomethylrhodol (MeRhoAz), which has been used to detect endogenous H₂S both in cells and also investigate H₂S in larval zebrafish.^[11] The azide motif in MeRhoAz locks the fluorophore in the closed non-fluorescent lactone form until the azide is reduced to an amine to generate the fluorescent quinoidal form of the dye. Notably, MeRhoAz showed a 1200-fold fluorescence turn-on with H₂S and minimal unwanted photoactivation when compared to other common fluorogenic platforms. Building from the MeRhoAz platform, we report here a cell trappable methylrhodolazide probe (CT-MeRhoAz) by addition of an acetoxymethyl ester to the 5(6)-position of the benzene ring. The CT-MeRhoAz fluorescent probe for H₂S shows a high fluorescence response for H₂S, excellent selectivity against other reactive species, and enhanced cell trappability when compared to the parent MeRhoAz platform.

Results and Discussion

To prepare CT-MeRhoAz, we first methylated 5(6)-carboxyfluorescein with methyl iodide to form the trimethylated precursor 1 (Figure 2). Basic hydrolysis of 1 removed the methyl esters to afford 2, and the carboxylic acid was converted to the desired acetoxymethyl group using bromomethyl acetate under basic conditions to form 3. Treatment of 3 with PhN(Tf)2 afforded triflate 4, which was converted to CT-MeRho using a Buchwald-Hartwig amination using Ph₂C=NH as the initial coupling partner followed by acidic hydrolysis. Subsequent azidification using tert-butyl nitrite followed by trimethylsilyl azide addition afforded CT-MeRhoAz. Each step of the CT-MeRhoAz synthesis is moderate to high yielding (42–98%), which allows for relatively simple preparation of the final probe in good overall yield.

Figure 2.

Synthesis of CT-MeRhoAz. Structures are shown in the closed lactone tautomer for simplicity.

Having prepared CT-MeRhoAz, we next measured the spectroscopic properties of the probe in 50 mM PIPES buffer (100 mM KCl, pH 7.4). As expected, prior to reduction of the azide, CT-MeRhoAz is only minorly absorptive (λ_ex = 286 nm, ε = 15,400 M⁻¹ cm⁻¹) and non-fluorescent in the visible region due to the fluorescein unit being locked in the closed lactone tautomer (Figure S1). By contrast, CT-MeRho shows a prominent absorbance band in the visible region (λ_ex = 480 nm, ε = 26,800 M⁻¹ cm⁻¹) with a strong fluorescence (λ_em = 514 nm, θ = 0.45), exemplifying the fluorescence turn-on of this system (Figure S2). Treatment of CT-MeRhoAz (5 μM) in 50 mM PIPES buffer (100 mM KCl, pH 7.4) with excess H₂S (NaSH, 250 μM, 50 equiv.) at 37 °C and monitoring from 490–650 nm over 90 minutes resulted in a 2500-fold fluorescence turn-on (Figure 3). The detection limit of the probe was also calculated as 160 ± 30 nM using the blank + 3σ method (Figure S3, Table S1). We also tested the fluorescent response of CT-MeRhoAz in the presence of both H₂S and porcine liver esterase (PLE). When CT-MeRhoAz was treated with only PLE (10 U mL⁻¹) for 90 minutes, a minimal 16-fold enhancement was observed, which confirmed that PLE alone does not lead to a significant false signal increase (Figure S4). Treatment of CT-MeRhoAz with H₂S (NaSH, 250 μM, 50 equiv.) in the presence of PLE (10 U mL⁻¹) resulted in a 3000-fold turn-on response over the course of 90 minutes (Figure S4), which highlights the overall dynamic range of the carboxylate form of the probe.

Figure 3.

Fluorescence turn-on of CT-MeRhoAz (5 μM) in response to H₂S (250 μM NaSH, 50 equiv.) in 50 mM PIPES buffer (100 mM KCl, pH 7.4) at 37 °C. λ_ex = 480 nm, λ_em = 490–650 nm. Slit width: 0.4 mm. The experiments were performed in triplicate and results are expressed as mean ± SD (n = 3).

