Aviii 700 mhz instrument

S. coelicolor M1154/pAMX4/pGP6738/pIJ10257-sl was inoculated in TSBY medium (supplemented with 50 μg/mL kanamycin, 25 μg/mL thiostrepton, and 50 μg/mL hygromycin) and grown at 30 °C, 250 rpm for 48–60 h. Then 1 mL of the seed culture was inoculated into 50 mL SAM medium in 250 mL flasks, and incubated at 30 °C, 250 rpm for 6 days. 150 mL of SAM culture was collected and lyophilized. The dried material was washed with 50% MeOH (4×) and extracted with DMSO (3×), resulting in ~70 mg pure GP6738. GP6738 was dissolved into d6-DMSO (20 mg/mL) and analyzed by 1D and 2D NMR recording on a Bruker AVIII 700 MHz instrument equipped with a cryoprobe. GP6738 was analyzed by high-resolution mass spectrometry (HRMS) using an Agilent 6550 iFunnel Q-TOF mass spectrometry with an inline Agilent 1290 HPLC system using electrospray ionization in positive mode, and mass scan range from 50–2000 Da. MS/MS spectrum of GP6738 was recorded on the same system using a collision-induced dissociation (CID) energy of 50 V. A Symmetry Shield RP 8 column (100 Å, 3.5 µm, 4.6 mm × 150 mm column, Waters, WAT094269) was used with the following gradient: t = 0–1 min, 5% solvent B; t = 20–22 min, 100% solvent B; t = 23–30 min, 5% solvent B (solvent A: 0.1% v/v formic acid in water and solvent B: 0.1% v/v formic acid in ACN); flow rate 0.6 mL/min, 40 °C.

ApmA-catalyzed acetylated apramycin was produced from 50-ml in vitro reactions (50 mM HEPES, pH 7.5) consisting of 500 μM aminoglycoside, 500 μM acetyl-CoA, and 1 μM ApmA. Reaction mixtures were incubated at room temperature until acetylated products (mass increase of 42.0 Da) were detected by liquid chromatography (LC)/ESI-MS. Enzymes were removed by centrifugation using an Amicon Ultra-15 centrifugal filter and the flowthrough subsequently concentrated. Acetyl-apramycin was purified from the concentrate using AG50W-X8 strong cation resin. The resin was preequilibrated with 1% NH₄OH and washed with H₂O until a neutral pH was obtained. Fractions containing acetylated products were identified by LC/ESI-MS, followed by detailed analysis with NMR and HR-ESI-MS. LC/ESI-MS data were acquired using a QTrap 2000 (Applied Biosystems) system equipped with an Agilent 1100 LC interface. HR-ESI-MS data were acquired using an Agilent 1290 ultraperformance liquid chromatography (UPLC) separation module and quadrupole time of flight (Q-TOF) G6550A mass detector in positive ion mode. NMR analysis was completed using a Bruker AVIII 700 MHz instrument in deuterated water as the solvent. The chemical shifts are reported in parts per million.