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

Abstract

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.

Bioorthogonal Chemistry and Its Applications

“…In most cases, tetrazole ligations require UV light for effective labeling (though the use of two-photon lasers with near-IR light and of 365 nm light has been reported), and such light can cause damage to organisms and has limited penetration into tissue. Recent developments such as the in situ generation of tetrazines from dihydrotetrazines using near-IR light may help to address this limitation …”

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 ).…”

A Photosensitized Singlet Oxygen (1O2) Toolbox for Bio-Organic Applications: Tailoring 1O2 Generation for DNA and Protein Labelling, Targeting and Biosensing

“…A careful evaluation of the photo-oxidation conditions was performed in a recent work of C. Wang et al [ 108 ]. In 2016, they described the application of the strong PSs MB ( 1 O 2 quantum yield (QY): 0.52 in water [ 109 ]) and RB ( 1 O 2 QY: 0.76 in water [ 109 ]) as a turn-on catalyst for tetrazine ligation.…”

“…This was illustrated in a proof-of-concept experiment, in which the protein thioredoxin (Trx, 12 kDa) was exposed to the MB photo-oxygenation conditions applied in their ligation protocol. As LC-MS experiments clearly showed protein damage ( Figure 8 C), they decided to adopt a milder PS, such as a Si-Rhodamine derivative [ 108 ]. Although, the 1 O 2 -generation by these dyes can be more controlled, oxidative damage is still observed.…”

“…( B ) Turn-on tetrazine ligation method, introduced in [ 107 ]. ( C ) ESI mass spectrometry illustration of the oxidative damage to the protein thioredoxin (Trx) caused by the presence of MB and irradiation, used with permission from [ 108 ]. Copyright 2021 American Chemical Society.…”