Antifouling And Fungicidal14-membered Resorcylic Acid Lactones From Marine Fungi Cochliobolus Lunatus

Marine invertebrates in the coral reef ecosystems have formed unique chemicaldefensive system in the competitive environments, and many chemical defensivecompounds produced by them have shown potentials to be exploited as antifouling,fungicidal, and insecticidal agents. As part of our ongoing investigation on newnatural bioactive compounds from marine invertebrates in the South China Sea, aseries of potent antifouling, fungicidal, and insecticidal compounds were obtained.However, obtaining more antifouling compounds is usually unfeasible because theirisolations from natural sources often furnish very small sample amounts. The ecologystudies found that the microorganisms isolated from the marine invertebrates may beinvolved in the biosynthesis of chemical defensive compounds in the host. In thisstudy, we attempt to find antifouling and fungicidal compounds from marinemicroorganisms, which provided foundations for the screening and discovery ofantifouling and fungicidal agents.In this study, two marine-derived fungal strains including Cochliobolus lunatus(M351) and C. lunatus (TA26-46) were investigated for their bioactive secondarymetabolites under the guidance of the chemical and bioactive screenings. Fourteenresorcylic acid lactones (RALs)(1–14), including seven new compounds and one newnatural compound, and thirty derivatives were obtained. The isolated RALs andderivatives were evaluated their antifouling and fungicidal activities, and fourteenantifouling compounds and one fungicidal compound were obtained. The preliminarystructure-activity relationships and the mode of action were also discussed.1. Isolation, Elucidation, and Antifouling Activity of RALs Isolated from C.lunatus (M351)In our previous research, some potent antifouling briarane diterpenoids were isolated from a gorgonian Dichotella gemmacea, which prompt us to find antifoulingcompounds from the gorgonian derived fungi. We isolated the epiphytic fungi fromthe gorgonian D. gemmacea, and a fungus C. lunatus (M351) were selected to studyunder the guidance of antifouling activity against the larval settlement of barnacleBalanus amphitrite. Seven14-membered resorcylic acid lactones (1–7), includingthree new compounds, two with a rare natural acetonide group and one with a5-chloro-substituted lactone, were isolated from the fungus by columnchromatography on silica gel, Sephadex LH-20and preparative reversed phase HPLC,and identified by spectroscopy of NMR, MS, IR, UV as well as chemical methods.The isolated resorcylic acid lactones were evaluated against the larval settlement ofbarnacle B. amphitrite, and the results revealed that compounds1,4,5, and7hadpotent antifouling activities at nontoxic concentrations with EC50values of1.2,5.0,5.3, and17.9μg/mL, respectively, which were lower than the standard requirement ofan EC50of25μg/mL established by the U.S. Navy program as an efficacy level fornatural antifouling agents. The active compounds1,4, and5have high therapeuticratios (LC50/EC50>15), suggesting that they might be useful as environmentallybenign antifouling agents.2. Isolation, Elucidation, Structural Modification, and Antifouling andFungicidal Activity of RALs Isolated from C. lunatus (TA26-46)The unique structural characteristics and potent antifouling activities ofresorcylic acid lactones merit further investigation as models for the discovery of newantifouling molecules. However, further research was hindered by strain-specificvariation regarding the quantity of metabolite production as well as the seeminglycapricious behavior of this fungal strain, especially altering metabolite profiles whenre-cultured. Consequently, another metabolically stable strain with the ability toproduce resorcylic acid lactones was expected to be identified from the marine fungilibrary in our laboratory. Fortunately, C. lunatus (TA26-46), another fungal strainisolated from the sea anemone Palythoa haddoni, also collected in the South ChinaSea, was found to produce resorcylic acid lactones, and at last this fungal strain wascultured by rice solid medium according to fermentation condition optimization. Four new RALs, cochliomycins D–G (8–10and12), and eight known analogues, wereisolated from the fungus C. lunatus (TA26-46). Compounds8–11are diastereomersdiffering from each other by the absolute configurations of the4′,5′-diol chiral centers.The absolute configurations of8–11were established by the CD exciton chiralitymethod and TDDFT ECD calculations. Compound12has a hydroxy group attached atC-8′, and the absolute configuration of4′,5′-OH was confirmed by the considerationof biogenetic synthesis, and the absolute configuration of8′-OH was not determinedbecause of its limited quantity. Thirty RAL derivatives were obtained by a series ofchemical reactions, such as acylation, acetonidation, and halogenatingreaction.The isolated RALs and derivatives were evaluated their antifouling andfungicidal activities. Compounds1a,1b,4j,6,6d,6f,8,10,11, and13exhibitedpotent antifouling activity at nontoxic concentrations with the EC50values of15.4,12.5,9.0,1.82,3.85,10.5,17.3,6.67,18.1,22.5μg/mL (EC50<25μg/mL)，andLC50/EC50>15, suggesting that they might be useful as environmentally benignantifouling agents. The preliminary structure-activity relationships suggested that thehydroxy groups, cis-enone and acetonide functionalities in the structures may playedan apparent role on the antifouling activity. On glass-house-based whole plant assays,compound6displayed good activity against P. infestans and Plasmopara viticola. Onthe preventative foliar applications against P. infestans (potato),6showed potentrate-for-rate activity, with disease control rate (DCR) of98,98, and92%at200,60,and20ppm, respectively, comparable to the positive control mefenoxam (98,98, and98%) in the initial assessment. Compound6also showed promising activity on the P.infestans drench test (DCR of86%at6ppm). More remarkably, the DCR of6was91%at6ppm on the preventative foliar applications against P. viticola (grapevine)compared to the positive control mefenoxam (6ppm, DCR=0), highlighting thepromising activity of6against P. viticola. In this study, the mode of action ofantifungal activity of6could be attributed to the observed thiol activity between6andfree thiol groups.The innovation of this study: Finding environmentally benign antifouling andfungicidal agents from the marine derived fungi under the guidance of chemical defensive system of the marine invertebrates and their microorganisms; seven newRALs and thirty derivatives were obtained, enriching the structures of this class ofmetabolites; the CD exciton chirality method and TDDFT ECD calculations wereused to establish the absolute configurations of new compounds, having referencevalue for elucidating the absolute configurations of RALs; finding some potentantifouling and fungicidal compounds, providing compound sources for the researchand development of efficient, non-toxic, and natural marine antifouling and fungicidalagents.