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. 2010 Apr 6;49(16):2869-72.
doi: 10.1002/anie.200906120. Epub 2010 Mar 19.

Bioorthogonal turn-on probes for imaging small molecules inside living cells

Neal K Devaraj  1 , Scott Hilderbrand, Rabi Upadhyay, Ralph Mazitschek, Ralph Weissleder
Affiliations

Bioorthogonal turn-on probes for imaging small molecules inside living cells

Neal K Devaraj et al. Angew Chem Int Ed Engl. .

Abstract

Glowing tags: a series of activatable ("turn-on") tetrazine-conjugated fluorescent probes was developed, which react rapidly in an inverse-electron-demand [4+2] cycloaddition with strained dienophiles such as trans-cyclooctene, thereby strongly increasing the fluorescence intensity. The novel turn-on probes were applied for intracellular live-cell imaging of a microtubuli-binding trans-cyclooctene modified taxol.

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Figures

Figure 1
Figure 1
a) Tetrazine-BODIPY FL (1) reacts rapidly with trans-cyclooctenol (2) via an inverse electron demand Diels-Alder cycloaddition to form isomeric dihydropyrazine products (3). b) Emission spectra of various tetrazine probes (black lines) and the corresponding dihydropyrazine products (dashed blue lines). Inset images compare the visible fluorescence emission of the tetrazine probes (left cuvettes) to their corresponding dihydropyrazine products (right cuvettes) under excitation from a handheld UV lamp.
Figure 2
Figure 2
a) Structure of taxol (1) and Trans-cyclooctene modified taxol (2) (TCO-taxol). b) Comparison of the ability of 10 µM taxol, trans-cyclooctene taxol, and a DMSO control to polymerize tubulin in the absence of GTP (polymerization assayed via absorbance at 350nm). Note that trans-cyclooctene taxol promotes polymerization similar to taxol and significantly better than a DMSO control. c) microtubule bundles formed in the presence of trans-cyclooctene taxol treated with tetrazine-BODIPY FL and visualized by fluorescence microscopy.
Figure 3
Figure 3
a) Confocal microscopy of a PtK2 cells after treatment with 1µM trans-cyclooctene-taxol followed by 1µM tetrazine-BODIPY FL (green). Scale bar: 30 µm. Expansion of the section indicated by the dashed white line (b) reveals that tubular structures are clearly stained. c) Confocal microscopy of PTK2 cells after treatment with 1µM taxol followed by 1µM tetrazine-BODIPY FL. Scale bar: 50 µm. d) Confocal microscopy of PTK2 cells after treatment with 1µM trans-cyclooctene-taxol followed by 1µM tetrazine-VT680. Scale bar: 50 µm.
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