title: Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition.
abstract: The 1,3-dipolar cycloaddition of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomolecule labeling in complex biological systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddition (SPAAC). In this study, a subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, we show that thiol-yne addition with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR analysis. Moreover, we show that undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins containing reduced cysteines with IAM.
authors.affiliation: Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials and Netherlands Proteomics Centre, Radboud University Nijmegen, The Netherlands.
authors.forename: Remon
authors.initials: R
authors.lastname: van Geel
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.forename: Floris L
authors.initials: FL
authors.lastname: van Delft
authors.forename: Wilbert C
authors.initials: WC
authors.lastname: Boelens
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003500:Cyclization; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000480:Alkynes; D001386:Azides; D011506:Proteins; D013438:Sulfhydryl Compounds