Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins

A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivatives were first oxidized to the corresponding quinonoidal spirolactories followed by ceric Ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene-Alanine derivatives exhibited efficient trans/cis photoswitching upon irradiation With a blue (448 nm) or green (530 nm) LED light Moreover, nine superfolder green fluorescent protein (sfGFP) Mutants carrying the atobenzene-alanine analogues Were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/Py1RS pair, and one of the mutants showed durable photoswitching with the LED light.