Synthesis of MVL5

Nα,Nδ,Nδ-Tris(2-cyanoethyl)ornithine (1).

To a solution of 4.00 g (100 mmol) of sodium hydroxide in 120 mL of methanol, a total of 16.9 g (100 mmol) of L-ornithine hydrochloride (Sigma) was added with stirring. After 2 min, a total of 26.3 mL (21.2 g, 400 mmol) of acrylonitrile (Fluka) was added in one portion. Stirring was continued for 24 h at room temperature, and a total of 100 mL of methanol was added. The reaction mixture was then acidified slightly by the addition of concentrated hydrochloric acid (about 13 mL) and was filtered immediately. The pH of the filtrate was adjusted to 6 using 25% sodium hydroxide solution. After standing at 4 °C for 16 h, the mixture was filtered to yield 20.3 g (62 mmol, 62%) of 1 (hydrochloride form) as white crystals. 

Nα,Nδ,Nδ-Tris(3-aminopropyl)ornithine (2).

To a mixture of 9.6 g (36 mmol) of 1 and 80 mL of 1.4 M sodium hydroxide in 95% ethanol in a Parr hydrogenator flask, a total of 5.00 g of a supension of Raney nickel in water (Fluka) was added. The mixture was agitated for 16 h under 60 psi (4 bar) of hydrogen pressure at room temperature. The Raney nickel was removed by filtration, and the solvent was evaporated. The resulting oil (15.5 g) was used for the next step without further purification. Pure hydrochloride for analytical purposes was obtained by adding concentrated hydrochloric acid to the reaction solution and collecting and drying the precipitate.

Nα-tert-Butylcarbamoyl-Nα,Nδ,Nδ-tris(3-[tert-butylcarbamoylamino] propyl)ornithine (3).

The raw product from the previous step (15.5 g; assay calculated for 36 mmol ) 100% yield from previous step) was dissolved in water/THF (1:1, v/v). Over a period of 1 h, a solution of 34.5 g (158 mmol) of Boc2O (Novabiochem) in 70 mL of THF and 40 mL of 4 M sodium hydroxide in water were added in four portions with stirring. The reaction mixture was kept at room temperature by cooling with a water bath, and stirring was continued for 6 h. Most of the THF was then evaporated. After addition of 250 mL of diethyl ether, the mixture was acidified using half-concentrated hydrochloric acid. The phases were separated, and the aqueous phase was extracted two more times with 150 mL of ether. The combined organic phases were washed twice with 250 mL of water, dried (Na2SO4), and evaporated. The residue was purified by flash chromatography on 800 g of silica gel using chloroform/methanol (19:1) as the eluent41 to yield 22.5 g (32.0 mmol, 89%) of 3 as a colorless solid.

N1-(2-aminoethyl)-3,4-di(oleyloxy)-benzamide (5).

To a solution of 4.00 g (6.11 mmol) of 4 in 10 mL of dichloromethane, 0.77 g (6.72 mmol) of N-hydroxysuccinimide (Bachem) and, after stirring for 5 min, 1.39 g (6.72 mmol) of N,N`-dicyclohexylcarbodiimide (DCC, Sigma) in 2 mL of dichloromethane were added. The reaction mixture was stirred for 2 h and then filtered. To the filtrate, a total of 4.1 mL (3.7 g, 61 mmol) of ethylenediamine was added quickly with stirring. After being stirred for 3 h, the reaction mixture was filtered and 200 mL of chloroform, 50 mL of methanol, and 100 mL of water were added. The phases were separated after thorough mixing. The organic phase was then extracted two more times with water to which NaCl and some NaOH had been added, dried (Na2SO4), and evaporated. The resulting residue was purified by flash chromatography on 250 g of silica gel using chloroform/methanol (9:1) as the eluent to yield 3.66 g (5.25 mmol, 86%) of 5 as an off-white, waxy solid.

N1-[2-((1S)-1-[tert-Butylcarbamoylamino(3-[tert-butylcarbamoylamino] propyl)amino]-4-[di(3-[tert-butylcarbamoylamino] propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (6).

To a solution of 615 mg (0.88 mmol) of 5, 619 mg (0.88 mmol) of 3, and 135 mg (0.88 mmol) of 1-hydroxybenzotriazole (HOBt, Peptides International) in 5 mL of dichloromethane, a total of 197 mg (0.955 mmol) of DCC in 0.5 mL of dichloromethane was added. After being stirred for 6 h, the reaction mixture was filtered and evaporated. The resulting residue was dissolved in 40 mL of diethyl ether, and the solution was washed with 10% citric acid, twice with saturated NaHCO3, and with water. The ether solution was dried (Na2SO4) and evaporated, and the residue was purified by flash chromatography on 60 g of silica gel using chloroform/methanol (15:1) as the eluent41 to yield 1.095 g (0.792 mmol, 90%) of 6 as a colorless solid.

N1-[2-((1S)-1-[(3-Aminopropyl)amino]-4-[di(3-aminopropyl) amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5).

To a concentrated solution of 1.00 g (0.72 mmol) of 6 in ethyl acetate, a total of 10 mL of a freshly prepared saturated solution of HCl in ethyl acetate was added. The mixture was stirred for 30 min at room temperature, during which time a white precipitate formed, and evaporated. To the residue, a total of 5 mL of ethyl acetate was added with stirring and was evaporated. This was repeated to yield 830 mg (0.71 mmol, 99%) of MVL5 as a white powder

GMO: glycerol monooleate: cat #: M-239 from Nu-Chek  www.nu-chekprep.com
100mg 35$
1g 60$
10g 500$

Maybe also from VWR

DOTAP and DOPC can be purchased as solutions in chloroform from Avanti Polar Lipids (Alabaster, AL). Cholesterol and GMO can be purchased from Sigma-Aldrich and Nu-Check-Prep,  and dissolved in chloroform to prepare stock solutions. MVL5, TMVLBisG1, TMVLBisG2 and TMVLG3 (Fig. 2) are dissolved in chloroform/methanol (9:1, v/v) to prepare stock solutions.

To prepare liposomes, lipid stock solutions were mixed at the appropriate ratios in glass vials and then dried, first under a stream of nitrogen and then in a vacuum (rotary vane pump) for 8-12 h. To the resulting thin lipid film, sterile high-resistivity (18.2 MΩ cm) water was added, and the mixture was incubated at 37 °C for at least 12 h. The final total lipid concentration was 1 mM. All aqueous lipid solutions were sonicated (tip sonicator) prior to use and stored at 4 °C.

GMO was obtained from Nu-Check-Prep. DOTAP and DOPC were purchased from Avanti Polar Lipids. All lipids were used as received. Liposomes were prepared using the solvent evaporation method by dissolving lipid powders in chloroform/methanol (4:1), combining these solutions at the desired ratios and evaporating the solvent, first under a stream of nitrogen and then in a vacuum overnight. The lipid films were hydrated at 37 °C overnight to achieve a final lipid concentration of 1 mM