Synthesis Lipophilic Polyamines

Synthesis of Lipophilic Oleoyl-TREN

Chemicals

oleoyl chloride: http://www.sigmaaldrich.com/catalog/product/aldrich/367850

367850-100ML, $65.60; 367850-500ML, $227.50

A: N,N′-Bis(3-aminopropyl)ethylenediamine

Add 662 mL of oleoyl chloride to 184 mL of N,N′-Bis(3-aminopropyl)ethylenediamine   in 8 mL of dioxane.

Stir the mixture  for 16 h at 70 °C.

Evaporate the dioxane under vacuum

Dissolve the residue  in 2 mL of chloroform.

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

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

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).

Dissolve the intermediate in tetrahydrofurane (THF)

Add slowly  to a stirring suspension of LiAlH4,

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

Filter.

Wash the precipitate two times with THF.

Dry the the filtrate and washings of the precipitate  (with MgSO 4 ) and evaporate.

Purify by silica gel column-chromatography (eluent: CHCl3 : CH3OH 96:4)

Yield is 7.6%.

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

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).

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).

C: Tetraethylenepentamine (TEPA)

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).

D: 1,4-Bis(3-aminopropyl)piperazine

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 (40% 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),
2.0 (8H), 2.35 (8H), 2.45 (4H), 2.60 (8H) and 5.35 (4H).

E: Tris-(2-aminoethyl)amine, TREN

Mix:

• 150 mL ul  (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.
Remove/discard the precipitate
Cool down-->form another precipitate
Collect the precipitate
Re-crystallize the precipitate two times from dioxane.
The yield is 57%. R f was 0.8 on TLC on Silica Gel 60 F254 in chloroform/methanol (3:0.5).
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).
The intermediate is reduced with LiAlH4:
dissolve the intermediate in tetrahydrofurane (THF)
Add slowly to a stirring suspension of LiAlH4,
Stir the reaction mixture for 72 h at 55 °C.
Add ethyl acetate (1 mL) and 1 N NaOH (0.8 mL) to the mixture
Filter
Wash the precipitate two times with THF.
Dry the filtrate and washings of the precipitate with MgSO4 and by evaporation.

The yield should be 97%.
R f was 0.57 on TLC on Cellulose Polygram Cell 300 in ethanol/water/HCl (100:60:2).
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).

F: 1,4,7-Triazacyclononane

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 :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).

G: 1,5,9-triazacyclododecane

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 :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).

H: 1,4,8,14-tetraaracyclotetradecane

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)
and 5.35 (8H).

Other lipids

Oleyl bromide, very expensive, $389.50/g

Polyamidoamine Cholesterol

  +-->

Add cholesteryl chloroformate to excess ethylenediamine to prepare EDA-Chol (ethylenediamine-cholesterol):

PAM-Chol (polyamidoamine-cholesterol, generation 0.5) is synthesized by Michael addition of the primary amine of ethylenediamine to double bonds of two methyl acrylate molecules,   use methyl acrylate/MeOH

Add ethylenediamine to PAM-Chol:  the resulting two ester groups of PAM-Chol (generation 0.5) is attacked by ethylenediamine to produce PAM-Chol (generation 1), use ethylenediamine/MeOH

To avoid side reaction and to obtain a perfect dendritic lipid, excess methyl acrylate and ethylenediamine should be added in each step

Methyl acrylate:

Synthesis

Synthesis of EDA-Chol (A)

Dissolve 80 g (1.3 mol) of ethylenediamine and 4 g (8.9 mmol) of cholesteryl chloroformate  in 10 mL of chloroform .

Warm the mixture to 35 oC and stirred for 48 hours.

Wash the reaction mixture with brine (5M NaCl in water) three times.

Collect organic layer

Dry over anhydrous sodium sulfate, and evaporate.

Yield is 2.8g of colorless powder.

Synthesis of PAM-Chol g =0.5 (B)

Dissolve 2.1g (4.5 mmol) of EDA-Chol and 300 mL (3.3 mol) methyl acrylate in 300 mL MeOH.

Warm the mixture to 35 oC and stirred for 48 hours.

Evaporate the organic solvent and excess methyl acrylate.

PAM-Chol g = 0.5 (B) is obtained as 3.0 g of yellow viscous oil.

