Comparative Study of Click Handle Stability in Common Ligation Conditions

Click chemistry efficiently ligates molecular building blocks in a robust and high-yielding manner and has found major application in the rapid modification of important molecular actors in biological systems. However, the high reactivity of click handles often correlates with decreased stability, which presents a significant challenge in the practical application of these systems. In the current study, we describe a survey of the stability of commonly deployed click manifolds across a range of widely used ligation conditions. Incompatible click handle and ligation condition combinations are identified, with kinetic half-lives and side products of each undesired reaction determined, including the assessment of stability over extended periods and in a protein environment. This data set provides researchers with a roadmap to expediently determine the most appropriate click reaction conditions for any given bioorthogonal application, thus elevating the probability of success of procedures that utilize click chemistry.