Fig. 3. Modification of the library with a diversity of ∼10⁹ peptides displayed on phage by the TSL-6. (A and B) M13 phage-displayed disulfide library was oxidated and ligated with TSL-6. (C) The TSL-6 ligated peptides were further converted into bicyclic peptides. Reaction conditions: (i) 0.06 mM NaIO₄, pH 7.9, 9 min, ice. 0.5 mM Met, 20 min, r.t. (ii) 1 mM TSL-6, 10% MeCN, 0.1% TFA, 1 h, r.t. (iii) Zeba™ column, elute with 10 mM NaAc buffer, pH 4.6 (iv) 1 mM TCEP in 10 mM NaAc buffer, pH 4.6, 30 min. Increase the pH to 10 by adding 1 M NaHCO₃ and incubate for 3 h, r.t. (D) Quantification of the phage with thiol-reactive groups before and after cyclization. Control incubation of TSL-6-ligated phages in pH 10 buffer for 3 h did not lead to a significant decrease of thiol-reactive group content. (E) Chemical structure of the biotin-thiol (BSH) probe.