Finally we asked whether a Lewis acid-catalyzed Friedländer macrocyclization reaction was compatible with the challenging conditions required for post-translational modifications of in vitro translation (IVT) products. First, the cyanomethyl ester of kynurenine (Kyn-CME) and the flexizyme eFx were used to acylate tRNA AsnE2#2 GCU , a previously reported engineered tRNA 78 (Supplementary Figs. &). The resulting Kyn-tRNA GCU was added to the IVT reaction while excluding serine and sequestering native tRNA GCU with an antisense oligonucleotide (M5-1) to suppress near cognate read-through . We confirmed that β-ketoacid 2 and kynurenine (Kyn) could independently and effectively recode methionine at position 1 and serine at position 6, respectively, of a peptide prepared using IVT (Supplementary Fig. ). Using an identical strategy, we then used IVT to generate tetrakaidecapeptide ivt.LP5, which contains both an N-terminal β-keto amide as well as an internal kynurenine residue (Fig. ). Subsequent reaction of ivt.LP5 with 100 mM Yb(OTf 3 ) in MeCN at 60 °C for 24 h yielded quinoline-cyclized peptide ivt.CP5 (Fig. ) whose retention time and mass spectrum were identical to that of an authentic standard produced by SPPS (Supplementary Fig. ). This result confirms that mild, post-translational Lewis acid-catalyzed Friedländer reactions can effectively generate macrocyclic quinoline-peptide hybrids even at very low peptide concentrations and in the presence of the high concentrations of salts and biomolecules that are intrinsic to in vitro translation reactions. Preparative scale intramolecular Friedländer reactions to generate peptide macrocycles with an embedded backbone quinoline. Each linear peptide contains monomer 2 at the N-terminus and a single internal kynurenine residue. b-e, Structures and total ion chromatograms of crude reaction mixtures for synthetic peptides CP1-4 after Friedländer macrocyclization reactions. The peak containing the cyclic peptide is highlighted in teal. Atom count of the macrocycle backbone, HPLC 254nm yield, and isolated yield (in parentheses) are provided. All peptides were prepared using solid phase methods and purified as described in Supplementary Methods.