In-depth analysis of the S. defensii MSr10575 metabolome by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) revealed a group of three peaks at m/z 541.45, 555.47 and 569.48 in positive ESI ionization mode. The three corresponding secondary metabolites were named sandacrabins A-C. According to sum formula predictions based on HRMS data, their sum formula lacks oxygen atoms commonly present in polypeptide and polyketide NPs, which suggested their classification among the alkaloid family of terpenoid NPs. Sandacrabin A was purified by semi-preparative reversed phase (RP) HPLC from the dried hexane layer obtained by partitioning of a S. defensii MSr10575 crude extract between methanol and hexane. The high lipophilicity of sandacrabin B and C, along with their structural similarity, however, required a four-step purification process (see Figure ). After separation of sandacrabin A from the crude extract, the remaining methanol layer was dried and partitioned between water and chloroform. The dried chloroform layer was first subjected to centrifugal partitioning chromatography (CPC). Separation of sandacrabin B and C from the respective CPC fractions was subsequently achieved by supercritical fluid chromatography (SFC) prior to isolation of the secondary metabolites by semipreparative RP-HPLC. HRESI-MS analysis of sandacrabin A showed an [M+H] + signal at m/z 541.4514 (calc. 541.4516 Δ = 0.4 ppm) consistent with the sum formula of C38H57N2 containing 12 double bond equivalents (DBEs). 1D and 2D NMR spectra of sandacrabin A (tables see SI) suggested a benzimidazole core structure of the molecule. The splitting pattern of the two aromatic protons in line with their COSY correlations furthermore revealed a 5,6-dimethyl substitution of this unit. COSY and HMBC correlations showed arrangement of the remaining methylene and methyl groups in two farnesyl moieties on the 5,6dimethylbenzimidazole (DMB) core. HMBC correlations of the Figure Phylogenetic classification of S. defensii MSr10575 as well as BGCs found in its genome using AntiSmash. aliphatic double bond proton at δ( 1 H) = 6.22 ppm, as well as its downfield shift, showed that the second farnesyl side chain substitutes the 2-position of DMB (see Figure ). The shielded shift of all methyl carbons indicates an all-E configuration of sandacrabin A. HRESI-MS analysis of sandacrabin B and C showed an [M] + signal at m/z 555.4514 (calc. 555.4673 Δ = 0.2 ppm) corresponding to the sum formula of C39H59N2 and 569.4825 (calc. 569.4829 Δ = 0.7 ppm) corresponding to the sum formula of C40H61N2, respectively, with both containing 12 DBEs, comparable to sandacrabin A. 1D and 2D NMRspectra of sandacrabin B and C (tables see SI), revealed a C2vsymmetry (see Figure ) and a bisfarnesylated DMB core structure for both derivatives. The C2v-symmetry of the molecules, as well as their permanent positive charge, indicated a 1,3-substitution of the DMB unit. In contrast to sandacrabin B, 1D and 2D NMR-spectra of sandacrabin C revealed a methylation in the 2-position of the DMB core. Tautomerization of the imidazole double bond as well the symmetry axis passing through this part of the molecule however hindered a detection of the corresponding proton signal at this position in sandacrabin B, wherefore HRESI-MS 3 spectra of the three sandacrabins were used to provide additional proof for their structures (see Figure ).