Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells

Abstract

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule–protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule–protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule–protein interactions.

Protein‐Metabolite Interactions: Discovery and Significance

“…A terminal alkyne‐containing “minimalist” diazirine linker (Figure 7b; left) developed by the Yao laboratory [48] has been conjugated to a wide variety of cellular metabolites and drugs for proteome profiling applications [49] . Recently, other diazirine linkers have been reported, such as a “minimalist fully‐functionalized” diazirine tag developed by the Parker group (Figure 7b; middle) that contains a smaller linker length than the one developed by the Yao group, [50] and PALBOX, a cyclobutane‐containing diazirine linker developed by the Woo group (Figure 7b; right) that employs ring strain to destabilize the linear diazo functional group formed upon diazirine photolysis, yielding an unstable carbene resulting in faster photo‐crosslinking than traditional alkyl diazirines [51] …”

“…[49] Recently, other diazirine linkers have been reported, such as a "minimalist fullyfunctionalized" diazirine tag developed by the Parker group (Figure 7b; middle) that contains a smaller linker length than the one developed by the Yao group, [50] and PALBOX, a cyclobutane-containing diazirine linker developed by the Woo group (Figure 7b; right) that employs ring strain to destabilize the linear diazo functional group formed upon diazirine photolysis, yielding an unstable carbene resulting in faster photo-crosslinking than traditional alkyl diazirines. [51] Research from the Cravatt laboratory has contributed significantly towards the discovery of metabolite-interacting proteomes by employing photoaffinity labeling, including those for S-adenosyl homocysteine, sterols, and arachidonic acid. [49a,52] The interactome of other fatty acids, [53] sphingosine, [54] and phosphatidic acid (PA) [49b] have also been determined in cells by using the bifunctional diazirine modified analogs of these lipids or their precursors.…”

Photoaffinity Labeling Chemistries Used to Map Biomolecular Interactions

“…We additionally developed a tag, dubbed PALBOX, which carries a diazirine embedded to a cyclobutyl ring (DOI: 10.1021/jacs.2c08257). [100] PALBOX has limited pH‐dependent reactivity and reduces labeling of proteins through the diazo intermediate, presumably due to destabilization of the diazo isomer from ring strain. With further evaluation, PALBOX may serve as a PAL tagging strategy with higher labeling specificity that is well‐suited for cellular target identification experiments.…”

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins

“…Selective labeling of proteins with small molecules is critical for structural and functional study of proteins and comprehensive investigation of protein/molecule interactions. − Among the well-studied approaches, photoaffinity labeling (PAL) has been proved as a powerful chemical tagging method to capture biomolecular targets and to study ligand–receptor interactions in biological systems by using photoactive probes to covalently capture the interacting targets. − Conventional photoactive probes typically consist of a molecule (ligand), a photoactive group, and a bioorthogonal handle (Scheme a). In the mechanism, the photoactive group will be converted into a highly reactive intermediate upon UV irradiation, which can react with many amino acid residues, forming covalent bonds between the molecule and target (Scheme a).…”

“…Herein, we proposed a novel photoaffinity labeling approach using UCNP-based photoactive probes, and Scheme b illustrates the proof-of-concept design for this work. Diazirine, one of the most commonly used photoactive groups, ,, and ligands are separately loaded onto the surface of Tm/Yb co-doped NaYF 4 core–shell nanoparticles via hydrophilic poly­(ethylene glycol) (PEG) and disulfide linkage. Upon NIR irradiation (980 nm), UV light was emitted via a multiphoton process known as upconversion, which can activate diazirines to carbenes via an intersystem transformation.…”