CN110003185A - Macrocyclic polyamine class amphipathic compound and its preparation method and application based on green fluorescent protein chromophore BI - Google Patents
Macrocyclic polyamine class amphipathic compound and its preparation method and application based on green fluorescent protein chromophore BI Download PDFInfo
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Abstract
The present invention provides a kind of Macrocyclic polyamine class amphipathic compound based on green fluorescent protein chromophore BI and preparation method thereof and purposes.Compound disclosed by the invention is mainly synthetically prepared by the condensation of carbonyl ammonia, 1,3 dipole-diople interactions, esterification and Click reaction.Compound provided by the present invention has the property of good two-photon fluorescence, there is the advantages that launch wavelength, 3D high resolution and low self-luminous；The present invention manually simulates the chromogenic mechanism of green fluorescent protein；Compound provided by the invention can form the small molecule non-viral gene vector of pH response with dioleoylphosphatidylethanolamine (DOPE), to promote the release of DNA, have high transfection efficiency and can target nucleus.
Description
Technical field
The present invention relates to non-viral gene vectors, and in particular to a kind of big ring based on green fluorescent protein chromophore BI
Polyamines class amphipathic compound and its preparation method and application.
Background technique
Gene therapy be by into sick cell import normal gene (for example, DNA, siRNA, mRNA and shRNA) with
This repairs or substitutes defective gene.Nearest decades, gene therapy are drawn as a kind for the treatment of means with fine prospect
The extensive concern of researcher is played.
Carrier is divided into viral genetic vector and non-viral gene vector two major classes.Viral genetic vector can be to low dosage
DNA carries out very efficient transfection, but viral genetic vector also has the carcinogenicity and immunogenicity of certain risk, has and carries
The limitation (usually 2-3kb) and operation of body capacity are more demanding.In order to solve these problems, researcher develops a variety of
Substitute of the non-viral gene vector as virus.Non-viral gene vector has carrier-free capacity limit, no infectiousness, chemistry
Structure understands adjustable, is easy to largely synthesize and the advantages such as easy to use, thus its have in gene therapy it is important
Effect.But there are still many problems, such as compound to accumulate in vivo in clinical treatment for traditional non-viral delivery system
It is poly-, the problems such as inner body escape, performance is single, and toxicity is big and transfection efficiency is low.In order to solve these problems, researcher turns
To learning naturally, masterpiece of the virus as the Nature can carry out the transfection of extreme efficiency by complex mechanism.It
Contain different functional components, molecule gradually can be carried out according to the variation of microenvironment and converted, to carry out efficient
Transfection.In entire transfection process, gene delivery system will receive pH variation, oxidation-reduction potential, the influence of enzymatic activity etc..Cause
This, design synthesis can be a kind of heavy of raising non-viral gene vector transfection efficiency to the genophore of microenvironment stimuli responsive
Want method.
Therefore, synthesize it is a kind of have it is multi-functional, can stimuli responsive and the non-viral gene with high transfection efficiency carry
Body has biggish researching value.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems 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 a kind of Macrocyclic polyamine class amphiphilics based on green fluorescent protein chromophore BI
Compound, it is involved in the present invention to compound be using BI as core, the head of BI unit connect hydrophobic long alkyl chain with
And hydrophilic Macrocyclic polyamine unit is connected in tail portion, form the amphiphilic compound of hydrophilic and oleophilic；This kind of compound can be in water
In with DNA be self-assembled into nano particle；The compound in conjunction with DOPE and DNA after the ternary complex that is formed can issue green
Fluorescence simulates the luminescence mechanism of green fluorescent protein；This kind of compound in conjunction with DOPE and DNA after have two-photon effect,
It can be excited by long wave, have many advantages, such as high 3D resolution ratio and low self-luminous；After this kind of compound and DOPE and DNA self assembly,
Gene transfection is entered into nucleus, makes it have hyperfluorescence, can be used as a kind of non-viral gene vector simultaneously using this feature
And tracer can be carried out to the transfection process of gene.The compound can carry out stimuli responsive to pH, be more advantageous to releasing for DNA
It puts.
It is a still further object of the present invention to provide the Macrocyclic polyamine class amphiphilic chemical combination based on green fluorescent protein chromophore BI
The purposes of the preparation method of object and the compound.
In order to realize these purposes and other advantages according to the present invention, provides one kind and added lustre to based on green fluorescent protein
The Macrocyclic polyamine class amphipathic compound of group BI, the structure formula (I) of the compound are as follows:
In formula (I), R is alkyl radical structure unit.
Preferably, wherein R is straight chained alkyl structural unit.
