*3.1. Extraction and Isolation*

Freeze-dried *Limnoraphis* CCNP1324 cells were homogenized using mortar and pestle. The ground cyanobacterial biomass (10 mg) was extracted with 75% methanol in MilliQ water (1 mL) by vortexing (5 min). The sample was then centrifuged (10,000× *g*; 15 min; 4 ◦C) and the content of aeruginosamides in the obtained supernatant was analyzed using LC-MS/MS.

For fractionation and isolation of aeruginosamides, the homogenized biomass (20 g) was extracted twice with 75% methanol in MilliQ water (2 × 500 mL) by vortexing (20 min). After centrifugation (4000× *g*; 15 min; 4 ◦C), the supernatants were combined and diluted with MilliQ water, so that the final concentration of MeOH in the extract was <10%. For flash and preparative chromatography a Shimadzu HPLC system model LC-20AP (Shimadzu, Canby, OR, USA) equipped with isocratic and binary pumps, a fraction collector and photodiode array detector (PDA) was used. PDA operated in a range from 190 nm to 500 nm and, during all chromatographic runs, the absorbance at 210 nm and 280 nm was recorded.

To perform flash chromatography, the aqueous methanol extract (MeOH < 10%) was loaded onto a preconditioned 120 g SNAP KP-C18-HS cartridge (Biotage Uppsala, Sweden) using an isocratic pump, at a flow rate of 15 mL min<sup>−</sup>1. Components of the extract were separated with a mixture of a mobile phase composed of MilliQ water (A1) and 100% MeOH (B1). The gradient started at 10% B1 and went to 30% B1 within 20 min. After 90 min, the content of B1 increased to 70% and was kept at that level for 10 min before increasing to 100% B1 within the next 30 min. The flow rate of the eluent was 20 mL m−<sup>1</sup> and 50 mL fractions were collected.

AEGs-containing flash fractions, eluted with 86–93% B1 (Prep1), and 58–68% B1 (Prep2), were combined and concentrated in a centrifugal vacuum concentrator (MiVac, SP Scientific, Ipswich, UK). Dried samples (Prep1 and Prep2) were first solubilized using 0.6 mL of 60% MeOH, followed by 0.6 mL of 20% MeOH. After centrifugation, the supernatants were loaded onto a preparative column using the Rheodyne injector. The sample components were separated on a Jupiter Proteo C12 column (250 × 21.2 mm; 4 μm; 90 Å) (Phenomenex, Aschaffenburg, Germany) with a mobile phase composed of 5% acetonitrile in MilliQ water (A2) and acetonitrile (B2), both with the addition of 0.1% formic acid. During the separation process the flow rate was 20 mL m−<sup>1</sup> and 4 mL fractions were collected.

In the case of fractions 86–93% (Prep1), the gradient started at 20% B2, then went to 30% B2 in 25 min, after 10 min B2 reached 90%. After another 2 min, B2 increased to 100% and was kept at that level for 13 min. Fractions eluted with 25–27% B2 (vials 75–103) and containing an isolated single peak were pulled, vacuum concentrated and marked as sample A (1 mg). Fractions eluted with 23–25% B2 (vials 40–74), which also corresponded to a single peak in HPLC-PDA chromatogram, were pulled, evaporated to dryness and marked as sample B (0.9 mg).

The preparative separation of flash fractions 58–68% B1 (Prep2) started at 15% B2 and went to 30% B2 in 20 min, after 10 min B2 reached 90%. After another 2 min, B2 increased to 100% and was kept at that level for 8 min. Fractions eluted with 24–27% B2 (with AEGs) were prepared as described above and subjected to further separation. In the subsequent run, the gradient started at 5% B2 and went to 40% B2 in 20 min, after 5 min B2 reached 100% and was kept at that level for 5 min. Fractions eluted with 27–37% B2, containing a single peak were pulled evaporated to dryness and marked as sample C (1.2 mg). The samples A, B and C were subjected to LC-MS/MS analyses and cytotoxicity assays. For sample A, the NMR analyses were additionally performed.
