We also investigated whether fluorescent amino acids 3, 4 could be directly incorporated into a model linear sequence P2 by mischarging onto the tryptophan tRNA by tryptophanyl-tRNA synthetase. Amino acids 3, 4 were supplemented to a Trp-free FIT system. MALDI-TOF MS analysis showed successful expression of the 4-cyanotryptophan containing peptide 4CNW-P2, indicating good tolerance of 3 with tryptophanyl-tRNA synthetase to allow efficient tRNA charging and subsequent translation (Figure ). However, consistent with a previous report, attempts at incorporating AzAla 4 were unsuccessful, presumably due to failed recognition by tryptophanyl-tRNA synthetase owing to ring expansion and removal of the indole amine.