Synthesis of Hybrid Cyclopeptides through Enzymatic Macrocyclization

Abstract

Natural products comprise a diverse array of molecules, many of which are biologically active. Most natural products are derived from combinations of polyketides, peptides, sugars, and fatty‐acid building blocks. Peptidic macrocycles have attracted attention as potential therapeutics possessing cell permeability, stability, and easy‐to‐control variability. Here, we show that enzymes from the patellamide biosynthetic pathway can be harnessed to make macrocycles that are hybrids of amino acids and a variety of manmade chemical building blocks, including aryl rings, polyethers, and alkyl chains. We have made macrocycles with only three amino acids, one of which can be converted to a thiazoline or a thiazole ring. We report the synthesis of 18 peptide hybrid macrocycles, nine of which have been isolated and fully characterized.

Roads to Rome: Role of Multiple Cassettes in Cyanobactin RiPP Biosynthesis

“…Several studies have examined selectivity of RSIII using the AYD/SYD motif and synthetic variants thereof. 26,28,33–35 Here, for the first time we examine both the RSIII motif that is appended to the second cassette and the RSIII-RSII hybrid motif that is appended to the first cassette. We show that both TruG and PatG essentially equally accept these two natural recognition sequences, and that there is no preferred order of circularization.…”

Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

“…This means that not only can multiple different non-natural amino acids easily be accommodated but hybrid molecules (peptide and nonpeptide) that are impossible to be produced in vivo can be generated. 13 To date, the majority of reports of in vitro macrocyclization have used the macrocyclase domain from the patellamide pathway (PatGmac). Although extremely promiscuous, the enzyme has extremely slow catalytic rates in vitro , a major drawback that limits its application on a large scale.…”

The Biochemistry and Structural Biology of Cyanobactin Pathways: Enabling Combinatorial Biosynthesis

“…In addition, they recognize slightly longer sequences resulting from cleavage of multiple cassettes, such as X-AYDGLEAS-COOH (Lee et al, 2009). Thus far, they have not cleaved sequences with C-terminal longer peptides, although interestingly 8-amino-3,6-dioxaoctanoicacid has been appended to the end of the X-AYD sequence leading to successful macrocyclization (Oueis, Nardone, Jaspars, Westwood, & Naismith, 2017). This may prove useful in improving solubility of otherwise challenging substrates.…”

“…The minimal substrate requirement for macrocyclization by PatG (i.e. minimal three residue follower sequence preceded by a heterocycle) has prompted several efforts focused on utilizing the isolated protease domain as a biotechnology tool (Alexandru-Crivac et al, 2017; McIntosh, Robertson, et al, 2010; Oueis et al, 2016; Oueis, Nardone, et al, 2017; Oueis, Stevenson, et al, 2017; Sardar, Lin, et al, 2015). For example, the macrocyclase was predicted to be useful in producing discrete compounds such as peptides that mimic hormones or neuropeptides, or libraries of natural unnatural products.…”

“…PatG tolerated D-amino acids in the interior of the peptide, but not at the N-terminal or two C-terminal amino acids of the core peptide. The semi-synthetic approach was further refined to carry out the macrocyclic peptide hybrids, which incorporated synthetic chemical building blocks such as aryl rings, alkyl chains, sugars, and polyethers (Oueis, Nardone, et al, 2017). The ring size synthesized by G enzymes is very flexible, ranging from 5–22 amino acids for PatG and 6–30 for OscG (Alexandru-Crivac et al, 2017; Sardar, Lin, et al, 2015).…”