Preferably, wherein R is
The purpose of the present invention can also be further by the Macrocyclic polyamine class amphiphilic based on green fluorescent protein chromophore BI
The preparation method of object is closed to realize, comprising the following steps:
1) BI derivative is prepared；
2) cyclen derivatives are prepared；
3) BI derivative is reacted with cyclen derivatives is prepared the big ring based on green fluorescent protein chromophore BI
Polyamines class amphipathic compound (I).
Preferably, wherein preparation BI derivative specifically includes in step 1): Step 1: by nucleophilic substitution system
Standby formula (II) compound represented；Step 2: esterification occurs by formula (II) compound represented and double azidobenzoic acids
Prepare formula (III) compound represented；
Preferably, wherein the step 1 specifically includes: compound shown in the formula (IV) by 1.0 equivalents and 2.0 equivalents
Bromoalkane and the potassium carbonate of 4.0 equivalents be dissolved in 20mL acetone, heating reflux reaction 48 hours, filtrate, column is collected by filtration
Chromatographic purifying obtains formula (II) compound represented；
Preferably, wherein the step 2 specifically includes: being added shown in the formula (II) of equivalent in the DCM of 30mL
Compound and 3, bis- (azido-methyl) benzoic acid of 5-, the DMAP of catalytic amount, the DCM that the DCC of 1.5 equivalents is then slowly added dropwise are molten
Liquid is stirred at room temperature reaction 2 days, is purified by column chromatography, obtain formula (III) compound represented.
The purpose of the present invention can also be further by the Macrocyclic polyamine class amphiphilic based on green fluorescent protein chromophore BI
Application of the object in non-viral gene vector is closed to realize.
The purpose of the present invention can also be further by the Macrocyclic polyamine class amphiphilic based on green fluorescent protein chromophore BI
Object is closed as the application of DNA tracer to realize.
The present invention is include at least the following beneficial effects:
1, the compound of the present invention is centered on BI unit, and hydrophobic long alkyl chain is modified on head, and tail portion connects hydrophilic
Macrocyclic polyamine [12] aneN of property3Amphiphilic compound is formed, DNA, formed nano particle can be effectively agglomerated；
2, the ternary complex after the compound of the present invention and DOPE and DNA self assembly has two-photon effect, has length
Wave excitation, low self luminous feature.Manual simulation's luminescence mechanism of green fluorescent protein；
3, the compound of the present invention can carry out stimuli responsive to pH, and the DNA of cohesion can be discharged when pH is 5；
4, the compound of the present invention can be used as non-viral gene vector, the compound BI-B wherein provided in implementation example figure
Its transfection efficiency is considerably beyond commercialized transfection reagent Lipofectamine 2000, its transfection efficiency is in the cell of part
10 times or more of Lipofectamine 2000；
5, the process that the compound of the present invention can transfect gene carries out single photon and two photon imaging tracer respectively, just
Mechanism is transfected in research, is laid the foundation for the research and development of novel transfection reagent.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 the compounds of this invention and Ago-Gel resistance for the cationic-liposome that is prepared by the compound of the present invention
Stagnant lab diagram；
Wherein, Figure 1A is Ago-Gel retardation experiment figure of the compound BI-A to 18 DNA of pUC；Figure 1B is compound
Ago-Gel retardation experiment figure of the BI-A/DOPE to 18 DNA of pUC；Fig. 1 C is fine jade of the compound BI-B to 18 DNA of pUC
Sepharose retardation experiment figure；Fig. 1 D is Ago-Gel retardation experiment figure of the compound BI-B/DOPE to 18 DNA of pUC；
Fig. 1 E is Ago-Gel retardation experiment figure of the compound BI-C to 18 DNA of pUC；Fig. 1 F is BI-C/DOPE pairs of compound
The Ago-Gel retardation experiment figure of 18 DNA of pUC；
Fig. 2 is the ctDNA fluorescence titration result figure of the cationic-liposome prepared by the compound of the present invention；
Wherein, Fig. 2A is the ctDNA fluorescence titration result figure of cationic-liposome BI-A/DOPE；Fig. 2 B is cationic lipid
The ctDNA fluorescence titration result figure of plastid BI-B/DOPE；Fig. 2 C is the ctDNA fluorescence titration of cationic-liposome BI-C/DOPE
Result figure；Fig. 2 D is the ctDNA fluorescence titration result figure of cationic-liposome BI-D/DOPE；
Fig. 3 is the Ago-Gel electricity that the cationic-liposome prepared by the compound of the present invention discharges 18 DNA of pUC
Swimming lab diagram；
Fig. 4 is compound BI-B in the present invention and its cationic-liposome formed with DOPE in Hela cell
Luciferase expression result figure；
Fig. 5 is the expression knot of luciferase of the compound BI-A/DOPE~BI-D/DOPE in different cells in the present invention
Fruit figure；
Wherein, Fig. 5 A is luciferase expression result of the liposome in HeK293T cell；Fig. 5 B is the liposome
Luciferase expression result in Hela cell；Fig. 5 C is luciferase expression knot of the liposome in HepG2 cell
Fruit；Fig. 5 D is luciferase expression result of the liposome in A549 cell；
Fig. 6 is that the compounds of this invention BI-A/DOPE~BI-D/DOPE transfects the red fluorescent protein of pERFP-N1 gene
Expression figure；
Fig. 7 is in different time sections, cellular uptake subject cationic liposome BI-B/DOPE and Cy5-DNA compound
Single photon be total to focused view；
Fig. 8 is in different time sections, cellular uptake subject cationic liposome BI-B/DOPE and Cy5-DNA compound
Two-photon be total to focused view.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
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.
