De Novo Construction of Fluorophores via CO Insertion-Initiated Lactamization: A Chemical Strategy toward Highly Sensitive and Highly Selective Turn-On Fluorescent Probes for Carbon Monoxide

Abstract

Extensive studies in the last few decades have led to the establishment of CO as an endogenous signaling molecule and subsequently to the exploration of CO's therapeutic roles. In the current state, there is a critical conundrum in CO-related research: the extensive knowledge of CO's biological effects and yet an insufficient understanding of the quantitative correlations between the CO concentration and biological responses of various natures. This conundrum is partially due to the difficulty in examining precise concentration-response relationships of a gaseous molecule. Another reason is the need for appropriate tools for the sensitive detection and concentration determination of CO in the biological system. We herein report a new chemical approach to the design of fluorescent CO probes through de novo construction of fluorophores by a CO insertion-initiated lactamization reaction, which allows for ultra-low background and exclusivity in CO detection. Two series of CO detection probes have been designed and synthesized using this strategy. Using these probes, we have extensively demonstrated their utility in quantifying CO in blood, tissue, and cell culture and in cellular imaging of CO from exogenous and endogenous sources. The probes described will enable many biology and chemistry labs to study CO's functions in a concentration-dependent fashion with very high sensitivity and selectivity. The chemical and design principles described will also be applicable in designing fluorescent probes for other small molecules.

Figure 1.

Comparison of the CO sensing mechanisms and representative probes reported in the literature (A) and the probes described in this study (B).

Figure 2.

Design of phthalimide-based CO probes (A) and naphthalimide-based CO probes (B).

Figure 3.

CO detection profiles of CODP-10x series. (A and B) Turn-on fluorescence response of CODP-102 (500 μM) to CO gas (0–100 nmol) in a headspace vial at 1 h (λ_Ex = 394 nm, bandwidth = 5 nm); (C) Regression of SNR vs. concentrations of CODP-102 at 1, 10, and 100 μM in PBS; (D) Selectivity of 20 μM CODP-102 in pH 7.4 PBS (species: 1: vehicle; 2: Cys; 3: GSH; 4: GSSG; 5: H₂O₂; 6: H₂S; 7: H₂S₂; 8: HClO; 9: NO₂⁻; 10: CN⁻; 11: 1% CO gas in air at 1 atm (concentration of other species was 100 μM) after 1h incubation (λ_Ex = 395 nm, bandwidth = 5 nm); insert: image of incubation solutions; (E) CO detection kinetics of CODPs. 800 μL 12.5 μM CODPs was mixed with 200 μL 1 mM CO saturated PBS at T_0, and the fluorescence intensity at 509 nm or 499 nm was recorded every second at 25 °C; insert: expanded range of 0–240 s.

Figure 4.

CO detection SNR of CODP-202. (A) CODP-202 at concentrations of 1, 10, 25, and 50 μM in PBS incubated with or without CO gas for 1 h; (B) linear regression of SNR against probe concentration in PBS (bandwidth: ex=3 nm, em=5 nm).

Figure 5.

Application of CODPs to determine CO in biological samples (Figure created with BioRender.com).

Figure 6.

Spectra (A) and a calibration curve (B) of mouse blood with various COHb levels determined by 10 mM CODP-102 (λ_EX = 395 nm); (C) blood COHb levels of mice dosed with or without AC-306 (50 mg/kg) determined with CODP-102 and a CO-oximeter; (D) blood COHb levels of mice dosed with or without CO-306 (200 mg/kg) determined with CODP-103 and a CO-oximeter; (E) relative CO levels of HeLa cells treated with 0.3 μM CDDO-Me (6 h) or 250 ppm CO gas (2 h); (E) Western blot of HO-1 in HeLa cells treated with 0.3 μM CDDO-Me (6 h), β-actin was probed as the loading control. For C-D, results shown as average ± SD (n=3), ****P<0.0001, ns: not significant (P>0.05), t-test.

Figure 7.

(A) Fluorescence spectra of 1 mM CODP-106 in DMA incubated with 10–100 ppm CO calibration gas; insert: calibration curve of CO concentration (ppm) vs. fluorescence intensity at 499 nm (λ_EX = 385 nm); CO concentrations of the liver (B) and kidney (C) tissues of mice dosed with or without CO-306 (200 mg/kg) determined by CODP-103 and methanizer-FID-GC; (D) CO concentrations in HeLa cells treated with CO gas or a CO prodrug, CO-111 (50 μM), for 2 h and tested with CODP-106. For B-D, results shown as average ± SD (n = 3), ***P<0.001, ns: not significant (P>0.05), t-test.

Figure 8.

Fluorescence microscopy image of CO in live cells. HeLa cells were treated with 0.5% DMSO vehicle control (A), 250 ppm CO gas (B), or 50 μM CO-201 (C) for 1 h followed by addition of 20 μM CODP-202 and incubation for 1 h (scale bar: 20 μm); HeLa cells were treated with 0.5% DMSO vehicle (D), or 0.3 μM CDDO-Me (E) for 6 h followed by incubation with 20 μM CODP-202 for 1h (scale bar: 50 μm). (F) Background-normalized maximum signal intensity of the cells in the image (*P < 0.05, n=3, ROI is shown in Figure S21).

Scheme 1.

Proposed CO detection mechanism of CODP-102.