Silica gel 60 f254 aluminum plates

All starting materials, reagents, and solvents were commercially available and purchased from VWR, Abcr, or Carl Roth. Unless otherwise stated, the starting materials were used as provided. Thin-layer chromatography on an analytical scale was performed using silica gel 60 F₂₅₄ aluminum plates supplied by Merck, and visualization was accomplished using UV light (254 nm). Flash column chromatography was carried out using an Interchim Puriflash XS 520Plus system and the appropriate 25 g silica gel cartridges available from Interchim (30-SI-HP). NMR analyses were run on a Bruker Biospin Avance III instrument at 400 MHz (¹H) and 101 MHz (¹³C) using DMSO-d₆ as the solvent. Chemical shifts are given relative to the internal standard tetramethylsilane and are reported in parts per million (ppm).

All starting materials and reagents were commercial products. They were used without further purification. Melting points of the compounds were determined in open capillaries on a Thermo Scientific 9100 Series and are uncorrected. ¹H and ¹³C NMR spectra were recorded on a Bruker spectrometer (400 and 100 MHz, respectively) in DMSO-d₆ using residual DMSO signals (2.52 ppm and 40.21 ppm for ¹H and ¹³C NMR spectra, respectively) as internal standard. ¹⁹F NMR spectra were recorded on a Bruker spectrometer (376 MHz) with CFCl₃ as an internal standard. Proton, carbon and fluorine chemical shifts were expressed in parts per million (ppm) in the indicated solvent. Multiplicity was defined as s (singlet), d (doublet), t (triplet), q (quartet), dd (a doublet of doublets), ddd (a doublet of doublet of doublets), m (multiplet), br s (broad singlet). TLC was performed with silica gel 60 F₂₅₄ aluminum plates (Merck) and visualized with UV light. Column chromatography was performed using silica gel 60 (0.040–0.063 mm, Merck). High-resolution mass spectra (HRMS) were recorded on a Dual-ESI Q-TOF 6520 mass spectrometer (Agilent Technologies). The purity of final compounds was verified by HPLC to be >95% using the Agilent 1290 Infinity instrument with a Poroshell 120 SB-C18 (2.1 mm × 100 mm, 2.7 μm) reversed-phase column.

Solution of stearic acid N-hydroxysuccinimide ester (32 µmol, 12.2 mg) in 850 mL was added to a solution of peptide NH₂-(RL)₄G-C(O)NH₂·5CF₃COOH (3.2 µmol, 5.5 mg) in 50 µL of sterile deionized water. Reaction mixture was basified up to pH~10 by 10% NaOH (10 µL) and incubated overnight at room temperature. The reaction was monitored by TLC (isopropanol:water:NH₃·H₂O, 7:3:1 v/v/v). When all peptide was consumed, 2 mL of dichlorometane and 2 mL water was added to the reaction mixture and acidified with CF₃COOH up to pH~2. After the extraction, water phase and interphase were gathered, 2 mL of dichlorometane were added, and the mixture was back basified with DIPEA up to pH~9. Water phase was washed twice with dichlorometane (2 mL), and organic layers were gathered and evaporated to dryness to remove volatiles and excess of organic salts. Stearate-peptide conjugate was purified by preparative silica gel chromatography in ethanol:dichloromethane (4:1) on Silica gel 60 F254 aluminum plates (Merck). Yield: 3.6 mg (79%), Rf 0.6 (isopropanol:water:NH₃·H₂O, 7:3:1 v/v/v), Rf 0.4 (ethanol:dichloromethane, 4:1). The product [Str-(RL)₄G-NH₂] (P) were characterized by MALDI-TOF MS analysis (theoretical mass 1418.04 Da, measured mass 1418.67 Da).