16 | December 2021 www.facs.website Interestingly, the N-terminal ClAc group reacts with Cys thiol at almost any position, except for Cys at the adjacent downstream position to ClAc-initiator (i.e. at the second position). This is simply because Cys cannot sterically reach to the ClAc group. Thus, when there is a Cys residue at the second position, arbitrary sequence and length of peptide followed by a downstream Cys residue, the latter Cys thiol (generally the second Cys residue) selectively reacts with the N-terminal ClAc group to form thioether-macrocycle. This fact has allowed to build a strategy for ribosomal synthesis of tricyclic peptides (Figure 3B). In this scheme, a peptide contains a total of four Cys residues, where ClAc-Trp is followed by Cys and then the rest of peptide sequence has three Cys residues at various position. The second Cys spontaneously reacts with the N-ClAc group to afford a monocycle. Then, the treatment of TBMB crosslinks the remaining three Cys residues to form a topologically complex tricyclic peptide. This ClAc thioether strategy can be expanded to inter-sidechain cyclization by incorporating an Nγ-ClAc-α,γ-diaminobutylic acid (ClAc-Cab).85 Again, a downstream cysteine thiol reacts with the ClAc group to afford a macrocycle closed by the thioether bond. Application of this methodology was demonstrated by translating a known biologically active peptide human urotensin II which is a potent vasoconstrictor. Single disulfide bond between cysteine residues at position 5 and 10 was replaced with a thioether bridge between Cab at position 5 and a cysteine at position 10 (Figure 3C). The resulting peptide was shown to retain biological activity and remarkable stability towards proteinase K under reducing conditions.85 In 2014, Fasan et al. developed a strategy of producing thioether linked macrocyclic peptides inside living bacterial cells (E.coli) which can be utilized on phage display platform (Figure 3D).86, 87 In order to supress cross reactivity with many other nucleophiles in the cellular environment, they ribosomally incorporated a rather slow reacting nonproteinogenic amino acid (O-(2bromoethyl)-tyrosine) termed O2beY. For proteolytic release of the cyclized peptide, they also incorporated an intein-based protein splicing element. Both features combined together, resulted in ribosomal production of a linear precursor peptide having a cysteine reactive nonproteinogenic amino acid O2beY and an intein splicing element. Remarkably, another cysteine present in the intein element did not show any reactivity towards cyclization reaction due to being partially buried within the active site. Yet, the practice of this approach for the disovery of de novo macrocyclic peptides has not been reported. Michael Addition Nucleophilicity of thiolate can also be exploited in Michael type addition reactions to yield thioether linkage. In fact, many biologically active natural lanthipeptides utilize this strategy for cyclization. For such ribosomally synthesized and posttranslationally modified peptides, dehydratase enzymes recognize the N-terminus of the precursor leader peptide and convert serine and threonine residues in the core peptide to dehydroalanine (Dha) and dehydrobutyrine (Dhb) respectively. The α,β-unsaturated moieties in Dha and Dhb acts as the electrophile where enzyme assisted Michael addition reaction by cysteine thiol generates a thioether linkage. The most extreme case observed in natural products is biosynthesis of nisin. Inspired by this chemistry, Goto et al.88 used genetic code reprogramming to incorporate vinylglycine in translated peptides which was isomerized to dehydrobutyrine by simply heating the peptide at 95˚C for 30 minutes. This was followed by spontaneous Michael addition by a cysteine thiol to give methyllanthionine containing macrocyclic peptide. They later demonstrated the applicability of this reaction by synthesizing two ring segments of the natural bioactive peptide nisin (Figure 4). Due to high temprature requirement of this cyclization step, this approach is inapplicable to the display system; therefore, a better alternative approach is needed. Oxidative Coupling Genetic code reprogramming allows for incorporation of various nonproteinogenic amino acids including those with orthogonal reactive handles to accomplish click type ligation (vide infra). A practically useful application of this methodology was incorporation of benzylamine and 5-hydroxyindole.89 These functional groups are known to react instantly under oxidative conditions to yield a fluorescent heterocyclic moiety. This methodology (Figure 5), although not used for display technology yet, seems to offer immense practical utility and potential for application in display-based selection. Azide-Alkyne Coupling Copper catalyzed Azide-Alkyne Click (CuAAC) reaction90, 91 needs no introduction and remains one of the most versatile and practically useful bioconjugation reaction (for some reviews see92-97). It has been exploited widely for peptide cyclization in solid phase98 and solution phase peptide synthesis.99-101 Its underutilization in macrocyclization of peptides for display technologies, however, is, in part, due to the lack of compatibility with nucleotides102-104 (with RNA in particular). RNA is susceptible to oxidation and degrades quickly in presence of Cu in aqueous medium.105 Use of acetonitrile as cosolvent, Cu stabilizing ligands and degassing buffer solutions are some of the ways to prevent mRNA degradation when using CuAAC reaction. Additionally, since double incorporation of both azide and alkyne bearing unnatural amino acids is rather tedious and low yielding, the use of this strategy for preparing monocyclic peptide F NH2 HN O HO N H F N H O N O NH WOH 0.5 mM K3Fe(CN)6 pH8, RT, 5 min WOH C fM H N O N H G A L M G N G G C G A L M G N G fM G H N O N H fM G G A L M G N G C HN O NH S HS HS 95°C Figure 4. Benzyl amine and hydroxyindole incorporated in translated peptides react rapidly under oxidative conditions, yielding a unique fluorogenic aromatic linkage. Figure 5. Cyclization via Michaels addition. Model peptide with vinylglycine isomerising to dehydrobutyrine on heating to 95˚C and subsequent intramolecular Michael addition by cysteine thiol to give the macrocycle.
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