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Macrocyclisation of small peptides enabled by oxetane incorporation.

ABSTRACT: Cyclic peptides are an important source of new drugs but are challenging to produce synthetically. We show that head-to-tail peptide macrocyclisations are greatly improved, as measured by isolated yields, reaction rates and product distribution, by substitution of one of the backbone amide C[double bond, length as m-dash]O bonds with an oxetane ring. The cyclisation precursors are easily made by standard solution- or solid-phase peptide synthesis techniques. Macrocyclisations across a range of challenging ring sizes (tetra-, penta- and hexapeptides) are enabled by incorporation of this turn-inducing element. Oxetane incorporation is shown to be superior to other established amino acid modifications such as N-methylation. The positional dependence of the modification on cyclisation efficiency is mapped using a cyclic peptide of sequence LAGAY. We provide the first direct experimental evidence that oxetane modification induces a turn in linear peptide backbones, through the observation of d NN (i, i + 2) and d αN (i, i + 2) NOEs, which offers an explanation for these improvements. For cyclic peptide, cLAGAY, a combination of NMR derived distance restraints and molecular dynamics simulations are used to show that this modification alters the backbone conformation in proximity to the oxetane, with the flexibility of the ring reduced and a new intramolecular H-bond established. Finally, we incorporated an oxetane into a cyclic pentapeptide inhibitor of Aminopeptidase N, a transmembrane metalloprotease overexpressed on the surface of cancer cells. The inhibitor, cCNGRC, displayed similar IC50 values in the presence or absence of an oxetane at the glycine residue, indicating that bioactivity is fully retained upon amide C[double bond, length as m-dash]O bond replacement.

SUBMITTER: Roesner S

PROVIDER: S-EPMC6385813 | biostudies-other | 2019 Feb

REPOSITORIES: biostudies-other

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Macrocyclisation of small peptides enabled by oxetane incorporation.

Roesner Stefan S Saunders George J GJ Wilkening Ina I Jayawant Eleanor E Geden Joanna V JV Kerby Paul P Dixon Ann M AM Notman Rebecca R Shipman Michael M

Chemical science 20190103 8

Cyclic peptides are an important source of new drugs but are challenging to produce synthetically. We show that head-to-tail peptide macrocyclisations are greatly improved, as measured by isolated yields, reaction rates and product distribution, by substitution of one of the backbone amide C[double bond, length as m-dash]O bonds with an oxetane ring. The cyclisation precursors are easily made by standard solution- or solid-phase peptide synthesis techniques. Macrocyclisations across a range of c ...[more]

PMID: 30881675

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Project description:Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

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Macrocyclisation of small peptides enabled by oxetane incorporation.

Publications

Macrocyclisation of small peptides enabled by oxetane incorporation.

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