Varian inova 500

To produce sufficient quantities of the two PSG derivatives for the purpose of elucidating their chemical structure, multiple scale-up reactions were carried out as follows; the molar concentrations of PSs, UDP-Glc and MrSGT enzyme were maintained in the proportions described above, while scaling up the total volume of the reaction mixture to 1 mL. At the end of the reaction, reaction mixtures from more than five batches were pooled and extracted by ethyl acetate partitioning. The solvent layer obtained was evaporated to dryness using a centrifugal evaporator (EYELA, Tokyo, Japan) set at 40 °C, and reconstituted in 5 mL of the mobile phase used for the HPLC–MS/MS analyses. The extracts were immediately loaded onto a reverse-phase C₈ cartridge of the CombiFlash Rf medium-pressure liquid chromatography (MPLC) system (Teledyne ISCO, Lincoln, NE, USA), and the flow rate was set at 8 mL/min. The eluents passing through a UV detector were all automatically fractionated over a 50-min running time. The fractions confirmed to contain the PSG derivative with a good purity (>97 %) by the tracing HPLC–MS/MS analyses, were pooled and freeze-dried. Their structures were further confirmed by Varian INOVA 500 nuclear magnetic resonance (NMR, Varian Inc., Palo Alto, CA, USA) spectroscopic analysis together with high resolution (HR) LCT-premier XE MS (Waters, Milford, MA, USA) analysis.

The phase transition behavior of the prepared polymers was observed by temperature dependent transmittance change of the polymer solution. Polymer solution was prepared by dissolving the polymer in PBS at a concentration of 5 mg/mL. The transmittance of the polymer solution at 500 nm was measured using a UV-Vis spectrometer (V-630, JASCO, Tokyo, Japan) whilst heating the polymer solution at 0.1 °C/min. The lower critical solution temperature was defined as the temperature at which 50% transmittance was observed.
The molecular weight of the polymer was obtained by GPC (GPC-8020, Tosoh, Tokyo, Japan) using two serially connected TSK-Gel α-M columns (Tosoh, Tokyo, Japan). The columns were calibrated using PEG standards. The mobile phase was DMF containing 10 mM of LiCl. The molecular weight of P(NIPAAm-co-DMAPAAm) was also determined by ¹H NMR. The sample solution was prepared by dissolving copolymers in CDCl₃ at a concentration of 15 mg/mL. ¹H NMR spectra were obtained using a nuclear magnetic resonance spectrometer (Varian INOVA 500, Varian, Palo Alto, CA, USA). The molecular weight of P(NIPAAm-co-DMAAm) was also obtained by titration molecular weight. The polymer (25 mg) was dissolved in 2 mL of pure water. Titration of the terminal carboxyl group of the polymer was performed using 0.0025 mol/L NaOH solution.

HPLC-LCQ ion-trap MS/MS (ThermoFinnigan, USA) was performed using both acetonitrile:methanol:water:formic acid (40:40:19.8:0.2 v/v/v/v) mixture as isocratic mobile phase at a flow rate of 150 µl/min, and Acquity CSH C₁₈ reversed-phase column (Waters, 50 × 1.0 mm, 1.7 µm; USA) as the stationary phase. The column effluent was introduced into the MS/MS operated in the positive ion mode. Acquisition was performed using MS/MS operated in the selective reaction monitoring mode by choosing a pair of mass transitions specific to phenoxodiol and the corresponding glycosides to detect the transition of the protonated precursor ion to the dominant product ion (241.2 [M+H]⁺ > 223.2 [M-H₂O+H]⁺ as a dehydrated product ion for phenoxodiol and 403.4 > 241.2 [M-Glc+H]⁺ as an aglycone product ion for phenoxodiol glycoside). In addition, both high-resolution (HR) LCT-premier XE MS (Waters) and Varian INOVA 500 nuclear magnetic resonance (NMR, Varian Inc., USA) were employed for the structural elucidation of two kinds of phenoxodiol glycosides.