Avance dpx 300 nmr spectrometer

Metabolite isolation and purification was done by the method described by Breinholt et al. [27 (link)]. The solvent layer was evaporated in a rotary evaporator (Buchi 114, Germany) under high vacuum. Bioassay-guided fractionation was followed to pool out the fraction having antagonistic activity against C. albicans. The active compound was identified following analytical tools such as IR, NMR, GCMS, and UV. A Perkin Elmer System 2000 FTIR spectrometer was used to record the IR spectra. The UV absorption spectrum was measured with an Analytik Jena UV–Vis Specord200 spectrophotometer and the operating software was Aspect plus v1.7. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were obtained with a Bruker AVANCE DPX 300 NMR spectrometer in CDCl3 using TMS as the internal standard. Mass spectra were recorded on a Bruker Esquire 3000 system.

The content measurement of CorA was determined by ¹H NMR spectrometry at 300 MHz using a Bruker Avance DPX300 NMR spectrometer. CorA (10–30 mg) and the internal reference dimethyl sulfone (2–4 mg) were weighed accurately to 0.1 mg and dissolved in acetonitrile-d₃ (600 µL) and D₂O (100 µL). For the measurement, 128 scans at a relaxation time of 30 s and a line broadening factor of 0.3 Hz were conducted. Baseline and phase correction were performed and the singlet of the reference compound and the analyte singlet of H-5 of CorA were quantified. The content of the analyte was calculated according to Equation (1).
${\mathrm{C}}_{\mathrm{corA}}(\%)={\mathrm{C}}_{\mathrm{Ref}.}(\%)\times \frac{{\mathrm{I}}_{\mathrm{CorA}}\times {\mathrm{No}}_{\mathrm{Ref}.}\times {\mathrm{MW}}_{\mathrm{CorA}}\times {\mathrm{m}}_{\mathrm{Ref}.}}{{\mathrm{I}}_{\mathrm{Ref}.}\times {\mathrm{No}}_{\mathrm{CorA}}\times {\mathrm{MW}}_{\mathrm{Ref}.}\times {\mathrm{m}}_{\mathrm{CorA}}}$
Equation (1). Content quantification for CorA-batches. C = content, CorA = corallopyronin A, Ref. = internal reference (dimethyl sulfone), I = signal intensity, No = number of protons (dimethyl sulfone; δ 2.99, 6H; CorA: δ 6.06, 1H, MW = molecular weight (dimethyl sulfone: 94.13 g/mol; CorA: 527.65 g/mol), m = sample weight)

All commercial chemicals and solvents were reagent grade and were used without further purification. Completion of the reactions was monitored by analytical thin layer chromatography (TLC) using precoated glass-backed plates (E-Merck, silica gel 60 F₂₅₄ 0.25 mm). For normal pressure and flash column chromatography purifications, Merck silica gel 60 (size 70–230 and 230–400 mesh, respectively) was used. ¹H and 13C NMR spectra were recorded with an Avance DPX-300 NMR spectrometer (Bruker, Germany) and Jeol 600 MHz spectrometer (Jeol Resonance ECZ 600 R, USA). All the ¹H and ¹³ C NMR spectra were recorded in deuterated chloroform (CDCl₃) with tetramethylsilane (TMS) as an internal standard or deuterated dimethyl sulfoxide (DMSO)-d6 as solvents; chemical shifts are reported in δ values (ppm) relative to the residual solvent peak. Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); m (multiplet); dd (doublet of doublet); brs (broad singlet). Mass spectra were obtained on Waters Aquity UPLC/QTOF (Waters Corporation, USA). HPLC was used Agilent, 1200 series using capcellpak MGII (4.6 × 150 mm, 5 μm) eluted with a 30 min gradient from 20–70% acetonitrile in water.