Synthesis of PAM-Chol g = 1 (C)

Dissolve 3.0 g PAM-Chol g =0.5 (B) in 300 mL MeOH.
Add 300 mL (4.5 mol) of ethylenediamine to the solution dropwise at 0^oC.

Warm the mixture to 35 ^oC
Stir for 48 hours.

Evaporate the organic solvent and excess ethylenediamine.

Purify PAM-Chol g = 1.0 (C) on silica gel with an eluent of n-propanol: ammonia water: water (70:10:20 to 70:20:10 v/v/v).

 The transfection efficiency followed the order of PAM-Chol/DOPE g = 1.0 > DC-Chol/DOPE > PAMAM g = 6.0 (with chloroquine) > PAM-Chol/DOPE g = 2.0 > PAMAM g = 6.0 (without chloroquine).  The transfection efficiency of PAM-Chol/DOPE g = 1.0 at the optimal condition was about 3- and 100-fold more efficient than those of DC-Chol/DOPE and PAMAM g = 6.0 (without chloroquine), respectively. PAM-Chol g = 1.0, which requires only one Michael addition/amidation step, showed significantly higher efficiency than PAMAM g = 6.0, which
requires seven iterative Michael addition/amidation steps.

Liposome Preparation: 0.5 mg of DOPE (Sigma, St. Louis, MO) and 0.5 mg of synthetic lipids (PAM-Chol g = 1.0 and g = 2.0) in chloroform were dried under a stream of N2 gas and vacuum. After overnight hydration with 1 mL of distilled water at 4 oC, the mixture was sonicated using a bath-type sonicator

Conclusion:  PAM-Chol g = 1 is good enough for transfection, and easy to make

PAMAM G 5-Cho

Hydroxyl terminated poly(amidoamine) PAMAM G 5 dendrimers (G 5 -OH) in water (41.22% w/w, Mn of 28,400 g/mol by GPC)
were obtained from Dendritech, Inc.

Synthesis. G 5 -OH in water was lyophilized until it reached constant weight. 0.5g of solvent free G 5 -OH was dissolved in 15 ml of anhydrous
DMSO, then chloroformate and triethylamine were added sequentially. The stoichiometric relationship between the number of hydroxyl groups and chloroformate (dodecyl or cholesteryl) was 5:1. 10% excess of triethylamine based on the amount of chloroformate was used. The reaction was stirred in a glass flask under nitrogen at room temperature for 2 days. The reaction mixture was dialyzed in ethanol/ water (1:1) followed by extensive dialysis with deionized water using a Spectro/Pro ® RC dialysis membrane (MWCO 6,000-8,000). The resulting aqueous solutions were lyophilized and the obtained products were stored at –20 oC. The product from cholesteryl chloroformate was purified by washing with diethyl ether and dried.

Spermidine Cholesterol

1. Dissolve cholesteryl chloroformate (Aldrich) in methylene chloride.
2. Add the cholesteryl chloroformate solution dropwise to a solution of spermidine (Aldrich) in methylene chloride and N,N-diisopropylethylamine.
3. Stir at room temperature for 2 h.
Conjugation of cholesterol to spermine or spermidine can occur at either end of the polyamine. Because spermidine is an asymmetric molecule, a mixture of two products is expected from its conjugation to a single cholesterol.
4. Purify polyamine-(mono)cholesterol conjugates from the reaction mix by chromatography on silica gel columns using methanol:methylene chloride (1:1) as eluent.
The final products (white to yellow- white solids) is obtained by evaporation of the solvent.
The preparations should be approximately 95% pure, the remaining mass presumably consisting of polyamine conjugated to two cholesterols.
5. Keep the purified reagents desiccated at -20 °C for long term storage
the polyaminolipids is first solubilized to a concentration of 3–5 mg/ml at room temperature and then diluted as necessary in physiological buffers.

BGSC & BGTC

Preparation of guanidino spermidine cholesterol BGSC

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

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%).

Preparation of guanidino tren cholesterol BGTC

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
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 reacted with cholesteryl chloroformate in CH2Cl2. The unreacted amine is removed by washing with water, and the crude product was dissolved in tetrahydrofuran/MeOH (50:50). To this mixture was added 2 equivalents of 1H-pyrazole-1 car-boxamidine (7) and 2 equivalents of diisopropylethylamine. After stirring at room temperature for 18 h, diethylether was added and the resulting precipitate was separated by decantation. To get a pure sample, this crude compound was 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.

