Paul.xu at 19:35, 20 July 2016

2016-07-20T19:35:12Z

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[[File:Synthesis-of-the-dendritic-lipid-MVLBG2-starting-from-ornithine-methyl-ester.png|RTENOTITLE]]

== ==

i: Boc-L-ornithine(Boc), TBTU, HOBt, DIEA;

ii: TFA;

iii: R-COOH (7), TBTU, HOBt, DIEA;

iv: excess ethylenediamine (20x) in MeOH.

HOBt: 1-hydroxybenzotriazole hydrate;

DIEA: N,N-diisopropylethylamine;

TBTU: O-benzotriazol-1-yl-N,N,N`,N`-tetramethyluronium tetrafluoroborate;

TFA: trifluoroacetic acid.

== ==

== Synthesis of Nα,Nδ-bis(tert-Butyl-carbamoyl)ornithine ==

Over the course of 1 hour, 25 ml of 4 M NaOH and  a solution of 27.3 g (125 mmol) Boc2O (Novabiochem) in THF were added to a solution of 9.15 g (54.3 mmol) L-ornithine hydrochlorite (Aldrich) and 30 ml 4 M NaOH in 250 ml water in four portions.

 L-ornithine hydrochlorite: VWR: $92.77/100g A12111-22

Di-tert-butyl dicarbonate , Boc2O, VWR/Alfa Aesar Di-tert-butyl dicarbonate $119.44/100g

After stirring for 3 hour at room temperature, the THF was evaporated.

Following the addition of 100 ml of ethyl acetate, the resulting mixture was acidified using half-concentrated HCl (about 60 ml).

The phases were separated, and the aqueous phase were extracted two more times with 100 ml of EtAc each

The combined organic phases were washed twice with 200 ml of water, dried (Na2SO4) and evaporated.

The residue was dissolved in little EtAc, and 600 ml of cyclohexane were added.

After standing at room temperature for 14 hours, the mixture was filtered, and the residue was washed with cyclohexane and dried to yield 16.6 g (49.9 mmol, 92%) of Boc2Orn as colorless crystals.

== Synthesis of Nα,Nδ--bis(2-Cyanoethyl)ornithine (5). ==

To a solution of 474 mg (11.9 mmol) NaOH in 6 mL of methanol, were added 1.00 g (5.93mmol) of L-ornithine hydrochloride (Aldrich) and 15 mL of methanol. The mixture was stirred at room temperature, 1.56 mL (1.26 g, 23.7 mmol) of acrylonitrile (Fluka) was added, and stirring was continued for 2 h. The reaction mixture was acidified slightly by the addition of concentrated hydrochloric acid (about 1.5 mL) and filtered immediately. The residue was dried in vacuo to yield 1.4 g (5.66 mmol, 86%) of 5hydrochloride as colorless microcrystals

== Synthesis of Nα,Nδ-Bis(3-aminopropyl)ornithine (6). ==

A mixture of 29.8 g (109 mmol) of 5, 175 mL of 1.4 m NaOH in 95% ethanol, and 6.7 g of a supension of Raney-nickel (Aldrich) in water was prepared in a Parrhydrogenator flask and agitated for 24 h at room temperature under 60 psi (4 bar) hydrogen pressure. The Raney-nickel was removed by filtration over Celite (diatomaceous earth, acid washed; Sigma), and the solvent was evaporated. The resulting oil was used for the next step without further purification. Pure 6 hydrochloride for analytical purposes was obtained by adding concentrated hydrochloric acid to the reaction solution and collecting and drying the precipitate:

== Synthesis of Nα,Nδ-Bis(tert-butyl-carbamoyl)-Nα,Nδ-bis(3-[tert-butyl-carbamoyl-amino]propyl)ornithine (7). ==

The raw 6 (assay calculated for 100% yield from the previous step: 108 mmol) was dissolved in water/THF (5:1, v/v). Over the course of 1 h, 100 mL of 4 M NaOH and a solution of 104.2 g (477 mmol) of Boc2O (Novabiochem) in 200 mL of THF were added in four portions with stirring while the reaction mixture was kept at room temperature by cooling with a water bath. Stirring was continued for 6 h, and most of the THF was then evaporated. After addition of 600 mL of diethyl ether and 200 mL of water, the mixture was acidified using half-concentrated hydrochloric acid. The phases were separated, and the aqueous phase was extracted two more times with 200 mL of ether. The combined organic phases were washed twice with water, dried (Na2SO4), and evaporated. The residue was purified in portions of 44 g by flash chromatography on 930 g of silica gel using chloroform/methanol (19:1) as the eluent83 to yield 26.5 g (41mmol, 38%) of 7 as a colorless solid

== General Procedure for TBTU-Mediated Coupling (Amine Compound with n Amino Groups). ==

To a mixture of 1.1n mmol acid  component, 1.1n mmol O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU; Bachem), and 1.1n mmol 1-hydroxybenzotriazole (HOBt) hydrate (Peptides International) in dimethylformamide (DMF) (peptide synthesis grade, Applied Biosystems), was added 2n mmol N,N-diisopropylethylamine (DIEA; Aldrich). After 10 min, a mixture of 1 mmol amine compound (hydrochloride or hydrotrifluoroacetate form) and n mmol DIEA in DMF was added to the resulting solution with stirring. Stirring was continued at room temperature for 2 h, and chloroform and water were added with vigorous stirring after evaporation of most of the DMF.84 The phases were separated, and after extracting two more times with chloroform, the organic phases were combined and washed twice with 2 M HCl, twice with 2 M NaOH, and once with water. After drying (Na2SO4), the solvents were evaporated.

