An Efficient, Site‐Selective and Spontaneous Peptide Macrocyclisation During in vitro Translation

Abstract

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioetherbased approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.

p‐Chloropropynyl Phenylalanine, a Versatile Non‐Canonical Amino Acid for Co‐Translational Peptide Macrocyclization and Side Chain Diversification

“…pCPF charged by PheRS A294G was used for translation in a variety of contexts resulting in the efficient co-translational synthesis of rigid PMCs of various sizes. Few ncAAs have been shown to give to spontaneous, co-translational cyclization [31][32][33][34][35]41,42] , and relative to these other strategies, the use of pCPF leads to peptides with a unique geometry due to the rigid aryl alkyne within the bridge. The unique reactivity towards thiols also allows pCPF to be decorated after charging onto tRNA, giving rapid access to a variety of novel ncAAs for ChemBioChem testing in translation.…”

“…The pioneering work from Suga and co‐workers [30,31] for example, described the spontaneous cyclization using N‐terminal chloroacetamide analogs and internal cysteines using Flexizyme. Recently, both Hohsaka and Jongkees harnessed the nucleophilicity of cysteine towards cyanothiazole‐containing ncAAs creating PMCs [32,33] . We reported a spontaneous cyclization with the pheny‐ lalanine analog p‐cyanoacetylene‐Phe which reacts with cysteine through a Michael addition mechanism, [34] but the high reactivity of this monomer required a thiol‐free translation system with reduced efficiency, and the resulting peptide macrocycles contain E and Z ‐mixtures at the cyclization position.…”

“…Recently, both Hohsaka and Jongkees harnessed the nucleophilicity of cysteine towards cyanothiazole-containing ncAAs creating PMCs. [32,33] We reported a spontaneous cyclization with the pheny-lalanine analog p-cyanoacetylene-Phe which reacts with cysteine through a Michael addition mechanism, [34] but the high reactivity of this monomer required a thiol-free translation system with reduced efficiency, and the resulting peptide macrocycles contain E and Z-mixtures at the cyclization position. Fasan and co-workers, developed a spontaneous cyclization strategy compatible with phage display (MOrPH-PhD) in which the non-canonical amino acid (ncAA) O-(2bromoethyl)-tyrosine (O2beY) and subsequently AAs containing other thiol-reactive groups harnessed cysteine's nucleophilicity to generate a thioether bridge spontaneously.…”

The Cyanopyridine–Aminothiol Click Reaction: Expanding Horizons in Chemical Biology

“…14 Remarkably, an unconventional i, i + 8 stapling pattern resulted in enhanced helicity compared to the wild-type aurein. Overall, peptides constrained by the cyanopyridine-aminothiol click reaction were investigated as inhibitors of viral proteases, 10-13, 15 influenza hemagglutinin, 16 deubiquitinase USP15, 17 as well as for the generation of large DNA-encoded libraries (DEL). 18 Scheme 3 Biocompatible strategies for constraining peptides based on the cyanopyridine-aminothiol click reaction.…”

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis

“…This reaction can be efficiently exploited in peptide macrocyclisation by incorporation of meta -cyanopyridylalanine ( m CNP) at the C-terminus of a peptide. 21 Based on this, we also recently reported a method for translation of a cyanobenzothiazole-containing amino acid that gave efficient peptide macrocyclisation, 22 while others have exploited this same reactive group together with a second cysteine-reactive moiety for stapling of peptides displayed on phage, 23,24 with selectivity arising from either differences in rate or reversibility of the second reaction. However, the high reactivity of cyanobenzothiazoles meant that extra precautions needed to be taken during translation of this moiety to prevent premature hydration.…”