1,1′,2-Tris-norsqualenic acid chloromethyl ester. 1,1′,2-Tris-norsqualenic acid was synthesized by oxidation of 1,1′,2-tris-norsqualenic aldehyde by Jones reagent as previously reported (35, 36). A solution of KHCO3 (300 mg, 3.0 mmol) in water (2 ml) was added to a solution of 1,1′,2-tris-norsqualenic acid (400 mg, 1 mmol) and n-Bu4NHSO4 (34 mg, 0.1 mmol) in DCM (2 ml). The reaction mixture was vigorously stirred, and chloromethyl chlorosulfate (185 mg, 1.15 mmol) was added dropwise. After stirring for 1 hour, DCM (10 ml) was added to extract the product. The organic phase was separated, washed with brine, dried over magnesium sulfate, and concentrated under reduced pressure to afford a pale yellow oil that was used in the following step without further purification.

Alloc-Leu-enkephalin-squalene (Alloc-LENK-SQ-Diox). The 1,1′,2-tris-norsqualenic acid chloromethyl ester (200 mg, 0.445 mmol) was added into a mixture of Alloc-LENK (285 mg, 0.445 mmol) and NaHCO3 (37 mg, 0.4 mmol) in 3 ml of DMF. The reaction mixture was stirred at 40°C under argon for 4 days. The final reaction mixture was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel DCM/EtOH (100:0 to 97:3) to afford the title compound as a pale yellow oil (168 mg, 40% yield).

Leu-enkephalin-squalene with dioxycarbonyl linker (LENK-SQ-Diox). TES (1215 mg, 10 mmol) was added dropwise neat to a stirred solution of Alloc-LENK-SQ (110 mg, 0.1 mmol) and 10% Pd-C (20% by weight of Alloc-LENK-SQ-Diox) in MeOH (11 ml) under argon. When the reaction was completed, the mixture was filtered through celite to remove the Pd-C, and the residual TES and solvent were removed by evaporation. The residue was first purified by flash column chromatography on silica gel with DCM/EtOH (90:10). The resulting product was dissolved in 200 μl of EtOH before undergoing a second purification via the semi-preparative RP-HPLC system (Waters, MA, USA) on an Uptisphere C18 column (100 mm by 21.2 mm; pore size, 5 μm; Interchim, CA, USA) to obtain the pure product (23 mg; 23% yield). HPLC was then performed using a gradient elution with the mobile phase composed of an ammonium acetate buffer (20 mM) and ACN. Elution was carried out at a flow rate of 21 ml/min for 10 min with the linear gradient from 10 to 100% ACN, and then, the system was held at 100% ACN with isocratic flow for 10 min. Temperature was set at 30°C, and ultraviolet (UV) detection was monitored at 280 and 257 nm. The retention time was 15 min, and the total yield of the pure product, after coupling and deprotection steps, corresponded to 9.5%.

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