Waters 2487

Molecular masses were analyzed by SEC-MALS (DAWN EOS or Wyatt-925-H2HC; DAWN Heleos, Wyatt Technology Inc., Santa Barbara, CA), refractive index (Wyatt-215-TRXH; Optilab T-rEX, Wyatt Technology Inc.) and UV (Waters 2487, Waters Corporation, Milford, MA) detectors. Volumes of injection ranged from 100–150 µl. Typically 200 µg of total protein was mixed in a molar ratio of 1∶1 antigen to antibody. Individual proteins were injected as controls. The sample was centrifuged at 12,800 rpm for 4 min in an Eppendorf 5415 centrifuge and the supernatant applied to a pre-equilibrated Superdex-75 column (1.0×30 cm, GE Healthcare) at a flow rate of 0.5 ml/min at room temperature and eluted in buffer A, unless stated otherwise. Under these conditions, the eluting concentrations are expected to be 6–10 µM, near the concentrations used for native-PAGE. Molecular masses were calculated using the Astra software (version 6) provided with the instrument.

DNA restriction enzymes were purchased from Transgen Biotech Co., LTD (Beijing, China). Polymerase chain reaction (PCR) was performed using TransStart® Fastpfu Fly DNA Polymerase (Transgen Biotech, Beijing, China). Optical rotations were measured on a P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). ECD spectra were recorded on a JASCO J-815 spectropolarimeter (JASCO Corporation). UV spectra were recorded on Waters 2487 (Waters Corporation, Milford, MA, United States). HRESIMS and ESIMS spectra were measured on Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). NMR spectra were recorded on Agilent 500 MHz DD2 spectrometer (Agilent Technologies Inc., Santa Clara, CA, United States). Semi-preparative HPLC was performed using a YMC Pack ODS-A column (250 × 10 mm, 5 μm, 3 ml/min, YMC Co., Ltd., Kyoto, Japan). Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden).

Molecular mass of MPro^1–199 and ⁺²⁵MPro^{1–199(C145A)} was estimated by analytical SEC with in-line MALS (DAWN Heleos-II, Wyatt Technology Inc., Santa Barbara, CA), refractive index (Optilab T-rEX, Wyatt Technology Inc.) and UV (Waters 2487, Waters Corporation, Milford, MA) detectors. Sample (125 µl) was applied onto a pre-equilibrated Superose-12 column (1.0 × 30 cm, Cytiva) and eluted at a flow rate of 0.5 ml/min in buffer A at 25 °C. Molecular mass was calculated using the Astra software provided with the instrument.

Molar masses of 6HB and HR1 were analyzed by analytical SEC with inline MALS (Wyatt-925- H2HC, DAWN Heleos; Wyatt Technology Inc.), refractive index (Wyatt-215-TRXH; Wyatt Technology Inc.), and UV (Waters 2487; Waters Corporation) detectors. Samples were applied (125 μl) to a pre-equilibrated Superose-12 column (1.0 × 30 cm; GE Healthcare) and eluted at a flow rate of 0.5 ml/min at room temperature in 20 mM sodium phosphate at pH 6.0 and 30 mM NaCl. Molar masses were calculated using the Astra software provided with the instrument. Calculated masses of 6HB (65 μM injection) and HR1 (90 μM injection) are 29.4 and 19.6 kDa, respectively.

Optical rotation was measured using a Jasco DIP-1000 polarimeter (Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded using a 250-MHz Bruker NMR spectrometer (DMX 250) and 600-MHz Varian NMR spectrometer (VNS-600, Palo Alto, CA, USA) at the Core Research Support Center for Natural Products and Medical Materials (CRCNM). High-resolution electron ionization mass spectrometry (HR–EI–MS) was performed using a JMS-700 instrument (JEOL, Tokyo, Japan) coupled to a 6890 Series gas chromatography MS (GC–MS) system (Agilent Technologies, Santa Clara, CA, USA). Low-resolution electrospray ionization MS (LR–ESI–MS) was performed using an Agilent 6120 single quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column [Phenomenex Luna 3 μm C18(2) 100 Å, new column, 150 × 4.6 mm (Phenomenex, Torrance, CA, USA)]. Isolation of the compounds was carried out using a Waters 1525 binary HPLC pump with a Waters 996 photodiode array (PDA) (Waters Corp., Milford, MA, USA) with a reversed-phase HPLC [RS Tech, Hector-M 5 μm C18, 250 × 4.6 mm (RStech Corp., Cheongju, Republic of Korea)] and an Alltech 301 HPLC pump (Alltech, Lexington, KY, USA) with a Waters 2487 dual wavelength absorbance detector and a chiral HPLC column [CHIRALPAK AD-H 5 μm, 250 × 4.6 mm (Daicel, Osaka, Japan)].