Isolation and structure elucidation of microginins 789 (1). Given the apparent abundance of some microginins in the smallscale organic extract of M. aeruginosa LEGE 91341 (Fig. ), we focused next on the isolation of sufficient amounts these metabolites to enable NMR-based structure elucidation. Fermentation of M. aeruginosa LEGE 91341 in 120 L culture medium yielded 42 g of biomass (d.w.), which was extracted by repeated percolation with methanol. The resulting crude extract (7.8 g) was subjected to reversed-phase (C18) Vacuum Liquid Chromatogaphy (VLC) with a stepwise gradient of decreasing polarity from water to MeOH, to give seven fractions (A-G). LC-HRESIMS analysis indicated that fraction E, eluting with 100% MeOH, contained the dihalogenated 1 in high abundance. Global Natural Products Social Molecular Networking (GNPS) analysis of this fraction revealed what could be additional minor microginins (Fig. ) but their identification was not pursued in this study. Subsequent reversed-phase Flash Chromatogaphy and HPLC steps afforded pure microginin 789 (1, 2.8 mg). This compound was subjected to 1D and 2D-NMR based structure elucidation. The 1 H NMR data for 1 (Table ) showed the presence of two broad singlets resonating at approx. 9 ppm, two exchangeable amide protons doublets (dH 8.28 and 8.12), several protons in the aromatic/olefin region, as well as resonances around dH ~5.0-3.5 that could correspond to alpha protons. In addition, a methylene envelope was observable (dH ~1.4-1.2) and a terminal methyl group resonance for a saturated alkyl chain was not present. The 13 C NMR data (Table ) notably indicated the presence of five carbonyls (dC ~175-169) and two aromatic, likely phenol moieties (eight resonances in the dC ~160-110 range). These 1 H and C NMR data would thus be consistent with a lipopeptide containing two aromatic residues. Additional structural information was obtained by analysis of the 2D NMR data. A combination of HSQC, HMBC and COSY data (Table ) was used to establish several substructures of 1, as follows. Two very similar aromatic spin systems could be readily constructed. The first corresponded to a para-substituted phenol that further connected to an alpha proton (H-32, dH 3.96) through a diastereotopic methylene (CH2-33, dHa 3.04, dHb 2.68) and was attributed to a Tyr residue. The second also corresponded to a para-substituted phenol moiety which was HMBC correlated to a COSY-derived spin system composed of two methylene groups (CH2-19 and CH2-18) and an alpha position (CH-17), thereby establishing a homotyrosine (Htyr) moiety. The two amide protons were COSY-correlated to each of the aromatic residue alpha protons (Table , Fig. ). Two proline residues could also be established (Table , Fig. ) and these explained the absence of additional amide protons. A conspicuous methine (CH-10, dC 74.8, dH 6.25) was assigned as a dichloromethyl group, taking into account the particular resonances and the presence of two unassigned chlorine atoms in the molecule. This was correlated (HMBC, COSY) to an alkyl moiety that degenerated into the methylene envelope at CH2-7. A single carbonyl (dC 169.0) remained to be assigned, was correlated to an alkyl spin system containing a substituted methine (CH-3, dC 47.1, dH 3.29), which also degenerated into the methylene envelope at CH2-9. A single methylene group, part of the methylene envelope, remained to be assigned in order to satisfy the molecular formula of 1 and was considered to be the bridging position between the two alkyl chain spin systems (CH2-8). This established that 1 contains a dichlorinated 3-aminodecanoic acid (Ada) residue (Table , Fig. ). Inter-residue HMBC correlations supported that 1 corresponds to a linear peptide of sequence [Cl2-Ada]-Pro-Htyr-Pro-Tyr (Table , Fig. ). The stereochemistry of all the proteinogenic amino acid residues in 1 was deduced, from the absence of epimerase domains, as of l configuration, while the stereochemistry of the Htyr residue and of the β-amino group were not elucidated.