Salivary Hydrogen Sulfide Measured with a New Highly Sensitive Self-Immolative Coumarin-Based Fluorescent Probe

Abstract

Ample evidence suggests that H₂S is an important biological mediator, produced by endogenous enzymes and microbiota. So far, several techniques including colorimetric methods, electrochemical analysis and sulfide precipitation have been developed for H₂S detection. These methods provide sensitive detection, however, they are destructive for tissues and require tedious sequences of preparation steps for the analyzed samples. Here, we report synthesis of a new fluorescent probe for H₂S detection, 4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (1). The design of 1 is based on combination of two strategies for H₂S detection, i.e., reduction of an azido group to an amine in the presence of H₂S and intramolecular lactamization. Finally, we measured salivary H₂S concentration in healthy, 18⁻40-year-old volunteers immediately after obtaining specimens. The newly developed self-immolative coumarin-based fluorescence probe (C₁₅H₁₅N₃O₄) showed high sensitivity to H₂S detection in both sodium phosphate buffer at physiological pH and in saliva. Salivary H₂S concentration in healthy volunteers was within a range of 1.641⁻7.124 μM.

Keywords: Ellman’s Reagent; assay; azide; biological systems; fluorescent probe; halitosis; hydrogen sulfide; saliva.

Figure 1

Mechanism of H₂S detection for the self-immolative probe.

Figure 2

HRMS confirmed formation of piperidin-2-one in the reaction of the compound 1 with NaHS.

Figure 3

Time course experiment of compound 2 (0.1 mM, A) and 3 (0.1 mM, B) reacting with NaHS in sodium phosphate buffer (pH = 7.4) at room temperature. Time points represent time range from 1 to 10 min after addition of NaHS (0.1 mM). Fluorescence spectra of compound 1 (C), 2 (A) and 3 (B) were recorded for 10 min after addition of NaHS (0.1 mM).

Figure 4

Comparison of fluorescence responses for the compound 1, 2 and 3 (0.1 mM). Data were acquired at room temperature after addition of NaHS (0.1 mM) in sodium phosphate buffer (pH = 7.4, 20% CH₃CN) with excitation at 365 nm. Means ± SE from three measurements of the fluorescence responses are presented.

Figure 5

The synthesis of 3-azidopropanonic (a) acid and 5-azidopentanonic acid (b).

Figure 6

The synthesis of compounds 1 (a) and 2 (b).

Figure 7

The synthesis of 4-methyl-2-oxo-2H-chromen-7-yl propionate (3).

Figure 8

Fluorescence response of the self-immolative probe/compound 1 (0.1 mM) to 0.1 mM NaHS. Data were acquired at room temperature in sodium phosphate buffer (pH = 7.4) with excitation at 365 nm. Emission was collected in the time range 1–10 min after the addition of 0.1 mM NaHS. The spectrum at t = 0 min was acquired from a 0.1 mM solution of the compound 1 without the addition of NaHS.

Figure 9

Time course experiment of the compound 1 (0.1 mM) reacting with NaHS (0.1 mM) in sodium phosphate buffer (pH = 7.4) at room temperature. Time points represent time range from 1 to 60 min after addition of NaHS (0.1 mM). Means ± SE from three measurements of the fluorescence responses are presented.

Figure 10

The correlation between fluorescence intensity and NaHS concentration determined using a fluorometer: the compound 1 (0.1 mM) with NaHS (20–100 μM) in sodium phosphate buffer (λ_ex = 365 nm) at room temperature. The points represent the mean fluorescence responses at 10 min after the addition of NaHS.

Figure 11

Time-dependent UV-vis absorption spectra of SH-free DTNB solution (green line-background) and its mixture with NaHS (0.1 mM, orange line) in the reaction buffer (pH 8.0) at room temperature. The absorbance at 412 nm was recorded 15 min after addition of NaHS.

Figure 12

The correlation between absorbance and NaHS concentration determined by the DTNB assay in sodium phosphate buffer (pH 8.0) at room temperature. The absorbance at 412 nm was recorded for 15 min after addition of various concentrations of NaHS from 20 to 100 μm. Values of absorbance are given as means obtained from 3 measurements. Background values were subtracted from the sample values.

Figure 13

The correlation between fluorescence intensity and NaHS concentration determined using a fluorometer: the compound 1 (0.1 mM) with NaHS (1–10 μM) in sodium phosphate buffer (λ_ex = 365 nm) at room temperature. The points represent the mean fluorescence responses at 10 min after the addition of NaHS.