Photocatalytic Diazirine Labeling of Proteins

We first tested the feasibility of visible light-sensitized N₂ elimination from diazirines by screening a variety of photocatalysts with increasing triplet energies (Fig. 2B). Although catalysts with triplet energies below 60 kcal/mol did not sensitize model diazirine 1, a catalyst with a triplet energy (E_T) exceeding this threshold {Ir[dF(CF₃)ppy]₂(dtbbpy)}PF₆ (2) (E_T = 60.1 kcal/mol) (28 ) promoted consumption of 1 under mild conditions (15 min, 25°C, 100 μM H₂O/dimethyl sulfoxide, 450 nm irradiation) in >97% yield. No reaction was observed with diazirine 1 in the absence of photocatalyst or light. We then redesigned this catalyst for biomolecular applications by increasing its water solubility through the addition of polyethylene glycol, carboxylic acid, and alkyne functional groups (3) (Fig. 2B). These modifications did not negatively affect its ability to sensitize N₂ elimination from 1 (supplementary materials). Diazirine sensitization could be extended to a variety of p- and m-substituted aryltrifluoro-methyl diazirines bearing valuable payloads for microscopy and proteomics applications, including free carboxylic acid, phenol, amine, alkyne, carbohydrate, and biotin groups (fig. S1). The extinction coefficient of the photocatalyst (2) is five orders ofmagnitude larger than that of the diazirine (1) at the wavelength emitted by the blue LEDs used for sensitization (450 nm), explaining the absence of a noncatalyzed background reaction (fig. S2). Last, we assigned a short-range (Dexter) energy transfer mechanism rather than a longer-range Förster energy transfer mechanism on the basis of a lack of overlap between the absorption band of diazirine 1 and the emission band of iridium catalyst 2 even at high concentrations of 1 (0.1 M) (fig. S3). Energy transfer was highly efficient, with a rate constant of 7.9(5) × 10⁷ M⁻¹s⁻¹ (measured through Stern-Volmer analysis; number in parentheses indicates standard deviation in trailing digit) (table S1 and figs. S4 and S5).
We sought to demonstrate that carbenes generated through photocatalytic diazirine sensitization could label proteins (Fig. 2C). When a solution of bovine serum albumin (BSA) (10 μM) and a biotinylated diazirine probe 4 (100 μM) were irradiated with 375-nm light, biotinylation of BSA was detected through Western blot. When irradiated with lower-energy visible light at 450 nm, the degree of biotinylation was <0.5% of the level observed through UV irradiation, establishing that the diazirine presents minimal background signal at this wavelength. However, in the presence of water-soluble iridium catalyst 3 (1 μM), catalyst-dependent biotinylation of BSA was observed. Photocatalytic labeling of BSA was further confirmed through intact protein mass spectrometry (fig. S6). Unlike other enzyme-based labeling methodologies, this approach requires continuous delivery of visible light to sustain diazirine sensitization for protein labeling. Accordingly, we exploited this feature to demonstrate how turning the light source on or off affords fine temporal control over the labeling process (Fig. 2C, right).
With an efficient photocatalytic system for carbene-based protein labeling in hand, we prepared a secondary antibody-photocatalyst conjugate as a general entry point for spatially targeted photocatalytic proximity labeling on cell surfaces. A goat anti-mouse (Gt/α-Ms) antibody was first decorated with azide groups through reaction with azidobutyric acid N-hydroxysuccinimide ester and then conjugated to alkyne-bearing iridium catalyst 3 by means of click chemistry, resulting in an antibody-photocatalyst ratio of 1:6. Next, to address protein-targeted labeling on a surface, we prepared a model system containing human Fc-tagged vascular endothelial growth factor receptor 2 (VEGFR2) and epidermal growth factor receptor (EGFR) proteins attached to α-human immunoglobulin G (IgG) agarose beads (Fig. 3A). These beads were sequentially incubated with a Ms/α-VEGFR2 antibody and Ir-Gt/α-Ms to position the iridium catalyst close to the VEGFR2 proteins on the bead surface. Irradiation of these beads with 450-nm light in the presence of a diazirine-biotin probe afforded selective labeling of VEGFR2 over EGFR. When Ms/α-EGFR was used as the primary antibody, the selectivity of labeling was reversed. An analogous experiment, using peroxidase-based labeling, was incapable of differentiating between EGFR or VEGFR2 (fig. S7).