Improved\nLarge-Scale Synthesis of Acridonylalanine\nfor Diverse Peptide and Protein Applications

Fluorescent unnatural amino acids give biochemists, biophysicists,\nand bioengineers new ways to probe the properties of proteins and\npeptides. Here, the synthesis of acridon-2-ylalanine (Acd) is optimized\nfor large-scale production to enable ribosomal incorporation through\ngenetic code expansion (GCE), and fluorenylmethoxycarbonyl (Fmoc)-protected\nAcd is prepared for solid-phase peptide synthesis (SPPS). We demonstrate\nthe utility of Acd in several applications: first, Acd quenching by\nTyr is used in the design of fluorescent protease sensors made by\nSPPS. Second, we demonstrate Acd incorporation into a lanthanide-binding\npeptide that is generated either by GCE or by SPPS and demonstrate\nthe utility of Acd for sensitizing the emission of Eu<sup>3+</sup>. Finally, Acd is inserted into the intrinsically disordered protein,\nα-synuclein, using GCE and used to study ion binding and aggregation.