(567at) Optimization of the Fluorogenic Copper(I)-Catalyzed 1,3-Dipolar Cycloaddition ("Click") Reaction in a Crude Cell Lysate

In recent years, the copper(I) 1,3-dipolar cycloaddition (?click?) reaction has become a widely used reaction for labeling and characterizing biological molecules. It is favored above other reactions because of its stability in biological systems. It can be performed in aqueous environments, at relatively neutral pHs, while consuming otherwise non-reactive reagents. However, due to the presence of copper(I), the click reaction proves cytotoxic when performed in conjunction with in vivo transcription/translation systems.

We seek to assess the effectiveness of the copper(I)-catalyzed 1,3-dipolar cycloaddition reaction in a crude cell lysate. Performing the click reaction in a cell lysate is complicated by sulfhydryl groups and other entities which interact with the copper(I) catalyst. To rapidly assess a range of conditions for optimum click reaction efficiency, 3-azido-7-hydroxycoumarin, a reagent that fluoresces upon completion of the click reaction, was synthesized and characterized. Fluorescence was used to measure the conjugation efficiency of the 3-azido-7-hydroxycoumarin with an alkyne containing small molecule over a range of conditions. By enabling and optimizing the copper(I)-catalyzed 1,3-dipolar cycloaddition reaction in a crude cell lysate, we seek to streamline the method for click conjugation involving biological products by eliminating the need for biological product purification before click conjugation.