Targeted activation in localized protein environments via deep red photoredox catalysis

Abstract

State-of-the-art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high-energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small-molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red-light (λ = 660 nm) photoredox catalysis and its use in photocatalysed proximity labelling. We demonstrate that aryl azides are converted to triplet nitrenes via a redox-centric mechanism and show that its spatially localized formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labelling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumour-selective expression with EpCAM.

Fig. 1. Selective and targeted uses of aryl azides in chemical biology require low-energy light.

a. Contemporary methods for aryl azide photolysis in chemical biology use high energy light. b. Direct azide photolysis proceeds through a singlet nitrene intermediate. c. Leveraging electron- or energy transfer mechanisms associated with deep red and near-infrared photoredox catalysis for azide activation. d. Targeted aryl azide activation with red light for labeling in biological environments.

Fig. 2. Deep red photoredox catalysis overcomes fundamental photolytic limitations of aryl azides.

a. Mechanistic investigations into the photolysis and catalytic activation of azides show that the background reactivity is present when using blue light, with isolated yields in parentheses. b. Overlaid absorption spectra of unsubstituted azide 3, perfluorinated azide 8, and osmium photocatalyst 7. c. Azide-to-aniline reduction studies suggest an electron transfer pathway is operative. d. Broad substrate compatibility is observed for azide-to-aniline reduction (See the supplemental information for experimental conditions, ¹⁹F NMR yields in parentheses).

Fig. 3. Computational analysis reveals a redox-neutral, electron-transfer pathway for triplet nitrene formation.

a. A comparison of density functional theory calculated azide-to-triplet nitrene pathways via electron transfer or triplet Dexter energy transfer. B3LYP-D3/CPCM(water)/aug-cc-pVTZ level of theory. b. Computational models show that the electron transfer to the azide results in a “barrierless” dissociation of N₂ from the reduced azide. B3LYP/ma-def2-SVP level of theory. c. A putative mechanism for accessing triplet nitrenes via a redox-neutral photoredox cycle.

Fig. 4. Mechanistic differences exist between singlet and triplet perfluoroaryl nitrenes.

Distinct reaction pathways are observed for the singlet and triplet nitrenes generated from perfluoroaryl azides, as shown by the products of toluene C–H amination (top) and dimethyl sulfoxide imidation (bottom) (See the supplemental information for experimental conditions, ¹⁹F NMR yields are listed in parentheses).

Fig. 5. Deep red light-mediated protein labeling.

a. Schematic depicting labeling of carbonic anhydrase in the presence of osmium-alkyne photocatalyst, PFAA-biotin, and red light. b. Western blot image of carbonic anhydrase labeled with PFAA-biotin under different irradiation conditions over 15 min. Selective PFAA activation is only achievable when using a combination of red light and photocatalyst 33, as background protein biotinylation is observed when using blue light alone. Bar plots of replicate analysis of protein biotinylation measured by western blot. Error bars represent standard deviation of n = 3 experiments. c. Time course of protein biotinylation. Western blot analysis shows increased protein labeling over a 30-minute time course. Bar plots of replicate analysis of protein biotinylation measured by western blot. Error bars represent standard deviation of n = 3 experiments.

Fig. 6. Deep red light-mediated photocatalytic labeling of cellular environments.

a. Schematic depicting antibody-mediated proximity labeling of EpCAM on the cell surface of different HCT116 cell systems using the osmium photocatalyst system followed by mass spectrometry-based proteomic analysis to identify protein microenvironments for downstream bioinformatic analysis. b. Western blot analysis of EpCAM targeted labeling on the surface of HCT116 cells with an α-EpCAM or isotype primary antibody and a secondary antibody photocatalyst conjugate. Increased protein biotinylation is detected with increased DR-light exposure in the presence of the α-EpCAM antibody. Data are representative of n = 2 independent experiments with similar results. c. Venn diagram analysis comparing statistically enriched surface proteins identified by proximity labeling in the three different cell systems (total number of enriched proteins in each system is indicated in the circle). Proteins with known surface expression are highlighted in bold. d. STRING protein interaction network analysis of significantly enriched membrane proteins from EpCAM-targeted labeling in different HCT116 cell systems. Line thickness between nodes correlates with experimental evidence of interactions from the STRING database. e. Colon Adenocarcinoma primary tumor samples (TCGA, n = 290) vs Sigmoid / Traverse Colon normal tissue samples (GTEx, n = 308) log-fold change of the genes enriched in two or more cell systems highlighted in panel C. Boxplot bounds represent the 25th (Q1) and 75th (Q3) percentile of expression values, with the center indicating median expression. Boxplot length (IQR) equals Q3-Q1. Boxplot whiskers indicate minimum and maximum value with outliers shown as points, and defined as values lower than Q1 - 1.5* IQR and greater than Q3 + 1.5* IQR f. Heatmap showing log₂ fold-change between tumor vs normal gene expression across the indicated tumor types. g. Scatterplots showing co-expression between EpCAM and the indicated gene in primary tumor colon adenocarcinoma samples from TCGA (red, n = 290) and healthy colon and large intestine samples from GTEX (blue, n = 308). Co-expression is denoted as % tumor samples that expressed both genes at TPM > 10, indicating medium/high expression level (blue, dashed line). The effect size of tumor versus normal co-expression difference is provided as Cohen h for each gene pair.