Fluorogenic Cyclopropenones for Multicomponent, Real-Time Imaging

Abstract

Fluorogenic bioorthogonal reactions enable biomolecule visualization in real time. These reactions comprise reporters that "light up" upon reaction with complementary partners. While the spectrum of fluorogenic chemistries is expanding, few transformations are compatible with live cells due to cross-reactivities or insufficient signal turn-on. To address the need for more suitable chemistries for cellular imaging, we developed a fluorogenic reaction featuring cyclopropenone reporters and phosphines. The transformation involves regioselective activation and cyclization of cyclopropenones to form coumarin products. With optimal probes, the reaction provides >1600-fold signal turn-on, one of the highest fluorescence enhancements reported to date. The bioorthogonal motifs were evaluated in vitro and in cells. The reaction was also found to be compatible with other common fluorogenic transformations, enabling multicomponent, real-time imaging. Collectively, these data suggest that the cyclopropenone-phosphine reaction will bolster efforts to track biomolecule targets in their native settings.

Figure 1.

Fluorogenic reactions for biomolecule visualization. (A) “Dark” reporters form fluorescent adducts upon treatment with a bioorthogonal chemical trigger. (B) Cyclopropenones react with phosphine catalysts to produce ketenes. These intermediates can be trapped by various nucleophiles to form cyclic products. (C) In this work, fluorogenic cyclopropenones were developed. These reporters react selectively with phosphine nucleophiles to form fluorescent hydroxycoumarins.

Figure 2.

Asymmetric CpOs can form two ketene-ylides upon phosphine treatment. Both intermediates react with pendent phenol nucleophiles to yield cyclized adducts. However, only phosphine addition at C2 yields the desired coumarin product; addition at C3 affords the undesired and non-fluorescent benzofuran-2(3H)-one adduct.

Figure 3.

Distinct regioisomers are formed upon CpO treatment with various phosphines. (A) Phosphine activation of sterically modified CpOs can provide two cyclized adducts. (B) Structures of the phosphine probes examined. (C) Product distributions from treatment of CpO 1a–c with phosphines 4–6. ^aYields measured by ¹H-NMR spectroscopy.

Figure 4.

Regioselective phosphine addition to CpOs yields fluorescent adducts. (A) Sterically occluded CpO 7 reacts with phosphine probes 6 or 8 to form two distinct products (9–10). (B) Photophysical properties of CpO 7 and adducts 9–10 (measured in 50% EtOH/PBS, pH 7.40). The fluorescence properties of cyclized product 9 are comparable to 7-hydroxycoumarin, an established fluorophore. (C) Fluorescence spectra of CpO 7 and products 9–10 (10 μM, 50% EtOH/PBS, pH 7.40). Excitation spectra are shown in dashed lines, and emission spectra are shown in solid lines. (D) Fluorescence is dependent on the reaction of CpO 7 and phosphine 8. (E) Kinetic analysis of the fluorogenic reaction. CpO 7 (100 μM) was reacted with phosphine probe 8 (5 mM) in PBS (pH 7.40), and coumarin fluorescence was monitored over time. ^aMeasured in water.

Figure 5.

The fluorogenic CpO-phosphine reaction labels protein targets in vitro. (A) FluorCpO-HEWL (1–6 modifications, Figure S11) was incubated with CyDPPDS to label protein conjugates. (B) SDS-PAGE analysis of FluorCpO-HEWL (40 μM) incubated with CyDPPDS (1 mM) for 5–120 min in PBS (pH 7.4). Fluorescent products were observed within 5 min. (C) SDS-PAGE analysis of FluorCpO-HEWL (40 μM) incubated with CyDPPDS (100–1000 μM) for 90 min in PBS (pH 7.4). Low concentrations of CyDPPDS (100 μM) were sufficient for fluorophore formation. Data are representative of three replicate experiments.

Figure 6.

The fluorogenic CpO-phosphine reaction can be performed on live cells. (A) A549 cells were non-specifically functionalized with CpO 21. CpO-tagged biomolecules were then visualized upon treatment with phosphine 8. (B) Coumarin fluorescence was only observed in the presence of both reagents. Cells surfaces were stained with a membrane marker to aid visualization (green channel, WGA-fluorescein conjugate). Data are representative of three replicate experiments. (Scale bar: 20 μm.)

Figure 7.

Dual bioorthogonal fluorogenic reactions to simultaneously visualize protein targets. (A) Protein targets bearing FluorCpO or TCO handles were visualized in “one pot” fashion via reaction with complementary phosphine or tetrazine probes. (B) Gel analysis of FluorCpO-HEWL (40 μM) and TCO-BSA (1 μM) reacted with 8 (10 mM) and Tz-BODIPY (100 μM) for 90 min in bacterial cell lysate (300 μg). Selective fluorophore formation was observed when both reactions were performed separately or together. Data are representative of three replicate experiments.

Figure 8.

Simultaneous two-color imaging using orthogonal fluorogenic chemistries in live cells. (A) HEK cells expressing a mitochondrial localized SNAP-tag protein were labeled with a TCO-SNAP-tag ligand (20 μM, 30 min, 37 °C). These cells were also nonspecifically conjugated with fluorogenic CpO 21 (1 mM, 15 min, 37 °C). To visualize modified biomolecules, Tz-BODIPY and CyDPPDS (8) probes were added (0.25 μM, 1 mM, respectively). The cells were imaged immediately after probe addition. (B) No background fluorescence was observed in the absence of the fluorogenic triggers, Tz-BODIPY and CyDPPDS (8). (Scale bar: 200 μm.) (C) Modified cells were reacted with Tz-BODIPY and CyDPPDS (8), enabling two-color visualization over 1 h. (Scale bar: 200 μm.) (D) High magnification images acquired after 90 min incubation. Data are representative of three replicate experiments. (Scale bar: 30 μm.)

Figure 9.

Dual metabolite imaging with fluorogenic bioorthogonal chemistries. (A) Cer-TCO and DCA-FluorCpO were incubated with HeLa cells, prior to bioorthogonal detection. (B) No background labeling was observed in the absence of matched bioorthogonal pairs. (C) Cer-TCO and DCA-FluorCpO were visualized upon incubation with Tz-BODIPY and CyDPPDS. Data are representative of three replicate experiments. (Scale bars: 50 μm.)

Scheme 1.

Synthesis of a fluorogenic CpO probe for bioconjugation.