It should be noted that experimental method described in following embodiments is unless otherwise specified conventional method, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
<embodiment 1>
A kind of Macrocyclic polyamine class amphipathic compound based on green fluorescent protein chromophore BI, the structural formula of the compound
(I) as follows:
In formula (I), R is alkyl radical structure unit.
Wherein, when R is straight chained alkyl structural unit, and R isWhen, synthesis is added lustre to based on green fluorescent protein
The Macrocyclic polyamine class amphipathic compound of group BI is denoted as BI-A；R isSynthesis based on green fluorescent protein chromophore BI
Macrocyclic polyamine class amphipathic compound be denoted as BI-B；R isSynthesis based on green fluorescent protein chromophore BI's
Macrocyclic polyamine class amphipathic compound is denoted as BI-C；R isSynthesis based on the big of green fluorescent protein chromophore BI
Ring polyamines class amphipathic compound is denoted as BI-D.Specific synthetic route is as follows:
In formula, (i) bromoalkane RBr, potassium carbonate, acetone, reflux, 48 hours；(ii) bis- (azido-methyl) benzoic acid of 3,5-,
DCC, DMAP, DCM, 48 hour；(iii) propargyl [12] aneN3, cuprous bromide, chloroform, room temperature, 24 hours；(iv) salt
The ethyl acetate solution of acid, room temperature, 2 hours.
Specific synthesis step is as follows:
(1), the bromoalkane of the equivalent of compound 1 and 2.0 of 1.0 equivalents and the potassium carbonate of 4.0 equivalents are dissolved in 20mL third
Ketone is heated to reflux 48 hours.Filtrate is collected by filtration, passes through column chromatographic purifying, eluant, eluent are as follows: PE:EA=1:1 obtains purifying and closes
Object 2~5, yield 61~84%；
Compound 2:1H NMR(600MHz,CDCl3): δ 8.09 (d, J=8.8Hz, 2H)), 7.07 (s, 1H), 6.93 (d,
J=8.9Hz, 2H), 4.01 (t, J=6.5Hz, 2H), 3.84 (d, J=4.9Hz, 2H), 3.77 (t, J=5.1Hz, 2H),
2.40 (s, 3H), 1.81-1.76 (m, 2H), 1.60 (s, 1H), 1.52-1.48 (m, 2H), 0.98 (t, J=7.4Hz, 3H)13C
NMR(151MHz,CDCl3):δ171.52, 161.29,161.15,136.39,134.28,128.17,126.86,114.90,
67.90,61.23,43.73, 31.27,19.29,15.92,13.90.HRMS(ES+)calcd.for C17H22N2O3(M+H)+:
303.1703,found:303.1705.
Compound 3:1H NMR(600MHz,CDCl3): δ 8.08 (d, J=8.5Hz, 2H), 7.07 (s, 1H), 6.92 (d, J
=8.4Hz, 2H), 3.99 (t, J=6.6Hz, 2H), 3.84 (t, J=4.8Hz, 2H), 3.77 (t, J=4.8Hz, 2H), 2.41
(s, 3H), 1.82-1.74 (m, 2H), 1.57 (s, 1H), 1.47-1.41 (m, 2H), 1.33-1.25 (m, 8H), 0.88 (t, J=
6.6Hz,3H).13C NMR(101MHz, CDCl3):171.41,161.46,161.14,136.33,134.28,128.16,
126.83,114.89,68.22, 61.00,43.71,31.88,29.42,29.30,29.23,26.08,22.73,15.89,
14.18.HRMS(ES+) calcd.for C21H30N2O3(M+H)+:359.2329,found:359.2332.
