Halogenation-Guided Chemical Screening Uncovers Cyanobacterin Analogues from the Cyanobacterium Tolypothrix sp. PCC9009

Halogenated specialized metabolites show high chemical diversity and exhibit a range of biological activities. A targeted screening of a cyanobacteria extract library for halogenated specialized metabolites using HPLC-HRMS combined with MassQL and Haloseeker indicated that several of the extracts contained halogenated compounds, among them an extract of Tolypothrix sp. PCC9009. This freshwater cyanobacterium has been known since the early 1980s for producing the chlorinated specialized metabolite cyanobacterin, containing a γ-lactone core structure with a hydroxy group that is essential for its herbicidal activity against cyanobacteria and green algae. Mass-spectrometry-based molecular networking was employed to explore the chemical space of natural cyanobacterin analogues. This analysis led to the identification of 15 previously unknown compounds structurally related to cyanobacterin, most of which are related to anhydrocyanobacterin, including a dimer formed by [2 + 2] photocycloaddition. Two further analogues, previously reported following heterologous expression of the cyanobacterin biosynthetic gene cluster in E. coli as the nonchlorinated precyanobacterin I and II, were now isolated from the natural cyanobacterin producer. Cytotoxicity assays of cyanobacterin, anhydrocyanobacterin and one further isolated analogue showed only modest activity of the compounds against HCT116 cells.