Colorimetric Carbonyl Sulfide (COS)/Hydrogen Sulfide (H2 S) Donation from γ-Ketothiocarbamate Donor Motifs

Abstract

Hydrogen sulfide (H₂ S) is a biologically active molecule that exhibits protective effects in a variety of physiological and pathological processes. Although several H₂ S-related biological effects have been discovered by using H₂ S donors, knowing how much H₂ S has been released from donors under different conditions remains challenging. Now, a series of γ-ketothiocarbamate (γ-KetoTCM) compounds that provide the first examples of colorimetric H₂ S donors and enable direct quantification of H₂ S release, were reported. These compounds are activated through a pH-dependent deprotonation/β-elimination sequence to release carbonyl sulfide (COS), which is quickly converted into H₂ S by carbonic anhydrase. The p-nitroaniline released upon donor activation provides an optical readout that correlates directly to COS/H₂ S release, thus enabling colorimetric measurement of H₂ S donation.

Keywords: anti-inflammation; carbonyl sulfide; colorimetry; hydrogen sulfide; γ-ketothiocarbamate.

Figure 1.

(a) Time-dependent UV-Vis spectra of γ-KetoTCM-1 and PNA. (10 mM PBS, pH 7.4, 25 μg CA/mL, 37 ºC). Inset: color of the solution at t = 0 (left) and t = 24 h (right). (b) Time-dependent absorbance of γ-KetoTCM-1 (333 nm) and PNA (381 nm) (10 mM PBS, pH 7.4). (c) pH dependence of PNA formation from of γ-KetoTCM-1 in PBS at pH 8.0 (blue), pH 7.4 (red), and pH 6.0 (black) at 37 ºC. (d) PNA formation (red) and H₂S release (blue) upon γ-KetoTCM-1 (50 μM) activation in PBS (pH 7.4, 10 mM) containing BSA (5 mg/mL) at 37 ºC. Inset: Correlation between measured [H₂S] and PNA formation. A_P: PNA absorbance (381 nm); A_D: γ-KetoTCM-1 absorbance (333 nm). Experiments were performed in triplicate. Results are expressed as mean ± SD (n=3).

Figure 2.

Measurement of PNA formation after compound activation. A_P: PNA absorbance (381 nm); A_D: compound absorbance (at λ_max). Experiments were performed in triplicate. Results are expressed as mean ± SD (n = 3)

Figure 3.

PNA formation after incubating γ-KetoTCM-1 (50 μM) in PBS (pH 7.4, 10 mM) only (1), or PBS containing 250 μM of Cys (2), NAC (3), Hcy (4), GSH (1.0 mM, 5), Lys (6), Ser (7), Gly (8), or GSSG (9) for 4 h at 37 ºC. Experiments were performed in triplicate. Results are expressed as mean ± SD (n = 3).

Figure 4.

H₂S Delivery from γ-KetoTCM-1 in HeLa cells. HeLa cells were treated with SF7-AM (5 μM) for 30 min, washed, and incubated with FBS-free DMEM only (left), with 100 μM γ-KetoCM-1 (middle), or with γ-KetoTCM-1 (right) for 2 h. Cells were then washed and imaged in PBS. Scale bar: 100 μm.

Figure 5.

Effects of γ-KetoTCM-1 on LPS-induced NO₂^– formation. RAW 264.7 cells were pretreated with γ-KetoTCM-1 (25 μM) or control compounds for 6 h, followed by LPS (1.0 μg/mL, 18-h). Results are expressed as mean ± SD (n = 4). ***P < 0.001 vs the control group; ^###P < 0.001 vs vehicle-treated group; and ^$$$P < 0.001 between γ-KetoTCM-1-treated and γ-KetoCM-1-treated groups.

Scheme 1.

(a) Previous design of COS-based H₂S donors; (b) colorimetric COS-based H₂S donors and proposed COS/H₂S releasing pathway; (c) control compound activation; (d) activation of CO₂-releasing control compounds; (e) synthesis of γ-KetoTCM-based COS/H₂S donors and control compounds.