== Methyl (2S)-2,5-Di[(1S)-1,4-di(tert-butyl-carbamoyl-amino)butyl]-carboxamidopentanoate (1). ==

As described in the general procedure for TBTU-mediated coupling (n ) 2), 14.6 g (44.0 mmol) of Nα,Nδ- bis(tert-Butyl-carbamoyl)ornithine was reacted with 4.38 g (20 mmol) of ornithine methyl ester dihydrochloride (Bachem) to yield 14.1 g (18.2 mmol, 91%) of 1 as a colorless solid:

== Methyl (2S)-2,5-Di[(1S)-1,4-diaminobutyl]carboxamidopentanoate(8). ==

About 70 mL of TFA (Acros) was saturated with nitrogen, cooled on ice, and added to 10.0 g (12.9 mmol) of 1. The mixture was evaporated after stirring for 1 h at room temperature, and diethyl ether (300 mL) was added. The ether was decanted, and the solid product was triturated with ether and finally dried in vacuo to yield 4.54 g (12.1 mmol, 94%) of 8 (trifluoroacetate) as a colorless solid:

== Methyl (2S)-2,5-Di((1S)-1,4-di[((1S)-1,4-di(tert-butyl-carbamoyl)-(3-[tert-butyl-carbamoyl-amino]propyl)aminobutyl)carboxamido]-butylcarboxamido)pentanoate (2). ==

As described in the general procedure for TBTU-mediated coupling (n ) 4), 5.83 g (9.01 mmol) of 7 was reacted with 1.70 g (2.05 mmol) of 8 to yield 5.15 g (1.78 mmol, 87%) of 2 as a colorless solid: Rf ) 0.53 (CHCl3/MeOH 9:1,1% concentrated NH4OH); 0.80 (CHCl3/MeOH 6:1, 1% concentrated NH4OH); For characterization purposes, a small sample was deprotected by adding cold TFA, stirring for 1 h and subsequent evaporation in vacuo:

== N1-(2-Aminoethyl)-(2S)-2,5-di((1S)-1,4-di[((1S)-1,4-di(tert-butylcarbamoyl)(3-[tert-butyl-carbamoyl-amino]propyl)aminobutyl)carboxamido] butylcarboxamido)pentanamide (3). ==

To a solution of 2.00 g (0.69 mmol) of 2 in methanol, was added 927 íL (830 mg, 13.8 mmol) of ethylenediamine in one portion, and the mixture was stirred at room temperature for 10 days. The solvent was evaporated, and water and chloroform were added. After agitation, the phases were separated, and the organic layer was washed two more times with water, dried (Na2SO4), and evaporated. The residue was purified by flash chromatography on 120 g of silica gel using chloroform/methanol (15:1, changed to 6:1 once the product started to elute) as the eluent to yield 1.33 g (0.45 mmol, 66%) of 3 as a colorless solid:

== N-1-2-[(1S)-1,4-Di((1S)-1,4-di[(tert-butyl-carbamoyl)(3-[tert-butylcarbamoyl-amino]propyl)amino]butyl)carboxamido]butylcarboxamido) butylcarboxamido]ethyl-3,4-di[(Z)-9-octadecenyloxy]benzamide(9). ==

A solution of 301 mg (460 ímol) of DOB60,39 in dichloromethane was added to a mixture of 148 mg (460 ímol) of TBTU and 71 mg (460 ímol) of HOBt hydrate in DMF. Following the addition of 216 íL (160 mg, 1.24 mmol) of DIEA, the mixture was left to stand for 10 min, and a solution of 1.22 g (418 ímol) of 3 in DMF was added. After standing for 4 h, the solvents were evaporated. Following the addition of chloroform and 5% citric acid and agitation, the phases were separated, and the organic phase was washed one more time with 5% citric acid, twice with 10% NaHCO3, and once with water. After drying (Na2SO4), the solvent was evaporated, and the residue was purified by flash chromatography on 100 g of silica gel using chloroform/methanol (25:1) as the eluent83 to yield 1.29 g (364 ímol, 87%) of 9 as a colorless solid:

== N-1-2-[((1S)-1,4-Di[(1S)-1,4-di((1S)-1,4-di[(3-aminopropyl)amino]-butylcarboxamido)butyl]carboxamidobutyl)carboxamido]ethyl-3,4-di[(Z)-9-octadecenyloxy]benzamide (MVLBG2, 4). ==

9 (1.30 g (366mmol)) was dissolved in 10 mL of 95% TFA (5% water), which had been saturated with nitrogen and cooled on ice. The mixture was left to stand at room temperature for 25 min with occasional swirling, and most of the TFA was then evaporated in vacuo. A mixture of diethyl ether/pentane (1:1, 30 mL/g protected lipid) was added with stirring, and the precipitate was isolated by decanting. This was repeated twice, and the precipitate was dried in vacuo to yield 1.31 g (348 ímol, 95%) of MVLBG2 as a colorless solid:
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Synthesis-of-dendritic-lipid - Revision history

Paul.xu at 19:35, 20 July 2016