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Lanthipeptides are natural products that belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain characteristic lanthionine (Lan) or methyllanthionine (MeLan) structures that contribute to their diverse biological activities. Despite its structurally diverse set of 30 substrates, the highly substrate-tolerant lanthipeptide synthetase ProcM is shown to display high selectivity for formation of a single product from selected substrates. Mutation of the active site zinc ligands to alanine or the unique zinc ligand Cys971 to histidine resulted in a decrease of the cyclization rate, especially for the second cyclization of the substrates ProcA1.1, ProcA2.8, and ProcA3.3. Surprisingly, for ProcA3.3 these mutations also altered the regioselectivity of cyclization resulting in a new major product. ProcM was not able to correct the ring topology of incorrectly cyclized intermediates and products, suggesting that thermodynamic control is not operational. Collectively, the data in this study suggest that the high regioselectivity of product formation is governed by the selectivity of the initially formed ring. [ABSTRACT FROM AUTHOR]

Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide—alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the a-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering. [ABSTRACT FROM AUTHOR]

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these cross-links, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) and a unique 5-chloro-l-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD as genes encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses, we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH 2 -dependent Trp halogenase. Most FADH 2 -dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.88-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase.

Cytolysin, a two-component lanthipeptide comprising cytolysin S (CylL S ″) and cytolysin L (CylL L ″), is the only family member to exhibit lytic activity against mammalian cells in addition to synergistic antimicrobial activity. A subset of the thioether cross-links of CylL S ″ and CylL L ″ have ll stereochemistry instead of the canonical dl stereochemistry in all previously characterized lanthipeptides. The synthesis of a CylL S ″ variant with dl stereochemistry is reported. Its antimicrobial activity was found to be decreased, but not its lytic activity against red blood cells. Hence, the unusual ll stereochemistry is not responsible for the lytic activity., Competing Interests: Notes The authors declare no competing financial interest.

Lanthipeptides are natural products that belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain characteristic lanthionine (Lan) or methyllanthionine (MeLan) structures that contribute to their diverse biological activities. Despite its structurally diverse set of 30 substrates, the highly substrate-tolerant lanthipeptide synthetase ProcM is shown to display high selectivity for formation of a single product from selected substrates. Mutation of the active site zinc ligands to alanine or the unique zinc ligand Cys971 to histidine resulted in a decrease of the cyclization rate, especially for the second cyclization of the substrates ProcA1.1, ProcA2.8, and ProcA3.3. Surprisingly, for ProcA3.3 these mutations also altered the regioselectivity of cyclization resulting in a new major product. ProcM was not able to correct the ring topology of incorrectly cyclized intermediates and products, suggesting that thermodynamic control is not operational. Collectively, the data in this study suggest that the high regioselectivity of product formation is governed by the selectivity of the initially formed ring.