*2.4. Additional Structure Hypothesis Generation*

The GNPS-produced LC-MS/MS molecular network highlights molecules with a potential structural similarity as "molecular families", based on fragmentation patterns. The cluster that contained wenchangamide A (**1**) suggested the presence of further analogue molecules in the extract. One of these compounds presented an *m*/*z* value of 1297, which is 86 Da higher than that of **1** in the same experiment. Upon closer examination of the MS/MS spectra and fragmentation ions related to the "y" ions produced from amide bond backbone cleavages, it was determined that the entire difference of 86 Da between the *m*/*z* 1297 molecule and **1** was located on the FA residue. Since this same mass difference corresponds to one additional repeating unit of a PKS-like moiety present 3x in **1,** akin to the 4x in minnamide A, the corresponding "hybrid" molecule, wenchangamide B, is here hypothesized (Figure 9). While the structure of this molecule contains the same FA moiety from minnamide A, it has the same polypeptide core as **1**, and is accordingly assigned the trivial name, wenchangamide B. Although fragmentation data from mass spectrometry cannot distinguish between configurational isomers of peptides, and even some constitutional isomers (e.g., Ile, Leu, and *N*-Me-Val), the structure of wenchangamide B is proposed as drawn, based on biosynthetic logic. This molecule was not able to be isolated in pure form in sufficient quantity for empirical structural characterization in this study, and is suggested for targeted isolation, structure elucidation, and more accurate pharmacological characterization in future research. This strategy of structure proposal based on MS/MS fragmentation analysis and molecular networking for compounds beyond isolation in the initial study, later followed by targeted isolation or synthesis for confirmation and structure-activity relationship (SAR) study, has been recently exemplified by Gerwick, Luesch, and coworkers in the expansion of the cyanobacterial natural product family of doscadenamides [47–49].

**Figure 9.** GNPS-based LC-MS/MS molecular network analysis of fraction C3 and active sub-fractions. Enlarged network highlights the structural similarity based on MS/MS fragmentation patterns of the discovered molecule **1** (wenchangamide A; node circled in black) and the proposed analogue (wenchangamide B; circled in red).

The reported activities of minnamide and **1** have been demonstrated in discrete cell lines, with different conditions, and using alternative temporal end points for measurement. In terms of activity, minnamide A was reported to be a potent inhibitor of HeLa cells that led to necrosis in a 72 h incubation assay (IC50 0.17 μM); it was also suggested to act via the generation of lipid ROS facilitated by specific metal ions including copper and manganese [46]. Wenchangamide B remains of particular interest because the parent subfraction (C3-7) was here found to inhibit HCT116 cells in vitro (Figure S24). Future research on new natural products and synthetic analogues may contribute relatable SAR data for the growing class of cytotoxic minnamide and wenchangamide lipopeptides.
