Facile Method for Determining Lanthipeptide Stereochemistry

Abstract

Lanthipeptides make up a large group of natural products that belong to the ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides contain lanthionine and methyllanthionine bis-amino acids that have varying stereochemistry. The stereochemistry of new lanthipeptides is often not determined because current methods require equipment that is not standard in most laboratories. In this study, we developed a facile, efficient, and user-friendly method for detecting lanthipeptide stereochemistry, utilizing advanced Marfey’s analysis with detection by liquid chromatography coupled with mass spectrometry (LC-MS). Under optimized conditions, 0.05 mg of peptide is sufficient to characterize the stereochemistry of five (methyl)­lanthionines of different stereochemistry using a simple liquid chromatography setup, which is a much lower detection limit than current methods. In addition, we describe methods to readily access standards of the three different methyllanthionine stereoisomers and two different lanthionine stereoisomers that have been reported in known lanthipeptides. The developed workflow uses a commonly used nonchiral column system and offers a scalable platform to assist antimicrobial discovery. We illustrate its utility with an example of a lanthipeptide discovered by genome mining.

Expression and Subcellular Localization of Lanthipeptides in Human Cells

“… 68 Bis-derivatization of Lan/MeLan residues from a hydrolyzed lanthipeptide using the advanced Marfey’s reagent N α -(5-fluoro-2,4-dinitrophenyl)- l -leucinamide (L-FDLA) leads to diastereomers with masses [M – H] = 795.2373 Da (for Lan) and [M – H] = 809.2530 Da (for MeLan). 69 The extracted ion chromatogram of CylL L ″-S15T produced in Expi293F cells was near identical to that of WT CylL L ″ produced in E. coli , showing LL-Lan, DL-Lan, and LL-MeLan, whereas no DL-MeLan was observed ( Figure 3 D). This observation, in combination with the tandem MS data ( Figure 2 A), strongly suggests that the CylL L ″-S15T variant has the same stereochemistry and cyclization patterns as the WT CylL L ″.…”

“…WT CylL L ″ contains one LL-MeLan, one LL-Lan, and one DL-Lan, , whereas nisin contains one DL-Lan and four DL-MeLan . Bis-derivatization of Lan/MeLan residues from a hydrolyzed lanthipeptide using the advanced Marfey’s reagent N α -(5-fluoro-2,4-dinitrophenyl)- l -leucinamide (L-FDLA) leads to diastereomers with masses [M – H] = 795.2373 Da (for Lan) and [M – H] = 809.2530 Da (for MeLan) . The extracted ion chromatogram of CylL L ″-S15T produced in Expi293F cells was near identical to that of WT CylL L ″ produced in E.…”

“…Commercial nisin from L. lactis (Sigma N5764-1G) containing 5% nisin (w/w) was further purified by HPLC . The absolute stereochemistry of the Lan cross-links was determined as previously described, with slight modifications .…”

“… 38 Commercial nisin from L. lactis (Sigma N5764-1G) containing 5% nisin (w/w) was further purified by HPLC. 69 The absolute stereochemistry of the Lan cross-links was determined as previously described, with slight modifications. 69 Briefly, 50 μg of dried peptides was subjected to hydrolysis by resuspending in 0.8 mL of 6 M DCl in D 2 O in a 4 mL amber glass vial, followed by sparging with a N 2 stream for 30 s. The mixture was heated at 120 °C for 16 h with stirring.…”

Initial Characterization of the Viridisins’ Biological Properties

“…Stereochemical analysis of the VdsA1-P1A and VdsA1-P1B isomers was performed according to a recently described method . Briefly, 0.05 mg samples of VdsA1-P1A and VdsA1-P1B were hydrolyzed under acidic conditions and derivatized with 1-fluoro-2,4-dinitrophenyl-5- l -leucine amide as described.…”