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.

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.

Preparation of Liposomes
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.
The final lipid concentration is 1.2 mg/ml.
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.
The resulting dispersion is again "sonicated" (Sonifier Branson 450) for 5 to 10 mm to form liposomes.
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
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).

Synthesis of Polyamine Conjugated Peptides

NI,NII,NIII,NIV -tetra(9-fluorenylmethoxycarbonyl)-5-carboxyspermine (Fmoc-carboxyspermine)

Dissolve 5-Carboxy spermine (11.0 g) in 100 ml water.

Chill the solution on ice and dilute with 200 ml dioxane and the flushe with argon.

Add a solution of 50 g of 9-fluorenylmethyl chloroformate (Fmoc-Cl) in 200 ml of dioxane  added to the chilled carboxy spermine.

Stir the reaction mixture  under argon at 4° C. for an hour and at room temperature overnight under argon.

Monitor the reaction by TLC (silica Gel, CHCl3 /MeOH::9/1).

Pour the reaction mixture into 1.5 L ice cold water (1.5 L) and extract with ethyl acetate (2 L).

Separate the organic layer and sequentially extract with 400 ml of 1 N HCl (2×) and 300 ml saturated NaCl.

Dry the organic layer  over Na2SO4 and concentrated on a rotary evaporator.

Purify the resultant gummy material by flash chromatography (silica, CHCl3 /MeOH::95/5) to yield 25.5 g of the desired material as a white fluffy solid.

TRENL for siRNA

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374058/

Cholesterol,

oleic acid,

linoleic acid

and linolenic acid,

tris(2-aminoethyl)amine (TREN) and 95% sodium hydride: Sigma-Aldrich (St. Louis, MO).

(1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol) (DMG-PEG):   NOF America Corporation (White Plains, NY).

Alkyl mesylates: Nu-Chek Prep, Inc. (Elysian, MN, USA).

mesylate:

Oleyl Mesylate:  http://www.trc-canada.com/detail.php?CatNum=O532965, 1g For $150(US)

Synthesis

TREN is first alkylated by linoleic mesylate (LM) at different TREN:LM ratios, and the remaining amines of TREN can be methylated by a mixture of formic acid (HCOOH) and formaldehyde (HCHO) to obtain TRENL series lipids.

Add linoleyl mesylate (3.1g, 9 mmol) and 95% sodium hydride (0.075 g, 3 mmol) to a solution of TREN (0.44 g, 3 mmol) in 15 mL anhydrous chloroform.

Stir the mixture on a hot plate at 60°C for 12 h under nitrogen.

Cool the reaction mixture in an ice bath.

Add  ethanol slowly.

Spin the reaction mixture at 2000 rpm for 5 min.

Transfer the organic phase  to a new flask and evaporate under vacuum.

Dissolve the crude product of TREN-LM (1:3) in ethanol, filter to remove precipitates.

Add formic acid (2 g)  add to a stirred aqueous solution of 1.5 g of TREN-LM (1:3) and 4 g of formaldehyde for 5 min in an ice bath.

Reflux under nitrogen for 24 h.

After cooling, the reaction mixture is neutralized by NaOH (10 N)

Wash twice using 30 ml of ethyl acetate.

Discard the aqueous layer and the organic phase is dried over magnesium sulfate at room temperature for 2 h.

Concentrate organic phase by rotary evaporation under vacuum.

Dissolve the oily residue in ethanol and evaporate again to get the final product TRENL3

Liposome Preparation

1. Mix cationic lipid and DOPE (molar ratio 3:2) in CHCl3.
2. Evaporate under vacuum and resuspend under N2 atmosphere in a 20 mM Hepes buffer solution (pH 7.4) to the final lipid concentration 1.2 mg/ml.
3. Votex the mixture for 5 min, then sonicate for 5 min in a bath sonicator (Branson Ultrasonics 2210).
4. Store at 4°C for 24 h for hydration.
5, sonicate athe resulting dispersion using a probe (Branson Sonifier 450) for 5-10 min to form liposomes.
6. Spin, filter the solution through a 0.22 micron filter (Millex GS; Millipore)
7. Store liposome at 4°C.

