Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Abstract

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.