Dc alufolien kieselgel 60 f254

Progress of reactions and purity of products obtained during syntheses and biotransformation were performed on silica gel-coated aluminium plates (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany) with a mixture of hexane and acetone. GC analysis of all compounds was carried out on an Agilent Technologies 6890N instrument (Varian, Agilent Technologies, Santa Clara, CA, USA) using a DB-17 column (cross-linked methyl silicone gum, 30 m × 0.32 mm × 0.25 μm). The enantiomeric excess of the product obtained during biotransformation was determined by GC analysis using the chiral column CP-cyclodextrin-B-225 (30 m × 0.25 mm × 0.25 μm) under the following conditions: injector 200 °C, detector (FID) 250 °C, column temperature: 140 °C (hold 1 min), 140–160 °C (rate 0.5 °C/min), and 160–200 °C (rate 10 °C/min) and 200 °C (hold 1 min).
¹H NMR spectra were recorded in a CDCl₃ solution on a Bruker Avance DRX 300 MHz spectrometer (Bruker, Billerica, MA, USA) or on a Bruker Avance^™ 600 MHz spectrometer (Bruker, Billerica, MA, USA). The molar mass of the new product was confirmed by high resolution mass spectrometry analysis using a Waters LCT Premier XE instrument (ESI ionisation) (Waters Division, Milford, MA, USA). Optical rotation was determined on a P-2000 polarimeter (Jasco Easton, PA, USA) in chloroform solutions.

Initial tests were carried out using TLC plates (SiO₂, DC Alufolien Kieselgel 60 F₂₅₄ (0.2 mm thick), Merck, Darmstadt, Germany). The mobile phase contained a mixture of chloroform and methanol in 9:1 (v/v) relation. The plates were observed using a UV lamp (254 and 365 nm).
The scale-up biotransformation products were separated using 1000 µm preparative TLC silica gel plates (Anatech, Gehrden, Germany). The mobile phase contained a mixture of chloroform and methanol in a 9:1 (v/v) ratio. Separation products were scraped out and extracted twice using ethyl acetate.

The course of biotransformations was monitored using TLC plates (SiO₂, DC Alufolien Kiesel gel 60 F254 (0.2 mm thick), Merck, Darmstadt, Germany). Products were separated using preparative TLC plates (Silica Gel GF, 20 × 20 cm, 500 μm, Analtech) and a cyclohexane: ethyl acetate mixture (9:1, v/v) as an eluent, according to a method described previously [29 (link),42 (link)]. The product was observed (without additional visualisation) under the UV lamp at a wavelength of 254 nm.
NMR analysis was performed using a DRX 600 MHz Bruker spectrometer (Bruker, Billerica, MA, USA). The prepared samples were dissolved in deuterated chloroform CDCl₃. The performed analyses include ¹H NMR, ¹³C NMR, HMBC (two-dimensional analysis), HMQC (heteronuclear correlation) and COSY (correlation spectroscopy) (Supplementary Materials).

The biomass of strain SCA2-4^T was collected by centrifugation at 8000 rpm for 20 min after growing in a shaken flask of the TSB broth (15.0 g of tryptone, 5.0 g of soytone, 30.0 g of sodium chloride, 1 L of sterile water, pH 7.1–7.5) at 25°C for 5 d. Respiration quinone was extracted from the lyophilized mycelia (0.5 g) by a reverse-phase partition high-performance liquid chromatography (HPLC) (Sasser, 1990 ). The cellular fatty acids were analyzed according to the standard Microbial Identification System (Sherlock Version 6.1, MIDI database) by a gas chromatography (56890N, Agilent Technologies) (Komagata and Suzuki, 1987 ). The polar lipids were extracted from dry mycelia and individually separated by a two-dimensional thin-layer chromatography (TLC) on silica gel plates (Art 5554, DC-Alufolien Kieselgel 60F 254, Merck, Germany) (Minnikin et al., 1979 (link)).

Commercially available thiourea 1 and acyl chlorides a-l were purchased by Alfa-Aesar and Sigma-Aldrich. DMF, DMA and pyridine were reagent grade and were dried on molecular sieves (5 Å 1/16" inch pellets). Unless otherwise stated, all commercial reagents were used without further purification. Organic solutions were dried over anhydrous sodium sulphate. Thin-layer chromatography (TLC) system for routine monitoring the course of parallel reactions and confirming the purity of analytical samples employed aluminium-backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254). DCM or DCM/methanol (9:1) were used as a developing solvent and detection of spots was made by UV light and/or by iodine vapours. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Varian Gemini, Bruker DPX-300 or JEOL JNM-ECZR instrument; chemical shifts were reported in d (ppm) units relative to the internal reference tetramethylsilane and the splitting patterns were described as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). The first-order values reported for coupling constants J were given in Hz. Elemental analyses were performed by an EA1110 Analyzer, Fison Instruments (Milan).

Thin-layer chromatography (TLC) was carried out employing aluminum-backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254, Merck, Washington, DC, USA), and detecting spots by UV light (254 nm or 365 nm), using a handheld UV lamp, LW/SW, 6W, UVGL-58 (Science Company^®, Lakewood, CO, USA). The chromatographs were eluted in a closed glass developing chamber to keep the environment saturated with solvent vapors, using a mixture of EtOAc/MeOH/AcOH 9/1/0.2 v/v/v (mixture A) or 100% MeOH (mixture B).

Progress of all biotransformations and purity of isolated products were checked by TLC on silica gel-coated aluminium plates (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany) and also by GC analysis performed on a CP03380 instrument (Varian, Agilent Technologies, Santa Clara, CA, USA) using an DB-1 column (dimethylpolysiloxane, Agilent, 30 m × 0.25 mm × 0.25 µm). Temperatures during GC analysis were as follows: injector 250 °C, detector (FID) 300 °C, column temperature: 75 °C (hold 3 min), 75–80 °C (rate 2 °C/min), 80–150 °C (rate 17 °C/min), 150–300 (rate 40 °C/min), 300 °C (hold 1 min). The enantiomeric excess of the products obtained during biotransformation were determined by GC analysis using chiral column Gamma DEX^TM 325 (30 m × 0.25 mm × 0.25 µm, Supelco, Bellefonte, PA, USA) under the following conditions: injector 150 °C, detector (FID) 250 °C, column temperature: 80 °C (hold 20 min), 80–107 °C (rate 1 °C/min), 107–200 °C (rate 30 °C/min), 200 °C (hold 1 min). All products were purified using preparative column chromatography on silica gel (Kieselgel 60, 230–400 mesh). NMR spectra were recorded with a Bruker Avance DRX-500 spectrometer in CD₃OD solution. Optical rotations were measured on P-2000 polarimeter (Jasco, Easton, PA, USA).