CO Sense and Release Flavonols: Progress toward the Development of an Analyte Replacement PhotoCORM for Use in Living Cells

Abstract

Carbon monoxide (CO) is a signaling molecule in humans. Prior research suggests that therapeutic levels of CO can have beneficial effects in treating a variety of physiological and pathological conditions. To facilitate understanding of the role of CO in biology, molecules that enable fluorescence detection of CO in living systems have emerged as an important class of chemical tools. A key unmet challenge in this field is the development of fluorescent analyte replacement probes that replenish the CO that is consumed during detection. Herein, we report the first examples of CO sense and release molecules that involve combining a common CO-sensing motif with a light-triggered CO-releasing flavonol scaffold. A notable advantage of the flavonol-based CO sense and release motif is that it is trackable via fluorescence in both its pre- and postsensing (pre-CO release) forms. In vitro studies revealed that the PdCl₂ and Ru(II)-containing CORM-2 used in the CO sensing step can result in metal coordination to the flavonol, which minimizes the subsequent CO release reactivity. However, CO detection followed by CO release is demonstrated in living cells, indicating that a cellular environment mitigates the flavonol/metal interactions.

Figure 1

Types of fluorescent sensors (a–c) for detection of CO or CORMs in biological systems. The coloration of the circles indicates the wavelength of the fluorescent emission.

Figure 2

Analyte replacement probes for (a) H₂S and (b) formaldehyde (c) CO sense and release molecule developed in this work. The coloration of the spheres indicates the wavelength of the fluorescent emission.

Figure 3

Representation of the X-ray structure of 1.

Scheme 1. Synthesis of CO-Sensing Flavonol Derivatives

Figure 4

Absorption (a) and emission (b) spectral data for 1–3 and Flav-1 in DMSO.

Figure 5

Changes in the emission spectra of 1 ((a,b)) and 2 ((c,d)) in 40% DMSO in PBS or 8% DMSO in DMEM upon incubation at 37 °C for 90 min (λ_ex = 370 nm).

Figure 6

Emission (top) and absorption (bottom) spectra produced upon treatment of Flav-1 (50 μM) with PdCl₂ (100 μM) or CORM-2 (500 μM) in (a,c) 8% DMSO in DMEM and (b,d) DMSO after 90 min at 37 °C (λ_ex = 411 nm). In each spectrum, the features produced in the CO-sensing Tsuji–Trost deprotection reaction of 1 are shown. A comparative control spectrum of Flav-1 is shown at the concentration (50 μM) anticipated for full conversion of 1 to Flav-1 in the CO sensing reaction.

Figure 7

(a) Fluorescent spectral changes produced upon reaction of 1 (50 μM) with PdCl₂ (100 μM) in DMSO upon addition of different concentrations of CORM-2 (5–200 μM). (b) Linear increase in fluorescence intensity produced upon reaction of 1 with PdCl₂ upon the addition of CORM-2 (5–25 μM). All spectra were measured 15 min after mixing in DMSO at 37 °C (λ_ex = 411 nm).

Figure 8

Fluorescent intensity increase at 603 nm (indicating Flav-1 formation) of a solution of 1 (50 μM) with PdCl₂ (100 μM) in DMSO upon addition of various analytes (100 μM). All spectra were measured 15 min after mixing in DMSO at 37 °C (λ_ex = 411 nm).

Figure 9

Emission (top) and absorption (bottom) spectra of Flav-1 (50 μM) and the Tsuji–Trost CO-sensing reaction of 1 in (a,c) 8% DMSO in DMEM and (b,d) DMSO (λ_ex = 411 nm). Spectra are shown in comparison with those resulting from illumination of the samples with visible light in the presence of O₂.

Figure 10

Emission (top) and absorption (bottom) spectra of solutions of Flav-1 in the presence of (a,c) CORM-2 or (b,d) PdCl₂. Spectral changes associated with illumination of the solutions in the presence of O₂ are shown.

Scheme 2. Tsuji–Trost-Type CO Sensing Reaction of 1

Formation of the Flav-1 anion results in coordination to CORM-2 fragments and Pd(II). Coordination of the flavonolato anion to these heavy metal ions results in quenching of the flavonol emission and loss of visible light-induced CO release reactivity.

Figure 11

Fluorescence microscopy images of HUVECs incubated for 24 h in DMEM/F-12K with 1. Row 1: media control cells. Row 2: cells exposed to 1. Blue channel: λ_ex = 310–390 nm, λ_em = 420–470 nm. Size of bar = 20 μm.

Figure 12

Fluorescent imaging of CO sensing in HUVECs using 1 (50 μM) with 100 μM PdCl₂ and 500 μM CORM-2. Row 1: 1 incubated for 4 h followed by the addition of PdCl₂ and CORM-2 and additional incubation for 4 h. Row 2: following illumination with 310–390 nm light. Green channel: λ_ex = 310–390 nm, λ_em = 500–550 nm. Scale bar = 40 μm.