Having confirmed the response of CT-MeRhoAz with H₂S, we next investigated the selectivity of the probe against other biologically relevant analytes over a 90-minute incubation period at 37°C. We tested the fluorescent response of CT-MeRhoAz against (250 μM, 50 equiv.) of L-cysteine, DL-homocysteine, and glutathione (5 mM) as well as the analytes S₂O₃ ^2–, SO₃ ^2–, SO₄ ^2–, H₂O₂, NO, O₂ ^–, and NO₂ ^– (Figure 4). No significant response was observed in the presence of any of these competing analytes, which is consistent with the significant body of literature reporting the selectivity of azide reduction for H₂S. Additionally, in the presence of 5.0 mM glutathione, only a 16-fold turn-on was observed, which further highlights the selectivity of this platform.

Figure 4.

Selectivity of CT-MeRhoAz (5 μM) in the presence of competing analytes (250 μM, 50 equiv.) including NaSH, L-cysteine, DL-homocysteine, glutathione, Na₂S₂O₃, Na₂SO₃, Na₂SO₄, H₂O₂, KO₂, DEA NONOate, NaNO₂, and 5 mM glutathione, respectively. λ_ex = 480 nm, λ_em = 490–640 nm. The experiments were performed in triplicate and results are expressed as mean ± SD (n = 3).

After confirming the response and selectivity of CT-MeRhoAz, we next investigated H₂S detection in live cells. We first incubated HeLa cells with either vehicle or aminooxyacetic acid (AOAA, 20 μM) for 45 minutes. AOAA is a CBS/CSE inhibitor and reduces endogenous production of H₂S.^[12] Next, cells were treated with 5 μM CT-MeRhoAz. After incubation for 45 minutes, the cells were treated with either the vehicle or 200 μM NaSH and incubated for 45 minutes and then imaged by fluorescence microscopy (Figure 5). As expected, treatment with AOAA resulted in a reduction in background fluorescence, which is consistent with detection of endogenous H₂S by the probe. Similarly, treatment with NaSH resulted in a significant increase in fluorescence, confirming that CT-MeRhoAz can image H₂S in live cells.

Figure 5.

Fluorescence imaging of CT-MeRhoAz in HeLa cells. Cells were incubated with 5 μM CT-MeRhoAz for 45 minutes with either (a) vehicle, (b) pre-treatment with 20 μM AOAA for 45 minutes, or (c) post-treatment with 200μM NaSH for 45 minutes and then imaged. Bar scale: 50 μm.

We next compared stability of signal from CT-MeRhoAz and the non-cell trappable MeRhoAz to confirm addition of the acetoxymethyl ester group imparted cell trappability to the probe. HeLa cells were first incubated with the H₂S donor AP39^[13] (100 nM) for 1 hour followed by incubation for 45 minutes with 5 μM of either CT-MeRhoAz or MeRhoAz (Figure 6). After the initial washing to remove any extracellular probe, the media was replaced every 10 minutes over the course of 1 hour. Cell images were acquired at 5, 30, and 60 minutes. Substantial loss of signal due to probe diffusion was observed for MeRhoAz, with minimal signal remaining at the end of the 60-minute time period. By contrast, using the same washing procedures, the signal from CT-MeRhoAz is retained after 60 minutes, confirming that the probe was trapped inside of the cells. Quantification of the cellular fluorescence further emphasized the attenuation of fluorescent signal CT-MeRhoAz displayed over MeRhoAz, with 53.5% of the initial fluorescent signal being retained for CT-MeRhoAz whereas MeRhoAz retains <0.01% of the original signal (Figure S9). Taken together, these cell experiments establish that CT-MeRhoAz is a cell trappable probe and can be used to detect H₂S in a cellular environment.

Figure 6.

Cell trappability experiments of (a) CT-MeRhoAz and (b) MeRhoAz in HeLa cells. Cells were incubated with AP39 (100 nM) for 1 hour and then incubated with the probe (5 μM) for 45 minutes. Cells were washed with fresh media every 10 minutes and imaged at 5, 30, and 60 minutes. Bar scale: 50 μm.

Conclusion

We demonstrated that addition of an acetoxymethyl ester to the bright MeRhoAz scaffold imparts cell trappability to the CT-MeRhoAz probe. When treated with excess H₂S, CT-MeRhoAz generates a 2500-fold turn-on response over the course of 90 minutes, and a 3000-fold response with concurrent cleavage of the ester group with PLE. When compared to other cell trappable fluorescence probes for H₂S, CT-MeRhoAz has a greater fluorescence turn-on and is synthetically easier to prepare.^[10] The selectivity profile against other biologically-relevant sulfur, nitrogen, and oxygen species is excellent, with a maximal turn-on of 16-fold observed for 5.0 mM GSH, which is significantly lower than the response to H₂S. Additionally, cell imaging experiments demonstrated that CT-MeRhoAz can respond to H₂S in a cellular environment and also confirmed enhanced cell trappability when compared to the parent MeRhoAz probe. In conclusion, CT-MeRhoAz is a bright, cell trappable fluorescent probe for H₂S with excellent selectivity that should be useful to advance imaging work related to H₂S detection in complex biological environments.

