Editing Synthesis Lipophilic Polyamines (section)

== BGSC &amp; BGTC<br>  ==

[[Image:BGSC BGTC.png]]<br> 

=== Preparation of guanidino spermidine cholesterol BGSC <br><br>  ===

'''Preparation of the Di-Boc Carbamate (1)''' 

A solution of cholesteryl chloroformate (0.9 g, 2 mmol) and triethylamine (0.214 g, 2 mmol) in CH2Cl2 (40 ml) is added to a solution of 1N,8N-Boc2-spermidine (0.690 g, 2 mmol) in CH2Cl2 (20 ml). After refluxing for 5 hours, the mixture is washed with water (2×50 ml), dried over Na2SO4 and the solvent is evaporated off. The crude product is purified by chromatography on a silica gel using CH2Cl2/MeOH 95/5 as eluent to give pure carbamate 

<br>'''2. Preparation of the Aminocarbamate (2)''' 

Compound (1) (1.3 g) is dissolved in 20 ml of CH2Cl2 and 2 ml of freshly distilled Trifluoroacetic acid (CF3CO2H) are added to this cooled solution (ice bed) in order to release the protective BOC groups. After stirring at room temperature for 3 hours, 50 ml of 1N NaOH are added and the mixture is extracted with CH2Cl2 The organic layers are washed with water, dried over Na2SO4 and evaporated so as to obtain the crude aminocarbamate (2) (0.945 g, 98%). 

The crude carbamate (2) (0.94 g) is dissolved in 30 ml of the Tetrahydrofuran(THF)/MeOH 85/15. Next, 1H-pyrazolecarboxamidine (0.495 g, 3.4 mmol) and diisopropylethylamine (0.436, 3.4 mmol) in 30 ml of Tetrahydrofuran(THF)/MeOH 85/15 are added. After stirring at room temperature for 18 hours, 250 ml of diethyl ether are slowly added and the precipitate obtained is separated by decantation. The crude compound is suspended three times in diethyl ether and separated by decantation to give pure GSC (0.570 g, 47%). 

=== <br>Preparation of guanidino tren cholesterol BGTC<br><br>  ===

'''1. Preparation of Aminocarbamate (5)''' 

A solution of cholesteryl chloroformate (1.8 g, 4 mmol) in 100 ml of CH2Cl2 is slowly added to a saturation of tris(2-aminoethyl)amine (TREN) (11.68 g, 80 mmol) in 400 ml of CH2Cl2. After stirring at room temperature for two hours, the inert amine is released by washing with water (3×100 ml) and after drying over Na2SO4, the solvent is evaporated off. (5) is isolated in the trihydrochloride form in the following manner: the crude product is dissolved in 10 ml of MeOH and a saturated methanolic HCl solution (5 ml) is added dropwise; the precipitate obtained is separated by decantation and, to obtain a pure compound, is suspended three times in MeOH and separated by decantation. After drying under vacuum, the pure trihydrochloride (6) is obtained (1.71 g, 64%). 

'''2. Preparation of BGTC'''<br> A solution of '''cholesteryl chloroformate''' (2.24 g, 5 mmol) in 100 ml of CH2Cl2 is slowly added to a broad saturation of '''tris(2-aminoethyl)amine''' (TREN) (29.2 g, 200 mmol) in 250 ml of CH2Cl2. After stirring at room temperature for 2 hours, the inert amine is released by washing with water (3×100 ml) and after drying over Na2SO4, the solvent is evaporated off. The crude product (3.2 g) is dissolved in THF/MeOH 50/50 (20 ml); next, '''1H-pyrazole-1-carboximidine''' (1.465 g, 10 mmol) and '''diisopropylamine '''(1.3 g, 10 mmol) are added to the mixture. After stirring at room temperature for 18 hours, diethyl ether is added and the precipitate obtained is separated by decantation. To obtain a pure sample, the crude compound is suspended three times in diethyl ether and separated by decantation (2.15 g), 60% after drying under vacuum. 



A large excess of TREN is&nbsp;reacted with cholesteryl chloroformate in CH2Cl2. The unreacted amine is removed by washing with water, and the crude&nbsp;product was dissolved in tetrahydrofuran/MeOH (50:50). To&nbsp;this mixture was added 2 equivalents of 1H-pyrazole-1 car-boxamidine (7) and 2 equivalents of diisopropylethylamine.&nbsp;After stirring at room temperature for 18 h, diethylether was&nbsp;added and the resulting precipitate was separated by decantation. To get a pure sample, this crude compound was&nbsp;suspended three times in diethylether and separated by decantation (60% after drying under vacuum).

Flash Chromatography: Merck silica gel 60 of particle siZe distribution 0.040—0.063 mm.<br> 

'''Diisopropylamine as non-nucleophilic base''' 

N,N-Diisopropylethylamine, or Hünig's base, DIPEA or DIEA, is an organic compound and an amine. It is used in organic chemistry as a base. Because the nitrogen atom is shielded by the two isopropyl groups and an ethyl group only a proton is small enough to easily fit. Like 2,2,6,6-tetramethylpiperidine, this compound is a good base but a poor nucleophile, which makes it a useful organic reagent. Hünig's base is named after the German chemist Siegfried Hünig. The compound is a colourless liquid.<br> 

Hünig's base was investigated for its use as a selective reagent in the alkylation of secondary amines to tertiary amines by alkyl halides. This organic reaction is often hampered by a quaternization reaction to the quaternary ammonium salt but this side-reaction is absent when Hünig's base is present. <br> 

<br> 

[[Image:Hunig s base for alkylating secondary amines.png]] 

'''Preparation of Liposomes'''<br> A mixture of cationic lipid and DOPE (in a molar ratio of 3/2) in chloroform (CHCl3) is evaporated under vacuum and resuspended in a 20 mM HEPES buffer solution at PH 7.4, under an N2 atmosphere.<br>The final lipid concentration is 1.2 mg/ml.<br>The mixture is vortexed for 5 min and then "sonicated" for 5 min with a sonicator (Branson Ultrasonic 2210), finally stored at +4° C. for 24 h for hydration.<br>The resulting dispersion is again "sonicated" (Sonifier Branson 450) for 5 to 10 mm to form liposomes.<br>After centrifugation, the solution is filtered on a filter of pore diameter of 0.22μ (Millex GS, Millepore) and stored at +4° C. 

Ref: US6143729<br>Lipofectin is a liposome formulation having, as neutral lipid, DOPE and, as cationic lipid, DOTMA. (N[1(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride). <br> 

<br>