CN105461646B - Containing Macrocyclic polyamine [12] aneN3Cation lipid, transgene carrier and preparation method thereof - Google Patents
Containing Macrocyclic polyamine [12] aneN3Cation lipid, transgene carrier and preparation method thereof Download PDFInfo
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- CN105461646B CN105461646B CN201610021249.XA CN201610021249A CN105461646B CN 105461646 B CN105461646 B CN 105461646B CN 201610021249 A CN201610021249 A CN 201610021249A CN 105461646 B CN105461646 B CN 105461646B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D255/00—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00
- C07D255/02—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00 not condensed with other rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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Abstract
The present invention provides a kind of containing Macrocyclic polyamine [12] aneN3Cation lipid and transgene carrier containing the cation lipid.The invention also discloses the preparation methods of the cation lipid and transgene carrier.Cation lipid provided by the invention contains fatty acid chain, the cohesion concentration of DNA can effectively be reduced, and it forms nano particle using its hydrophobic function and DNA and is entered in cell by endocytosis, conducive to cross-film, there is low cytotoxicity using the transgene carrier that cation lipid is formed, there is higher transfection efficiency；The preparation method of cation lipid and transgene carrier is simple, maturation, is easy to control.
Description
Technical field
The present invention relates to cation lipid and transgene carriers, and in particular to a kind of [12] containing Macrocyclic polyamine simultaneously
aneN3Cation lipid, the transgene carrier and preparation method thereof containing such cation lipid.
Background technology
Gene therapy is that the normal gene of external source is imported target cell by certain method, instead of or compensate some and lack
It falls into or abnormal gene is to achieve the purpose that treatment.Gene therapy provides possibility for the treatment of many diseases, such as：Cancer
Disease, diabetes, cystic fibrosis, AIDS, angiocardiopathy etc..
The key of gene therapy is therapeutic gene efficiently to be imported into the cell, however exposed DNA is generally to relax
The line style spiral form of exhibition exists, and volume is larger, and DNA molecular sheet is as negatively charged anion, into can be with when cell
Electrostatic repulsion is generated between the anion of cell membrane outer layer.These unfavorable factors all make DNA molecular not by other
In the case of technological means, cell membrane can not be independently passed through, therefore it is before gene therapy is successful to develop efficient gene carrier
Put forward condition.
Genophore includes two class of viral vectors and non-virus carrier.Viral vectors is a kind of efficient transfection reagent, but
It is it there are disadvantage, such as immunogenicity, carcinogenicity, the size of gene is easy to be limited while depositing by the size that virus is accommodated
In the possibility of variation, therefore its safety is troubling.Viral vectors non-immunogenicity is convenient for large-scale production, but is transfected
Efficiency is less than viral vectors.Therefore the efficient non-viral gene vector of low toxicity is developed to be of great significance.
Invention content
As various extensive and careful research and experiment as a result, it has been found by the inventor that more in big ring
Amine [12] aneN3Fatty acid chain is introduced in cationoid lipid, can effectively reduce the cohesion concentration of DNA, and hydrophobic using its
Sexual function forms nano particle with DNA and is entered in cell by endocytosis, is conducive to cross-film, is formed using the cation lipid
Transgene carrier has low cytotoxicity, has higher transfection efficiency.Based on this discovery, the present invention is completed.
It is excellent it is an object of the invention to solve at least the above and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide one kind having multi-functional Macrocyclic polyamine [12] aneN3Cation lipid
Transgene carrier not only has low cytotoxicity, but also has higher transfection efficiency simultaneously, to promote the hair of gene therapy
Exhibition.
It is a still further object of the present invention to provide containing Macrocyclic polyamine [12] aneN3Cation lipid preparation method and
The preparation method of transgene carrier containing the cation lipid.
In order to realize these purposes and other advantages according to the present invention, provide a kind of containing Macrocyclic polyamine [12] aneN3
Cation lipid, the structure formula (I) of the cation lipid is as follows：
In formula (I), R is long chain fatty acids.
Preferably, wherein R is
WithIn one kind.
The purpose of the present invention can also be further by Macrocyclic polyamine [12] aneN3Cation lipid preparation method come it is real
Existing, the synthetic route of this method is as follows：
Wherein, R is as defined in claim 1, and the method specific steps include：
Step 1: 1,3- dibromopropanes are added into the acetonitrile solution dissolved with 1 compound of formula, be stirred at reflux reaction 8~
10 hours, removing solvent is concentrated under reduced pressure and obtains yellow solidliquid mixture, ethyl alcohol and hydrobromic acid are added thereto, is stirred at reflux reaction
It 7~8 hours, after reaction, stands, extracts organic layer with water, combining water layer is concentrated under reduced pressure to give light yellow or white solid
Solid is dried in vacuo to obtain 2 compound of formula, 2 compound of formula is dissolved in tetrahydrofuran, three second are added in ice-water bath by body
Amine and tertbutyloxycarbonyl acid, react at room temperature 8~10 hours, reaction product obtains 3 compound of formula through column chromatography for separation；
Step 2: 3 compound of formula that the step 1 obtains heats instead with sodium azide in N,N-dimethylformamide
It answers 8~10 hours, after reaction, saturated salt solution is added into system, is then extracted with ethyl acetate, merge organic phase,
With saturated common salt water washing organic phase, concentrated solvent obtains 4 compound of formula after drying organic phase with anhydrous sodium sulfate；
Step 3: 4 compound of formula that the step 2 obtains is dissolved in anhydrous tetrahydrofuran and methyl alcohol mixed liquor,
In, the volume ratio of anhydrous tetrahydrofuran and methanol is 1:1, catalyst is added under stirring into system, under an atmosphere of hydrogen instead
It answers 8~10 hours, after reaction, decompression filters, and filtrate decompression is concentrated to give 5 compound of formula；
Step 4: 5 compound of formula that the step 3 obtains is stirred to react to obtain 6 compound of formula with long chain fatty acids；
Step 5: the formula 6 that the step 4 obtains is stirred to react to obtain 7 compound cation fat of formula with trifluoroacetic acid
Matter.