Compound 4:1H NMR(600MHz,CDCl3): δ 8.09 (d, J=8.8Hz, 2H), 7.08 (s, 1H), 6.93 (d, J
=8.9Hz, 2H), 4.00 (t, J=6.6Hz, 2H), 3.85 (t, J=5.0Hz, 2H), 3.78 (t, J=5.0Hz, 2H), 2.42
(s, 3H), 1.83-1.75 (m, 2), 1.58 (s, 1H), 1.49-1.42 (m, 2H), 1.34-1.22 (m, 16H), 0.88 (t, J=
7.0Hz,3H).13C NMR(151MHz, CDCl3):δ171.51,161.29,161.16,136.37,134.28,128.19,
126.85,114.91,68.23, 61.22,43.72,31.99,29.73,29.71,29.67,29.64,29.46,29.42,
29.23,26.08,22.76, 15.91,14.19.HRMS(ES+)calcd.for C25H38N2O3(M+H)+:415.2955,
found: 415.2951.
Compound 5:1H NMR(400MHz,CDCl3): δ 8.06 (d, J=8.9Hz, 2H), 7.03 (s, 1H), 6.92 (d, J
=8.9Hz, 2H), 3.99 (t, J=6.6Hz, 1H), 3.80 (s, 2H), 3.73 (d, J=4.9Hz, 2H), 2.36 (s, 3H),
1.81-1.75 (m, 2H), 1.50-1.41 (m, 2H), 1.26 (s, 24H), 0.88 (t, J=6.9Hz, 3H)13C NMR
(101MHz,CDCl3):δ171.39,161.47,161.15, 136.31,134.28,128.18,126.83,114.88,
68.23,60.96,43.71,32.00,29.78,29.77, 29.76,29.74,29.73,29.72,29.68,29.66,
29.47,29.44,29.24,26.08,22.77,15.88, 14.19.HRMS(ES+)calcd.for C29H46N2O3(M+H)+:
471.3581,found:471.3578.
(2) in the DCM of 30mL be added 0.25mmol compound 2~5 and 3, bis- (azido-methyl) benzoic acid of 5-, and
Then the DCM solution of the DCC of 1.5 equivalents is slowly added dropwise in the DMAP of catalytic amount, reaction 2 days is stirred at room temperature.It is chromatographed by column
Purifying, eluant, eluent are as follows: PE:EA=1:1 obtains pure compound 6~9, yield 52~63%；
Compound 6:1H NMR(400MHz,CDCl3): δ 8.08 (d, J=8.8Hz, 2H), 7.91 (s, 2H), 7.48 (s,
1H), 7.09 (s, 1H), 6.91 (d, J=8.88Hz, 2H), 4.52 (t, J=5.5Hz, 2H), 4.43 (s, 4H), 4.03-3.98
(m, 4H), 2.39 (s, 3H), 1.75 (s, 2H), 1.31 (s, 2H), 0.96 (d, J=8.2Hz, 3H)13C NMR(151MHz,
CDCl3):δ170.71,165.51,161.18,160.18, 137.09,134.27,132.20,131.89,129.04,
128.22,126.84,126.18,114.92,67.90, 62.81,54.13,39.72,35.00,25.54,15.73,
13.89.HRMS(ES+)calcd.for C26H28N8O4(M+H)+:517.2306,found:517.2301.
Compound 7:1H NMR(400MHz,CDCl3):δ8.11(s,2H),7.92(s,2H),7.49(s, 1H),7.12
(s, 1H), 6.93 (d, J=5.9Hz, 2H), 4.54 (s, 2H), 4.42 (d, J=15.9Hz, 4H), 4.03 (s, 4H), 2.44
(s, 3H), 1.79 (s, 2H), 1.45 (s, 2H), 1.27 (d, J=12.1Hz, 8H), 0.89 (s, 3H)13C NMR(151MHz,
CDCl3):δ170.70,165.51,161.18, 160.20,137.09,134.27,132.21,130.77,129.03,
128.20,126.83,126.19,114.92, 68.23,62.81,54.12,39.71,31.87,29.40,29.29,29.22,
26.07,22.72,15.73,14.16. HRMS(ES+)calcd.for C30H36N8O4(M+H)+:573.2932,found:
573.2930.
Compound 8:1H NMR(400MHz,CDCl3):δ8.09(s,2H),7.91(s,2H),7.48(s, 1H),7.09
(s, 1H), 6.92 (s, 2H), 4.52 (t, J=5.52Hz, 2H), 4.43 (s, 4H), 4.02-3.97 (m, 4H), 2.39 (s,
3H), 1.89 (s, 2H), 1.74-1,71 (m, 2H), 1.27 (d, J=13.5Hz, 16H), 0.87 (t, J=6.86Hz, 3H)13C
NMR(151MHz,CDCl3):δ170.71,165.51, 161.19,160.17,137.10,134.27,132.20,130.78,
129.04,128.23,126.83,126.18, 114.93,68.24,62.81,54.13,39.72,35.00,31.99,
29.72,29.70,29.63,29.45, 29.41,29.22,26.07,22.76,15.74,14.18.HRMS(ES+)
calcd.for C34H44N8O4 (M+H)+:629.3558,found:629.3562.
