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the microbial incubation

the antiperiplanar arrangement that the isomers A undergo, which is not achieved by the isomers B can lead to particular physicochemical interactions with the stationary phase and likely cause earlier elution of the diasteroisomer A prior to B in reversed-phase chromatography. Based on this, we propose that the new dichlorinated rugulovasines A and B follow the same pattern and we thus assign the relative configuration of the first peak to A and the second to B, according to their retention in the reversed phase chromatographic systems used in our work.In order to isolate the 8-chlororugulovasines A and B and the new metabolites to confirm their chemical structures based on NMR and HRMS data analysis, the fungal strain was cultivated in large scale. The scaled-up extract was submitted to medium pressure chromatography, giving rise to enriched fractions in the target compounds. The final purification of the target alkaloids showed to be a laborious task due to the low yield of these metabolites when compared to the polyketides co-produced by the strain, and in particular the occurrence of signal broadening favored by the interconvertion of the isomers A and B. To overcome this, the most enriched fraction in the halometabolites was submitted to an automated coupled HPLC-SPE system with the sequential analysis by NMR with a cryogenic probehead. Along the chromatographic run, the compounds were trapped in polydivinylbenzene SPE cartridges, in a total of 30 sequential injections.Afterwards, the isolated compounds were then eluted from the SPE cartridges with methanol-d4 before 1D and 2D NMR spectroscopy analysis. The NMR spectroscopic data allowed the identification of the known 8-chlororugulovasines