Experimental Section

Methods and Materials:

Reagents were purchased from Sigma-Aldrich, Tokyo Chemical Industry (TCI), Fisher Scientific, Combi-Blocks, and VWR and used directly as received. AP39^[14] and MeRhoAz^[11] were prepared as described in the literature. Silica gel (SiliaFlash F60, Silicycle, 230−400 mesh) was used for column chromatography. Deuterated solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, Massachusetts, USA). ¹H and ¹³C{¹H} NMR spectra were recorded on Bruker 500 MHz NMR or Bruker 600 MHz NMR instrument at the indicated frequencies. Chemical shifts are reported in ppm relative to residual protic solvent resonances. Compounds 1–4, CT-MeRho, and CT-MeRhoAz are a combination of the 5- and 6-carboxyfluorescein isomers, which is reflected in the ¹H and ¹³C{¹H} NMR spectra. Mass spectrometric measurements were performed by the University of Illinois, Urbana Champaign MS facility or on a Xevo Waters ESI LC/MS instrument. UV−visible spectra were acquired on a Cary 60 spectrometer and fluorescence spectra were obtained on a Quanta Master 40 spectrofluorometer (Photon Technology International).

Synthesis of CT-MeRhoAz:

Compound 1.

5(6)-Carboxyfluorescein (1.02 g, 2.71 mmol, 1.0 equiv.) and potassium carbonate (1.12 g, 8.13 mmol, 3.0 equiv.) were added to 3 mL of DMF, after which methyl iodide (0.67 mL, 11 mmol, 4.0 equiv.) was added dropwise. The reaction mixture was stirred at room temperature for 21 hours, diluted with a saturated sodium bicarbonate solution (50 mL), and then extracted into ethyl acetate. The organic phase was washed with brine and then dried with MgSO₄. After removal of solvent under reduced pressure, the crude product was purified by column chromatography (1–2% methanol in DCM as the eluent) to afford 1 as an orange solid (1.06 g, 93.3%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.72 (s, 1 H), 8.38 (d, J = 7.9 Hz, 1 H), 8.33 (m, 3 H), 8.00 (s, 1 H), 7.69 (d, J = 7.9 Hz, 1 H), 7.26 (s, 2 H), 6.88 (s, 4 H), 6.83 (dd, J = 9.8 Hz, 2 H), 6.40 (d, J = 9.7 Hz, 2 H), 6.27 (s, 2 H), 3.98 (s, 3 H), 3.93 (s, 6 H), 3.90 (s, 3 H), 3.65 (s, 3 H), 3.62 (s, 3H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 184.2, 184.1, 165.5, 165.4, 165.1, 164.8, 164.6, 158.8, 158.8, 154.2, 154.1, 149.5, 139.0, 134.7, 133.9, 133.9, 133.9, 132.1, 131.9, 131.7, 131.6, 131.5, 131.0, 130.9, 130.8, 130.6, 129.8, 129.4, 129.4, 117.4, 117.0, 114.7, 114.4, 114.2, 105.2, 105.1, 101.1, 56.8, 53.3, 53.2, 53.2, 53.1. HRMS m/z [M + H]⁺ calcd. for [C₂₄H₁₉O₇]⁺ 419.1125; found 419.1136.

Compound 2.

