14 | December 2021 www.facs.website Cys or amino groups of lysine (Lys) ε-sidechain (or occasionally N-terminus), which are present at fixed positions in the translated peptide that conjugate with a small organic motif added after translation. Thus, this strategy has been applied for the majority of phage display works. Thioether bond formation This has been a popular strategy due to its simplicity and the ability to yield macrocyclic peptides with more than one loop. Cys thiols at fixed positions react with organohalides forming thioether bonds in a SN2 reaction. The libraries have vast diversities consisting of proteinogenic amino acids only. Using bis/tris/tetrakis (bromomethyl) benzenes Inspired by some naturally occurring peptides with multiple fused rings and loops and having interesting biological activities,8-10 many groups have tried to develop methods to create peptide libraries having similar topologies. Beginning of this was the report of Timmerman (Figure 1) that treating di-, tri-, and tetra-Cys containing peptides with bis-, tris-, and tetrakis(bromomethyl) benzene derivatives in aqueous ACN results in fast, one-step chemical synthesis of single-, double-, and triple loop peptides.11 In 2009, this reaction was later utilized by Winter et al.12 to produce bicyclic peptide libraries for phage display. They designed peptide libraries with three reactive Cys residues, each separated by several random amino acids and conjugated with tris(bromomethyl)benzene (TBMB) in aqueous solvents (Figure 2A). The conjugation reaction however posed several challenges including cross reactivity of TBMB with the disulfide bridges D1 and D2 domain in the phage PIII and a loss in phage infectivity due, probably, to the crosslinking of the phage coat protein through lysine side chains. The problems were, however, solved by using a disulfide free gene-3-protein phage and using low concentration of TBMB. The phage display selection was successfully carried out to find an inhibitor ligand to human plasma kallikrein. This elegant approach represents that the appropriate engineering of the phage system allows to control selective crosslinking of Cys residues only appeared in the random library of displayed peptides. In 2012, Szostak group also utilized a similar strategy to cyclize highly modified peptides having two flanking cysteine residues using dibromoxylene.13 The peptide libraries having several non-proteinogenic amino acids were used for in vitro selection based on mRNA display against the target protease thrombin with successful isolation of binders with low nanomolar affinity. Using perfluoroarenes Perfluoroarenes react with a reactive thiol in peptide via nucleophilic aromatic substitution reaction SNAr, which has been used extensively for polymer arylation and bioconjugation.14-18 Derda et al.19 used decafluoro-diphenylsulfone (DFS) to crosslink Cys thiols yielding cyclic peptides in one of the fastest Cys conjugation reactions (Figure 2B). They improved the previously reported SNAr reagents such as 1-chloro-2,4-dinitrobenzene,20 perfluorobenzene21, 22 and perfluorobiphenyl22 which show low reactivity and poor solubility in aqueous systems. The group has demonstrated this reaction to be biocompatible and faster than most Cys conjugation reactions with the reaction rates up to 180 M-1S-1, although the rate is largely sequence dependent; e.g. positively charged residues such as arginine accelerated it while negatively charged aspartate supressed the rate. This unique reaction is fairly selective for Cys, but with large excess and prolonged exposure to DFS showed some cross-reactivity with amine groups. As for applicability of the reaction in phage display, a clone of M13 phage could be 60–70% modified with DFS in 5% DMF as cosolvent. The modification efficiency was decreased to 35% when a whole library containing 109 peptides was used. Interestingly, the crosslinked peptides generally exhibit higher oxidative resistance compared with the traditional α,α’-dibromo-meta-xylene. Using Dichloro-oxime In 2015, Dawson et al. reported side chain linking of cysteine or homocysteine thiols using dichloroacetone (DCA) to give stapled (macrocyclic) peptide with an acetone bridge.23 This linking not only stabilized the secondary structure of the peptides but also provided a ketone moiety to link various molecular tags through oxime ligation. Building further on this concept, Derda et al. used pre-formed dichloro-oxime (DCO) derivatives (Figure 2C) to cyclize phage displayed glycopeptide libraries.24 Reaction went on to completion giving approximately C C S S C C HS SH Br Br C C C Br Br Br C C C S S S HS SH SH C C C C S S S S C C C C SH HS SH HS A B C Br Br Br Br F F S F S F F O O F F S F F F S F F F F O O F F F F Br Br Br Cl Cl N R S S N R C C C S S S C C SH HS C SH C SH HS C C C C C R = biotin mono-glycoside A B C H N H N H N H N Figure 1. Formation of peptide loops by reacting Cys-containing peptides with di-, tri-, or tetra-Cys reacting to bis-, tris-, or tetrakis-(bromomethyl)benzene as a crosslinking agent. Figure 2. Examples of peptide cyclizations using bromomethyl benzenes, amenable to display technologies. (A) Bicyclic peptide library using 1,3,5-tris(bromomethyl) benzene reported by Winter group. (B) Decafluoro-diphenylsulfone (DFS) cyclizaton and (C) Dichloro-oxime cyclization reported by Derda group.
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