Compound 9:1H NMR(400MHz,CDCl3): δ 8.09 (d, J=8.8Hz, 2H), 7.96-7.91 (m, 2H),
7.49 (s, 1H), 7.09 (s, 1H), 6.92 (d, J=8.9Hz, 2H), 4.53 (t, J=5.5 Hz, 2H), 4.43 (s, 4H),
4.06-3.96 (m, 4H), 2.40 (s, 3H), 1.81-1.76 (m, 2H), 1.44 (m, 2H), 1.25 (s, 24H), 0.86 (d, J=
7.0Hz,3H).13C NMR(101MHz, CDCl3):δ170.71,165.52,161.18,160.20,137.09,134.27,
132.23,130.75, 129.04,128.24,126.81,126.21,114.91,68.23,62.82,54.11,39.72,
34.05,32.01, 29.78,29.77,29.74,29.67,29.64,29.46,29.44,29.23,26.08,25.70,
25.03,22.77, 15.75,14.21.HRMS(ES+)calcd.for C38H52N8O4(M+H)+:685.4184,found:
685.4188.
(3) under an argon, the compound 6-9, propargyl [12] N of 2.5 equivalents of 1 equivalent is added3, catalytic amount
CuBr and 5mL chloroform is spin-dried for obtaining crude product, column chromatography (eluant, eluent: DCM:MeOH=after room temperature reaction 24 hours
10:1) obtain Boc (tertbutyloxycarbonyl) the protection product of respective compound BI-A~BI-D, yield: 51-60%.By above-mentionedization
Close object be dissolved in 4mL saturation hydrogen chloride ethyl acetate solution, after being stirred at room temperature 2 hours, be obtained by filtration respective compound BI-A~
BI-D, yield: 72%~90%.
Wherein, propargyl [12] aneN3Preparation method bibliography Bioorganic&Medicinal Chemistry
20(2012)801–808。
Compound BI-A:1H NMR (600MHz, DMSO): δ 8.54 (s, 2H), 8.34 (s, 1H), 8.16 (d, J=
8.6Hz,1H),7.87(s,2H),7.53(s,1H),7.12–6.92(m,1H),5.77(s,4H), 4.47(s,2H),4.01
(q, J=7.0Hz, 4H), 3.56 (s, 4H), 3.52-3.35 (m, 12H), 3.26 (s, 4H), 3.07 (s, 4H), 2.19 (s,
8H),2.05(s,4H),1.97(s,3H),1.70(s,4H),1.59(s, 2H),1.39(s,2H),1.22(s,4H),0.83
(t, J=6.8Hz, 3H)13C NMR(151MHz, DMSO):δ172.46,166.00,165.39,161.23,157.30,
137.75,134.66,131.08, 130.86,129.14,128.80,126.69,126.46,115.47,115.44,68.12,
63.49,56.57,53.09, 48.09,47.15,41.75,33.81,31.19,25.87,24.95,21.72,19.26,
14.29.HRMS(ES+) calcd.for C50H74N14O4(M+H)+:935.6090,found:935.6082.
Compound BI-B:1H NMR (400MHz, DMSO): δ 8.42 (s, 2H), 8.16 (d, J=8.7 Hz, 2H), 7.80
(s,2H),7.55–7.44(m,1H),7.37(s,1H),7.05–6.92(m,2H),5.69 (s,4H),4.42(s,4H),
4.09-3.92 (m, 4H), 3.81 (d, J=32.5Hz, 4H), 3.38 (d, J=7.1Hz, 6H), 3.13 (d, J=39.3Hz,
12H), 2.67 (s, 3H), 2.43-2.33 (t, J=14.6Hz, 8H), 2.18 (s, 4H), 2.07 (s, 4H), 1.91 (s, 4H),
1.72 (s, 2H), 1.41 (s, 2H), 1.26 (s, 8H), 0.86 (t, J=6.5Hz, 3H)13C NMR(151MHz,DMSO):δ
172.47,166.01, 165.41,163.31,161.17,137.77,134.61,132.72,130.86,129.02,
128.64,126.59, 126.52,115.46,115.39,68.30,65.43,63.37,60.28,52.73,46.98,
41.48,32.86, 31.74,29.24,29.15,29.10,26.00,22.59,21.62,18.02,15.69,14.47.HRMS
(ES+) calcd.for C54H82N14O4(M+H)+:991.6716,found:991.6730.
