The amino acid acridon-2-ylalanine (Acd) can be a valuable fluorescence probe for studying protein conformational change because it is long lifetime, high quantum yield, visible wavelength fluorophore, that is small enough to be incorporated into proteins of interest during ribosomal protein synthesis. Acd can be synthesized from tyrosine in a few steps in over 85% overall yield.  However, there is a decarbonylated by-product (<i>N-</i>phenyl-aminophenylalanine, NpF), which is produced in trace amounts, but can be incorporated into proteins by the permissive first generation aminoacyl tRNA synthetase selected for Acd incorporation (AcdRS1). Here, we have screened three generations of AcdRS (1, 2, and 3) and investigated the efficiency of Acd-specific incorporation to proteins of interest. AcdRS3 showed the highest specificity. It can tolerate up to 10% contamination by Npf, as confirmed by MALDI-MS of full-length and trypsin digested proteins. This improved AcdRS allows us to use a simple, efficient synthesis of Acd on the multi-gram scale with very limited chromatography.  Furthermore, our studies of Npf contamination provide a cautionary tale to those evolving novel tRNA synthetases for the incorporation of unnatural amino acids. The analogs of Acd containing electron withdrawing and donating groups, or extended aromatic system have also been synthesized and characterized their photophysical properties.

Development of intrinsically fluorescent unnatural amino acids for <i>in vivo</i> incorporation into proteins

252nd National Meeting (2016)

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Development of intrinsically fluorescent unnatural amino acids for in vivo incorporation into proteins