18 | December 2021 www.facs.website and HSFCl are installed by the genetic code reprogramming in the FIT system. Since the thioester bond originating from the incorporation of HSFCl acts as a thioester donor, the intramolecular trans-thioesterification occurs at the Cys residue in SPCG to afford an intermediate of cyclic thiolactone; and then rapidly rearranges into the CDP (Figure 8B). In-depth studies on mutants of the SPCG motif have revealed that this motif can be relaxed to SXCX (X can be nearly any amino acids). Since this method of CDP synthesis can proceed in one-pot and also on a wide variety of peptide sequences between the SPCG and HSFCl, it is applicable to the RaPID system to screen bioactive CDPs against protein targets of interest. Conclusions It becomes evident that so far, majority of chemical space for peptide macrocyclization on display platform is occupied by either bi/tri/ tetra functional crosslinker induced cyclization or thioether bond formation by incorporating N-chloroacetyl group at the initiator position using genetic code reprogramming. The later one, in par ticular, has been tremendously successful in producing selection resul ts against a variety of interesting targets yielding extremely strong binding macrocyclic peptide ligands against a wide range of intracellular and extracel lular targets. The success of FIT and other genetic code reprogramming approaches have empowered researchers to explore newer and more challenging approaches for peptide macrocyclization in order to diversify the field and break through various barriers and limitations. Many valuable bioconjugation reactions like Michael addition to α,β-unsaturated systems and various ‘click’ type reactions remain underutilized on this platform and offer great opportunity for expanding the scope and pushing boundaries of this active field. ◆ Acknowledgements This work was suppor ted by the Japan Soc i et y for the Promot i on of Sc i ence (JSPS) Grant-in-Aid for Scientific Research S (26220204) and Spec i a l l y Promoted Research (20H05618), and Japan Agency for Medical Research and Development (AMED), Pl at form Pro j ec t for Suppor t i ng Dr ug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research, JP19am0101090) to H.S. References 1. G. Luca, M. Rossella De and C. Lucia, Current pharmaceutical design, 2010, 16, 3185-3203. 2. D. Mathur, S. Prakash, P. Anand, H. Kaur, P. Agrawal, A. Mehta, R. Kumar, S. Singh and G. P. S. Raghava, Scientific Reports, 2016, 6, 36617. 3. H. Huang, J. Damjanovic, J. 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