Preferably, wherein in the step 4, specific steps include：Long chain fatty acids are dissolved in chloroform
In, dicyclohexylcarbodiimide is added, is stirred under ice-water bath, when white precipitate to be generated, 5 compound of formula and 4- diformazans is added
Aminopyridine, after mixture is stirred 24 hours under ice bath, decompression filters, and obtains that acetic acid second is added after filtrate decompression is spin-dried for
Ester, after 0.5 hour is again stirring under ice bath, decompression again filters, and obtains carrying out column chromatography after filtrate is spin-dried for, the column chromatography
The eluant, eluent used is ethyl acetate and methanol, wherein the volume ratio of ethyl acetate and methanol is 30: 1.
Preferably, wherein in the step 5, specific steps include：6 compound of formula that step 4 is obtained is molten
In anhydrous methylene chloride, under ice bath, it is added dropwise to the dichloromethane solution dissolved with trifluoroacetic acid thereto, stirs 6 at room temperature
Hour, after reaction, solvent and the decompression of excessive trifluoroacetic acid are spin-dried for, formula 7 is recrystallized to give with dichloromethane-n-hexane
Compound cation lipid.
Preferably, wherein in the step 2, the molar ratio of 3 compound of formula and sodium azide is 1: 1.5, heating
The temperature of reaction is 68 DEG C.
Preferably, wherein in the step 3, the catalyst be palladium-carbon catalyst, wherein 4 compound of formula with
The mass ratio of palladium-carbon catalyst is 10: 1.
In order to realize these purposes and other advantages according to the present invention, a kind of transgene carrier is additionally provided, described turn
Genophore is by Macrocyclic polyamine [12] aneN3Cation lipid, dioleoylphosphatidylethanolamine and high-purity sterilizing buffer solution group
At, wherein the molar concentration of the cation lipid is 2.0mM, the cation lipid and the dioleoyl phospholipid acyl ethyl alcohol
The molar ratio of amine is 1:2.
The purpose of the present invention can also further be realized that this method specifically includes step by the preparation method of transgene carrier
Suddenly：The cation lipid, dioleoylphosphatidylethanolamine and anhydrous chloroform are added in the flask of high-temperature sterilization, after dissolving
It is concentrated under reduced pressure to give transgene carrier film, the transgene carrier film is dried in vacuo removal residual chloroform, turns base after drying
It is made into the solution of required concentration because carrier film is mixed with the tris-HCI buffer for being preheating to 70 DEG C in advance,
Ultrasound obtains the transgene carrier, wherein the molar concentration of tris-HCI buffer is 10mM, and pH is
7.4。
The purpose of the present invention can also be carried further by the cation lipid of Macrocyclic polyamine [12] aneN3 in prepare transgenosis
Application in body is realized.
The present invention includes at least following advantageous effect：
1, cation lipid containing aliphatic chain is provided, can effectively reduce the cohesion concentration of DNA, and utilize its hydrophobicity
The nano particle that function forms suitable size with DNA is entered by endocytosis in cell；
2, the transgene carrier cytotoxicity formed by the cation lipid containing aliphatic chain is relatively low, shows well
Transfection, some Lipofectamine 2000 being even more than commercialized, this is containing [12] aneN3Unit compound is made
Good place mat has been made for the further research of genophore；
3, the preparation method of cation lipid and transgene carrier is simple, maturation, is easy to control；
4, the compatibility that transgene carrier and cell have had has the potentiality as non-virus carrier in gene therapy；
5, transgene carrier can all wrap up DNA, can be used as DNA cross-films, transhipment good carrier；
6, the grain size after transgene carrier and DNA agglomerate.The particle of 100-250nm sizes is formed with DNA, this is successfully to wear
Enter cell membrane and realizes that gene transfection has great importance.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the agarose gel electrophoresis experimental result schematic diagram of the transgene carrier and DNA compounds of the present invention；
Cells of the Fig. 2 for transgene carrier of the present invention and the compound of Plasmid DNA in HepG2 and A549 cells
Toxicity figure；
Fig. 3 is the grain size shape appearance figure of transgene carrier of the present invention and the compound of Plasmid DNA；
Fig. 4 is transfection picture of the compound of transgene carrier of the present invention and DNA in A549 cells.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
<Example 1>
A kind of cation lipid of Macrocyclic polyamine [12] aneN3, the structural formula of the cation lipid are as follows：
In formula (I), R is long chain fatty acids.
Wherein, R is
WithIn one kind.
