Isolera one flash chromatography

Silica gel 60 (230–400 and 100–200 mesh), pre-coated Kieselgel-60 F-254-TLC plates silica gel (Merck, Darmstadt, Germany), and Sephadex LH-20 (Pharmacia Co. Ltd) were used as packing materials for column chromatography. Isolera-one flash chromatography (Biotage; Suite C Charlotte, NC; USA) was used for flash chromatographic analysis as well as the isolation and purification processes. The compounds were purified using HPLC with a Jasco-pump (PU-980) equipped with a Jasco UV-970 intelligent detector (UV/VIS) at 210 nm. A HPLC semi-preparative Supelco C18-RP-column (250 × 10 mm, 5 μm) was also used. The optical rotation of the isolated compounds was measured by a JASCO (P-2300) polarimeter (Tokyo, Japan). A Bruker 500 Hz (MA, USA) spectrometer was used for recording the 1D NMR, including ¹H, ¹³C, and DEPT-135, and the 2D NMR, including HSQC, HMBC, ¹H ¹H COSY, and NOESY. All NMR analyses were measured in deuterated methanol (CD₃OD) at room temperature. The chemical shifts and coupling constants were given in delta (δ, ppm) and Hertz (Hz), respectively. CD₃OD was referenced at δ_H 4.77 and δ_H 3.31 ppm in ¹H NMR, and at δ_C 49.14 in ¹³C NMR. The experimentally measured electronic circular dichroism (ECD) of the compounds were measured in CH₃OH by a JASCO-810 spectrometer. The mass spectral data including, LR and HR-MS, were derived by a JEOL (JMS-700) instrument (Tokyo, Japan).

Crude peptides and TSC-conjugates were purified by Reverse-Phase Flash Liquid Chromatography (RP-HPLC) on Isolera One Flash Chromatography (Biotage, Uppsala, Sweden) using a SNAP Ultra C18 column (40 g) at 20 ml/min flow. Eluent systems: 0.1% TFA in H₂O (A), 0.1% TFA in ACN (B). Due to the poor solubility in water of final compounds, the samples for purification were dissolved in the mixture of solvents A and B at the initial percentage of the purification gradient and treated with ultrasounds. The linear gradient applied for each purification was: 35–65% B in A in 30 min.