Avance 400 mhz instrument

Analytical separation and quantification were performed on a Dionex Ultimate 3000 system (Thermo Scientific) coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific). Electrospray ionization was performed in positive mode ionization, with the following parameters: capillary temperature, 380 °C; spray voltage, 3.000 V; sheath gas flow, 60 arbitrary units; and aux gas flow, 20 arbitrary units. The LC separation column was a SeQuant ZIC-HILIC column (5 µm, 2.1 × 150 mm, SeQuant), equipped with a SeQuant ZIC-HILIC guard column (5 µm, 2.1 × 20 mm). NMR spectra were recorded on a Bruker Avance 400 MHz instrument.

¹H and ¹³C NMR spectra were recorded on a Bruker Avance 400 MHz instrument. HR-ESI-MS was taken on BrukerBioApex-FTMS with electron spray ionization. Solvents used in this work, e.g., n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), methanol (MeOH), and ethanol (EtOH), were purchased from Fisher Scientific, USA. Deuterated solvents purchased from Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA, including methanol-d₄ (CD₃OD), chloroform-d₃ (CDCl₃), and pyridine-d₅ (C₅H₅N-d₅), were used for nuclear magnetic resonance (NMR) spectroscopic analyses. Acetonitrile, methanol, and formic acid of HPLC-certified grade were used for LC-MS analysis, and water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Column chromatography (CC) was performed using silica gel 60 (Merck, Darmstadt, Germany; 70–230 mesh). Thin-layer chromatography (TLC) analyses were carried out using pre-coated silica G plates w/UV254 (Sorbent Technologies, USA; 20 × 20 cm, 200 µm in thickness). An ultraviolet lamp (Spectroline ENF-240C, Spectronics Corporation, New York, NY, USA) was used for visualization of spots on thin-layer chromatograms at 254 and/or 365 nm. Spots were visualized by spraying with 2% vanillin (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) in sulfuric acid–ethanol followed by heating at 110 °C.

TLC was performed on silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Compounds were visualized by irradiation with UV light or by treatment with 0.05 g/mL ninhydrin in ethanol or potassium iodide reagent. ¹H-NMR and ¹³C-NMR were recorded on a Bruker Avance-400 MHz instrument (Bruker BioSpin AG, Fällanden, Switzerland). HRMS were obtained with a LC-ESI-Q-TOF-MS apparatus (Waters, Milford, MA, USA). HPLC analysis of GLYX-13 was performed on an Agilent LC 1100 system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a diode array detector. Chiral analysis of intermediate were carried out using a Shimadzu LC 20AD system (SIMADZU, Kyoto, Japan) with a SPD-20A UV detector or an Agilent LC 1100 system equipped with a diode array detector. Single crystal diffraction analysis of 7 was performed on a MicroMax-003 (Rigaku Corporation, Tokyo, Japan) X-ray single crystal diffractometer and powder diffraction analysis of 7 was performed by a Bruker D8 FOCUS powder diffractometer (Bruker BioSpin AG, Fällanden, Switzerland). The L-amino acids (protected or free) were obtained from GL Biochem Ltd. (Shanghai, China). Other reagents were provided by Aladdin (Shanghai, China). The organic solvents were commercially available products (Lingfeng Chemical Reagent Co., Ltd., Shanghai, China) and were used without further purification.

General procedures: ¹H-NMR spectra were recorded on a Bruker Avance 400 MHz instrument in CDCl₃, CD₃OD, or D₂O as solvent at 400 MHz. ¹³C-NMR spectra were recorded in CDCl₃, CD₃OD, or D₂O as solvent at 101 MHz. Coupling constants are expressed in Hertz and are rounded to the nearest 0.1 Hz. LC-MS data were collected with a Waters Acquity^TM Ultra performance LC equipped with an Acquity UPLC^TM HSS T3 column (2.1 × 50 mm, 1.8 µm) and a SQD detector. Purifications were carried out either by flash chromatography on silica gel (particle size 60 μm, 230–400 mesh), on Kieselgel, or by Biotage^TM flash chromatography [Biotage columns Si-25-M (150 × 25 mm; silica gel (40–63 μm), flow rate 25 mL/min)], or by Biotage^TM C₁₈ reverse phase chromatography [Biotage column C₁₈HS (150 × 25 mm; KP-C₁₈-HS (35–70 μm), flow rate 25 mL/min)]. Some final compounds were purified by C₁₈ reverse phase semi-preparative HPLC using a Waters X-Bridge column (19 mm × 15.0 cm, 5 μm). Melting points were determined with a Stuart Scientific SMP3 melting point apparatus. Solvents were distilled and dried according to standard procedures, and reactions requiring anhydrous conditions were performed under nitrogen or argon atmosphere.