Editing Synthesis Lipophilic Polyamines (section)

== Synthesis of Lipophilic Oleoyl-TREN<br>  ==

=== Chemicals<br>  ===

'''oleoyl chloride''': http://www.sigmaaldrich.com/catalog/product/aldrich/367850<br> 

[[Image:Oleoyl chloride.gif]]<br> 

367850-100ML, $65.60; 367850-500ML, $227.50<br> 

[[Image:Lipopolyamines.png]]<br> 

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=== A: N,N′-Bis(3-aminopropyl)ethylenediamine<br>  ===

<br>Add 662 mL of oleoyl chloride to 184 mL of N,N′-Bis(3-aminopropyl)ethylenediamine &nbsp; in 8 mL of dioxane. <br> 

Stir the mixture&nbsp; for 16 h at 70 °C.<br> 

Evaporate the dioxane under vacuum<br> 

Dissolve the residue&nbsp; in 2 mL of chloroform. <br> 

Purify on silica gel column-chromatography (eluent:chloroform/methanol 98:2) <br> 

Yield: 180 mg (15.4% yield) of the unreduced intermediate. <br> 

R f is 0.85 on thin-layer chromatography (TLC) on Silica Gel 60 F254 (Alltech Associates, Deerfield, IL, USA) in chloroform/methanol (3:0.6). Proton nuclear magnetic resonance (NMR) (in CDCl 3 ): d 0.87 (12H), 1.25 (88H), 1.7 (4H), 2.00 (16H), 2.2 (8H), 3.4 (12H), 5.35 (8H) and 6.65 (2H). <br> 

Dissolve the intermediate in tetrahydrofurane (THF)<br> 

Add slowly&nbsp; to a stirring suspension of LiAlH4,<br> 

Stir the reaction mixture for 72 h at 55 °C.<br>Add ethyl acetate (1 mL) and 1 N NaOH (0.8 mL) <br> 

Filter.<br> 

Wash the precipitate two times with THF. <br> 

Dry the the filtrate and washings of the precipitate&nbsp; (with MgSO 4 ) and evaporate.<br> 

Purify by silica gel column-chromatography (eluent: CHCl3&nbsp;: CH3OH 96:4)<br> 

Yield is 7.6%. <br> 

R f was 0.7 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.5). <br> 

Proton NMR (in CDCl 3 ): d 0.87 (12H), 1.25 (96H), 1.87 (2H), 2.00 (16H), 2.50 (4H), 2.8 (20H) and 5.38 (8H). 

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=== B: Tetraethylenepentamine (TEPA)  ===

The unreduced intermediate for this compound was synthesized by adding 1.046 mL of diisopropylethylamine and 2 mL of oleoyl chloride to a solution of 190 mL of tetraethylenepentamine (TEPA) in 10 mL of dimethylformamide (DMF). The reaction mixture was stirred at 56 ° C for 16 h. Two layers formed. The upper layer containing the product was collected and washed two times with 2 mL of DMF. The residue was dissolved in CHCl 3 . After column-chromatography (eluent:CHCl 3 /CH 3 OH- 98:2), 233 mg ('''16.2%''' yield) were obtained. R f was 0.9 using CHCl 3 /CH 3 OH- 3:0.6. Proton NMR (in CDCl 3 ): d 0.88 (15H), 1.27 (110H), 2.01 (20H), 2.25 (10H), 3.44 (16H), 5.34 (10H) and 6.37 (2H). The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. After column chromatography (eluent: CHCl 3 /CH 3 OH- 96:4), 45.9 mg were obtained ('''yield = 20.7%'''). R f was 0.78 using CHCl 3 /CH 3 OH- 3:0.5. Proton NMR (in CDCl 3 ): d 0.87(15H), 1.3(120H), 2.00 (20H), 2.6(26H) and 5.37(10H). 

