Shim pack prep ods column

All peptides used in this study were synthesized by the standard solid phase technique (ANYGEN Co., Ltd.). 5-Iodoacetamido fluorescein (5-IAF)-labeled peptides were prepared as previously described^{68 (link)}. Briefly, peptides containing a single cysteine residue were incubated with 5-IAF (Sigma-Aldrich) at a 1:2 molar ratio in 100 mM HEPES–NaOH pH 7.5 for 2 h at RT. The 5-IAF-labeled peptide was separated from the native peptide and excess 5-IAF by Shim-pack PREP-ODS column-equipped reversed-phase high-performance liquid chromatography (Shimadzu 10Avp). The extent of 5-IAF labeling was confirmed by mass spectrometry.

Mass spectra were obtained using a Bruker micro-TOFQ mass spectrometer (Bruker Daltonics, Bremen, Germany). Nuclear magnetic resonance spectra were acquired with a Bruker AV-500 FT-NMR spectrometer operating at 500.1 MHz for ¹H and at 125.8 MHz for ¹³C at 25 °C; chemical shifts are expressed in δ referring to the residual solvent signals δ_H 2.50 and δ_C 39.5 for DMSO-d₆, coupling constants, J., are in hertz. All chemical shifts are given in ppm. Column chromatography was performed over a RP-18 reversed-phase silica gel (S-50 μm; YMC, Kyoto, Japan). Analytical HPLC (Agilent technologies, Santa Clara, CA, USA) was performed on an Agilent 1260 system equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A thermostated column compartment, and a G1314F variable wavelength detector coupled with an analytical workstation. Semipreparative HPLC was performed on an Agilent ProStar SD-1 pump connected with an Agilent ProStar 320 UV–Vis detector (at 360 nm), utilizing a Shim-Pack PREP-ODS column (250 mm × 21.2 mm, i.d., 10 μm, Shimadzu, Kyoto, Japan). HPLC-grade water was purified using a Milli-Q system (Millipore, Boston, MA, USA). All solvents used for the chromatographic separations were distilled before use.

HPLC conditions were as follows: Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d., 5 μm); gradient elution performed using an A eluent (MeOH) and a B eluent (0.3% acetic acid in water, v/v) with the following linear gradient combinations: at t = 0, 70% B; at t = 20 min, 40% B; at t = 22.5 min, 40% B. Flow rate was 1.0 mL/min, and 10 μL portion was injected into the column. The effluent was monitored at 254 nm. Prep-HPLC separations were performed on a Shim-pack PREP-ODS column (20.0 mm × 250 mm, 15 μm) (Shimadzu, Kyoto, Japan); The mobile phase, a solution of eluent A (MeOH) and eluent B (0.3%, v/v, acetic acid in water), was monitored at 254 nm. The processing conditions for the linear gradient elution of F1, F4 and F5 are listed in Table 3.