posted on 2016-02-21, 16:53authored byRemon van Geel, Ger J.
M. Pruijn, Floris L. van Delft, Wilbert C. Boelens
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.