Fluorescein derivative 1 (1.00 g, 2.44 mmol, 1.0 equiv.) was added to a mixture of 40 mL of methanol and 40 mL of 1.0 M NaOH. The reaction mixture was stirred for 18 hours, and then the methanol was removed under vacuum. This solution was then acidified to a pH 4.0 with 1.0 M HCl and then extracted into ethyl acetate. The organic phase was washed with brine and then dried with MgSO₄. After removal of solvent under reduced pressure, compound 2 was isolated as a yellow solid (910 mg, 97.5%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 10.22 (s, 2 H), 8.41 (s, 1 H), 8.31 (d, J = 8.1 Hz, 1 H), 8.24 (d, J = 8.0 Hz, 1 H), 8.13 (d, J = 8.0 Hz, 1 H), 7.67 (s, 1 H), 7.41 (d, J = 8.0 Hz, 1 H), 6.96 (s, 2 H), 6.72 (s, 5 H), 6.64 (dd, J = 2.0 Hz, 2 H), 6.58 (dd, J = 9.0 Hz, 2 H), 3.83 (s, 6 H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 168.3, 166.8, 161.7, 160.2, 156.6, 153.2, 152.4, 152.3, 152.2, 137.8, 136.6, 133.4, 131.5, 129.8, 129.8, 129.7, 129.7, 129.6, 127.1, 126.0, 125.8, 125.1, 124.9, 113.4, 112.5, 110.8, 110.8, 109.3, 109.2, 102.7, 101.3, 83.5, 56.2. HRMS m/z [M + H]⁺ calcd. for [C₂₂H₁₅O₇]⁺ 391.0812; found 391.0813.

Compound 3.

Fluorescein derivative 2 (910 mg, 2.31 mmol, 1.0 equiv.) was added to 3 mL of DMF, and then bromomethyl acetate (216 μL, 2.31 mmol, 1.0 equiv.) followed by DIPEA (403 μL, 2.31 mmol, 1.0 equiv.) were added dropwise. The reaction mixture was stirred at 40 °C for 25 hours and then diluted with water, extracted into ethyl acetate, washed with brine, and then dried with MgSO₄. After removal of solvent under reduced pressure, the crude product was purified by column chromatography (5090% ethyl acetate in DCM as the eluent) to afford 3 as a yellow solid (792 mg, 75.8%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 10.22 (s, 2 H), 8.44 (s, 1 H), 8.33 (d, J = 8.0 Hz, 1 H), 8.27 (d, J = 8.0 Hz, 1 H), 8.19 (d, J = 8.1 Hz, 1 H), 7.72 (s, 1 H), 7.47 (d, J = 8.1 Hz, 1 H), 6.96 (s, 2 H), 6.72 (m, 8 H), 6.58 (m, 2 H), 6.00 (s, 2 H), 5.88 (s, 2 H), 3.83 (s, 6 H), 2.14 (s, 3 H), 2.07 (s, 3 H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 169.9, 169.8, 168.0, 163.9, 163.8, 161.7, 160.3, 157.4, 153.2, 152.4, 152.3, 152.2, 136.8, 131.7, 131.1, 130.8, 129.9, 129.8, 129.7, 129.7, 127.4, 126.5, 126.2, 125.5, 125.1, 113.4, 112.5, 110.6, 110.5, 109.1, 109.0, 102.8, 101.3, 83.7, 83.6, 80.6, 80.5, 56.2, 21.0. HRMS m/z [M + H]⁺ calcd. for [C₂₅H₁₉O₉]⁺ 463.1024; found 463.1029.

Compound 4.

Fluorescein derivative 3 (500 mg, 1.08 mmol, 1.0 equiv.) was added to 3.5 mL DMF and then DIPEA (755 μL, 4.33 mmol, 4.0 equiv.) followed by the PhN(Tf)₂ (386 mg, 1.08 mmol, 1.0 equiv.) were added dropwise. The reaction mixture was stirred at room temperature for 24 hours and then diluted with water, extracted into ethyl acetate, washed with brine, and then dried with MgSO₄. After removal of solvent under reduced pressure, the crude product was purified by column chromatography (25% ethyl acetate in hexanes as the eluent) to afford 4 as a white solid (532 mg, 85.7%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.48 (s, 1 H), 8.36 (d, J = 8.1 Hz, 1 H), 8.30 (d, J = 8.1 Hz, 1 H) 8.24 (d, J = 8.0 Hz, 1 H), 7.93 (s, 1 H), 7.73 (d, J = 6.5 Hz, 2 H), 7.59 (d, J = 8.1 Hz, 1 H), 7.26 (m, 2 H), 7.15 (m, 2 H), 7.01 (s, 2 H), 6.84 (m, 2 H), 6.78 (m, 2 H), 6.01 (s, 2 H), 5.88 (s, 2 H), 3.85 (s, 6 H), 2.14 (s, 3 H), 2.07 (s, 3 H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 169.9, 169.8, 167.8, 167.7, 163.7, 162.0, 162.0, 156.8, 152.6, 151.9, 151.8, 151.7, 151.7, 150.3, 150.3, 137.0, 135.8, 132.1, 131.5, 131.4, 131.3, 130.4, 130.0, 129.9, 129.9, 127.0, 126.8, 126.5, 125.6, 125.5, 123.4, 120.0, 120.0, 119.4, 117.9, 117.8, 117.4, 113.2, 113.2, 111.2, 111.2, 110.1, 109.9, 101.1, 82.0, 81.8, 80.6, 80.5, 56.3, 56.3, 21.0, 21.0. HRMS m/z [M + H]⁺ calcd. for [C₂₆H₁₈F₃O₁₁S]⁺ 595.0516; found 595.0522.

