Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials and Netherlands Proteomics Centre, Radboud University Nijmegen, The Netherlands.
Bioconjug Chem. 2012 Mar 21;23(3):392-8. doi: 10.1021/bc200365k. Epub 2012 Mar 8.
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.
叠氮化物与环张力炔烃的 1,3-偶极环加成反应是少数几种适合于复杂生物体系中特定生物分子标记的生物正交反应之一。然而，在一定程度上，应变炔烃可以非依赖于叠氮化物的方式对蛋白质进行标记，从而限制了基于应变促进的叠氮化物-炔烃环加成（SPAAC）的分析方法的灵敏度。在这项研究中，使用了一组三种环辛炔，二苯并环辛炔（DIBO）、氮杂二苯并环辛炔（DIBAC）和双环[6.1.0]壬炔（BCN），来评估蛋白质在体外的非依赖于叠氮化物的标记。对于所有三种环辛炔，我们表明，还原肽基半胱氨酸的硫醇-炔加成反应是导致大多数非依赖于叠氮化物的多肽标记的原因。通过 LC-MS 和 NMR 分析确认了反应产物的身份。此外，我们表明，通过用碘乙酰胺（IAM）对肽基半胱氨酸硫醇进行烷基化，可以防止不需要的硫醇-炔反应。由于 IAM 与 SPAAC 兼容，因此通过预先用 IAM 孵育含有还原半胱氨酸的蛋白质，可以实现更特异的依赖于叠氮化物的标记。
