Fluorogenic, two-photon-triggered photoclick chemistry in live mammalian cells

Abstract

The tetrazole-based photoclick chemistry has provided a powerful tool to image proteins in live cells. To extend photoclick chemistry to living organisms with improved spatiotemporal control, here we report the design of naphthalene-based tetrazoles that can be efficiently activated by two-photon excitation with a 700 nm femtosecond pulsed laser. A water-soluble, cell-permeable naphthalene-based tetrazole was identified that reacts with acrylamide with the effective two-photon cross-section for the cycloaddition reaction determined to be 3.8 GM. Furthermore, the use of this naphthalene-tetrazole for real-time, spatially controlled imaging of microtubules in live mammalian cells via the fluorogenic, two-photon-triggered photoclick chemistry was demonstrated.

Figure 1

Design of naphthalene-tetrazoles for fluorogenic, two-photon-triggered photoclick chemistry. (a) General features of the naphthalene-tetrazoles and scheme of fluorogenic, two-photon-triggered photoclick chemistry. (b) Chemical structures of the naphthalene-tetrazoles 1–6 used in this study.

Figure 2

Determining tetrazole two-photon absorption cross section and two-photon cycloaddition cross-section. (a) UV-Vis (dash line) and fluorescence spectra (solid line) monitoring of the cycloaddition reaction between 25 μM tetrazole 6 and 2.5 mM acrylamide in acetonitrile/PBS (1:1) under 700 nm femtosecond pulsed laser irradiation. For fluorescence measurement, λ_ex = 405 nm. (b) Plot of the pyrazoline cycloadduct formation over 120 min of 700 nm femtosecond pulsed laser irradiation. Tetrazole 3 was not included because of its insufficient solubility in acetonitrile/PBS (1:1). (c) Plot of the time course of photo-decaging of BHC-OAc upon 740 nm femtosecond pulsed laser irradiation. (d) Kinetic and photophysical data for naphthalene-tetrazoles in the two-photon triggered photoclick chemistry. The two-photon cross section of the cycloaddition reaction, δ_cT, and two-photon absorption cross section, δ_aT, were measured in GM (10⁻⁵⁰ cm⁴s/photon).

Figure 3

Selective femtosecond pulsed laser induced fluorogenic labeling of sfGFP-S2AcrK by tetrazole 6 in vitro. (a) Reaction scheme. A solution of 10 μM sfGFP-S2AcrK and 200 μM tetrazole 6 in PBS buffer in a quartz cuvette was irradiated with a 700 nm femtosecond laser for 4 h. (b) Coomassie blue stain (top panel) and in-gel fluorescence (bottom panel) of the same protein gel after the reaction mixtures were resolved by SDS-PAGE. (c) De-convoluted mass spectra of sfGFP-S2AcrK (in red) and the product (in blue) showing the formation of the pyrazoline adduct.

Figure 4

Fluorescence microscopy of the fluorogenic, two-photon triggered photoclick chemistry in CHO cells. (a) Fluorescence/DIC overlap image before photoirradiation (panel 1), time-lapsed fluorescence images (panels 2–9) and fluorescence/DIC overlap image after photoirradiation (panel 10). CHO cells were treated with 3 μM tetrazole 6 in the presence (top row) or absence (bottom row) of 40 μM IPFAD followed by photoillumination of the red rectangle area with a 700 nm femtosecond pulsed laser. Scale bar = 61.3 μm. (b) Time courses of fluorescence development in ten cytosolic regions in selected CHO cells; red curve denotes the regions in the IPFAD-treated cells while blue curves denote the regions in the untreated cells. The concentrations of tetrazole 6 and IPFAD were 3 μM and 40 μM, respectively. (c) Effect of tetrazole 6 concentration on fluorescence intensity monitored by confocal microscopy. See Figure S21 for details.