Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins

J Med Chem. 2018 Dec 27;61(24):11199-11208. doi: 10.1021/acs.jmedchem.8b01302. Epub 2018 Dec 10.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / chemistry
  • Azides / chemistry*
  • Binding Sites
  • Cysteine / chemistry*
  • Deuterium Exchange Measurement
  • Ligands
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Molecular Probes / chemistry*
  • Peptides / analysis
  • Peptides / chemistry
  • Receptor, Cannabinoid, CB2 / agonists
  • Receptor, Cannabinoid, CB2 / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Tandem Mass Spectrometry
  • Ultraviolet Rays

Substances

  • Amino Acids
  • Azides
  • CNR2 protein, human
  • Ligands
  • Membrane Proteins
  • Molecular Probes
  • Peptides
  • Receptor, Cannabinoid, CB2
  • Cysteine