2.4.2. PEHE\_5 (TSRR0002\_F-08) *Haliclona* sp.

Based on the results of the primary screening against *S. aureus*, the corresponding crude extract (1 mg/mL in MeOH) was fractionated in 2 and 5 μL injection volume replicates and rescreened against the same indicator strain. Only the 5 μL replicate showed active fractions, namely fractions 47–48 and fraction 108 (Figures S7 and S8).

Activity of fractions 47-48 was assigned to a compound of *m*/*z* 398.9449 [M + H]<sup>+</sup> showing the specific isotope pattern of a dibrominated compound (Figure S7b) corresponding to a molecular formula of C13H13Br2N4O1. The compound shows UV absorption maxima at 220 and 292 nm. The fragmentation pattern is indicative of a dibrominated triptamine framework as structural subunit. A substructure search on SciFinder retrieved no hits corresponding to the assigned molecular formula, however, one candidate, namely 5,6-dibromo-2 -demethylaplysinopsin (C13H11Br2N4O1 - one additional degree of unsaturation compared to the compound in the extract), was found to fit the fragmentation pattern observed for the compound in the extract. Therefore, the active compound was putatively assigned the structure of 5,6-dibromo-1 ,8-dihydro-2 -demethylaplysinopsin [12] (Figure 5), which is in agreement with the molecular formula, fragmentation pattern, and observed UV spectrum. Aplysinopsins are a family of indole alkaloids isolated from sponges [13], scleractinian corals [14,15] and sea anemones [16]. The tentative 5,6-dibromo-1 ,8-dihydro-2 -demethylaplysinopsin is, to our knowledge, not reported in literature.

Activity of fraction 108 was assigned to a mixture of co-eluting compounds of *m*/*z* 627.4414 [M + H]+, *m*/*z* 315.2321 [M + H]+, *m*/*z* 329.2115 [M + H]<sup>+</sup> (Figure S8b–d; proposed structures in Figure 5), corresponding to the molecular formulae C42H58O4, C21H30O2, and C21H28O3, respectively. The compounds showed UV absorption maxima at 223 and 299 nm. The MS/MS fragmentation pattern

of all three compounds showed a base peak ion of *m*/*z* 191.1798, corresponding to the molecular formula of C14H23<sup>+</sup>, which was assigned to the retro Diels–Alder fragmentation product ion of sesquiterpene hydroquinone frameworks. Based on the MS/MS data for C21H30O2, no distinction between the two literature known isomeric sponge metabolites, aureol and chromazonarol [17,18], could be made. The same holds true for C42H58O4 (putatively 6 -aureoxyaureol or 6 -aureoxychromazonarol). The bissesquiterpene 6 -aureoxyaureol was reported together with several dibrominated aplysinopsin derivatives in *Smenospongia* sp., whereas the hypothetical chromazonarol stereoisomer was not described [19]. Finally, literature query of C21H28O3, produced a range of hits corresponding to algal metabolites, while only one compound, chondrosine (a.k.a. puupehenone) [20], was previously reported from sponges. The structures of 6 -aureoxyaureol, chondrosine, and chromazonarol (Figure 5) were chosen as representative examples for each of the ions of *m*/*z* 627.4414, *m*/*z* 329.2115, and *m*/*z* 315.2321, respectively.

The same extract was fractionated against *S. tritici* (injection volume 2 and 5 μL), however, no active fractions could be observed.

### 2.4.3. ULU\_16 (TSRR0002\_H-07) *Neopetrosia* sp.

To investigate the observed antifungal activity of ULU\_16, the extract was fractionated in 2 and 5 μL injection volume replicates and rescreened against *S. tritici*. The activity zone was reproduced in the two replicates and could be assigned to a compound of *m*/*z* 385.9249 [M + H]<sup>+</sup> showing the specific isotope pattern of a dibrominated compound (Figure S9) with the molecular formula C11H9Br2N5O1. The compound showed UV absorption maxima at 220 and 340 nm. Based on the MS/MS fragmentation pattern, the compound was dereplicated as stevensine, also known as odiline (Figure 5), a metabolite reported in various sponge species [21].
