Structure Prediction and Synthesis of Pyridine-Based Macrocyclic Peptide Natural Products

Abstract

The structural and functional characterization of natural products is vastly outpaced by the bioinformatic identification of biosynthetic gene clusters (BGCs) that encode such molecules. Uniting our knowledge of bioinformatics and enzymology to predict and synthetically access natural products is an effective platform for investigating cryptic/silent BGCs. We report the identification, biosynthesis, and total synthesis of a minimalistic class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with the responsible BGCs encoding a subset of enzymes known from thiopeptide biosynthesis. On the basis of the BGC content, these RiPPs were predicted to undergo enzymatic dehydration of serine followed by [4+2]-cycloaddition to produce a trisubstituted, pyridine-based macrocycle. These RiPPs, termed “pyritides”, thus contain the same six-membered, nitrogenous heterocycle that defines the thiopeptide RiPP class but lack the ubiquitous thiazole/thiazoline heterocycles, suggesting that thiopeptides should be reclassified as a more elaborate subclass of the pyritides. One pyritide product was obtained using an 11-step synthesis, and the structure verified by an orthogonal chemoenzymatic route using the precursor peptide and cognate pyridine synthase. This work exemplifies complementary bioinformatics, enzymology, and synthesis to characterize a minimalistic yet structurally intriguing scaffold that, unlike most thiopeptides, lacks growth-suppressive activity toward Gram-positive bacteria.

Nocathioamides, Uncovered by a Tunable Metabologenomic Approach, Define a Novel Class of Chimeric Lanthipeptides

“…Notably,the co-occurrence of asplit lanB with lanC was singly reported for class Il antibiotics of the antifungal pinensins despite its frequent existence in LAPs and thiopeptides. [4,28] It is also noteworthy to mention the thioamidation, which exclusively occurs at the His residue.Incontrast to the few so far discovered thioamide containing RIPPs,r ecent genome mining efforts commenced to leverage and expand this chemical space with new thioamidated candidates either as thioviridamide-similar entities [29] or as thiopeptides,exemplified by saalfelduracin and thiopeptin. [12] Interestingly,t he associations of lanthionine synthetases with either F-dependent and/or TfuA-paired YcaOs were bioinformatically Angewandte Chemie projected before by the teams of Mitchell and van der Donk to chart novel multi-biosynthetic systems [30] which were followed up by their engineering efforts through combinatorial biosynthesis to mix and match new-to-nature hybrid RiPPs as thiazolinyl-lanthipeptides( I, II).…”

Mining and unearthing hidden biosynthetic potential

“…Using this method, a number of synthetic-bioinformatic natural products (syn-BNPs) were discovered, including the peptide humimycin (1) with potent antimethicillin-resistant Staphylococcus aureus (MRSA) activity 22 , the antibiotic paenimucillins (e.g., paenimucillin A, 2) 23,24 , and an antifungal peptide 23 . Similar methodologies were applied to identify novel RiPPs with antibacterial activity 25 and a new class of RiPPs, the pyritides 26 . Based on the architecture of a BGC in Micromonospora rosaria, the corresponding natural products were predicted to undergo a formal, enzymatic [4 + 2]cycloaddition with subsequent elimination of the leader peptide and water to produce a pyridine-based macrocycle (pyritide A2, 3) 26 .…”

“…Similar methodologies were applied to identify novel RiPPs with antibacterial activity 25 and a new class of RiPPs, the pyritides 26 . Based on the architecture of a BGC in Micromonospora rosaria, the corresponding natural products were predicted to undergo a formal, enzymatic [4 + 2]cycloaddition with subsequent elimination of the leader peptide and water to produce a pyridine-based macrocycle (pyritide A2, 3) 26 . Chemical synthesis of the predicted structures and chemoenzymatic reconstitution of the pathway confirmed the validity of the hypothesis and demonstrated the combined power of bioinformatics and chemical synthesis for investigating cryptic gene clusters.…”

Nocathioamides, Uncovered by a Tunable Metabologenomic Approach, Define a Novel Class of Chimeric Lanthipeptides

“…Notably,the co-occurrence of asplit lanB with lanC was singly reported for class Il antibiotics of the antifungal pinensins despite its frequent existence in LAPs and thiopeptides. [4,28] It is also noteworthy to mention the thioamidation, which exclusively occurs at the His residue.Incontrast to the few so far discovered thioamide containing RIPPs,r ecent genome mining efforts commenced to leverage and expand this chemical space with new thioamidated candidates either as thioviridamide-similar entities [29] or as thiopeptides,exemplified by saalfelduracin and thiopeptin. [12] Interestingly,t he associations of lanthionine synthetases with either F-dependent and/or TfuA-paired YcaOs were bioinformatically Angewandte Chemie Forschungsartikel projected before by the teams of Mitchell and van der Donk to chart novel multi-biosynthetic systems [30] which were followed up by their engineering efforts through combinatorial biosynthesis to mix and match new-to-nature hybrid RiPPs as thiazolinyl-lanthipeptides( I, II).…”

“…[3] Thet ypical introduction of sulfur and oxygen heterocycles within the core peptide comprising thazol(in)e and (methyl)oxazol(in)e moieties has also been perceived in several RiPP variants,such as linear azoline scaffolds (LAPs), head-to-tail monocyclic skeletons (cyanobactins), and multicyclic frameworks with an itrogen-containing heterocycle (pyritides). [4] Among the rare alterations of RiPPs,t hioamitides arise in which thioamide(s) can be exceptionally incorporated into the peptide backbone to leverage their structural diversification and bioactive potential. [5] With the explosion of affordable sequencing technologies in orchestration with improved bioinformatics algorithms for biosynthetic gene cluster (BGC) identification and defined motif annotation, [6] theq uest has been paved towards the discovery of ah idden arsenal of novel RiPP-based molecules across various bacterial (meta)genomes.N evertheless,c onnecting the RiPPs genetic locus of interest to the cognate metabolite-(s) is yet ac hallenging task [7] due to the production of complex metabolite mixtures,t he silence of many BGCs under laboratory cultivation conditions or if the source organism is considered uncultivable.T he latter two hurdles are nowadays overcome by the application of heterologous expression and synthetic biology,w hile analytical problems are foremost addressed by the integration of mass spectrometry (MS)-based metabolomics techniques.…”