1,1′,2-Tris-norsqualenol was synthesized from squalene via 1,1′,2-tris-norsqualenic aldehyde according to previously reported methods (35, 36). Diglycolic anhydride (150 mg, 1.29 mmol) was added to a solution of 1,1′,2-tris-norsqualenol (200 mg, 0.52 mmol) in 3 ml of dry pyridine. The reaction was stirred overnight at room temperature. The solvent was removed and the residue was extracted with DCM from dilute hydrochloric acid and brine. Conversion to the squalene–diglycolic acid, monitored by TLC, was approximately 100%. The resultant product was dried under vacuum and used in the following step without further purification. Ethyl chloroformate (10.8 mg, 0.1 mmol) was added to a solution of squalene–diglycolic acid (50 mg, 0.1 mmol) and TEA (12 mg, 0.12 mmol) in 1 ml of anhydrous THF under argon at 0°C. The reaction was stirred for 1 hour at room temperature, and a solution of LENK (55 mg, 0.1 mmol) in 1 ml anhydrous DMF was added. The mixture was maintained at 40°C for 2 days with stirring under argon. The solvents were removed in vacuo, and the crude product was purified using silica gel chromatography (purified with gradient eluent DCM/EtOH: 100:0 to 90:10). Then, ammonium salt was eliminated by simple filtration on silica using EtOH/AcOEt (40:60) as solvents. The pure bioconjugate was obtained with 69% yield.

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