Endogenous Production and Vibrational Analysis of Heavy‐Isotope‐Labeled Peptides from Cyanobacteria

Abstract Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass‐sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/ 13 C‐enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC–MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM‐IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin‐LR (MCLR), cyanopeptolin‐A (CYPA), and aerucyclamide‐A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy‐labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy‐labeled nutrients provides an efficient means of producing heavy‐labeled natural products for mass‐sensitive imaging studies.