**3. Conclusions**

The isolation and structural elucidation of bacillimidazoles A–F, six imidazoliumcontaining heterocycles from marine *Bacillus* sp. WMMC1349 represent the discovery of a new class of marine-derived heterocyclic natural products. Isotopic labeling of **1**–**6** using 13C enriched culture media, biomimetic synthetic approaches and bioinformatic analyses were performed in order to gain insights into the biosynthetic assembly of these interesting compounds. To the best of our knowledge, there are only a few reports of natural products containing 1,3-difunctionalized imidazolium moieties, and this is the first report of naturally occurring imidazolium-containing heterocycles. We contend, based on these findings, that the discovery of **1**–**6** contributes to our advancing knowledge of significantly underexplored biosynthetic pathways, specifically those that intersect primary and secondary metabolic pathways. We anticipate that the lessons learned here will help to expedite efforts to more fully understand and exploit the full biosynthetic potential of *Bacillus* spp.

#### **4. Materials and Methods**

#### *4.1. General Experimental Procedures*

UV spectra were recorded on an Aminco/OLIS UV-Vis spectrophotometer. IR spectra were measured with a Bruker Equinox 55/S FT–IR spectrophotometer. NMR spectra were obtained in CD3OD (*δ*H 3.34 ppm, *δ*C 49.0 ppm) with a Bruker Avance 600 III MHz (Billerica, MA, USA) spectrometer equipped with a 1H{13C/15N/31P} cryoprobe, a Bruker Avance III 500 MHz (Billerica, MA, USA) spectrometer equipped with a 13C/15N{1H} cryoprobe, and a Bruker Avance III HD 400 MHz (Billerica, MA, USA) spectrometer. HRMS data were acquired with a Bruker MaXis™ 4G ESI-QTOF (Billerica, MA, USA) mass spectrometer. RP HPLC was performed using a Shimadzu Prominence HPLC system and a Phenomenex Gemini C18 column (250 × 30 mm). UHPLC-HRMS was acquired using a Bruker MaXis™ 4G ESI-QTOF (Billerica, MA, USA) mass spectrometer coupled with a Waters Acquity UPLC system operated by Bruker Hystar software and a C18 column (Phenomenex Kinetex 2.6 μm, 2.1 mm × 100 mm). Bruker timsTOF Pro instrument (Billerica, MA, USA) was used for the trapped ion mobility MS analysis using direct infusion with 0.003 mL/min of flow rate and ESI+ ionization source. Nebulizer gas 0.4 bar, dry gas 3.5 L/min, source temperature 220 ◦C, ESI voltage 4200V (+). MS spectra were collected using the following parameters: tims ramp time = 350 ms, PASEF on, scan range (*m*/*<sup>z</sup>*, 20–1000; 1/k0, 0.70–1.00 <sup>V</sup>·s/cm2.