The method of cation lipid, synthetic route are as follows：
The method specific steps include：
Step 1: 1,3- dibromos are added into the 30.0mL acetonitrile solutions dissolved with 1 compound of formula (3.0g, 16.5mmol)
Propane (98.5mmol), is stirred at reflux reaction 8~10 hours, stops reaction when having a large amount of white solids to occur in bottle.By second
Nitrile vacuum rotary steam removes, and obtains yellow solidliquid mixture.Into obtained yellow solidliquid mixture be added 15.0mL ethyl alcohol and
15.0mL hydrobromic acids are stirred at reflux 7-8 hours；After reaction, it waits for that reaction temperature is reduced to room temperature, stands, reaction solution layering,
Water phase is collected, lower layer's organic phase water is extracted 3 times, combining water layer, water is largely removed using the decompression of ethyl alcohol revolving, is obtained
Light yellow or white solid, decompression suction filtration is carried out by solid, and white solid, which is put into vacuum, after being washed twice with cold ethyl alcohol does
It is dried overnight for 50 DEG C in dry case, obtains 2 compound 8.0g of white solid formula, yield：90%.Take above-mentioned white solid 7.2g
(13.5mmol) is added the dissolving of 80.0mL tetrahydrofurans, 8.0g triethylamines is added into mixed liquor under ice-water bath
(79.1mmol, 6.0e.q.) is sour (32.1mmol, 2.4e.q.) with 7.0g tertbutyloxycarbonyls, and reaction at room temperature is stayed overnight.Stop anti-
It answers, decompression filters, and filter cake is washed 3 times with tetrahydrofuran, merges organic phase, and the purification of crude product column chromatography for separation is obtained after concentration
(ethyl acetate:Petroleum ether=1:10) 3 compound of bright yellow solid formula, 5.4 g, yield 82%, are obtained；
Step 2: 3 compound of formula (0.5g, 1.0mmol) that the step 1 obtains is dissolved in N,N-dimethylformamide
In, sodium azide (0.1g, 1.5mmol) is added, reaction is heated to 68 DEG C, reacts 8~10 hours, after reaction, to system
Interior addition 20.0mL saturated salt solutions, are then extracted with ethyl acetate 3 times, merge organic phase, organic with saturated common salt water washing
Phase, concentrated solvent obtains 4 compound of formula (0.43g, 0.95mmol), yield after drying organic phase with anhydrous sodium sulfate：93%；
Step 3: 4 compound of formula (0.2g, 0.44mmol) that the step 2 obtains is dissolved in 14.0mL anhydrous four
Hydrogen furans and methyl alcohol mixed liquor, wherein the volume ratio of anhydrous tetrahydrofuran and methanol is 1:1, it is added into system under stirring
Palladium-carbon catalyst 0.02g, overnight, reaction mixture decompression filters, and filtrate decompression is concentrated to give yellow for reaction under an atmosphere of hydrogen
5 compound of oily formula (0.17g, 0.4mmol) yield：90%；
Step 4: 5 compound of formula that the step 3 obtains is stirred to react to obtain 6 compound of formula with long chain fatty acids,
Wherein, R takes different aliphatic acid, and synthetic method is similar, specially：Aliphatic acid (0.30 mmol) is dissolved in chloroform,
Dicyclohexylcarbodiimide (0.075g, 0.36mmol, 1.2 eq) is added, is stirred under ice-water bath, when white precipitate to be generated,
5 compound of addition formula (0.130g, 0.3 mmol), 4-dimethylaminopyridine (7.4mg, 0.2eq.), by mixture under ice bath
After stirring 24 hours, decompression filters, and obtains that ethyl acetate is added after filtrate decompression is spin-dried for, is again stirring under ice bath 0.5 hour
Afterwards, decompression filters again, obtains carrying out column chromatography after filtrate is spin-dried for, ethyl acetate: methanol=30: 1 obtains yellow oil formula
6 compounds, yield：40-55%；
Step 5: the formula 6 that the step 4 obtains is stirred to react to obtain 7 compound cation fat of formula with trifluoroacetic acid
Matter, wherein R takes different aliphatic acid, and synthetic method is similar, specially：By 6 compound of formula (0.14mmol) be dissolved in 3.0mL without
In water dichloromethane, under ice bath, it is added dropwise to the dichloromethane that 1.0mL trifluoroacetic acids are dissolved in 1.0mL thereto, stirs at room temperature
It mixes 6 hours, after reaction, solvent and the decompression of excessive trifluoroacetic acid is spin-dried for, are recrystallized to give with dichloromethane-n-hexane
7 compound of yellow solid formula, yield：90-95%.
Wherein, when R is
With
6 compound of definition is followed successively by 6a~6f, and 7 compound of formula is followed successively by 7a~7f.
The dosage of reactant and other reagents is not limited to above-described embodiment in above-mentioned synthetic method, for being familiar with ability
It for the personnel in domain, is easily implemented, as long as synthetic agent ratio and synthetic method are similar, belongs to the conjunction in the present invention
Within the scope of method.
The structural formula of 3 compound of formula, nuclear-magnetism (1H-NMR,13C NMR), infrared and mass spectrum (ESI-MS) is characterized as below：
Nuclear-magnetism1H NMR(400MHz,CDCl3) δ 3.34 (t, J=6.4Hz, 2H, BrCH2(CH2)2), 3.33 (dd, J=
11.3,6.0Hz,8H,Boc-N(CH2)2CH2(CH2)2), N-Boc 2.54 (t, J=6.4Hz, 2H, Br (CH2)2CH2-),2.41
(t, J=6.2Hz 4H ,-N (CH2CH2)2),1.94(m,2H, BrCH2CH2CH2-),1.84(m,2H,Boc-NCH2CH2CH2N-
Boc),1.81-1.75(m,4H, -NCH2CH2CH2CH2N-Boc),1.45(s,18H,2Boc).