CT-MeRho.

In a glovebox, compound 4 (265 mg, 0.446 mmol, 1.0 equiv.), Pd(OAc)₂ (10 mg, 0.045 mmol, 0.10 equiv.), rac-BINAP (41.6 mg, 0.067 mmol, 0.15 equiv.), and Cs₂CO₃ (436 mg, 1.34 mmol, 3.0 equiv.) were dissolved in toluene (10 mL) in a three-neck flask fitted with a reflux condenser. After the reaction vessel was sealed under N₂ and removed from the glovebox, benzophenone imine (89.8 μL, 0.535 mmol, 1.2 equiv.) was added via syringe. The reaction mixture was heated and stirred at 140 °C for 5 min, and then the temperature was reduced to 100 °C for an additional 8 hours. After heating, the reaction mixture was allowed to cool to room temperature and was filtered through a plug of Celite. After removal of the solvent under reduced pressure, the residue was dissolved in a solution of THF (20 mL) and 1.0 M HCl (5 mL) and stirred at room temperature for 1 hour. The THF was removed under reduced pressure, the crude product was diluted with water, and then the pH was neutralized. The aqueous solution was extracted into EtOAc, and the organic phase was washed with brine and dried using MgSO₄. The crude product was purified using column chromatography (20–80% ethyl acetate in hexanes as the eluent) to afford CT-MeRho as a yellow solid (87.2 mg, 42.4%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.41 (s, 1 H), 8.32 (d, J = 9.6 Hz, 1 H), 8.25 (d, J = 9.4 Hz, 1 H), 8.16 (d, J = 8.0 Hz, 1 H), 7.68 (s, 1 H), 7.44 (d, J = 8.1 Hz, 1 H), 6.92 (s, 2 H), 6.67 (m, 4 H), 6.44 (m, 4 H), 6.34 (m, 2 H), 6.00 (s, 2 H), 5.88 (s, 2 H), 5.71 (s, 4 H), 3.82 (s, 6 H), 2.14 (s, 3 H), 2.07 (s, 3 H). ¹³C{¹H} NMR (150 MHz, DMSO-d₆) δ (ppm): 169.1, 168.1, 136.8, 161.6, 157.5, 152.6, 152.1, 136.6, 130.9, 129.6, 129.2, 127.7, 126.3, 125.5, 112.1, 111.8, 110.9, 104.8, 101.3, 99.5, 84.6, 80.4, 56.1, 21.0. HRMS m/z [M + H]⁺ calcd. for [C₂₅H₂₀NO₈]⁺ 462.1183; found 462.1186.

CT-MeRhoAz.

CT-MeRho (20 mg, 0.043 mmol, 1.0 equiv.) was dissolved in 1 mL of acetonitrile and then cooled to 0 °C in an ice bath. tert-Butyl nitrite (7.7 μL, 0.065 mmol, 1.5 equiv.) was added dropwise, and the solution was stirred for 5 minutes. Trimethylsilyl azide (8.6 μL, 0.065 mmol, 1.5 equiv.) was then added dropwise, and the solution was stirred for an additional 10 minutes. The reaction mixture was removed from the ice bath and stirred at room temperature for 5 hours. The crude product was purified using column chromatography (30% ethyl acetate in hexanes as the eluent) to afford CT-MeRhoAz as a white solid (19.0 mg, 90.4%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.46 (s, 1 H), 8.34 (d, J = 8.1 Hz, 1 H), 8.29 (d, J = 9.4 Hz, 1 H), 8.22 (d, J = 8.1 Hz, 1 H), 7.77 (s, 1 H), 7.50 (d, J = 8.1 Hz, 2 H), 7.14 (s, 2 H), 6.97 (s, 2 H), 6.92 (m, 2 H), 6.91 (s, 2 H), 6.80 (t, J = 9.11 Hz, 2 H), 6.74 (m, 2 H), 3.84 (s, 6 H), 2.14 (s, 4 H), 2.06 (s, 3 H), 1.99 (s, 3 H). ¹³C{¹H} NMR (150 MHz, DMSO-d₆) δ (ppm): 169.9, 169.8, 168.0, 167.9, 163.9, 163.8, 161.9, 161.9, 157.2, 153.0, 152.0, 152.0, 151.9, 143.0, 142.9, 136.9, 135.6, 131.9, 131.3, 130.5, 129.8, 129.8, 127.1, 126.7, 126.4, 125.5, 125.2, 116.0, 115.2, 115.1, 112.9, 110.3, 110.2, 107.7, 107.7, 101.4, 101.4, 82.7, 82.6, 80.6, 80.5, 56.2, 56.2, 21.0, 21.0. HRMS m/z [M + H]⁺ calcd. for [C₂₅H₁₈N₃O₈]⁺ 488.1088; found 488.1094.

