Luna 5 m c8 2 100

1 gram of crude GM extract was applied to a column packed with 10 g silica and washed with chloroform (CHCl₃, Fisher Chemical, BPC298500). Sample was serially eluted into five fractions using 1 mL mobile phase comprised of CHCl₃ and an increasing gradient of MeOH. Each fraction was analyzed by LC/MS to identify fractions enriched for the mass features of interest. A CHCl₃:MeOH solution of 100:1 yielded the fraction with greatest enrichment. This fraction was further purified into eight fractions by reverse phase C8 chromatography (Luna 5 µm C8(2) 100 Å, 250 × 10 mm, Phenomenex) using a gradient elution of 20% H₂O and 80% MeOH, increasing to 100% MeOH over 20 min. Each fraction was analyzed by LC/MS to confirm the presence and relative yield of target compounds. Column temperatures and sample compartments were maintained at 20 °C and 4 °C, respectively. Data were acquired at a wavelength of 280 nm using a DAD detector.

Ammonium chloride (20 g, 0.374 mol) was added to a glass jar containing 2 L of deionized (DI) water and a magnetic stir bar. Once the solid dissolved, the pH of the mixture was adjusted to 9.0 using ~10 mL of ammonium hydroxide solution. Then 1,4-dihydroxy-2-naphthoic acid (DHNA - 1.0 g, 4.5 mmol) was added to the mixture. The reaction mixture was stirred for 24 hr at rt, at which point the mixture was acidified to pH five and then extracted with 2 L ethyl acetate (EtOAc) x 2. The organic layer was concentrated under vacuum. The crude material was then resuspended in 10 mL 50% ACN/H₂O, syringe filtered (Corning Inc, 0.2 μm), and purified using prep-HPLC. Pure ACNQ eluted between 33–35 min using Phenomenex Luna 5 µm C8(2) 100 Å preparative column (250 × 21.2 mm), with the following method: holding 20% ACN +0.1% FA/H₂O + 0.1% FA for 5 min then gradient to 55% ACN +0.1% FA/H₂O + 0.1% FA over 35 min at 10 mL/min flow rate. The overall reaction yield, through purification, is approximately 1%. Synthetic ACNQ was compared to the isolated material by back-to-back injection on the HRLCMS using a Phenomenex Kinetex 2.6 μm EVO C18 100 Å (100 × 2.1 mm) under the following LC method: holding 2.0% ACN +0.1% FA/H₂O + 0.1% FA for 1 min then gradient to 20% ACN +0.1% FA/H₂O + 0.1% FA over 39 min at 0.3 mL/min.