Building on previous advances in the development of covalent peptides using encoded libraries, we aimed to develop a covalent peptide discovery platform using RaPID in which an unmasked electrophile would be directly incorporated into the in vitro translated peptides. This first required successful ribosomal incorporation of an electrophile warhead into mRNA-displayed cyclic peptide libraries. We synthesised an unnatural amino acid version of 1, N-d-fluoroacetimidoyl ornithine (FAO, 2, Figure ). To allow flexizyme recognition for aminoacylation onto tRNA we activated the carboxylic acid as the 4-chlorobenzyl thioester (CBT) (FAO-CBT, 3, Figure ). This thioester was used because the more commonly used dinitrobenzyl ester of FAO was synthetically intractable. After confirming successful flexizyme-mediated loading onto a short tRNA mimic (Figure ), we tested for ribosomal compatibility. An elongator tRNA with the methionine anticodon (CAU) was aminoacylated with FAO. This aminoacylated tRNA was used to perform in vitro translation using the PURExpress Ò translation system with methionine omitted. The initiator methionine was reprogrammed to chloroacetyl-D-tyrosine. We translated a peptide template containing a single methionine in the elongator region. MALDI-TOF spectroscopy showed that ribosomal incorporation of FAO was successful (Figure ). As our initial translation efficiency was low, we optimised this by screening four different elongator tRNAs containing variable tRNA Tstems (Figure ). As the T-stem number increases (1-4) this increases the affinity of the tRNA for elongation factor thermo unstable (EF-Tu), which we observed to correlate with enhanced ribosomal FAO incorporation efficiency. Optimal translation was observed with T- stem 4 which we went on to use in all subsequent experiments. No warhead hydrolysis or reaction with components in the translation system or buffer was observed, confirming that, at least with this electrophile, covalency can be encoded for in mRNA display with genetic code reprogramming, without the need for masking and post-translational modification. Denaturing guanidium chloride washes have been used by us and others during the affinity panning step of peptide selections to remove non-covalent binders and only retain covalently binding peptides (Figure ). We confirmed that at concentrations up to 8 M, this did not disrupt the interaction between biotinylated PADI4 and streptavidin beads (Figure ).