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

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.