A 2-aza-Cope reactivity-based platform for ratiometric fluorescence imaging of formaldehyde in living cells

Abstract

Formaldehyde (FA) is a major reactive carbonyl species (RCS) that is naturally produced in living systems through a diverse array of cellular pathways that span from epigenetic regulation to the metabolic processing of endogenous metabolites. At the same time, however, aberrant elevations in FA levels contribute to pathologies ranging from cancer and diabetes to heart, liver, and neurodegenerative diseases. Disentangling the complex interplay between FA physiology and pathology motivates the development of chemical tools that can enable the selective detection of this RCS in biological environments with spatial and temporal fidelity. We report the design, synthesis, and biological evaluation of ratiometric formaldehyde probe (RFAP) indicators for the excitation-ratiometric fluorescence imaging of formaldehyde production in living systems. RFAP-1 and RFAP-2 utilize FA-dependent aza-Cope reactivity to convert an alkylamine-functionalized coumarin platform into its aldehyde congener with a ca. 50 nm shift in the excitation wavelength. The probes exhibit visible excitation and emission profiles, and high selectivity for FA over a variety of RCS and related reactive biological analytes, including acetaldehyde, with up to a 6-fold change in the fluorescence ratio. The RFAP indicators can be used to monitor changes in FA levels in biological samples by live-cell imaging and/or flow cytometry. Moreover, RFAP-2 is capable of visualizing differences in the resting FA levels between wild-type cells and models with a gene knockout of ADH5, a major FA-metabolizing enzyme, establishing the utility of this ratiometric detection platform for identifying and probing sources of FA fluxes in biology.

Scheme 1. Design of a ratiometric formaldehyde probe (RFAP) platform.

Scheme 2. Synthesis of RFAP-0. Reagents and conditions: (i) lithium bis(3-((tert-butyldimethylsilyl)oxy)propyl)copper, Et₂O, THF, –20 °C, 3 h; (ii) NH₃, MeOH, 0 °C, then allylboronic acid pinacol ester, rt, 10 h; (iii) AcOH, H₂O, rt, 14 h.

Fig. 1. FA response of RFAP-0. Data were acquired at 37 °C in 20 mM PBS (pH 7.4). Excitation spectra were collected between 400 and 500 nm with emission monitored at λ _em = 510 nm. (a) Excitation ratiometric response of 10 μM RFAP-0 to 100 μM FA. Excitation spectra are shown at 0, 30, 60, 90, and 120 min (red, yellow, green, blue, and purple traces, respectively) after the addition of FA. (b) Quantification of 470/420 nm excitation ratio over time.

Scheme 3. Installation of a geminal dimethyl group is designed to accelerate the 2-aza-Cope rearrangement and thermodynamically bias the reaction toward the desired product.

Scheme 4. Synthesis of RFAP-1. Reagents and conditions: (i) NH₃, MeOH, 0 °C, then prenylboronic acid, rt, 10 h; (ii) AcOH, H₂O, rt, 14 h.

Fig. 2. FA response and selectivity of RFAP-1. Data were acquired at 37 °C in 20 mM PBS (pH 7.4). Excitation spectra were collected between 400 and 500 nm with emission monitored at λ _em = 510 nm. (a) Excitation ratiometric response of 10 μM RFAP-1 to 100 μM FA. Excitation spectra are shown at 0, 30, 60, 90, and 120 min (red, yellow, green, blue, and purple traces, respectively) after addition of FA. (b) Quantification of 470/420 nm excitation ratio over time. (c) Excitation ratiometric response of 10 μM RFAP-1 to biologically relevant RCS and related molecules. Bars represent relative 470/420 nm excitation ratios at 0, 30, 60, 90, and 120 (black) min after the addition of a given analyte. The data shown are for a concentration of 100 μM for all species unless otherwise denoted. Legend: (1) PBS; (2) FA; (3) acetaldehyde; (4) pyruvate; (5) glucose, 1 mM; (6) 4-HNE; (7) dehydroascorbate; (8) oxaloacetate; (9) glucosone; (10) acrolein; (11) methylglyoxal; (12) methylglyoxal, 10 μM.