Compound BI-C:1H NMR (600MHz, DMSO): δ 8.50 (s, 2H), 8.11 (d, J=8.0 Hz, 2H), 7.82
(s,2H),7.52(s,1H),6.97(s,3H),5.73(s,4H),4.38(s,4H),4.03– 3.96(m,4H),3.51(s,
3H),3.37(s,12H),3.22(s,6H),3.04(s,6H),2.15(s,8H), 2.01(s,4H),1.85(s,3H),1.66
(d, J=6.4Hz, 6H), 1.56 (d, J=12.2Hz, 4H), 1.44 (d, J=12.0Hz, 2H), 1.39-1.36 (m, 2H),
1.21-1.15 (m, 4H), 1.03-0.99 (m, 4H), 0.88 (t, J=7.1Hz, 3H)1H NMR(101MHz,DMSO):δ
172.47,170.85, 166.00,165.39,156.92,137.74,136.99,134.75,132.86,132.78,
130.84,129.04, 128.70,115.45,115.43,68.91,68.37,60.33,56.53,52.91,48.14,
41.64,33.75, 31.80,29.54,29.52,29.49,29.27,29.22,25.98,25.84,24.91,22.61,
21.71,18.03, 14.66,14.49.HRMS(ES+)calcd.for C58H90N14O4(M+H)+:1047.7342,found:
1047.7337.
Compound BI-D:1H NMR(600MHz,DMSO):δ8.50(s,2H),8.26(s,1H),8.09 (s,1H),
7.85 (s, 2H), 7.78 (s, 1H), 7.32 (s, 1H), 6.93 (s, 2H), 5.73 (s, 4H), 4.37 (s, 4H), 3.97 (d, J=
6.7Hz,2H),3.81(s,6H),3.51(s,12H),3.36(s,6H),3.04(s,6H), 2.15(s,8H),2.01(s,
4H),1.85(s,3H),1.66(s,4H),1.56(s,4H),1.45(s,2H), 1.35(s,2H),1.17(s,16H),0.78
(s,3H).1H NMR(101MHz,DMSO):δ172.48, 170.85,167.59,165.97,161.13,137.94,
134.71,133.17,131.17,129.35,128.82, 126.87,126.54,115.70,115.30,68.42,60.47,
53.58,53.37,48.02,47.34,42.03, 33.85,31.80,30.86,29.52,29.51,29.30,29.29,
29.26,29.20,29.17,29.15,25.90, 25.00,22.63,21.87,21.60,18.29,14.83,14.61.HRMS
(ES+)calcd.for C62H98N14O4(M+H)+:1103.7968,found:1103.7983.
<embodiment 2>
The solution for preparing various concentration compound BI-A~BI-D respectively, by the BI-A~BI-D and pUC18 of various concentration
Plasmid DNA (9 μ g/mL) is placed in 37 DEG C of water-baths, is incubated for 1h, and then progress DNA Ago-Gel retardation experiment obtains different dense
Compound is spent to the gel blocking result of pUC18 DNA.
Figure 1A~1H is the compounds of this invention BI-A~BI-D and BI-A/DOPE~BI-D/DOPE (compound respectively
It is 1 ︰ 3 with DOPE ratio) to the Ago-Gel retardation experiment result of pUC18 DNA；The upper numerical value marked is in Figure 1A~1H
Test concentrations (μM)；Figure 1A~1H show compound BI-A~BI-D and BI-A/DOPE~BI-D/DOPE (compound with
DOPE ratio can block completely DNA for 1 ︰ 3) under low concentration and migrate in agarose.
By embodiment 2, it can be concluded that, the amphiphilic compound prepared by the present invention based on BI unit can effectively agglomerate DNA
Nano particle is formed, can be used as non-viral gene vector.
<embodiment 3>
To cationic-liposome BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE (compound and DOPE ratio
Example tests its fluorescence intensity and maps, obtain Fig. 2A~2D for ct DNA is added in the solution of 1 ︰ 3).
Fig. 2A~2D is ctDNA to compound BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE (chemical combination
Object and DOPE ratio are 1 ︰ 3) fluorescence titration result；Wherein, X-axis is the concentration of DNA, and Y-axis indicates fluorescence intensity.By implementing
Example 3 it can be concluded that, the present invention manually simulates the process of adding lustre to of green fluorescent protein.
<embodiment 4>
Certain density BI-A/DOPE~BI-D/DOPE (compound and DOPE ratio be 1 ︰ 3) is configured, by itself and pUC
18 Plasmid DNA form compound, and 37 DEG C are incubated for 2 hours, under conditions of pH is 5.2, carries out DNA agarose release experiment, obtain
The case where to BI-A/DOPE~BI-D/DOPE (compound and DOPE ratio be 1 ︰ 3) to sour stimuli responsive.
Fig. 3 is the compounds of this invention to acid response condition；It can be seen from the figure that BI-A/DOPE~BI-D/DOPE
(compound and DOPE ratio can be illustrated that the present invention can be stimulated in acid condition and be rung within 2 hours for 1 ︰ 3) with released dna
It answers.
