Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light

Abstract

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as "CABL". Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O 2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels−Alder kinetics exceeding k 2 of 10 6 M −1 s −1 . CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin, or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of "ordinary" tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

Fluorescence turn-on by photoligation – bright opportunities for soft matter materials

“…To avoid the use of metals, organic photosensitisers are an alternative and several suitable photosensitisers have been employed in photocatalytic activation of bioorthogonal ligation by red and far-red light (600-660 nm). [130][131][132][133] While tracing reaction loci in biological systems is perhaps to date the most exploited application of fluorogenic productgenerated photochemical reaction systems, opportunities in synthetic soft matter materials science abound, as exemplified by the extensive utilisation of tetrazole-ene photoligation within the last 15-20 years. In initial work in this realm, we have exploited the tetrazole ligation for counting chemical ligation points in polymer networks as a probe to quantitatively assess the formed bonds during network generation.…”

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

“…In that context, copper-free azide–alkyne cycloaddition as well as inverse electron-demand Diels–Alder (IEDDA) tetrazine ligations have seen frequent use in live-cell imaging. − Subsequently, there have been numerous bioconjugation applications as well as new synthetic procedures for accessing symmetrical and unsymmetrical tetrazines as well as novel reactive dienophiles such as cyclopropenes and trans -cyclooctene (TCO). − Despite the widespread adoption of tetrazine ligations in bioconjugation chemistry, a noted drawback is the challenge in gaining spatial and temporal control over the initiation of the IEDDA reaction. Furthermore, reactive tetrazines are often unstable in water or in the presence of strong nucleophiles, and degradation by hydrolysis constitutes a major issue for live-cell imaging. , One strategy to achieve stimuli responsive tetrazine ligations is to control the redox state of the tetrazine. Tetrazines can be reduced to relatively unreactive dihydrotetrazines.…”

Seeking Citius: Photochemical Access of Reactive Intermediates for Faster Bioorthogonal Reactions

“…More recently, Fox and coworkers reported the design of a cell-permeable DHTz-3, which can be efficiently oxidized into the tetrazine using fluorescein or SiRbased photocatalyst (Figure 3d). [28] The photogenerated, highly reactive tetrazine reacts with trans-5-oxocene (o-TCO) with a second-order rate constant of 1.13 × 10 6 M À 1 s À 1 . Notably, the cytocompatibility of photocatalysis has been improved by limiting the extracellular production of singlet oxygen.…”

“…Unfortunately, DHTz‐ 2 showed poor cell permeability, preventing its use inside live cells. More recently, Fox and coworkers reported the design of a cell‐permeable DHTz‐ 3 , which can be efficiently oxidized into the tetrazine using fluorescein or SiR‐based photocatalyst (Figure 3d) [28] . The photogenerated, highly reactive tetrazine reacts with trans‐5‐oxocene (o‐TCO) with a second‐order rate constant of 1.13×10 6 M −1 s −1 .…”