Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins

Upon ultraviolet
activation, cannabinergic aliphatic azido (N3) ligands
covalently label cannabinoid receptors, prominent
G-protein-coupled receptor (GPCR) drug targets. We report here the
mechanism of covalent attachment to selected substrates of the high-affinity
CBR inverse agonist AM1335 and its deuterated analog AM1335­(d10),
arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized
the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6)
peptide and an N-acyl-protected cysteine (NAC). The
photochemical reaction products of model substrates with AM1335 and
AM1335­(d10) were analyzed with tandem electrospray ionization mass
spectrometry fragmentation and deuterium exchange mass spectrometry.
The nitrene initially formed after photoreaction undergoes rearrangement
to an imine which then interacts with the cysteine sulfhydryl group,
resulting in ligand attachment. Our results demonstrate that covalent
probes carrying aliphatic azides behave more selectively than originally
thought and can be used to label protein cysteine residues preferentially.