Ligand-Directed Photocatalysts and Far-Red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells.

Described are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red light (660 nm) photocatalysis is used to initiate a cascade of DHTz oxidation, intramolecular Diels-Alder reaction, and elimination to release phenolic compounds. Silarhodamine (SiR) dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially available conjugates of Hoechst dye (SiR-H) and docetaxel (SiR-T) are used to localize SiR to the nucleus and microtubules, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM SiR and 40 µM photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate-determining elimination step. In cellular studies, this uncaging process is successful at low concentrations of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as a photocatalyst and the fluorescent reporter for microtubule depolymerization, and with confocal microscopy, it was possible to visualize microtubule depolymerization in real time as the result of photocatalytic uncaging in live cells.

Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.

Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of trans-cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by trans-cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and trans-cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation.

Rapid bioorthogonal reactivity can be induced by controllable, catalytic stimuli using air as the oxidant. Methylene blue (4 µM) irradiated with red light (660 nm) catalyzes the rapid oxidation of a dihydrotetrazine to a tetrazine thereby turning on reactivity toward trans-cyclooctene dienophiles. Alternately, the aerial oxidation of dihydrotetrazines can be efficiently catalyzed by nanomolar levels of horseradish peroxidase under peroxide-free conditions. Selection of dihydrotetrazine/tetrazine pairs of sufficient kinetic stability in aerobic aqueous solutions is key to the success of these approaches. In this work, polymer fibers carrying latent dihydrotetrazines were catalytically activated and covalently modified by trans-cyclooctene conjugates of small molecules, peptides, and proteins. In addition to visualization with fluorophores, fibers conjugated to a cell adhesive peptide exhibited a dramatically increased ability to mediate contact guidance of cells.