General Procedure for Fluorescence and Selectivity Measurements:

A stock solution of CT-MeRhoAz (15 μL, 1.0 mM in DMSO) was added to 3.00 mL of 50 mM PIPES buffer (100 mM KCl, pH 7.4) under N₂ at 37 °C in a setup-capped cuvette. To start the experiment, a stock solution of the analyte (15 μL, 50 mM) was added. Scans were taken at 0, 1, 5, 10, 15, 30, 45, 60, and 90 minutes after injection. DEA NONOate was used to generate NO, and stock solutions were prepared in degassed 0.01 M NaOH. λ_ex = 480 nm, λ_em = 490–640 nm. Slit width: 0.4 mm. The experiments were performed in triplicate.

Quantum Yield Measurement:

Quantum yields were determined according to published standard methods and are presented relative to fluorescein in 0.1 M NaOH (θ = 0.95).^[15]

Determination of Detection Limit:

A stock solution of CT-MeRhoAz (15 μL, 1.0 mM in DMSO) was added to 3.00 mL of 50 mM PIPES buffer (100 mM KCl, pH 7.4). To start the experiment, the cuvettes were treated with various concentrations (10, 7.5, 5.0, 2.5, 0.1 μM) of NaSH and incubated at 37 °C for 90 minutes. The background-corrected fluorescence was fit to a linear regression, and the detection limit was determined as the point at which [a + 3σ] where a is the y-intercept and σ is the standard deviation of the blank.

Cell Culture and Imaging Experiments:

HeLa cells (ATCC CCL-2) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) at 37 °C under 5% CO₂. Cells were seeded in 35 mm imaging dishes at 1.0×10⁵ cells per dish and allowed to adhere for 24 hours prior to fluorescence imaging studies. Cells were then washed with 1× Phosphate Buffered Saline (2 × 2.0 mL) and treated with 2.0 mL of DMEM containing the vehicle or 20 μM AOAA for 45 minutes. Next, cells were washed with 1× Phosphate Buffered Saline (2 × 2.0 mL) and treated with 2.0 mL of DMEM containing the vehicle or 5 μM CT-MeRhoAz. After incubation for 45 minutes, the cells were washed again with 1× Phosphate Buffered Saline (2 × 2.0 mL) and treated with 2.0 mL of DMEM containing either the vehicle or 200 μM NaSH and incubated for 45 minutes. After incubation, cells were washed with 1x Phosphate Buffered Saline (2 × 2.0 mL) and then the media was replaced with 2.0 mL of FluoroBrite DMEM. Cell imaging was performed on a Zeiss LSM 880 confocal microscope using a 40× objective with standard DAPI and GFP filter cubes equipped for imaging of Hoechst 33342 and CT-MeRhoAz, respectively. All images were processed using LasX.

For cell trappability experiments, HeLa cells were incubated with 100 nM AP39 for 1 hour followed by incubation with 5 μM CT-MeRhoAz or MeRhoAz for 45 minutes at 37 °C under 5% CO₂ in 35 mm imaging dishes as described above. After incubation, the cells were washed with 1× PBS (2 × 2.0 mL), and then the media was replaced with 2.0 mL of FluoroBrite DMEM. The media was exchanged every 10 minutes for 60 minutes, and the same section of cells was imaged at 5, 30, and 60 minutes after the initial media exchange. All images were processed using LasX and ImageJ was used to quantify cellular fluorescence.