Fig. 3. Representative ratiometric confocal microscopy images of FA detection in live HEK293T cells loaded with 10 μM RFAP-1. Images were taken 60 min after the addition of (a) vehicle, (b) 25 μM FA, (c) 50 μM FA, (d) 100 μM FA, and (e) 200 μM FA. (f–j) Bright-field images of the cells in (a–e). Scale bar represents 40 μm in all images. (k) Mean 488/405 excitation ratios of the HEK293T cells treated with varying concentrations of FA for 60 min relative to the mean 488/405 excitation ratios before FA addition; error bars denote SEM, n = 5. ***P < 0.001.

Scheme 5. Synthesis of RFAP-2. Reagents and conditions: (i) Pd-PEPPSI-IPr, K₃PO₄·H₂O, THF, rt, 10 h; (ii) NH₃, MeOH, 0 °C, then prenylboronic acid, rt, 10 h; (iii) LiOH, THF/MeOH/H₂O, rt, 6 h; (iv) 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine, HATU, DIPEA, DMF, rt, 12 h.

Fig. 4. FA response and selectivity of RFAP-2. Data were acquired at 37 °C in 20 mM PBS (pH 7.4). Excitation spectra were collected between 400 and 500 nm with emission monitored at λ _em = 510 nm. (a) Excitation ratiometric response of 10 μM RFAP-2 to 100 μM FA. Excitation spectra are shown at 0, 30, 60, 90, and 120 min (red, yellow, green, blue, and purple traces, respectively) after the addition of FA. (b) Quantification of 470/420 nm excitation ratio over time. (c) Excitation ratiometric response of 10 μM RFAP-2 to biologically relevant RCS and related molecules. Bars represent relative 470/420 nm excitation ratio at 0, 30, 60, 90, and 120 (black) min after addition. Data shown are for a concentration of 100 μM for all species unless otherwise denoted. Legend: (1) PBS; (2) FA; (3) acetaldehyde; (4) pyruvate; (5) 1 mM glucose; (6) 4-HNE; (7) dehydroascorbate; (8) oxaloacetate; (9) glucosone; (10) acrolein; (11) methylglyoxal; (12) 10 μM methylglyoxal; (13) H₂O₂; (14) 5 mM glutathione.

Fig. 5. Representative ratiometric confocal microscopy images of FA detection in live HEK293T loaded with 10 μM RFAP-2. Images were taken 60 min after the addition of (a) vehicle, (b) 50 μM FA, (c) 100 μM, and (d) 200 μM FA. (e–h) Bright-field images of the cells in (a–d). Scale bar represents 40 μm in all images. (k) Mean 488/405 excitation ratios of the HEK293T cells treated with varying concentrations of FA for 60 min relative to the mean 488/405 excitation ratios before FA addition; error bars denote SEM, n = 4. ***P < 0.001.

Fig. 6. Representative ratiometric confocal microscopy images of FA detection in various cell lines using RFAP-2. Images were taken 30 min after the addition of either vehicle or 200 μM FA. Scale bar represents 40 μm in all images. (a) Ratio and bright-field images; (b) the mean 488/405 excitation ratios of the cells treated with vehicle or 200 μM FA for 30 min relative to the mean 488/405 excitation ratios before vehicle or FA addition; error bars denote SEM, n = 2 (3 fields per condition).

Fig. 7. FA detection in ADH5 –/– vs. WT HAP1 cells with 500 nM RFAP-2. (a) Representative ratiometric confocal microscopy images taken 60 min after treatment with vehicle or 100 μM FA. Scale bar represents 25 μm in all images. (b) Normalized mean 488/405 excitation ratios of the ADH5 –/– and WT HAP1 cells treated with vehicle or 100 μM FA for 60 min by confocal microscopy; error bars denote SEM, n = 5. (c) Representative histograms obtained via flow cytometric analysis of the ADH5 –/– and WT HAP1 cells treated with either vehicle or 100 μM FA for 60 min. (d) Normalized median 488/405 excitation ratios of the ADH5 –/– and WT HAP1 cells treated with vehicle or 100 μM FA for 60 min by flow cytometry; error bars denote SEM, n = 5. *P < 0.05, **P < 0.01, ****P < 0.0001.