P 200 polarimeter

Optical rotations were obtained on a JASCO P200 polarimeter. Melting points were determined using a Büchi^® B-540 melting point apparatus. IR spectra were measured on a Perkin-Elmer FTIR Spectrum 2. NMR spectra were recorded on a Bruker Avance III - 500 MHz NMR spectrometer equipped with a multi-nucleus probe BBFO (5 mm). UV spectra were recorded on the PDA detector of the Varian LC-920 system. LC-HRESIMS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF hyphenated to an Agilent 1200 system equipped with a Zorbax Agilent C18 column (50 mm × 2.1 mm, 1.8 μm). Compounds were purified by semi-preparative HPLC with a Gilson 322 system equipped with an Axia C18 column (21.2 mm × 100 mm, 5 μm). HPLC analyses were performed on a Varian LC-920 system with a Kinetex column C18 100 Å (100 × 3.0 mm, 2.6 μm). Solid-phase extractions were performed on Chromabond^® SPE cartridges (Macherey-Nagel). The reading of the microplates was performed on an ELISA Versamax plus^® (Molecular Devices) plate reader. Solvents for extraction, fractionation and HPLC were supplied by Sigma-Aldrich. The bioassay material was purchased as following: GLP-1 ELISA kit (Active GLP-1, EGLP-35K Millipore), DMEM and PBS (Gibco), BSA (Sigma-Aldrich) and MTS mother solution CellTiter 96 (Promega), compounds standards pregnenolone and pregnenolone sulphate (Sigma-Aldrich).

Retimycin (200 μg) was hydrolyzed (6 M HCl, 160°C, 5 min) and dried under nitrogen. The resulting solid was re-dissolved in 1 M sodium bicarbonate (200 μl) followed by addition of 1 mL of 1-fluoro-2,4-dinitrophenyl-D-alanine amide (D-FDAA) in acetone (1.5 mg/ml). The reaction was stirred at 50°C for 1 h, quenched with 1 M HCl (200 μL), and dried under nitrogen. The resulting solid was re-dissolved in 1:1 H2O:CH₃CN (200 μl) and filtered. The resulting solution was analyzed (20 μL) by LC-MS (positive mode) on a Luna C₁₈ column, 250×4.6 mm, 5u (Phenomenex) with a gradient from 0% CH₃CN to 40% CH₃CN in H₂O over 95 minutes (0.4 ml/min). The mass for Thr-FDAA (m/z: 372.1) was extracted and determined to elute at 53.28 min. This was compared to the retention times for all 4 threonine isomers, derivatized with D-FDAA (D-Thr: 52.90 min, D-allo-Thr: 53.20 min, L-allo-Thr: 56.70 min, L-Thr: 61.50 min). The optical rotation was determined on a Jasco P200 polarimeter (c: 0.2, CHCl₃).

Optical rotations were measured using a Jasco P-200 polarimeter. UV spectra were measured using a Beckman Coulter DU-800 spectrophotometer. ECD spectra were recorded on a Jasco J-715 spectrophotometer using a 1-mm cell. NMR spectra were determined on Varian Unity Inova spectrometers at 700 MHz; chemical shifts were referenced to the residual solvent signal (CD₃OD: δ_H 3.31, δ_C 49.00). Other NMR data were recorded using a Bruker 800 MHz NMR and a Varian 500 MHz NMR; chemical shifts were referenced to the residual solvent signal (DMSO-d₆: δ_H 2.51, δ_C 39.5). For an accurate measurement of the coupling constants, the one-dimensional ¹H NMR spectra were transformed at 64 K points (digital resolution: 0.09 Hz). Through-space ¹H connectivities were evidenced using a ROESY or NOESY experiment with a mixing time of 450 ms. The HSQC spectra were optimized for ¹J_CH = 142 Hz, and the HMBC experiments for ^{2 (link),3} J_CH = 8.3 Hz. HRESIMS analysis of the new conulothiazoles was performed using an AB SCIEX TripleTOF 4600 mass spectrometer with Analyst TF software. High performance liquid chromatography (HPLC) separations were achieved on an Agilent 1260 Infinity Quaternary LC apparatus equipped with a Diode-Array Detector (DAD). The new conulothiazoles were isolated using a Dionex UltiMate 3000 HPLC system each equipped with a micro vacuum degasser, an autosampler, and a DAD.