Fluorogenic label for biomolecular imaging

Abstract

Traditional small-molecule fluorophores are always fluorescent. This attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single user-designated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

Figure 1

Spectra of Rh₁₁₀ and its derivatives. a) Absorption spectra of Rh₁₁₀ and derivatives 1–5 (7.5 μM). b) Fluorescent emission spectra of Rh₁₁₀ and 1–3 (λ_ex = 450 nm, not to scale).

Figure 2

Hammett plot of extinction coefficient (○) and quantum yield (●) versus σ_p for Rh₁₁₀, urea 1, and amide 2.

Figure 3

Stability of pro-fluorophore 8 and fluorescein diacetate in aqueous solution. a) Time course for the generation of fluorescence (λ_ex 496 nm, λ_em 520 nm) of pro-fluorophore 8 (25 nM) and fluorescein diacetate (25 nM) in PBS. b) Time course of the generation of fluorescence (λ_ex 496 nm, λ_em 520 nm) of pro-fluorophore 8 (25 nM) and fluorescein diacetate (25 nM) in DMEM containing FBS (10% v/v).

Figure 4

Kinetic traces (λ_ex 496 nm, λ_em 520 nm) and Michaelis–Menten plot (inset) of a serial dilution of pro-fluorophore 8 (0.5 μM → 2 nM) with PLE (2.5 μg/mL).

Figure 5

Unmasking of pro-fluorophore 8 in live human cells. a) Unwashed HeLa cells incubated for 1 h with pro-fluorophore 8 (10 μM) at 37 °C in DMEM and counter-stained with Hoechst 33342. b) Washed HeLa cells incubated for 1 h with pro-fluorophore 8 (10 μM) at 37 °C in DMEM and counter-stained with Hoechst 33342 and LysoTracker Red (5% v/v CO₂(g), 100% humidity). Scale bar: 20 μm.

Figure 6

Live-cell imaging experiments with protein conjugates. a) Unwashed HeLa cells incubated for 1 h with Oregon Green–RNase A conjugate (10 μM) at 37 °C in DPBS and counter-stained with Hoechst 33342. b) Washed HeLa cells incubated for 1 h with Oregon Green–RNase A conjugate (10 μM) at 37 °C in DPBS and counter-stained with Hoechst 33342. c) Unwashed HeLa cells incubated for 1 h with fluorogenic label 13–RNase A conjugate (10 μM) at 37 °C in DPBS and counter-stained with Hoechst 33342. d) Washed HeLa cells incubated for 1 h with fluorogenic label 13–RNase A conjugate (10 μM) at 37 °C in DPBS and counter-stained with Hoechst 33342 and LysoTracker Red (5% v/v CO₂(g), 100% humidity). Scale bar: 20 μm.

Scheme 1

Synthetic Route to Pro-Fluorophore 8

Scheme 2

Synthetic Route to Fluorogenic Label 13

Scheme 3

Modules in Fluorogenic Label 13