A Fluorogenic Reaction for Monitoring Cross-Coupling with Turn-On Ratio Greater than a Hundred Thousand

Abstract

Fluorogenic reactions convert nonfluorescent reactants into fluorescent products and are ubiquitous in molecular sciences, allowing discovery, quantification, and imaging of a range of chemical and biological processes. However, the "non-fluorescent" reactants typically have substantial residual fluorescence, reducing the turn-on ratio of the reaction. Limits on the turn-on ratio then impose constraints on the fluorogenic reaction's application, such as lowering the maximum allowable concentration of reactants or increasing the minimum detectable amount of product. Here, we report a design scheme producing exceptionally high turn-on ratios of up to 207,000. The scheme relies on a condensation reaction uniting electron-rich and electron-deficient reactants, resulting in a product molecule with "push-pull" character and significantly altered color and photophysical behavior. This approach is demonstrated using the Suzuki cross-coupling to produce a highly fluorescent dicyanomethylenedihydrofuran (DCDHF) product. The product has a peak absorption nearly 1 eV redder than the nearest reactant, while also exhibiting a large Stokes shift of nearly 0.5 eV. As a result, the reactants are negligibly excited, leading to the exceptionally high turn-on ratio, and enabling reactions to be performed at high concentrations of starting material without drowning out the product signal. The reaction is demonstrated in bulk conditions and in microdroplets, where differences in reaction rate are observed.

Keywords: DCDHF; donor−acceptor; fluorescence microscopy; fluorogenic reaction; in situ monitoring; microdroplet; push−pull chromophore; turn-on ratio.

1

Production of a DCDHF fluorophore via Suzuki coupling.

2

Optical characterization of key compounds. (a) UV–vis absorption spectra of DMAPBA. (b) UV–vis absorbance and fluorescence emission spectra (excitation at 351 nm) of reactant 1. The small peak at 700 nm is from residual scatter of the excitation light at the second order of the diffraction grating. (c) UV–vis absorbance and fluorescence emission spectra (excitation at 488 nm) of fluorophore 2. (d) Picture of reactants (left) and fluorophore 2 (right) upon UV excitation. All spectra are in toluene.

3

Monitoring production of compound 1 from a Suzuki reaction over time by fluorimetry. Spectra were recorded before catalyst addition (t = −1 min), immediately after catalyst addition (t = 0 min), and every 10 min thereafter for 120 min. (a) Fluorescence emission spectra of the reaction at various time points when excited at 532 nm. (b) Peak fluorescence intensity over time. (c) Qualitative color change of the fluorogenic Suzuki reaction over time.

4

Fluorescence from reactions with varying catalyst concentration (left) indicative of reaction yield can be used to measure turnover number (right) of the catalyst at varying concentrations, down to 0.023 mol percent.

5

Fluorogenic Suzuki coupling preformed in microscopic emulsion droplets. The brightfield of the droplets (a) and their fluorescence emission (b) can be overlaid (c) with fluorescence in false color (slightly magnified), showing the reaction is localized to the organic droplet phase and does not extend into the aqueous continuous phase. In emulsions with catalyst present, the fluorescence of the system increases on the time scale of hours in individual droplets (d, thin gray) and for the mean of all drops in the field of view (d, color). In control experiments (d, black), emulsions were made with both starting materials but without catalyst, and no increase in fluorescence was observed.