Transfection

1. Dilute plasmid DNA (1 ug) in  50 ul of DMEM without FCS.

2. Dilute lipisome in  50 ul of DMEM without FCS. 

3. Mix two solutions and  incubate for 15 min at room temperature.

4. Add the transfection mixture to 12 well plate.

Other Lipophilic Polyamines

PAMAM dendrimer generation 0: 

http://www.sigmaaldrich.com/catalog/product/aldrich/412368

412368-5G, $58.50; 412368-25G, $256.50

Mix  ethylenediamine (EDA) with a three-fold molar amount of methyl aerylate (MA, http://www.sigmaaldrich.com/catalog/product/aldrich/m27301, M27301-250ML, $31.00, M27301-1L $38.00) at 25° C for about 48 hours under N2 -->generation -0.5 PAMAM tetra ester.

Add excess EDA, react under inert nitrogen in methanol for 48 hours at 0 oC --> generation 0 PAMAM tetramine.

2-bromoacetamido-olean

 

2-bromoacetamido-olean:

1. Haloacetamido-alkane or alkenes: Oleylamine (18C, 油胺,十八烯胺, MW 267.49, 70% from Aldrich,0.813g/ml,  O-7805-500g), palmitylamine (16 C), stearylamine (18C) is reacted with chloroacetic chloride or chloroacetic bromide or bromoacetic chloride or bromoacetic bromide (MW 201.84, 2.317g/ml) in the presence of a triethylamine (TEA) base in Dichloromethane CH2Cl2. The resulted N-haloacetamido-alkane or alkenes is purified by chromatograph on silica gel (Hexane:ethyl acetate 15:85). 

Inside a chemical hood because Bromoacetyl bromide is a lachrymator. Excessive amount of moisture in the glassware or reactants will result in a low yield.

Add Oleylamine (267.49/(0.7*0.813*1000)==0.47 ml==> 1 mmole) to dry dichloromethane (2 ml), add triethylamine (0.14 ml, 1 mmol) on ice.

Add bromoacetyl bromide (201.84/(0.98*2.317*1000), 89 ul--> 1 mmole)  in 0.5 ml of dichloromethane (ice-cold) to Oleylamine solution on ice dropwise over 20min using a glass microsyringe

Stire for a further30 min  on ice.

React o/n at room temperature.

Transfer the mixture to a separatory funnel. Add 20 ml of dichloromethane and wash the organic phase with two 10 ml portions of 1 M aqueous HCl, then with 10 ml of a saturated solution of NaHCO3 and finally with 10 ml of water.

Dry the organic phase in an Erlenmeyer flask with anhydrous sodium sulfate. Add approx 5 g of anhydrous sodium sulfate to the solution in the flask, swirl it and then allow to stand for 5 min. Remove sodium sulfate by gravity filtration using a glass funnel and fluted filter paper.

Concentrate under reduced pressure. Evaporate the solvents under vacuum at 40 °C using a rotary evaporator.

Purify the crude product  by chromatography on silica gel: Pack a chromatography column (3 cm i.d. times 30 cm length) with silica gel (30 g) and wash with dichloromethane under pressure until no air is present in the silica gel. Load the product dissolved in a small amount of dichloromethane (1–2 ml) on top of the column and allow it to soak into the column. Then elute the column with dichloromethane under a slight pressure and collect 10 ml fractions.

Identify fractions containing the product using thin-layer chromatography (TLC; retention factor of the product using dichloromethane as an eluent (Rf) = xx).

Reaction type: Nucleophilic Acylation

+ -->

Bromoacetyl bromide: http://www.sigmaaldrich.com/catalog/product/aldrich/b56412, B56412-5G, $36.20, B56412-100G, $64.70, B56412-500G $143.00

Alfa Aesar BROMOACETYL BROMIDE 98+% 500 g bottle A19580-36 $86.98 https://us.vwr.com/store/catalog/product.jsp?catalog_number=AAAA19580-36, 

Oleylamine: http://www.sigmaaldrich.com/catalog/product/aldrich/o7805, O7805-5G, $32.60, O7805-100G, $36.10, O7805-500G, $111.50

TCI America : https://us.vwr.com/store/catalog/product.jsp?catalog_number=TCO0059-500ML 500 mL O0059-500ML TCO0059-500ML (500ml) $59.93

2. Acryloylamido alkane or alkenes: Oleylamine is treated with acryloyl chloride in the presence of TEA in CH2Cl2. The resulted acryloylamido-olean is purified by chromatograph on silica gel (Hexane:ethyl acetate 10:90).

