Integrating molecular networking and 1H NMR spectroscopy for isolation of bioactive metabolites from the persian gulf sponge axinella sinoxea

Abstract

The geographic position, highly fluctuating sea temperatures and hypersalinity make Persian Gulf an extreme environment. Although this unique environment has high biodiversity dominated by invertebrates, its potential in marine biodiscovery has largely remained untapped. Herein, we aimed at a detailed analysis of the metabolome and bioactivity profiles of the marine sponge Axinella sinoxea collectedfromthenortheast coast of thePersianGulf inIran. The crude extract anditsKupchansubextracts were tested in multiple in-house bioassays, and the crude extract and its CHCl3-soluble portion showed in vitro antibacterial activity againstMethicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium (Efm). A molecular networking (MN)-based dereplication strategy by UPLC-MS/MS revealed the presence of phospholipids and steroids, while 1H NMR spectroscopy indicated the presence of additionalmetabolites, such as diketopiperazines (DKPs). IntegratedMN and 1H NMR analyses on both the crude and CHCl3 extracts combinedwith an antibacterial activity-guided isolation approach afforded eight metabolites: A new diketopiperazine, (-)-cyclo(L-trans-Hyp-L-Ile) (8); a known diketopiperazine, cyclo(L-trans-Hyp-L-Phe) (7); two known phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphocholine (1) and 1-O-octadecanoyl-sn-glycero-3-phosphocholine (2); two known steroids, 3-hydroxycholest-5-ene-7,24-dione (3) and (22E)-3β-hydroxycholesta-5,22-diene-7,24-dione (4); two known monoterpenes, loliolide (5) and 5-epi-loliolide (6). The chemical structures of the isolateswere elucidated by a combination of NMR spectroscopy, HRMS and [α]D analyses. All compounds were tested againstMRSA and Efm, and compound 3 showedmoderate antibacterial activity againstMRSA (IC50 value 70 μg/mL). This is the first study that has dealtwith chemical and bioactivity profiling of A. sinoxea leading to isolation and characterization of pure spongemetabolites.