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ156.38,79.42,51.47,50.09,45.12,43.89, 32.19,
29.25,28.60,27.41,26.68.
IR(KBr,cm-1):3120,2976,2928,2796,1688,1470,1407,1368,1248,1164, 1056,
981,865,768,694.
Mass spectrum ESI-MS:M/z=492.8 ([M+H]+).
The structural formula of 4 compound of formula, nuclear-magnetism (1H-NMR,13C NMR), infrared and mass spectrum (ESI-MS) is characterized as below：
Nuclear-magnetism1H NMR(400MHz,CDCl3)δ3.38-3.28(m,10H,N3CH2(CH2)2-, Boc-N(CH2)2(CH2)2), N-Boc 2.48 (t, J=7.0Hz, 2H, N3(CH2)2CH2-),2.4(m,4H, -N(CH2CH2)2), 1.85 (tt, J=14.0,
7.1Hz,2H,N3CH2CH2CH2-),1.80-1.72(m,4H, -N(CH2CH2)2), 1.68 (tt, J=13.7,6.9Hz, 2H,
Boc-NCH2CH2CH2N-Boc),1.45(s, 18H,2Boc).
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ156.42,79.47,50.51,50.03,49.81,45.15, 43.96,
28.63,27.52,26.64,25.60.
IR(KBr,cm-1):3114,2958,2096,1688,1460,1398,1254,1158,873,804,613.
Mass spectrum ESI-MS:M/z=455.8 ([M+H]+).
The structural formula of 5 compound of formula, nuclear-magnetism (1H-NMR,13C NMR), infrared and mass spectrum (ESI-MS) is characterized as below：
Nuclear-magnetism1H NMR(400MHz,CDCl3)δ3.33(m,10H,NH2(CH2)2CH2-),Boc-N (CH2)2(CH2)2N-
), Boc 2.70 (t, J=7.0Hz, 2H, NH2CH2CH2CH2-),2.43(m,6H, Boc-NCH2CH2CH2N-Boc,-N
(CH2CH2)2),1.88-1.82(m,2H,NH2CH2CH2CH2-), 1.78-1.71(m,4H,-N(CH2CH2)2),1.45(s,
18H,2Boc).
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ154.12,79.87,51.80,51.61,47.01,46.73, 39.86,
29.83,29.10,28.53.
IR(KBr,cm-1):3401,2972,2365,1692,1477,1415,1366,1249,1167,863,772.
Mass spectrum ESI-MS:M/z=429.8 ([M+H]+).
The structural formulas of formula 6a-6f compounds, nuclear-magnetism (1H-NMR,13C NMR) and mass spectrum (ESI-MS) be characterized as below：
6a compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3) δ 6.21 (t, J=5.2Hz, 1H), 3.26 (dd, J=
11.3,6.0Hz, 8H), 3.18 (dd, J=12.7,6.6Hz, 2H), 2.38 (m, 6H), 2.08 (t, J=7.6 Hz, 2H), 1.79
(m,2H),1.73-1.64(m,4H),1.59-1.51(m,4H),1.38(s,18H), 1.25-1.16(m,12H),0.80(t,J
=6.8Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ172.20,155.28,78.33,50.51,48.86, 44.09,
43.10,37.22,35.84,30.84,28.37,27.48,25.37,24.87,21.64,13.10.
Mass spectrum ESI-MS:M/z=583.6 ([M+H]+).
6b compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3) δ 6.28 (t, J=5.0Hz, 1H), 3.26 (dd, J=
11.3,5.9Hz, 8H), 3.18 (dd, J=12.6,6.6Hz, 2H), 2.38 (m, 6H), 2.08 (t, J=7.6Hz, 2H),
1.83-1.75(m,2H),1.73-1.65(m,4H),1.56(m,4H),1.38(s,18H), 1.19(m,16H),0.80(t,J
=6.8Hz, 3H)
6c compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3) δ 6.23 (t, J=5.3Hz, 1H), 3.35- 3.21 (m,
8H), 3.18 (dd, J=12.7,6.6Hz, 2H), 2.38 (dd, J=13.1,6.6Hz, 6H), 2.14-2.01 (m, 2H), 1.79
(m, 2H), 1.73-1.64 (m, 4H), 1.59-1.51 (m, 4H), 1.38 (s, 18H), 1.19 (m, 20H), 0.80 (t, J=
7.0Hz,3H).
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ172.21,155.28,78.33,50.51,48.88, 44.11,
43.11,37.21,35.85,30.91,28.48,27.48,24.93,21.67,13.11.
Mass spectrum ESI-MS:M/z=639.8 ([M+H]+).
6d compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3)δ6.11(s,1H),3.31-3.23(m,8H), 3.18(dd,
J=12.5,6.5Hz, 2H), 2.38 (dd, J=12.3,6.1Hz, 6H), 2.07 (t, J=7.6Hz, 2H), 1.83-1.75 (m,
2H), 1.73-1.65 (m, 4H), 1.56 (d, J=6.2Hz, 4H), 1.38 (s, 18H), 1.18 (m, 24H), 0.81 (t, J=
6.7Hz,3H).
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ172.24,155.24,78.26,76.67,76.35,76.04,
50.37,48.98,44.13,43.04,37.13,35.77,30.91,28.69,28.55,28.44,28.34,27.49,
26.66,25.20,24.90–24.73,21.67,13.12.