3. 1,2-epoxy-3-cis-9-octadecenoxypropane (or oleyl glycidyl ether): Oleyl alcohol in DMSO is treated with crushed KOH pellet and two molar excess of epichlorohydrin at 60° C. for 48 hrs. The resulted epoxypropy oleyl ether is purified by chromatograph on silica gel (Hexane:ethyl acetate 10:90).

4. Other lipid derivatives with a nucleophilic reacting group could also be readily prepared including: reacting iodoacetic chloride, chloroethyl chloroformate, or 2-chloroethyl isothiocyanate with an alkyl or alkenyl amine;

5. Alternatively, 2-chloroethylamine or 2-bromoethylamine reacts with a long chain fatty acid, such as oleic acid, either with the activated acyl chloride or by a dehydrating agent. Commercially available alkyl iodine, bromide, or chloride can also been used.

6.  Synthesis of Lipophilic Polyamines Using Haloacetamido Olean

Add Polyamine (0.1 mmole) in ethanol to haloacetamido alkane or alkene at varied molar ratio of amino nitrogen to alkylating lipid derivative, followed by 1 equivalent of Na2CO3 to alkylating agents dissolved in small amount of water. Heat the solutions to 70° C. The progress is monitored by TLC test and is completed at the end of 24-48 hrs based on the total consumption of the alkylating agents. The resulted polyamine derivatives are obviously a mixture of analogous with different numbers of lipid chains linked to the polyamine. After evaporation of the solvent, the resulted lipidic polyamines is dissolved in methanol-CHCl3 (1:1 vol/vol) at a concentration of 20 mM according to the original polyamine and stored at −20° C.

7. Synthesis of Lipophilic Polyamines Using Oleyl Glycidyl Ether

Polyamine (0.1 mmole) in ethanol is added with oleyl glycidyl ether at varied molar ratio of amino nitrogen to alkylating lipid derivative. The solutions are heated to 70° C. The progress is monitored by TLC test and is completed at the end of 48 hrs based on the total consumption of the alkylating agents. The resulted polyamine derivatives are obviously a mixture of analogous with different numbers of lipid chains linked to the polyamine. After evaporation of the solvent, the resulted lipidic polyamines are dissolved in methanol-CHCl3 (1:1 vol/vol) at a concentration of 20 mM according to the original polyamine and stored at −20° C.

8. Synthesis of Lipophilic Polyamines Using Acryloylamido-Olean

Polyamine (0.1 mmole) in ethanol is added with acryloylamido-olean at varied molar ratio of amino nitrogen to alkylating lipid derivative. The solutions are heated to 70° C. The progress is monitored by TLC test and is completed at the end of 48 hrs based on the total consumption of the alkylating agents. The resulted polyamine derivatives are obviously a mixture of analogous with different numbers of lipid chains linked to the polyamine. After evaporation of the solvent, the resulted lipidic polyamines are dissolved in methanol-CHCl3 (1:1 vol/vol) at a concentration of 20 mM according to the original polyamine and stored at −20° C.

9. Bromoacetyl lipid-derivative can also be conjugated to the polyamines at room temperature, for example 20 oC to 35 oC, at a ratio of lipid to polyamine of 1:1 to 6:1 in methanol for example. Reaction can be completed after 48 hours for example. No additional base is needed.

In terms of reactivity, the haloacetamido compounds are more reactive than epoxy or acryloylamido lipid derivative, as the later two require considerable longer reaction time to reach completion at the same reaction temperature than the haloacetamido compounds. Both bromo and chloride derivative are reactive enough under the condition. The bromo compound yielded darker color than the chloro compound, both as lipid intermediates or after reaction to produce lipopolyamines.

the reaction by alkylation at elevated temperature is expected to be random modification and the end products should be a mixture of lipopolymers with varied numbers of lipid chains conjugated to the amino groups. Some NH2 may be unmodified; while others may be conjugated with two or three lipid chains. However, it is expected that under the condition that was used, the majority of the lipopolymer should contain a total number of the lipid chains close to the initial ratio of amine to lipid intermediate. As it is difficult to purify individual species of the lipopolycations, the end product was used as is, without purification. However, using known purification methods, such as phase partition, vacuum distillation, size exclusion, ionic exchange or hydrophobic interaction chromatography.

