Monitoring Protein−Polymer Conjugation by a Fluorogenic Cu(I)-Catalyzed Azide−Alkyne 1,3-Dipolar Cycloaddition

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Abstract
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has recently proven to be a powerful synthetic tool in various fields of chemistry, including protein-polymer conjugation. In this article, we describe a fluorogenic CuAAC, which allows for efficient monitoring of protein-polymer conjugation. We show that profluorescent 3-azido coumarin-terminated polymers can be reacted with an alkyne-functionalized protein to produce a strongly fluorescent triazole-linked conjugate. Upon formation of the product, the evolution of fluorescence can accurately be determined, providing information about the course of the CuAAC. As a proof of concept, we synthesized several 3-azido coumarin terminated poly(ethylene glycol) (PEG) chains and investigated their conjugation with alkyne-functionalized bovine serum albumin (BSA) as a model protein. CuAAC conjugation was shown to be very efficient and proceeded rapidly. Conversion plots were constructed from measuring the fluorescence as function of reaction time. An additional benefit of the fluorogenic CuAAC is the in situ labeling of bioconjugates. We envision that the fluorogenic protein-polymer conjugation is not restricted to the reaction system reported in this work, but may also be ideal to screen for optimal reaction conditions of various other systems.