Mass spectrum ESI-MS:M/z=667.7 ([M+H]+).
6e compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3) δ 5.98 (s, 1H), 3.34 (dd, J=14.4,6.8 Hz,
8H), 3.27 (t, J=6.6Hz, 2H), 2.47 (m, 6H), 2.18-2.12 (m, 2H), 1.90-1.84 (m, 2H), 1.81-1.73
(m, 4H), 1.62 (d, J=7.4Hz, 4H), 1.46 (s, 18H), 1.25 (m, 28H), 0.88 (t, J=6.8Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ173.29,156.23,79.29,51.32,49.75,45.10,
44.04,38.02,36.73,31.90,29.68,29.54,29.40,29.34,28.46,25.90,22.66,14.11.
Mass spectrum ESI-MS:M/z=695.0 ([M+H]+).
6d compounds：Nuclear-magnetism1H NMR(400MHz,CDCl3)δ5.86(s,1H),5.38-5.33(m,2H), 3.54-
3.28 (m, 8H), 3.28-3.21 (m, 2H), 2.77 (t, J=6.4Hz, 2H), 2.45 (dd, J=11.0,6.4Hz, 4H),
2.18-2.11 (m, 2H), 2.03 (dd, J=15.6,7.0Hz, 4H), 1.91-1.81 (m, 2H), 1.81-1.70 (m, 4H),
1.62 (s, 4H), 1.46 (s, 18H), 1.32-1.24 (m, 20H), 0.90-0.87 (t, J=7.4Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,CDCl3)δ172.49,155.40,129.18,129.01,128.95, 128.72,
128.72,78.63,76.48,76.16,75.84,59.37,50.41,47.80,44.17,43.62, 36.81,35.75,
30.89,30.50,28.70,28.33,27.46,26.18,24.84,24.62,24.19,21.61, 20.01,13.13.
Mass spectrum ESI-MS:M/z=693.0 ([M+H]+).
The structural formulas of formula 7a-7f compounds, nuclear-magnetism (1H-NMR,13C NMR), infrared and mass spectrum (ESI-MS) characterization such as
Under：
7a compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ3.37-3.09(m,16H),2.20(m,8H), 1.99-1.86
(m, 2H), 1.53 (m, 2H), 1.23 (m, 12H), 0.83 (t, J=6.7Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ176.33,163.10,162.75,162.40,162.05,120.78,
117.87,114.97,52.37,47.24,42.12,41.12,35.91,31.83,29.47,29.13,27.97, 25.65,
23.89,22.51,20.58,17.65,13.72.
Infrared IR (KBr, cm-1):3423,2922,2851,1670,1642,1557,1468,1203,1131,837,
798,721,517,435.
Mass spectrum HR-MS:383.2534.
7b compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ3.23(m,14H),3.03(m,2H),2.19- 2.07(m,
6H), 1.84 (m, 2H), 1.51 (m, 2H), 1.36 (m, 2H), 1.21 (m, 16H), 0.80 (t, J=6.7Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ176.33,163.10,162.75,162.40,162.05,120.78,
117.87,114.97,52.37,47.24,42.12,41.12,35.91,31.83,29.47,29.13,27.97, 25.65,
23.89,22.51,20.58,17.65,13.72.
Infrared IR (KBr, cm-1):3423,2922,2851,1670,1642,1557,1468,1203,1131,837,
798,721,517,435.
Mass spectrum HR-MS:411.2277.
7c compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ3.31-3.17(m,16H),2.18(m,8H), 1.90(s,
2H), 1.53 (m, 2H), 1.24 (m, 20H), 0.84 (t, J=6.5Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ176.02,163.00,162.65,162.30,161.95,120.82,
117.92,115.01,112.11,48.42,42.82,42.50-41.92,36.13,35.81-35.24,31.90, 29.78,
29.40,29.19,25.76,22.88,20.95,18.94,13.73.
Infrared IR (KBr, cm-1):3441,2926,2854,1676,1638,1560,1467,1433,1202,1135,
836,799,721,519,473.
Mass spectrum HR-MS:439.2774.
7d compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ3.22-3.03(m,16H),2.03(m,8H), 1.77(s,
2H), 1.33 (m, 2H), 1.05 (m, 24H), 0.65 (t, J=8.0Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ176.55,162.53,162.17,161.81,161.45,120.40,
117.51,114.62,111.72,52.70,46.85,41.78,40.64,35.87,35.61,35.28,31.78, 29.69,
29.26,29.05,25.57,23.92,22.43,20.44,17.20,13.56.
Infrared IR (KBr, cm-1):3257,2922,2852,2657,1670,1642,1555,1487,1468,1436,
1372,1203,1130,1026,922,839,798,747,721,598,517,435.
Mass spectrum HR-MS:468.4435.
7e compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ3.40-3.16(m,16H),2.17(m,8H), 1.89(m,
2H), 1.51 (m, 2H), 1.24 (m, 28H), 0.83 (t, J=6.4Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ176.35,162.64,162.29,161.93,161.57,120.48,
117.58,114.68,111.79,52.57,46.71,41.74,40.55,35.75,31.84,29.82,29.34, 29.10,
25.59,23.91,22.48,20.45,17.12,13.62.
Infrared IR (KBr, cm-1):33424,2921,2852,1671,1642,1558,1468,1435,1372, 1203,
1131,837,798,721,598,517.
