Synthesis of DSPE-PEG2000-DVar7

DSPE-PEG₂₀₀₀-DVar7 was synthesized by the covalent conjugation of DSPE-PEG₂₀₀₀-maleimide with the single cysteine residue at the N-terminus of DVar7. Briefly, DSPE-PEG₂₀₀₀-MAL 3.53 mg (~1.2 μmol) was dissolved in chloroform and evaporated to form a thin film in a round flask. The film was hydrated with HBS solution (pH 7.0) for 20 min and sonicated at 37 °C for 5 min. After DVar7 3.065 mg (~1 μmol) was added to the mixture and oscillated until completely dissolved, the mixture was separated by the reversed-phase high-performance liquid chromatography (HPLC) system. A Sepax Bio-C4 column (10 × 250 mm) was used for DSPE-PEG₂₀₀₀-DVar7 purification with a flow rate of 3 mL/min. For analytical HPLC, a Sepax Bio-C4 (4.6 × 250 mm) was used with a flow rate of 1 mL/min. The monitor wavelength was 254 nm, and the injection volume was 20 µL. The mobile phase was changed from 60% solvent A (0.1% trifluoroacetic acid [TFA] in water) and 40% solvent B (0.1% TFA in acetonitrile [ACN]) (0–5 min) to 20% solvent A and 80% solvent B at 15 min, followed by a gradient mobile phase from 0% solvent A and 100% solvent B at 35 min to 60% solvent A and 40% solvent B at 40 min. The existence of the product was ensured by SELDI-TOF mass-spectrometry analysis.