www.asiachem.news December 2021 | 13 also structures consisting of one, two, three or even more cyclic motifs. Cyclization reactions become more complicated and challenging due to the presence of various reactive sidechains on proteinogenic amino acids. Even though there are various techniques for chemical synthesis of cyclic peptides on solid support based on traditional protection-deprotection chemistries4 and/or metal-catalyzed reactions,5 most of these reactions are not suitable for the use on display platforms because of the following reasons: they must be compatible to physiological-like conditions (e.g. at near-neutral pH) and high chemoselective to the aiming functional groups. This review deals with techniques of peptide cyclization as applied to in vitro display techniques, represented by the phage and mRNA displays. Challenges Display technologies6, 7 rely on the translation machinery consisting of ribosome, protein translation factors, various enzymes including aminoacyl-tRNA synthetases, amino acids, tRNAs, mRNAs, energy sources, and others. Thus, the cyclization chemistry needs to selectively work for the aimed peptides in the presence of all these bio- and small-molecules. Even a harder challenge is that their chemistry must efficiently take place regardless of peptide sequences originating from huge mRNA libraries and vast tertiary structures originating from the diverse peptide sequences. For the phage display, a classical and general method for generating cyclic peptides is disulfide bond formation via two cysteine (Cys) residues. This is simply because their genotype of mRNA or DNA sequence is packaged in the bacteriophage, the easiest way to cyclize the peptide sequences is to use the naturally occurring crosslinking bond(s) of disulfide. However, disulfide bond is a reducible bond, and therefore in consideration for physiological conditions this bond is not necessarily ideal for drug use. Even though such a disulfide bond can be elaborated to an alternative bond, but in such a case the activity of the parental peptide is often diminished. Thus, it is important to develop an alternative approach to produce macrocyclic peptides closed by a more physiologically stable bond from the initial library. For the mRNA display, the respective peptides are directly attached to the genotype sequences of mRNA via puromycin molecule. Occasionally, the mRNA sequence is reverse transcribed to cDNA sequence forming the noncovalent annealing pair. This means that the peptide-mRNA/cDNA fusion contains not only the peptide motif but also ‘naked’ nucleic acids, and thereby the chemistry for cyclization is even more challenging than the phage case, where the cyclization must take place without unwanted reactions with sidechains of peptide nor with nucleic acid’s nucleobases/phosphates. Cyclization strategies Traditionally, peptide cyclization has been categorized as taking place between two ends of the peptide (head-to-end), two sidechains (sidechainto-sidechain) or one end to a sidechain (head-tosidechain and sidechain-to-end). However, for the sake of this review which deals mainly with those methods applied to display technologies, we will broadly categorize the strategies in two, i.e., cyclization without using genetic code manipulation and cyclization using genetic code manipulation. Cyclization via chemical crosslinking This strategy usually takes advantage of inherent reactivity of a native amino acid side chain and an external organic motif. Majority of groups have exploited nucleophilicity of thiol groups of
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