Unsaturated cationic lipid derivative, preparation method, and application in plasmid delivery system

A technology of cationic lipids and cationic liposomes, applied in liposome delivery, cyanide reaction preparation, chemical instruments and methods, etc., can solve the problems of difficult DNA delivery and release

Active Publication Date: 2020-05-01
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the molecular weight of DNA is much larger than that of siRNA and its stronger negative charge, the interaction between DNA and cationic liposomes is stronge

Method used

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  • Unsaturated cationic lipid derivative, preparation method, and application in plasmid delivery system
  • Unsaturated cationic lipid derivative, preparation method, and application in plasmid delivery system
  • Unsaturated cationic lipid derivative, preparation method, and application in plasmid delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Preparation of Dioleyl Glutamate (OA 2 -Glu), the chemical structure is as follows:

[0076]

[0077] Dissolve L-glutamic acid (5.00g, 33.9mmol) in 200mL of anhydrous toluene, add p-toluenesulfonic acid (6.44g, 37.4mmol) with stirring, raise the temperature to 140°C, and reflux for 2h. After cooling to room temperature, oleyl alcohol (19.2 g, 71.4 mmol) was added, the temperature was raised to 150° C., and the mixture was refluxed overnight. After the reaction, the toluene was removed by rotary evaporation to obtain a dark brown oil. Dissolved in 300mL chloroform, washed with saturated aqueous sodium bicarbonate (200mL×2), washed with saturated brine (200mL×1), dried over anhydrous sodium sulfate, concentrated after suction filtration to obtain a dark brown oil, which was purified by column chromatography ( Petroleum ether: ethyl acetate = 10: 1) to obtain 6.40 g of a colorless transparent oil, yield: 54%.

[0078] 1 H NMR (300MHz, CDCl 3 ): δ(ppm)5.43–5.28(m,4H...

Embodiment 2

[0080] Preparation of Boc group-protected lysine glutamic acid dioleyl ester (OA 2 -Glu-Lys(Boc) 2 ), the chemical structure is as follows:

[0081]

[0082] Boc-Lys(Boc)-OH (484mg, 2.085mmol) was dissolved in 30mL of chloroform and placed at 0°C, EDCI (639mg, 3.335mmol) and HOBT (451mg, 3.335mmol) were added sequentially with stirring. After the addition was complete, transfer to room temperature and stir for 3 h to obtain reaction solution A; 2 -Glu (1.35 g, 2.085 mmol) was dissolved in 20 mL of chloroform, triethylamine (872 μL, 6.254 mmol) was added with stirring, and stirred at room temperature for 1 h to obtain reaction solution B. The reaction solution B was slowly dropped into the reaction solution A, and stirred overnight at room temperature. After the reaction, wash twice with an appropriate amount of water, twice with an appropriate amount of 10% citric acid aqueous solution, once with an appropriate amount of saturated saline, dry over anhydrous sodium sulfat...

Embodiment 3

[0085] Preparation of Dioleyl Lysine Glutamate (OA 2 -Glu-Lys), the chemical structural formula is as follows:

[0086]

[0087] will OA 2 -Glu-Lys(Boc) 2 (481mg, 0.493mmol) was placed at 0°C, and 30ml of hydrogen chloride-1,4-dioxane solution (4.0M concentration) was slowly added dropwise. After the reaction, the reaction solution was concentrated to obtain 335 mg of a yellow gel-like solid, yield: 80.2%.

[0088] 1 H NMR (500MHz, CDCl 3 ):δ(ppm)8.21(brs,2H,NH 2 ),7.87(brs,2H,NH 2 ),5.40–5.30(m,4H,CH 2 CHCHCH 2 ),4.55–4.39(m,1H,NH 2 CH),4.14–3.99(m,4H,COOCH 2 ),3.27–3.08(m,1H,NH 2 CH),2.68–2.40(m,2H,NH 2 CH 2 ),2.19–2.10(m,2H,CH 2 CO), 2.08–1.93 (m, 8H, CH 2 CHCHCH 2,2H,NHCHCH 2 ),1.76–1.55(m,4H,COOCH 2 CH 2 ,2H,NHCH 2 CH 2 CH 2 CH 2 ), 1.36–1.25 (m, 44H, CH 2(oleyl) ,2H,NH 2 CH 2 CH 2 CH 2 ,2H,NH 2 CH 2 CH 2 ), 0.88(t, J=7.1Hz, 6H, CH 2 CH 3 ). 13 C NMR (125MHz, CDCl 3 ): δ (ppm) 173.32 (1C, CH 2 COOCH 2 ), 171.44 (1C, NHCHCO), 169.47 ...

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Abstract

The invention relates to the field of chemistry and preparations, in particular to an unsaturated cationic lipid derivative, a preparation method thereof, and application in a plasmid delivery system.The cationic lipid takes electropositive lysine as a hydrophilic head group, amino acid as a skeleton and an unsaturated alkane chain as a hydrophobic tail chain, so that the cationic lipid has the advantages of good biocompatibility, high safety and the like. The synthesis method is simple and quick, low in synthesis cost and beneficial to large-scale production. The cationic liposome constructed on the basis of the unsaturated cationic lipid designed by the invention can stabilize the load plasmid without leakage, has better gene transfection efficiency on various tumor cells and primary cells than positive control Liponectamine2000, and has no obvious cytotoxic effect on cells. The invention provides a safe and efficient cationic lipid and non-viral vector platform for plasmid delivery.

Description

technical field [0001] The invention relates to the field of chemistry and preparation, in particular to an unsaturated primary amine cationic lipid derivative, a preparation method and an application in a plasmid delivery system. Background technique [0002] Gene therapy is the introduction of exogenous genes, such as plasmids (DNA), small interfering RNA (siRNA), mRNA, etc., into target cells to correct or compensate for diseases caused by gene defects or abnormal gene expression, including genetic diseases, malignant tumors, metabolic disease, cardiovascular disease, and autoimmune disease. Among them, the plasmid is a kind of genetic unit capable of autonomous replication. Compared with other gene therapy substances, it has advantages such as continuous expression, so it has been widely studied. [0003] However, due to the characteristics of water solubility, negative charge, easy to be degraded by nucleases, and the need for nuclear expression, direct injection of ep...

Claims

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Application Information

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IPC IPC(8): C07C227/18C07C229/24C07J9/00C07C229/08C07K5/068A61K9/127A61K47/18A61K48/00
CPCC07C227/18C07C229/24C07J9/00C07C229/08C07K5/06086A61K9/1271A61K47/183A61K48/005
Inventor 张灿莫希叶乐杜俊杰鞠曹云林子鸣薛玲静
Owner CHINA PHARM UNIV
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