Recently, non-canonical amino acids 4-cyanotryptophan (4CNW) and b-(1-azulenyl)-L-alanine (AzAla) have been shown to be efficient blue fluorescent emitters. Benefiting from their close structural similarity with tryptophan, incorporation of such molecules into cyclic peptides is potentially less disruptive to overall structure and permeability properties. Inspired by this, we set out to explore the introduction of 4CNW and AzAla into cyclic peptides by ribosomal translation. We selected the flexible in vitro translation system (FIT), which uses Flexizymes (small flexible tRNA acylation ribozymes) to prepare nonproteinogenic acyl-tRNAs using chemically activated amino acids. In a typical RaPID selection, the initiator is reprogrammed with an Nchloroacetylated amino acid, that readily undergoes peptide cyclisation with a downstream cysteine after in vitro translation of the linear peptide. We designed compounds ClAc-4CNW-CME 7 and ClAc-AzAla-CME 8 to explore the inclusion of fluorescent amino acids into in vitro translated cyclic peptides via initiator reprogramming. The chloroacetyl (ClAc) group facilitates peptide cyclisation with a cysteine residue following translation, and the cyanomethyl ester (CME) acts as an efficient leaving group for flexizyme-mediated tRNA charging. Amino acids 3 and 4 were obtained by enzymatically assembling 4cyanoindole (1) or azulene (2) with serine using a tryptophan synthase β-subunit (TrpB) variant (Tm9D8*) developed by Arnold and coworkers. Treatment of 3 and 4 with N-(chloroacetoxy)succinimide gave intermediates 5 and 6 in moderate yields. Finally, esterification of the carboxylic acids with chloroacetonitrile installed the CME functionality of 7 and 8 (Scheme 1). With compounds 7 and 8 in hand, we set out to assess their tRNA acylation efficiency by using a tRNA analogue, the microhelix RNA (FAM-MiHx_23b) (Figure ). The FAM label allows for visualisation through in gel fluorescence and loading yields were calculated by densitometric analysis of RNA bands. Initial studies were carried out with ClAc-4CNW-CME 5 at pH 7.5. We observed a gradual improvement in acylation yields (33-39%) as reaction time increased from 2 to 16 h. Altering the pH to 9 significantly improved MiHx charging, reaching 50% after 2 h, but prolonging incubation time under these conditions did not further increase yields. A similar reactivity profile was observed for ClAc-AzAla-CME 6, with the higher pH giving the highest MiHx loading (50%). Using the optimised conditions (pH 9.0, 2 h) from the MiHx loading assay, substrates 7, 8 were charged onto initiator tRNA fMet CAU through eFx mediated aminoacylation (Figure ). The resulting conjugates were then supplemented to a Met-free FIT system (PURExpress TM , Δaa, ΔtRNA, NEB). Synthesis of a model cyclic peptide P1 initiated with the fluorescent amino acids or ClAc-W as a control, was monitored by MALDI-TOF mass spectrometry (MS). The translated cyclic peptide products were detected for both fluorescent initiators (Figure ). We noted a minor mass peak corresponding to the incorporation of methionine as the initiator was present for all translations (including the control with Cl-W-CME), presumably arising from charged tRNA fMet present in the commercial PURExpress kit.