Mass spectrum HR-MS:495.3536.
7f compounds：Nuclear-magnetism1H NMR(400MHz,D2O)δ5.31(m,2H),3.50-2.94(m,16H), 2.16(m,
8H), 2.07-1.87 (m, 6H), 1.55 (s, 2H), 1.26 (s, 20H), 0.86 (t, J=3.5Hz, 3H)
Nuclear-magnetism13C NMR(101MHz,D2O)δ175.79,163.18,162.83,162.48,162.13,129.68,
129.42,127.85,120.80,117.89,114.99,112.09,48.51,43.18,36.49,35.95,31.89,
31.42,29.76,29.29,27.12,25.64,22.52,20.93,19.07,13.77.
Infrared IR (KBr, cm-1):3433,2928,2856,1677,1432,1204,1135,836,799,620, 517.
Mass spectrum HR-MS:493.4373.
<Example 2>
Preparation containing the transgene carrier of cation lipid formula 7a-7f compounds in embodiment 1
Raw material includes the cation lipid formula 7a-7f compounds for implementing 1 preparation, dioleoylphosphatidylethanolamine DOPE
(dioleoylphosphotidylethanolamine) and anhydrous chloroform.By the cation of synthesis in the flask of high-temperature sterilization
1 compound of lipid formula (0.005mmol) is with DOPE with molar ratio 1:2 are dissolved in 2.5mL anhydrous chloroforms, after being sufficiently mixed dissolving
Solvent is depressurized at room temperature and is spin-dried for obtaining transgene carrier film, mixture is put into drying in vacuum drying chamber, and (drying time 12 is small
When, 25 DEG C of temperature) removal residual chloroform, it is dry after transgene carrier film and be preheating to 70 DEG C in advance, trihydroxy methyl amino first
Alkane-hydrochloric acid (Tris-HCl) buffer solution (10mM, pH 7.4) mixes, and the amount that buffer solution is added makes the ultimate density of lipid
For 1.0mM.It is finally that mixture is 20 minutes ultrasonic in 60 DEG C of ultrasounds, it is placed in 4 DEG C of refrigerators and saves backup.
<Example 3>
The transgene carrier that embodiment 2 is prepared carries out the detection of following several respects to it：
(1) the agarose gel electrophoresis experiment transgene carrier of transgene carrier and pEGFP-N1 plasmid composites with
The preparation of pEGFP-N1 plasmid composites
At room temperature, the transgene carrier of 7a-7f preparations, wherein transgene carrier and pEGFP-N1 are separately added into PE pipes
N/P ratios be 0,2,4,8,16, pEGFP-N1 plasmid quality be 0.125 μ g, the ultra-pure water of respective volume is added, makes reaction solution
Final volume is maintained at 20 μ L, and centrifugation is uniformly mixed.It is put into 37 DEG C of waters bath with thermostatic control, after keeping the temperature 1 hour, the 10 of 2 μ L is added
× Loading Buffer sample-loading buffers terminate reaction.
The preparation of Ago-Gel
280mg agaroses are weighed, 40mL 1 × TAE buffer solutions are added, microwave heating makes agarose particle be completely dissolved,
Obtain colourless transparent liquid.It waits for that temperature is down to 60 DEG C or so, 2 μ L Goldview, II nucleic acid color developing agents is added, take advantage of after mixing
Heat is poured into the plastic tank with comb.After gel completely solidification, comb is carefully extracted, and will carry solidifying within cooling 40 minutes
The plastic tank of glue is put into electrophoresis tank, and 1 × TAE electrophoretic buffers are added and did not had glue surface slightly.
The agarose gel electrophoresis of compound is tested
Take the transgene carrier of above-mentioned preparation that the well on gel is added with 10 μ L of pEGFP-N1 plasmid composites respectively
It is interior.Electrophoresis lid is covered, electrophoresis stops after forty minutes under the conditions of voltage 80V, takes out gel and is exposed in gel electrophoresis is at phase system
Sampling.
As shown in Figure 1, being tested using agarose gel electrophoresis, we observe by cationic transgene carrier 7a-7f systems
The operative condition of standby transgene carrier and pEGFP-N1 plasmids.As seen from Figure 1, as the N/P of transgene carrier and pEGFP-N1
Major part pEGFP-N1 plasmids are all trapped in loading hole when than being equal to 2, and when N/P is equal to 4, all pEGFP-N1 plasmids are all detained
In loading hole, show that transgene carrier can all wrap up pEGFP-N1 plasmids.
(2) transgene carrier cytotoxicity experiment
The preparation of transgene carrier and pEGFP-N1 plasmid composites
At room temperature, the transgene carrier of 7a-7f preparations, wherein transgene carrier and pEGFP-N1 are separately added into PE pipes
N/P ratios be 2,4,8,16, pEGFP-N1 plasmid quality be 1.875 μ g, the DMEM of respective volume is added, makes the final of reaction solution
Volume is maintained at 300 μ L.It is put into 37 DEG C of waters bath with thermostatic control, keeps the temperature 1 hour.
