Development of strategies for the site-specific in vivo incorporation of photoreactive amino acids: p-azidophenylalanine, p-acetylphenylalanine and benzofuranylalanine

A major limitation of conventional site-directed mutagenesis is that substi tutions are restricted to the 20 naturally occurring amino acids. While thi s problem can be circumvented in vitro to allow the site-specific incorpora tion of non-canonical amino acids, no similar in vivo methodologies yet exi st. The main requirement for such a system is an aminoacyl-tRNA synthetase able to exclusively recognize a cion-canonical amino acid and a suppressor tRNA. The engineering of such activities in aminoacyl-tRNA synthetases has proven to be problematic due to their high substrate specificity. Here we r eport progress towards the development of an antibody-based methodology to screen large mutant aminoacyl-tRNA synthetase libraries for specific recogn ition of the non-canonical photoreactive benzofuran amino acid [3-(5'-benzo furanyl)-alanine]. We also report the chemical synthesis and enantiomeric r esolution of this amino acid. (C) 2000 Elsevier Science Ltd. All rights res erved.