Enzymatic Macrocyclization of 1,2,3‐Triazole Peptide Mimetics

Abstract

The macrocyclization of linear peptides is very often accompanied by significant improvements in their stability and biological activity. Many strategies are available for their chemical macrocyclization, however, enzyme‐mediated methods remain of great interest in terms of synthetic utility. To date, known macrocyclization enzymes have been shown to be active on both peptide and protein substrates. Here we show that the macrocyclization enzyme of the cyanobactin family, PatGmac, is capable of macrocyclizing substrates with one, two, or three 1,4‐substituted 1,2,3‐triazole moieties. The introduction of non‐peptidic scaffolds into macrocycles is highly desirable in tuning the activity and physical properties of peptidic macrocycles. We have isolated and fully characterized nine non‐natural triazole‐containing cyclic peptides, a further ten molecules are also synthesized. PatGmac has now been shown to be an effective and versatile tool for the ring closure by peptide bond formation.

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

“…After 16 h of reaction both 13mer and 14mer substrates produce similar amounts of macrocycle, but the 14mer generates less linear product. Linear peptide produced this may not be a significant drawback, as purification of macrocycles from liner peptides is straightforward 12 , 38 . Compared to PatG this represents a significant improvement, since biocatalytic reactions with PatG in vitro can require over 7 days and utilize up to stoichiometric amounts enzyme 12 , 38 .…”

Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase

“… 50 No macrocyclase enzyme has been engineered to improve or diversify function, although their natural substrate promiscuity has been exploited. 14 − 19 , 51 This is the first kinetic characterization of a macrocyclase and a necessary step toward designing a better catalyst for macrocyclization.…”

“… 10 PatGmac has been shown to be a highly versatile tool in the production of cyclic peptides. 14 − 16 However, PatGmac reactions are very slow in vitro , 11 although addition of reductant to Escherichia coli cultures improves production of the cyclic peptide patellin using the homologous tru enzymes by multiple orders of magnitude. 17 Butelase 1 is remarkably fast and effective as a tool for producing large peptide macrocycles, 18 − 20 but it cannot effectively cyclize peptide substrates containing nine or fewer amino acids.…”

Mechanisms and Evolution of Diversity-Generating RiPP Biosynthesis

“…Furthermore, the initial pathway enzymes are highly conserved in sequence despite the great diversity of core peptides [35,40]. These observations have been used to prepare non-natural variants starting from precursor peptides containing the conserved RSs flanking hypervariable core regions [34,35,[42][43][44][45][46][47]. The following sections discuss how the biosynthetic enzymes involved in the biosynthesis of the cyanobactins as well as a subclass of lanthipeptides called the prochlorosins achieve the very high level of chemical diversity in their pathway products.…”

“…The leader peptide engages a domain in these enzymes called the RiPP recognition element (RRE) that is found in about half of all RiPP biosynthetic gene clusters [88]. Binding of the leader peptide to the RRE allows enzymatic azoline formation that is nearly insensitive to flanking residues [43,44,46,87,89]. For both lanthipeptide and cyanobactin biosynthetic enzymes, it has been shown that attachment of the leader peptide to the enzyme results in the processing of leaderfree core peptide, suggesting that leader peptide binding serves as an allosteric regulation mechanism and can work trans-acting [43,87,90,91].…”