Cytotoxicity experiment
A549 the and HepG2 cells of exponential phase will be collected with pancreatin digestion to enter 96 holes every about 7000, hole cell kind
In plate, contain 5%CO in 37 DEG C224 hours of incubator culture, make the fusion for growing to 70%-80% of cell.Discard hole
Interior original culture medium, is washed 1-2 times with PBS buffer solution, and the transgenosis that the above-mentioned various concentration prepared is separately added into every hole carries
Three parallel holes are arranged with 100 μ L of pEGFP-N1 plasmid composites, each concentration in body；Using the DMEM without transgene carrier
Culture medium is used as positive control as blank control, using commercialization Lipofectamine 2000, there was only DMEM without cell
As blank control.All culture mediums are sucked out after 4 hours, 200 μ the L DMEM containing 10%FBS, Yu Pei is added per hole thereto
It supports after being cultivated 24 hours in case, 10 μ L MTT solution is added to every hole, all addition liquid, the hyacinthine of generation are sucked out after 4 hours
150 μ L DMSO dissolvings of crystallization, absorbance of each hole at 490nm is measured in being shaken on shaking table with microplate reader after ten minutes
Value.Cell survival rate (%)=[A490test- blank]/[A490control- blank] × 100 are calculated as follows.
As shown in Fig. 2, the cytotoxicity by measuring above-mentioned six kinds of transgene carriers in HepG2 and A549 cells, hair
This existing six kinds of cytotoxicities are all relatively low.In A549 cells, when N/P ratios are 2 and 4, the survival rate of cell is all 80%
Left and right.The rule totally presented is：With the increase of N/P ratio, natural death of cerebral cells number increases, this is because transgene carrier nitrogen
Atom can increase cytotoxicity；DNA amounts are certain, and with the increase of N/P ratio, transgene carrier concentration increases, and toxicity increases.
Therefore in order to achieve the purpose that cytotoxicity is small and transfection efficiency is high, select proper N/P ratio quite important.
(3) transgene carrier and the grain size of pEGFP-N1 plasmid composites are tested
The preparation of transgene carrier and pEGFP-N1 plasmid composites
At room temperature, transgene carrier is separately added into PE pipes, the N/P ratios of wherein transgene carrier and pEGFP-N1 are
4, pEGFP-N1 plasmid quality are 0.25 μ g, and the ultra-pure water of respective volume is added, the final volume of reaction solution is made to be maintained at 500 μ
L, centrifugation are uniformly mixed.It is put into 37 DEG C of waters bath with thermostatic control, keeps the temperature 1 hour.It (in scanning electron microscope experiment, need not dilute, directly
The compound of transgene carrier and DNA are added drop-wise on silicon chip, room temperature naturally dry).
Grain size and pattern test
With reaction solution rinse cuvette repeatedly, finally 500 μ L reaction solutions are transferred in cuvette and are measured.Setting is surveyed
It is 25.00 DEG C to try temperature, measures 10 groups of panel datas of collection every time and is averaged.
As shown in figure 3, using dynamic laser light scattering technology, we have studied the grains after transgene carrier and DNA cohesions
Diameter.The agglomeration reagent of formation can form the particle of 130-220nm sizes with DNA, this is successfully to penetrate cell membrane to realize gene
Transfection has great importance.
(4) the outer transfection experiment of the composite body of transgene carrier and pEGFP-N1
The preparation of transgene carrier and pEGFP-N1 compounds
At room temperature, transgene carrier is separately added into PE pipes, the N/P ratios of wherein transgene carrier and pEGFP-N1 are
4, pEGFP-N1 plasmid quality are 1.25 μ g, and the DMEM of respective volume is added, the final volume of reaction solution is made to be maintained at 200 μ L,
Mixing is gently blown and beaten, is put into 37 DEG C of waters bath with thermostatic control, half an hour is kept the temperature；And pass through the method prepare transgenosis carrier
The compound of Lipofectamine2000 and pEGFP-N1 plasmids is tested as a contrast.
In-vitro transfection is tested
The A549 cells that exponential phase is collected with pancreatin digestion will be every about hole 7.0 × 104-9.0×104A cell kind
Enter in the burnt culture dish of copolymerization, contains 5%CO in 37 DEG C224 hours of incubator culture, make cell grows to 70%-80%'s
Fusion.Original culture medium in hole is discarded, is washed 1-2 times with PBS buffer solution, the above-mentioned transgenosis prepared is separately added into every hole and is carried
Body and 200 μ L of pEGFP-N1 plasmid composites.All culture mediums are sucked out after 4 hours, 500 μ L are added per hole thereto contains
The DMEM of 10%FBS after being cultivated 24 hours in incubator, takes out the burnt culture dish of copolymerization and is observed under inverted fluorescence microscope
GFP protein expression situations.
According to the compound of above-mentioned method prepare transgenosis carrier Lipofectamine2000 and pEGFP-N1, according to
Transgene carrier Lipofectamine2000 and pEGFP-N1 compounds are added in A549 cells and carry out body by above-mentioned method
Outer transfection experiment.
As shown in figure 4, can be obtained by cell transfection assays：The transgene carrier prepared by 7a-7f can carry
Luciferase plasmid, which enters in cell, is expressed, and compound 7c, 7f show best transfection, and transfection has surpassed
The relatively good lipofectamine 2000 of the transfection efficiency of commercialization is crossed, is obtained by experiment, transgene carrier mistake is being formed
The addition of DOPE can make compound that DNA is condensed into the particle of suitable size in journey, while can improve certain transfection
Efficiency.It is obtained by a series of experiments：The cationic transgene carrier of experiment synthesis has cell compatibility, has and is used as gene
The potentiality of non-virus carrier in treatment.
