Editing Synthesis Lipophilic Polyamines (section)

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

For siRNA transfection 

Sigma: 

Dichloromethane (DCM) 

[[Image:Dichloromethane DCM.png]] 

<br> 

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

[[Image:Dicyclohexylcarbodiimide.png]] 

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

[[Image:1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine.png]] 

<br> 

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

[[Image:4-dimethylamino-pyridine.png]] 

<br> 

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

[[Image:N-Carbethoxyphthalimide.png]] 

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

[[Image:Spermine.png]] 

triethylamine (TEA),&nbsp;T0886-1L $94.30 

[[Image:Triethylamine.png]] 

and trifluoroacetic acid (TFA) 

[[Image:Trifluoroacetic acid.png]]<br> 

<br> 

hydrazine monohydrate&nbsp;225819-50G,$37.50 

[[Image:Hydrazine.jpg]] 

[[Image:Spermine-Based Fatty Acid Amide Guanidines.png]] 

<br> 

<br> 

[[Image:Spermine-Based Fatty Acid Amide Guanidines Synthesis.png]] 

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:&nbsp;TFA, DCM. 

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

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

Stir the solution at 20 °C for 3 h&nbsp;<br> 

Evaporate to dryness in vacuo<br> 

use&nbsp; the residue directly in the following step. <br> 

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

'''alternatively add fatty acid (2 mmol), DMAP (0.24 g, 2 mmol), and DCC (0.4 g,&nbsp;2 mmol) to the above solution.'''<br> 

Stir for 18 h under nitrogen atmosphere. <br> 

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

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

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

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

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

Treat the residue&nbsp; with&nbsp;'''hydrazine monohydrate''' (2 mL) in a mixture of DCM (15 mL) and THF (15 mL) and heat under reflux for 4 h. <br> 

Evaporate the solvent in vacuo to dryness<br> 

Purify the residue over silica&nbsp;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&nbsp;amides of spermine.<br> 

The N 1 ,N 12 -diamidino-N 4 ,N 9 -diacylated spermines is&nbsp; 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. <br> 

Evaporate the reaction mixture to dryness in vacuo <br> 

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

Collect desired fractions and concentrate.<br> 

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

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

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siRNA 

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