Ne-acryloyl-L-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a mutant pyrrolysyl-tRNA synthetase pair. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of biocompatible and unique reactions for protein modifications in vitro. These reactions include Michael addition, radical polymerization and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Ne-acryloyl-L-lysine is efficiently modified with thiol-containing Michael donors at slightly alkali conditions and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Ne-acryloyl-L-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.

Genetically encoded acrylamide functionality directs diverse bioorthogonal protein modifications

246th National Meeting (2013)

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