Chemical Investigation of the Mediterranean Sponge Crambe crambe by UHPLC-HRMS/MS via Manual and Computational Dereplication Approaches

The CH₂Cl₂-MeOH extract of the Mediterranean sponge <i>Crambe crambe</i> was investigated via UHPLC-HRMS/MS employing manual dereplication and in silico mass spectrometry tools. A deconvolution approach was implemented for the extensive metabolic characterization of the sample, resulting in the annotation of 53 compounds. The analysis of data-dependent HRMS/MS scans was conducted to establish fragmentation patterns characteristic of each crambescin A, B, and C sub-families. Among the 39 compounds identified from these groups, 22 analogues were reported for the first time including 4 new homologous series that differed by the ratio of methylene units in the upper (<i>n</i> + 2) and lower (<i>m</i> + 2) alkyl side chains. More specifically, crambescins presenting <i>m</i> = 5 or 6 and <i>n</i> = 5 (compounds <b>7</b>, <b>11</b>, <b>22</b> and <b>24</b>) as well as <i>m</i> = 5 or 6 and <i>n</i> = 4 (compounds <b>5</b>, <b>6</b>, <b>8</b>, <b>9</b>, 12 and 14) were characterized. Additionally, four new features, potentially corresponding to new crambescidin analogues (compounds <b>13</b>, <b>15</b>, <b>35</b>, and <b>39</b>), were also reported. The identity of the dereplicated features was further validated by studying crambescins' spectral similarities through a feature-based molecular networking approach. Overall, this study suggests UHPLC-HRMS/MS-through the integration of manual and computational dereplication approaches-as a valuable tool for the investigation and high-throughput characterization of the <i>C. crambe</i> metabolome.