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=== C: Tetraethylenepentamine (TEPA)<br><br>  ===

The unreduced intermediate for this compound was synthesized by dissolving 190 mL (1 mmol) TEPA in 6 mL of dioxane with 349 mL (2 mmol) of N,N-diiso-propylethylamine and then adding 662 mL (2 mmol) of oleoyl chloride. The reaction mixture was stirred 16 h at 56 ° C and dioxane was evaporated under vacuum. The residue was dissolved in chloroform and applied to a silica gel column. The column was washed with chloroform, the product was eluted by chloroform/methanol (96:4) and the solvent was evaporated. The yield was 150 mg ('''21%'''). R f was 0.8 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.5). Proton NMR (in CDCl 3 ): d 0.85 (6H), 1.28 (44H), 1.6 (4H), 1.75 (3H), 2.00 (8H), 2.25 (4H), 2.50 (8H), 3.4 (4H), 5.35 (4H) and 6.50 (2H). The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. The yield was 99 mg ('''88%'''). R f was 0.55 on TLC on Cellulose Polygram  Cell 300 (Brinkmann Instruments, Westbury, NY, USA) in ethanol/water/HCl (100:60:2). Proton NMR (in CDCl 3 ): d 0.88 (6H), 1.25 (48H), 2.00 (8H), 2.55 (20H) and 5.35 (4H). 

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=== D: 1,4-Bis(3-aminopropyl)piperazine<br>  ===

<br>Two hundred eighty milligrams (1 mmol) of 1,4-Bis(3-aminopropyl)piperazine was dissolved in 10 mL of 1,4-dioxane and mixed with 800 mg (2.2 mmol) of oleoyl chloride (J. Hagstrom, V. Gurevich, V. Budker and J. Wolff, unpublished). The reaction mixture was stirred 16 h at 70 ° C. The formed precipitate was filtered, dissolved in CHCl 3 and filtered again. After the CHCl 3 was evaporated, the residue was purified by silica gel column-chromatography (eluent CHCl 3 /methanol-9:1). The intermediate (314.6 mg) was obtained (4'''0% yield'''). The compound was dissolved in 5 mL CHCl 3 , and 5 mL H 2 O were added. NaOH was added to adjust the pH to 9.0, and the CHCl 3 layer was dried with MgSO 4 . The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. The yield was 216 mg ('''70%'''). R f was 0.575 on TLC on Cellulose Polygram Cell 300 in ethanol/water/HCl (100:60:2). Proton NMR (in CDCl 3 ): d 0.85 (6H), 1.25 (48H), 1.4 (2H), 1.65 (4H),<br>2.0 (8H), 2.35 (8H), 2.45 (4H), 2.60 (8H) and 5.35 (4H). 

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=== E: Tris-(2-aminoethyl)amine, TREN<br>  ===

<br>Mix:<br> 

*150 <strike>mL</strike> ul&nbsp; (1 mmol) of tris-(2-aminoethyl)amine (TREN, MW=146, 146 g=1 mole, 0.15 g=1 mmol, ~150 ul) dissolved in 5 mL of dioxane 
*400 mg of N,N-diisopropylethylamine 
*1080 mg (3.3 mmol) of oleoyl chloride (MW 300.9, purity 89%).

Stir the mixture for 16 h at 70 °C. <br>Remove/discard the precipitate<br>Cool down--&gt;form another precipitate<br>Collect the precipitate <br>Re-crystallize the precipitate two times from dioxane.<br>The yield is '''57%.''' R f was 0.8 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.5). <br>Proton NMR (in CDCl3 ): d 0.85 (9H), 1.25 (66H), 2.00 (12H), 2.25 (6H), 3.35 (6H), 3.65 (6H), 5.35 (6H) and 7.62 (3H). <br>The intermediate is reduced with LiAlH4:<br>dissolve the intermediate in tetrahydrofurane (THF) <br>Add slowly to a stirring suspension of LiAlH4, <br>Stir the reaction mixture for 72 h at 55 °C.<br>Add ethyl acetate (1 mL) and 1 N NaOH (0.8 mL) to the mixture<br>Filter<br>Wash the precipitate two times with THF. <br>Dry the filtrate and washings of the precipitate with MgSO4 and by evaporation.<br><br>The yield should be '''97%. '''<br>R f was 0.57 on TLC on Cellulose Polygram Cell 300 in ethanol/water/HCl (100:60:2). <br>Proton NMR (in CDCl 3 ): d 0.85 (9H), 1.25 (72H), 1.9 (3H), 2.00 (12H), 2.95 (12H), 3.25 (6H) and 5.35 (6H). 

