Jms t100lp

The porous hybrids were prepared using the freeze-drying method [17 (link)]. We used a planetary centrifuge (ARE-310, Thinky, Tokyo, Japan) at 2000 rpm (Program: MIX 5 min − DEFOAM 3 min) × 3, MIX 5 min) to yield the chitosan solution and mixtures with GPTMS.
The compositions were chitosan:GPTMS = 1:0.5 and 1:1 as the molar ratios, named ChG05 and ChG10, respectively. The obtained porous hybrids were cut into a square (10 mm × 10 mm, thickness = 2 mm) and sterilized with ethylene oxide gas (20% CAPOX, 45 °C, 50% humidity, Steri-Tech Inc., Saitama, Japan). Square specimens were soaked (0.3 mm³/mL) in ultrapure water (Life Technologies, Carlsbad, CA, USA) at 37 °C for 1 week. Extractions were filtered using a syringe filter (0.22 µm pore size) before the following analysis. The amount of Si(IV) and molecular weight of the degradation products in the extractions were measured using inductively coupled plasma emission spectrometry (ICP, ICPS-8000, Shimazu Corporation, Kyoto, Japan) and electron spray ionization TOF-MS spectrometry (JMS-T100LP, JEOL, Tokyo, Japan) [20 (link)]. The free amino groups in the extractions were measured using the ninhydrin method [21 (link)].

Optical rotations were recorded on a JASCO P-1020 polarimeter. NMR spectra were recorded on JEOL ECP600 and ECZ600 spectrometers in a deuterated solvent whose chemical shift was taken as an internal standard. ESI-MS were obtained on a JEOL JMS-T100LP. Silica gel 60 N (Kanto Chemical Co., Inc., Tokyo, Japan), Chromatorex ODS and Chromatorex PSQ100B (Fuji Silysia Chemical Ltd., Kasugai, Japan) were used for column chromatography. Luna Phenyl-Hexyl (φ 10 × 250 mm) (Phenomenex Torrance, CA) was used for Preparative HPLC. Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA) was used for protein concentrations measurement.

¹H-NMR spectra were recorded in CDCl₃ on a JEOL ECS-400 (400 MHz) spectrometer; tetramethylsilane was used as the internal reference. Electron ionization (EI) and electrospray ionization (ESI) mass spectrometry were performed using JEOL JMS-T100 GCv and JEOL JMS-T100 LP instruments, respectively. Elemental analyses were performed using MICRO CORDER JM10. UV-vis absorption spectra for ligand 1 and Eu(III) complex 2 were measured using a JASCO V-670 spectrophotometer. Emission spectrum, excitation spectrum, and emission lifetime for Eu(III) complex 2 were measured using a Horiba FluoroLog®3 spectrofluorometer. Emission spectrum and lifetime for Gd(III) complex 5 were measured using a FP-6300 spectrofluorometer with a nitrogen bath cryostat (Oxford Instruments, Optistat DN) and a temperature controller (Oxford Instruments ITC-502S). Emission spectrum for the ligand 1 was measured using a FP-6300 spectrofluorometer with a nitrogen bath cryostat (Oxford Instruments, Optistat DN) and a temperature controller (Oxford Instruments ITC-502S). Emission quantum yield for Eu(III) complex 2 was measured using a FP-6300 spectrofluorometer with an integration sphere (ILF-533).

Aliquots containing 5 μL of an amino acid mixture including Tau, (S)-glutamate, (S)-glutamine, (S)-arginine, and (S)-valine (each 100 µM) in phosphate-buffered saline (PBS) were added to 5 μL of 20 mM Ns-MOK-(S)-Pro-OSu in CH₃CN or 20 mM Ns-MOK-(S)-β-Pro-OSu in CH₃CN, and 5 μL of 10 mM DMAP in CH₃CN. The solutions were allowed to react at room temperature for 5, 15, 30, 60, 90, and 120 min. Each reacted solution was diluted with 35 μL of 0.2% HCO₂H in H₂O/MeOH (1:1, v/v) and subjected to LC-TOF-MS (JMS-T100LP, JEOL Ltd., Tokyo, Japan) (Supplementary Materials, Section 2.2). The observed peak areas were plotted against the derivatization times.

Unless otherwise noted, reagents and solvents were purchased at the highest commercial quality and used without further purification. LC-UV analysis was carried out with Agilent 1100 system (Agilent Technology, Inc.) under the following condition; column, Symmetry C18 (Waters Co., Ltd., 2.1 φ × 150 mm); UV detection, 210 nm; flow rate, 0.2 mL/min; mobile phase, MeCN-H₂O with 0.05% H₃PO₄, (5–100% linear gradient over 20 min). ODS column chromatography was carried out with Sep-Pak^® Plus C18 Short Cartridge (Waters Co., Ltd.) or CHROMATOREX^® (Fuji Silysia Chemical, Ltd.). ¹H NMR spectra were recorded on JEOL JNM-ECA-500 (500 MHz) and ¹³C NMR spectra were recorded on JEOL JNM-ECA-500 (125 MHz). Chemical shifts are expressed in ppm downfield from the internal solvent peaks for CD₃OD (¹H; δ = 3.31 ppm,¹³C; δ = 49.0 ppm) and J values are given in Hertz. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, and br = broad. High- and Low-resolution mass spectra were measured on JEOL JMS-AX505 HA, JEOL JMS-700 MStation and JEOL JMS-T100LP.