We next asked whether Friedländer reactions would also proceed efficiently when performed on IVT peptides initiated with tRNA fMet acylated with monomer 2. Performing reactions on in vitro translation products is challenging because the reaction must proceed cleanly at low peptide concentration (< 1 µM) and in the presence of high concentrations of biomolecules and salts. β-keto amide peptide 2-P1 was generated using IVT, isolated using anti-FLAG magnetic beads, lyophilized, and treated with 2-aminoarylcarbonyl substrates 6, 8, 9, or 12 in acetic acid at 40 °C for 24 h. The formation of Friedländer products Q6-P1, Q8-P1, Q9-P1, and Q12-P1 were monitored using LC-HRMS (Supplementary Fig. ). In each case, the post-translational Friedländer reaction of 2-P1 proceeded to generate peptides with quinoline derivatives at their N-termini (Fig. ). In theory, identical quinoline-peptide hybrids could be prepared ribosomally using tRNA fMet acylated with a pre-assembled quinoline. Yet treatment of tRNA fMet with eFx and dimethyl quinoline cyanomethyl ester 25-CME (Supplementary Fig. ) led to no detectable acyl-tRNA product under any condition tested (Fig. and Supplementary Table ). We conclude that post-translational Friedländer reactions represent a simple path to ribosomally synthesized peptides with diverse quinoline functionality at the N-terminus.