<embodiment 5>
By the compound BI-B of the various concentration and different proportion DOPE compound formed and PGL-3 DNA in 37 DEG C of conditions
Lower incubation is added in cultured Hela cell after 30 minutes, acts on 5 hours, and compound is sucked out, is rinsed, has been added with DMEM
Full culture medium is incubated for 40 hours.It is eventually adding cell pyrolysis liquid, by cell cracking, measures its luminous intensity and protein content,
It is standard specimen, the luminous intensity (RLU/mg protein) of every milligram of albumen with commercial transfection reagent lipofectamine 2000
The transfection efficiency of compound is indicated with the percentage (%of Lipo2000) of commercial transfection reagent lipofectamine 2000.
Fig. 4 is the compound that is formed of the compounds of this invention BI-B and different proportion DOPE as non-viral gene vector,
Transfected condition under the conditions of various concentration, to be commercialized transfection reagent Lipofectamine 2000 as reference；X-axis is in Fig. 4
Indicate the compound of different proportion and the compound that DOPE is formed, wherein the last item histogram graph representation Lipofectamine
2000；Y-axis indicates luciferase expression amount；
In first group of histogram, by it is left-to-right successively indicate compound BI-A concentration be 10 μM, 15 μM, 20 μM, 25
μM, 30 μM and luciferase expression amount at 35 μM；In second group of histogram, compound and DOPE are successively indicated by left-to-right
Luciferase expression when ratio is 1 ︰ 1, when concentration is 10 μM, 15 μM, 20 μM, 25 μM, 30 μM and 35 μM；In third group column
In figure, by it is left-to-right successively indicate that compound and DOPE ratio are 1 ︰ 2 when, be 10 μM, 15 μM, 20 μM, 25 μM, 30 μ in concentration
Luciferase expression amount at M and 35 μM；In the 4th group of histogram, it is by left-to-right successively expression compound and DOPE ratio
Luciferase expression amount when 1 ︰ 3, when concentration is 10 μM, 15 μM, 20 μM, 25 μM, 30 μM and 35 μM；In the 5th group of column
In figure, by it is left-to-right successively indicate that compound and DOPE ratio are 1 ︰ 4 when, be 10 μM, 15 μM, 20 μM, 25 μM, 30 μ in concentration
Luciferase expression amount at M and 35 μM；The last one indicates luciferase expression amount of Lipo2000 when with 10 μ g/mL；
The result shows that when compound BI-B/DOPE and DOPE ratio is 1 ︰ 3, best transfection efficiency is in Hela cell
10.0 times of Lipofectamine 2000.
<embodiment 6>
By compound BI-A/DOPE~BI-D/DOPE of various concentration (compound and DOPE ratio be 1 ︰ 3) and pGL-3
Plasmid DNA after acting on 30min, is added in HEK293T, Hela, HepG2 and A549 cell and cultivates under the conditions of 37 DEG C
5h；Then containing compound culture solution is changed into fresh complete culture solution culture 40h；After removing culture medium, 20 μ L are added
Cell pyrolysis liquid, then relative luminous intensity and protein content are measured respectively, finally with the relative luminous intensity of every milligram of albumen
The percentage (%of Lipo2000) of (RLU/mg protein) and commercial transfection reagent lipofectamine 2000 indicate multiple
Close the transfection efficiency of object BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE.
Fig. 5 be the compounds of this invention BI-A/DOPE~BI-D/DOPE compound (compound and DOPE ratio are 1 ︰ 3, under
It is used as non-viral gene vector, transfected condition under the conditions of various concentration, to be commercialized transfection reagent together)
Lipofectamine 2000 is reference；X-axis is to indicate different composite object in Fig. 5, wherein the last item histogram graph representation
Lipofectamine 2000；Y-axis indicates luciferase expression amount；
Fig. 5 A indicates luciferase expression result of the compound BI-A/DOPE~BI-D/DOPE in HEK293T；
Fig. 5 B indicates luciferase expression result of the compound BI-A/DOPE~BI-D/DOPE in Hela；
Fig. 5 C indicates luciferase expression result of the compound BI-A/DOPE~BI-D/DOPE in HepG2；
Fig. 5 D indicates luciferase expression result of the compound BI-A/DOPE~BI-D/DOPE in A549；
(1) the result shows that in HEK239T cell, the best transfection efficiency point of compound BI-A/DOPE~BI-D/DOPE
It is not 2.5 times, 11.1 times, 4.0 times and 1.4 times of Lipofectamine 2000.
(2) the result shows that in Hela cell, the best transfection efficiency of compound BI-A/DOPE~BI-D/DOPE is distinguished
It is 2.3 times, 10.0 times, 4.1 times and 2.6 times of Lipofectamine 2000.
