*Article* **Bacillimidazoles A***−***F, Imidazolium-Containing Compounds Isolated from a Marine** *Bacillus*

**Jia-Xuan Yan 1,†,‡, Qihao Wu 1,‡,§, Eric J. N. Helfrich 2,***-***, Marc G. Chevrette 3,¶, Doug R. Braun 1, Heino Heyman 4,\*\*, Gene E. Ananiev 5, Scott R. Rajski 1, Cameron R. Currie 3, Jon Clardy 2 and Tim S. Bugni 1,5,\***



**Abstract:** Chemical investigations of a marine sponge-associated *Bacillus* revealed six new imidazoliumcontaining compounds, bacillimidazoles A–F (**1**–**6**). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of **1**–**6**. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics sugges<sup>t</sup> that **1**–**6** result from a fascinating intersection of primary and secondary metabolic pathways in *Bacillus* sp. WMMC1349. Antimicrobial assays revealed that, of **1**–**6**, only compound **six** displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

**Keywords:** marine-derived *Bacillus*; antibacterial; biosynthetic gene cluster; isotopic enrichment; heterocycles; imidazolium
