Enabling in vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Si-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts

Abstract

<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>

Bioorthogonal Chemistry and Its Applications

“…Recent developments such as the in situ generation of tetrazines from dihydrotetrazines using near-IR light may help to address this limitation. 167 We hope that we have shown that bioorthogonal chemistry has been a useful enabling technology in biology and chemistry. It has enabled better understanding of biological structures, pathways, and organelles, may enable the development of more effective and selective disease treatments and diagnostic agents, and has been a broadly used technique in biology and chemistry.…”

Visible Light Chemical Micropatterning Using a Digital Light Processing Fluorescence Microscope

“…The absence of a thiol functionality from the beads, the alkene functionality from the fluorophore, or the addition of free-radical inhibitor TEMPO led to no observed patterning. Interestingly, experiments lacking eosin y did result in some patterning, indicating that the reaction could take place without this photoinitiator, perhaps mediated by the silicon rhodamine itself, 43 though not as efficiently (Figure S6). We also studied the effect of light intensity on the patterning contrast for on-bead thiol−ene micropatterning and RhBNN−PVA film micropatterning and found that increasing the LED intensity generally led to higher contrast under controlled conditions, but the contrast approached a plateau at higher intensities (Figure S6 and 7).…”

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

“…More recently we have shown that silarhodamine (SiR) dyes, initially developed as fluorophores for biological imaging, 74−77 can be repurposed as photocatalysts for DHTz oxidation. 78 The Janelia-SiR dyes 77 were found to be especially effective even at low catalyst loadings. With SiR-photocatalysts, DHTz oxidation is more rapid than the competing sensitization of singlet oxygen, and therefore the photocatalytic activation of tetrazine ligation can be applied to protein modification while minimizing oxidative damage.…”