(3) the result shows that in HepG2 cell, the best transfection efficiency of compound BI-A/DOPE~BI-D/DOPE is distinguished
It is 0.56 times, 4.1 times, 1.2 times and 0.82 times of Lipofectamine 2000.
(4) the result shows that in A549 cell, the best transfection efficiency of compound BI-A/DOPE~BI-D/DOPE is distinguished
It is 0.33 times, 5.2 times, 1.1 times and 0.27 times of Lipofectamine 2000.
<embodiment 7>
To take concentration respectively be 30 μM, 20 μM, 15 μM, 10 μM of compound and after pERFP incubation 30 minutes, is added into
It is cultivated into Hela cell, then the culture medium after administration is sucked out, the complete medium containing FBS is added and is incubated for for 24 hours；
Finally, culture medium is sucked out and is washed 3~5 times with PBS, taken pictures with laser confocal scanning microscope；With commercial transfection reagent
Lipofectamine 2000 is control group.
By embodiment 7 it can be concluded that, BI-B/DOPE transfection efficiency highest.
<embodiment 8>
After cationic-liposome BI-B/DOPE and Cy5-DNA is incubated for 0.5h, when cultivating different in addition Hela cell
Between, culture medium is sucked out, is washed 3-5 times with PBS, is taken pictures by laser confocal scanning microscope, carries out bio-imaging, observation
DNA transfection process.
Fig. 7 is that compound BI-B/DOPE cohesion Cy5-DNA is added in Hela cell, is then obtained in different time sections
Take cellular uptake figure.By embodiment 8, it can be concluded that, BI-B/DOPE cohesion DNA enters nucleus after 0.5h, as the time increases
Add, the compound into nucleus increases.
<embodiment 9>
Tested, taken pictures by two-photon Laser Scanning Confocal Microscope according to the method described above, to gene transfection process into
Row tracer.
Fig. 8 is that compound BI-B/DOPE cohesion Cy5-DNA is added in Hela cell, is obtained in different time periods
Cellular uptake figure.By embodiment 9, it can be concluded that, which can enter the intracorporal two photon imaging of biology.
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 (9)
1. a kind of Macrocyclic polyamine class amphipathic compound based on green fluorescent protein chromophore BI, the structural formula of the compound
(I) as follows:
In formula (I), R is alkyl radical structure unit.
2. as described in claim 1 based on the Macrocyclic polyamine class amphipathic compound of green fluorescent protein chromophore BI, wherein R
For straight chained alkyl structural unit.
3. as claimed in claim 2 based on the Macrocyclic polyamine class amphipathic compound of green fluorescent protein chromophore BI, wherein R
For
4. a kind of prepare the described in any item Macrocyclic polyamine classes two based on green fluorescent protein chromophore BI of claims 1 to 3
The method of close compound, comprising the following steps:
1) BI derivative is prepared；
2) cyclen derivatives are prepared；
3) BI derivative is reacted with cyclen derivatives is prepared the Macrocyclic polyamine based on green fluorescent protein chromophore BI
Class amphipathic compound (I).
5. method as claimed in claim 4, the middle preparation BI derivative of step 1) is specifically included: Step 1: passing through nucleophilic displacement of fluorine
Reaction preparation formula (II) compound represented；Step 2: ester occurs by formula (II) compound represented and double azidobenzoic acids
Change reaction preparation formula (III) compound represented；
6. method as claimed in claim 5, the step 1 is specifically included: compound shown in the formula (IV) by 1.0 equivalents and
The potassium carbonate of the bromoalkane of 2.0 equivalents and 4.0 equivalents is dissolved in 20mL acetone, heating reflux reaction 48 hours, is collected by filtration
Filtrate, column chromatographic purifying obtain formula (II) compound represented；
7. method as claimed in claim 5, the step 2 is specifically included: the formula (II) of equivalent is added in the DCM of 30mL
Then the DCC of 1.5 equivalents is slowly added dropwise in compound represented and 3, bis- (azido-methyl) benzoic acid of 5-, the DMAP of catalytic amount
DCM solution, be stirred at room temperature reaction 2 days, purified by column chromatography, obtain formula (III) compound represented.
8. the Macrocyclic polyamine class amphipathic compound described in claim 1 based on green fluorescent protein chromophore BI is in non-viral base
Because of the application in carrier.
9. the Macrocyclic polyamine class amphipathic compound described in claim 1 based on green fluorescent protein chromophore BI shows as DNA
The application of track object.
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| CN115073437A (en) * | 2021-03-11 | 2022-09-20 | 北京师范大学 | Macrocyclic polyamines based on dicyanopyrans [12]aneN 3 Compound and preparation method and application thereof |
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