In conclusion the introducing of the aliphatic chain in the present invention can effectively reduce the cohesion concentration of DNA, and it is hydrophobic using its
Sexual function forms nano particle with DNA and is entered in cell by endocytosis.Six compounds of 7a-7f for designing synthesis can be high
Effect ground cohesion DNA, the transgene carrier of formation show that cytotoxicity is very low to the MTT experiment of HepG2 and A549, and synthesis turns
Genophore has carried out gene transfection experiments to A549 cells with the DOPE compounds formed and has shown good transfection, has
The Lipofectamine 2000 of a little even more than commercializations, this is containing [12] aneN3Unit compound is as genophore
Further good place mat has been made in research, and preparation method is simple, maturation, is easy to control.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (1)
1. one kind containing Macrocyclic polyamine [12] aneN3Cation lipid transgene carrier preparation method, the method includes such as
Lower step：
1) contain Macrocyclic polyamine [12] aneN3Cation lipid preparation：
The structure formula (I) of the cation lipid is as follows：
In formula (I), R is R=(CH2)8CH3, (CH2)10CH3, (CH2)12CH3, (CH2)14CH3, (CH2)16CH3, (Z-) (CH2)7CH
=CH (CH2)7CH3In one kind；
Step 1: 1,3- dibromopropanes are added into the acetonitrile solution dissolved with 1 compound of formula, it is small to be stirred at reflux reaction 8~10
When, removing solvent is concentrated under reduced pressure and obtains yellow solidliquid mixture, ethyl alcohol and hydrobromic acid are added thereto, is stirred at reflux reaction 7~8
Hour, it after reaction, standing, organic layer is extracted with water, combining water layer is concentrated under reduced pressure to give light yellow or white solid,
Solid is dried in vacuo to obtain 2 compound of formula, 2 compound of formula is dissolved in tetrahydrofuran, triethylamine is added in ice-water bath
With tertbutyloxycarbonyl acid, react at room temperature 8~10 hours, reaction product obtains 3 compound of formula through column chromatography for separation；
Step 2: obtained 3 compound of formula of the step 1 heated in N,N-dimethylformamide with sodium azide react 8~
10 hours, after reaction, saturated salt solution is added into system, is then extracted with ethyl acetate, merges organic phase, with saturation
Brine It organic phase, concentrated solvent obtains 4 compound of formula after drying organic phase with anhydrous sodium sulfate, wherein 3 compound of formula
Molar ratio with sodium azide is 1: 1.5, and the temperature for heating reaction is 68 DEG C；
Step 3: 4 compound of formula that the step 2 obtains is dissolved in anhydrous tetrahydrofuran and methyl alcohol mixed liquor, wherein nothing
The tetrahydrofuran of water and the volume ratio of methanol are 1:1, catalyst is added under stirring into system, under an atmosphere of hydrogen react 8~
10 hours, after reaction, decompression filtered, and filtrate decompression is concentrated to give 5 compound of formula, wherein the catalyst is urged for palladium carbon
The mass ratio of agent, 4 compound of formula and palladium-carbon catalyst is 10: 1；
Step 4: long chain fatty acids is dissolved in chloroform, dicyclohexylcarbodiimide is added, is stirred under ice-water bath,
When white precipitate to be generated, 5 compound of formula and 4-dimethylaminopyridine is added, after mixture is stirred 24 hours under ice bath,
Decompression filters, and obtains that ethyl acetate is added after filtrate decompression is spin-dried for, and after 0.5 hour is again stirring under ice bath, decompression again is taken out
Filter obtains progress column chromatography after filtrate is spin-dried for and obtains 6 compound of formula, and the eluant, eluent that the column chromatography uses is ethyl acetate and first
Alcohol, wherein long chain fatty acids 0.30mmol, dicyclohexylcarbodiimide 0.36mmol, compound 5 are 0.3mmol and 4-
Dimethylamino naphthyridine is 7.4mg, and the volume ratio of ethyl acetate and methanol is 30: 1；
Step 5: 6 compound of formula that step 4 obtains is dissolved in anhydrous methylene chloride, under ice bath, it is added dropwise to thereto molten
There is the dichloromethane solution of trifluoroacetic acid, stirs 6 hours, after reaction, solvent and excessive trifluoroacetic acid are subtracted at room temperature
Pressure is spin-dried for, and 7 compound cation lipid of formula is recrystallized to give with dichloromethane-n-hexane；
2) contain Macrocyclic polyamine [12] aneN3Cation lipid transgene carrier preparation：Step 5 is obtained more containing big ring
Amine [12] aneN3Cation lipid, dioleoylphosphatidylethanolamine and anhydrous chloroform be added in the flask of high-temperature sterilization, it is molten
It is concentrated under reduced pressure to give transgene carrier film after solution, the transgene carrier film is dried in vacuo removal residual chloroform, after dry
Transgene carrier film mixes with the tris-HCI buffer for being preheating to 70 DEG C in advance and is made into the molten of required concentration
Liquid, ultrasound obtain the transgene carrier, wherein the molar concentration of tris-HCI buffer is 10mM, pH
It is 7.4.
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| [12]aneN3-based BODIPY as a selective and sensitive off–on sensor for the sequential recognition of Cu2+ ions and ADP;Ying Zhang et al.;《Chinese Chemical Letters》;20150528;第26卷;第894-898页 * |
| TACN-containing cationic lipids with ester bond: Preparation and application in gene delivery;Qin F.Z.et al.;《Bioorganic & Medicinal Chemistry Letters》;20110929;第21卷;第7045-7049页 * |
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