www.asiachem.news December 2021 | 17 libraries is virtually unreported. This strategy has been proven to be valuable for producing bicyclic libraries in particular. Suga lab in 2008 reported first double incorporation of azide and alkyne106 bearing unnatural amino acids azidohomoalanine (Aha) and propargylglycine (Pgl) respectively using Leu codon CUC for Aha and Thr codon ACC for Pgl. This orthogonal pair was expressed along with another reacting pair 4-(2-Chloroacetyl)aminobutyric acid (Cab) and cysteine to generate a bicyclic peptide scaffold (Figure 6). Hartman’s group utilized the CuAAC reaction generating bicyclic peptide library for mRNA display.107 β-azidohomoalanine (AzHA) and p-ethynyl phenylalanine (F-yne) were incorporated in place of methionine and phenylalanine, respectively. The second cycle was formed by two cysteine thiols reacting with dibromoxylene. They further carried out a competitive mRNA selection on streptavidin target using a library of linear, monocyclic and bicyclic peptides to investigate the effect of different ring sizes and topologies on selection results and they found all the selection winners were linear peptides. This raised the question as to why all selection winners were linear peptides with only µM KD values even though some cyclic peptides capable of exhibiting nM KD values were known. For a detailed discussion on this see.108 Backbone Cyclization Formation of backbone cyclized peptide libraries for in vitro display technologies coupled with ribosomal translation is not possible directly because the C-terminus of the peptide is involved in genotype-phenotype linkage and is not available for cyclization reaction. This intrinsic limitation had hindered devising a display strategy of backbone cyclized peptide. This means that a new strategy is required to covalently trap peptide phynotype to the cognate genotype via non-C-terminus. To break this technical hinderance, Takatsuji et al. has devised a two-step rearrangement strategy by utilizing genetic code reprogramming to incorporate three nonproteinogenic amino acids in the peptide.109 Peptide is expressed with a thiazolidine-Cys (Thz-Cys) dipeptide initiator charged onto tRNAfMet CAU and ClAc-Cab are installed in the N-terminal region of peptide by the genetic code reprogramming (Figure 7). Continuing the elongation of arbitrary sequence of peptide (p2-W8-SFCl9), an a-thio-p-chlorophenyl-lactic acid (HSFCl) is installed to form a thioester in the backbone and Cys residue at a downstream position (generally dipeptide, e.g. Ile-Gly, are inserted between HSFCl and Cys). Upon the completion of ribosomal peptide synthesis, the Cys thiol spontaneously exchanges with the thioester bond of HSFCl, to yield an intermediate (p2-W8-sC12). The thiol group of HSFCl then reacts with the ClAc group on Cab to give a covalent thioether linkage (tcp2-W8-SC12). In the second step, mild deprotection of the Thz group on the initiator Cys gives an N-methyl-Cys residue at the N-terminus (tcp2-W8-SC12:deprptected), whose thiol sidechain immediately undergoes intramolecular thioester exchange followed by transfer to the N-methyl-amino group on Cys (similar to native chemical ligation) to yield backbone-cyclized peptide (bcp2-W8). Most importantly, it was demonstrated that this entire process allows to maintain the C-terminal region of peptide covalently attaching to the backbone-cyclized peptide via the thioether linkage between ClAcCab and HSFCl groups (Figure 7). Since this leaves C-terminal peptide region remaining as carboxyl group, the strategy is compatible to the RaPID display via the puromycin molecule attached to cognate mRNA, as demonstrated in this work.109 Macrocyclic Depsipeptide Formation Cyclic depsipeptides (CDPs) are a class of naturally occurring peptides which contain one or more ester bonds and exhibit wide range of biological activities.110 The O-acyl isopeptide bond, usually formed between the hydroxyl sidechain of serine or threonine residues and the carboxyl group of the C-terminus amino acid is stable towards esterases and proteases. Because of biological significance of CDPs, several methods for the chemical synthesis of CDPs have been developed, but none of them is applicable to the conditions required for translation where the chemistry must work at near neutral pH and mild temperature. Nagano et al. conducted a selection campaign for self-esterifying peptide species from random peptide libraries using a thioester acyl-donor, and discovered a peptide containing a short SerProCysGly (SPCG) motif that can effectively esterify on the Ser residue. It turns out that trans-thioesterification between the acyl-donor and thiol sidechain of Cys residue in the SPCG motif firstly takes place, and the resulting acyl group on Cys rapidly transfers to the hydroxyl group of Ser (Figure 8A).111 This unique chemistry was then applied for CDP synthesis. Linear peptide is expressed, where the SPCG motif in the N-terminal region, arbitrary peptide sequence, Pgl C Aha Cab SH HN Cl O N3 Pgl C Aha Cab HN S O N N N TCEP, Cu(I) Figure 6. Cyclization of peptide via azide-alkyne coupling. Double incorporation of both azide and alkyne in translated peptides via genetic code reprogramming by Suga group and subsequent formation of bicyclic peptide in conjugation with thioether bond formation. Figure 7. Backbone cyclization amenable to the RaPID display. (A) Non-proteinogenic thioacid HSFCl. (B) mRNA sequence coding the arbitrary peptide p2-W8. (C) Thioester exchange followed by thioether bond formation between ClAc-Cab and HSFCl groups keeps the C-terminal peptide region to the sidechain of backbone-macrocyclic peptide. Deprotection of Thz-Cys followed by spontaneous thiol-thioester exchange leads to native chemical ligation which yields the backbone cyclized peptide attached to its genotype.
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