DCC-mediated esterification of fatty acids (Steglich esterification)

The Steglich esterification is a variation of an esterfication with dicyclohexylcarbodiimide as a coupling reagent and 4-dimethylaminopyridine as a catalyst. The reaction was first described by Wolfgang Steglich in 1978.[1] It is an adaptation of an older method for the formation of amides by means of DCC (dicyclohexylcarbodiimide) and 1-hydroxybenzotriazole (HOBT).[2][3]

This reaction generally takes place at room temperature. A suitable solvent is dichloromethane. Because the reaction is mild, esters can be obtained that are inaccessible through other methods for instance esters of the sensitive 1,4-dihydroxybenzoic acid. A characteristic is the formal uptake of water generated in the reaction by DCC, forming the urea compound dicyclohexylurea (DCU).

Add DCC (3.61g,11 mmole) and DMAP (1.22g, 10 mmole) in dichloromethane (50 mL) to a solution of fatty acid  ( e.g., 1.84g, 10 mmole), alcohol 2 or 3 (2.56g or 3.28g, 10 mmole),

Stir mechanically at room temp. The progress of the reaction is monitored by TLC.

After the completion of reaction the N,N ′ -dicyclohexylurea (DHC) is filtered off

Wash  the filtrate  with water (3 × 50 mL), 5% acetic acid solution (3 × 50 mL), again with water (3 × 50 mL)

Dry the liquid over anhydrous sodium sulphate.
Evaporate the solvent  under reduced pressure to give the crude product (liquid),

Purify lipid by flash chromatograph over a column of silica gel. Elution with petether-diethyl ether (98:2, v/v) to yield the ester.

Synthesis of N1 ,N12 -diamidino-N4 ,N9 -diacylated spermines

For siRNA transfection

Sigma:

Dichloromethane (DCM)

Dicyclohexylcarbodiimide (DCC), D80002-25G, $37.40; D80002-100G, $55.00; D80002-500G,$191.00; D80002-1KG, $389.00

1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, 15033-1G $156.50, 15033-5G $568.00

4-dimethylamino-pyridine (DMAP), 107700-5G $16.30; 107700-25G $48.00; 107700-100G $136.50; 107700-500G $522.00

N-carbethoxyphthalimide, C5459-25G, $36.00; C5459-100G, $128.00

spermine S4264-1G $56.10; S4264-5G$193.50

triethylamine (TEA), T0886-1L $94.30

and trifluoroacetic acid (TFA)

hydrazine monohydrate 225819-50G,$37.50

Synthesis of N 1 ,N 12 -diamidino-N 4 ,N 9 -diacylated spermines.

a: N-Carbethoxyphthalimide, DCM;

b: fatty acid, DCC, TEA;

c: hydrazine monohydrate,DCM/THF 1:1 mixture;

d: 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, TEA, DCM;

e: TFA, DCM.

he synthesis of the guanidinylated N 4 ,N 9 -diacylated spermine conjugates started with the synthesis
of the N 4 ,N 9 -difatty acids spermine derivatives (Figure 2). The symmetrical lipospermines; i.e., those
with the same fatty acid chains conjugated to positions N 4 and N 9 of the spermine chain was carried out
as described previously [23]. For the synthesis of the unsymmetrical N 4 -linoleoyl-N 9 -oleoylspermine,
the primary amine groups of spermine were selectively protected with the phthalimide protecting
group (2 eq. of N-carbethoxyphthalimide in CH 2 Cl 2 ). Then one oleoyl chain was conjugated to one of
the free secondary amine groups of spermine (1 eq. oleic acid, 1 eq. DCC, and 1 eq. DMAP).
Purification of the mono-acylated spermine was followed by flash chromatography. The second
linoleoyl chain was added using DCC coupling of linoleic acid to the 1,12-diphthalimido-N 9 -oleoyl-
4,9-diazadodecane (1 eq. linoleic acid, 1 eq. DCC, and 1 eq. DMAP). Deprotection of the phthalimide
protecting groups then followed by refluxing in hydrazine monohydrate in DCM/THF 1:1 mixture to
obtain N 4 -linoleoyl-N 9 -oleoylspermine, which was purified by flash chromatography [23]. The
guanidinylation of amines typically involves an electrophilic amidine group as part of the
guanidinylating reagent [25]. 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine was used as it can carry
out the guanidinylation of primary and secondary amines under mild conditions [25,26]. The
guanidinylation was carried out on the di-acylated spermine derivatives and deprotection of the Boc
protected guanidine group was carried out using TFA to obtain the trifluoroacetate salts of the
synthesized compounds (Figure 3).

