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.

No abstract

trans-Cyclooctenes (TCOs) are essential partners for the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, 'a-TCOs', that is prepared in high yield via stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on large scale in two steps from 1,5-cyclooctadiene. Computational transition state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analog. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to washout more rapidly than other TCOs. File list (2) download file view on ChemRxiv aTCO paper Jan 30 JMF no watermark.pdf (3.24 MiB) download file view on ChemRxiv aTCO SI Jan 30 no water mark.pdf (3.60 MiB)

<p><a></a>Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts for the generation of 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. Described here is the use of Si-Rhodamine (SiR) dyes 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 applications in biology, but have not previously been applied to catalyze chemical reactions. A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A series of SiR derivatives were evaluated, and the Janelia-SiR dyes were found to be especially effective in catalyzing rapid photooxidation at low catalyst loadings (typically 1 µM). A protein that was site-selectively modified by trans-cyclooctene was quantitively 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 crosslink hyaluronic acid derivatives that were functionalized by dihydrotetrazine and trans-cyclooctenes, enabling 3D culture of human prostate cancer cells. This photoinducible hydrogel formation could also be carried out in vivo in live mice through subcutaneous injection of a solution containing SiR photocatalyst and a Cy7-labeled hydrogel precursor, followed by brief in vivo irradiation with 660 nm light to produce a stable hydrogel material. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in the in vivo environment. <br></p>

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.