Openlab cds chemstation

All chemicals and solvents were obtained from commercial suppliers and used without further purification, unless specified. DO2A-tert-butyl ester and 2-chlorotrityl chloride resin were purchased from Chematech (Dijon, France) and Advanced Chemtech (Louisville, KY, USA), respectively. The peptide sequence was synthesized manually using standard solid phase synthesis protocols. ¹H NMR spectra were recorded at 600 MHz on Bruker AMX600 spectrometers (Delft, The Netherlands) and ¹³C NMR at 15 MHz on Nanalysis 60PRO (Calgary, Canada) at ambient temperature in CDCl₃ unless specified. The chemical shifts (δ) for ¹H and ¹³C are quoted relative to residual signals of the solvent on the ppm scale. Coupling constants (J values) are reported in Hertz (Hz) and are H-H coupling constants unless otherwise stated. Quality control was performed by LC-MS using an Agilent 1260 Infinity II LC/MSD XT system (Amstelveen, The Netherlands). Electrospray ionization in positive mode was used to confirm the identity of the obtained products. Purification of the synthesized ligands were performed by preparative HPLC on an Agilent 1290 Infinity II or by semi-preparative HPLC on a Waters 2695 system (Etten-Leur, The Netherlands) equipped with a diode array detector 2998. The LC-MS and the HPLC were controlled by Agilent OpenLab CDS Chemstation or Empower 3 software, respectively.

The HPLC analysis was performed as previously described with minor modifications [16 (link),17 ]. HPLC analysis used an HPLC 1260 Infinity II (Agilent Technologies, Inc., Santa Clara, CA, USA) system with an Eclipse C18 column (2.1 × 150 mm, 3.5 μm; Agilent Technologies) under 40 °C. Ellagic acid (>95% purity, Sigma-Aldrich, CA, USA) was used as a marker. The mobile phases were 0.1% formic acid (A) in DW and acetonitrile (B). A flow rate of 0.12 mL/min was used, with the gradient condition as flow with a of flow rate: 95–70% A, 0–30 min; 70–5% A, 30–56 min; 30–0% B, 56–57 min; 0–95% B, 57–58 min; and 95% A, 58–70 min. The injection volume was 2 μL, and the UV wavelength was set at 275 nm. Data acquisition was performed by using the Agilent OpenLab CDS ChemStation (version 2.15.26; Santa Clara, CA, USA).

Gas chromatography was performed on a 7890B GC System (Agilent, Santa Clara, CA, USA) with a HP88 column (112/8867, 60 × 0.25 mm × 0.2 µm) for 30 min with the following temperature gradient: 50 °C to 150 °C with 20 °C/min, 150 °C to 240 °C with 6°C/min and 240°C for 10 min. Nitrogen was used as carrier gas (constant flow 1 mL/min). A total of 1 µL of the samples was injected into the injector (splitless injection, 280 °C). Flame ionization detection was performed at 250°C with the following gas flows: hydrogen 20 mL/min, air 400 mL/min and make up 25 mL/min. Methylated fatty acids in the samples were identified by comparing the retention times with those of known methylated fatty acids of the Supelco^® 37 FAME Mix and single FAME standards purchased from Cayman Chemicals (Ann Arbor, MI, USA). Analysis and integration of the peaks was carried out with the OpenLAB CDS ChemStation (Agilent).

The analysis of enzymatic reactions was performed on an Infinity II 1260 liquid chromatography system (Agilent). Separation of the enzymatic reaction products occurred on a Triart C₁₈ column (YMC) in 30 mM ammonium acetate buffer system (pH 4.3) in a linear gradient of acetonitrile from 0 to 10% for 25 min. Processing of chromatograms was performed in OpenLab CDS ChemStation (Agilent). The elution profiles were exported in comma-separated value (.csv) format for visualization.