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=== F: 1,4,7-Triazacyclononane <br>  ===

<br>The unreduced intermediate for this compound was synthesized by adding 540.5 mL of N,N-diisopropylethylamine and 1.026 mL of oleoyl chloride to a solution of 100 mg of 1,4,7,-triazacyclononane (Aldrich Chemical) in 5 mL of dioxane. The reaction mixture was stirred for 16 h at 67 ° C. A precipitate was formed. The reaction mixture was filtered, the dioxane was evaporated and the residue was dissolved in 2 mL of chloroform. After silica gel column-chromatography (eluent CHCl 3&nbsp;:CH 3 OH 98:2), 87 mg of the unreduced intermediate were obtained. Yield was 12%. R f was 0.83 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.5). Proton NMR (in CDCl 3 ): d 0.9 (9H), 1.27 (66H), 2.00 (12H), 2.27 (6H), 3.4 (6H), 3.7 (6H) and 5.35 (6H). The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. Yield was 76%. R f was 0.65 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.6). Proton NMR (in CDCl 3 ): d 0.9 (9H), 1.25 (66H), 2.00 (12H), 2.75 (6H), 3.7 (12H) and 5.35 (6H). 

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=== G: 1,5,9-triazacyclododecane <br>  ===

<br>The unreduced intermediate for this compound was synthesized by adding 407.5 mL of N,N-diisopropylethylamine and 774 mL of oleoyl chloride to a solution of 100 mg of 1,5,9-triazacyclododecane (Aldrich Chemical) in 5 mL of dioxane. The reaction mixture was stirred for 16 h at 67°C. When the reaction mixture was cooled to room temperature, it formed a white precipitate, which was filtered. Dioxane was evaporated, and the residue was dissolved in 2 mL of chloroform. After silica gel column-chromatography (eluent CHCl 3&nbsp;:CH 3 OH 98:2) 124.5 mg of the intermediate were obtained. Yield was 22%. R f was 0.8 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.4). Proton NMR (in CDCl 3 ): d 0.9 (9H), 1.3 (66H), 2.00 (12H), 2.3 (6H), 3.42 (12H) and 5.35 (6H). The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. Yield was 32.3%. Proton NMR (in CDCl 3 ): d 0.87 (9H), 1.25 (68H), 1.7 (6H), 2.00 (12H), 2.27 (4H), 2.95 (6H), 3.66 (8H) and 5.75 (6H). 

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=== H: 1,4,8,14-tetraaracyclotetradecane <br>  ===

<br>The unreduced intermediate for this compound was synthesized by adding 418.5 mL of triethylamine and 957.3 mL of oleoyl chloride to a solution of 100 mg of 1,4,8,14-tetraaracyclotetradecane (Aldrich Chemical) in 5 mL of CHCl 3 . The reaction mixture was stirred for 30 min at room temperature. After the triethylamine chlorohydrate was extracted with water, the CHCl 3 was evaporated. After silica gel column-chromatography (eluent:CHCl 3 ), 230 mg of intermediate were obtained (yield of 36.6%). R f was 0.9 on TLC on Silica Gel 60 F254 in chloroform/methanol (8:1). Proton NMR (in CDCl 3 ): d 0.89 (12H), 1.27 (88H), 1.61 (4H), 2.0 (16H), 2.35 (8H), 3.5 (16H) and 5.35 (8H). The intermediate was reduced with LiAlH 4 as described for the synthesis of compound A. Yield was 137 mg (60.9%). R f was 0.7 on TLC on Silica Gel 60 F254 in chloroform/methanol (2:0.4). Proton NMR (in CDCl 3 ): d 0.87 (12H), 1.25 (92H), 1.7 (4H), 2.0 (16H), 2.37 (4H), 2.53 (12H), 3.72 (8H)<br>and 5.35 (8H).<br> 

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=== Other lipids<br>  ===

Oleyl bromide, very expensive, $389.50/g<br> 

[[Image:Oleyl bromide.png]]<br> 

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