3100 mass detector

Unless otherwise described, all commercial reagents and solvents were purchased from commercial suppliers and used without further purification. All reactions were performed under N₂ atmosphere in flame-dried glassware. Reactions were monitored by TLC with 0.25 mm E. Merck precoated silica gel plates (60 F254). Reaction progress was monitored by TLC analysis using a UV lamp, ninhydrin, or p-anisaldehyde stain for detection purposes. All solvents were purified by standard techniques. Purification of reaction products was carried out by silica gel column chromatography using Kieselgel 60 Art. 9385 (230 − 400 mesh). The purities of all compounds were shown to be over 95% by using Waters LCMS system (Waters 2998 photodiode array detector, a Waters 3100 mass detector, a Waters SFO system fluidics organiser, a Water 2545 binary gradient module, a Waters reagent manager and a Waters 2767 sample manager) using a SunFireTM C18 column (4.6 mm × 50 mm, 5 µm particle size): solvent gradient = 60% (or 95%) A at 0 min, 1% A at 5 min. Solvent A = 0.035% TFA in H₂O; solvent B = 0.035% TFA in MeOH; flow rate 3.0 (or 2.5) mL/min. ¹H and 13 C NMR spectra were obtained by using a Bruker 400 MHz FT-NMR (400 MHz for ¹H, and 100 MHz for 13 C) spectrometer. Standard abbreviations are used for denoting the signal multiplicities.

Four litres of fermentation broth of SUKA34::rapH/pKU503rapΔM9AT::M6ATm was centrifuged to obtain a mycelial cake, which was extracted twice with 500 ml of acetone. The acetone was removed in vacuo, and the residual aqueous layer was extracted twice with ethyl acetate (EtOAc). The resultant EtOAc layer was concentrated in vacuo to afford 1.9 g of crude extract. The crude extract was subjected to medium-pressure liquid chromatography (MPLC) on silica gel (SNAP Ultra 25 g, Biotage, Uppsala, Sweden) eluted with a gradient system of n-hexane–EtOAc (0-25% EtOAc) followed by a stepwise solvent system of chloroform (CHCl₃)–methanol (MeOH) (0, 1, 3, 5, 10, 50 and 90% MeOH). The 3% MeOH fraction (242 mg) was collected and subjected to silica gel MPLC (SNAP Ultra 25 g) with isocratic elution with 3% MeOH in CHCl₃. The fractions were monitored by UPLC analysis, and fractions containing 2 were collected (48.1 mg). The sample was further purified by preparative reversed-phase HPLC using a CAPCELL PAK MG-II C18 column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan). 2 was detected using a 2996 photodiode array detector (Waters) and a 3100 mass detector (Waters) following elution with 80% aqueous acetonitrile, and 4.4 mg of 2 was obtained.

Peptides were prepared as C-terminal carboxamides on NOVASYN® TGR resin and as C-terminal carboxylic acids on NOVASYN® TGA resin by standard Fmoc/tBu chemistry using HCTU/DIPEA in DMF on an automated peptide synthesizer (Prelude, Gyros Protein Technologies, USA). After deprotection and cleavage from the resin with a cocktail of TFA (95 % v/v), TIPS (2.5 % v/v), water (2.5 % v/v) for 3 h at RT, the peptides were precipitated in diethyl ether. The crude peptides were purified by reverse-phase HPLC (Varian) using a C8 column (Agilent Polaris, 21.2 × 250 mm, 5 μm) and a linear gradient elution of 5–50% acetonitrile containing 0.1 % TFA (v/v). To verify molecular masses against calculated theoretical values, purified peptides were characterized by single quadrupolar mass spectrometry using a Waters Mass Lynx 3100 platform. Positive electrospray ionisation (ESI) was used as the source. Analytes were loaded onto a C18 column (Waters X-Bridge, 4.6 × 100 mm, 3 μm), eluted using a linear gradient elution of 10–90 % acetonitrile with 0.1 % TFA over 10 min at 1.5 mL/min at RT and they were detected by both UV absorption at 210 nm and ionization using a Waters 3100 mass detector. Analytical RP-HPLC spectra were also recorded on an Agilent 1260 Infinity system using a linear gradient elution of 10–90 % acetonitrile with 0.1 % TFA over 15 min at 40 °C.