A CuSO/Bicinchoninic acid/Reducing sugar based stable and non-ROS catalyst system for the CuAAC reaction in bioanalysis.

The limitations of commonly used sodium ascorbate-based catalyst system for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction include excess production of reactive oxygen species and rapid catalyst deactivation. In this study instead of using a highly active reducing agent, such as, sodium ascorbate, we chose reducing sugar as a mild reducing agent to build up the catalyst system for CuAAC reaction. Interestingly, the bicinchoninic acid (BCA) assay system containing reducing sugar satisfies the essential elements of the catalyst system for CuAAC reaction. We found that CuSO/BCA/Reducing sugar system can catalyze the CuAAC reaction but with low yield. Rational analyses of various parameters in CuSO/BCA/Glucose catalyst system suggested storage at room temperature might enhance the catalytic activity, which was proven to be the case. Importantly, the system remains stable at room temperature and minimal HO was detected. Notably, our study showed that the coordination between the slow reduction of Cu(I) by reducing sugar and the selective chelation of Cu(I) by BCA is key to developing this system. The CuSO/BCA/Reducing sugar catalyst system was successfully applied to various CuAAC reaction based bioanalyses, and it is suitable for the CuAAC reaction based bioanalyses that are sensitive to ROS or request long reaction time.