____________

Add N-Carbethoxyphthalimide (0.44 g, 2 mmol)   to a solution of 1,12-diamino-4,9-diazododecane (spermine) (0.20 g, 1 mmol) in DCM (10 mL).

Stir the solution at 20 °C for 3 h 

Evaporate to dryness in vacuo

use  the residue directly in the following step.

Add fatty acid chloride (2 mmol) to a solution of 1,12-diphthalimido-4,9-diazadodecane  in DCM (10 mL) and TEA (0.28 mL, 2 mmol)

alternatively add fatty acid (2 mmol), DMAP (0.24 g, 2 mmol), and DCC (0.4 g, 2 mmol) to the above solution.

Stir for 18 h under nitrogen atmosphere.

To make 1,12-diphthalimido-N 4 -linoleoyl-N 9 -oleoylspermine:

prepare 1,12-diphthalimido-N 4 -oleoylspermine  by reacting 1,12-diphthalimido-4,9-diazadodecane (1 mmol) with 1 (mmol) oleic acid using DCC as described [23].

Purify the product over silica gel (DCM/MeOH 20:1 v/v then 10:1 v/v)

Conjugate 1,12-diphthalimido-N 4 -oleoylspermine to linoleic acid (1 mmol) using DCC as the coupling agent.

Evaporate the solvent (for all of the prepared compounds) to dryness in vacuo

Treat the residue  with hydrazine monohydrate (2 mL) in a mixture of DCM (15 mL) and THF (15 mL) and heat under reflux for 4 h.

Evaporate the solvent in vacuo to dryness

Purify the residue over silica gel (DCM/MeOH 10:1 v/v then DCM/MeOH/NH 4 OH 20:10:1 v/v/v) to get the N 4 ,N 9 fatty acid amides of spermine.

The N 1 ,N 12 -diamidino-N 4 ,N 9 -diacylated spermines is  prepared by reacting each of the prepared N 4 ,N 9 -diacylated spermine (1 mmol) with 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (2 mmol) and TEA (2 mmol) in DCM (10 mL) at 20 °C for 24 h.

Evaporate the reaction mixture to dryness in vacuo

Purify the residue over silica gel (DCM/MeOH 100:1 v/v then 100:2 v/v)

Collect desired fractions and concentrate.

Add DCM (6 mL), TFA (2 mL) to the residue
Stir at 20 °C for 4 h.

Evaporate the reaction mixture to dryness in vacuo --> N 1 ,N 12 -Diamidino-N 4 ,N 9 -dierucoylspermine 1

siRNA

Sense strand: 5'-GCAAGCUGACCCUGAAGUUCAUTT-3',
Anti-sense strand: 5'-AUGAACUUCAGGGUCAGCUUGCCG-3',
Target DNA sequence: 5'-CGGCAAGCTGACCCTGAAGTTCAT-3'.

Liposome Formulation, Transfection

Ten μl of lipidic polyamine solutions and 10 μl of dioleoylphasphotidyl ethanolamine (DOPE, 50 mg/ml in chloroform, Avanti Polar Lipids, Alabaster, Ala.) were mixed in a glass test tube, the organic solvents were removed by N2 stream followed by vacuum. This formed a thin lipid film deposited on the test tube. One ml of distilled water was added to the test tube and the tube was vortexed to suspend the lipid film to give liposomes. The resulted liposomes are stored at +4° C. till use.

To prepare liposome DNA complexes, Opti-MEM® medium (Invitrogen) with antibiotics is used to dilute DNA to 4-12 μg/ml, and liposomes to 2-40 μl/ml. Fifty μl of DNA solution and 50 μl of liposome solution were mixed.

The lipopolyamines prepared from polyamine and bromoacetamido olean at fixed 1:2 lipid to amine group ratio showed substantial transfection activity when co-formulated with DOPE at 1:3.5 mole ratios