CN108822052A - Non- peptides polyguanidine small molecule and protein import technology based on non-peptides polyguanidine small molecule-protein matter couplet - Google Patents
Non- peptides polyguanidine small molecule and protein import technology based on non-peptides polyguanidine small molecule-protein matter couplet Download PDFInfo
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Abstract
The present invention provides the non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt that one kind can carry bioactive protein molecule penetrating cell film, and the non-peptides polyguanidine small organic molecule has structure shown in Formulas I：Wherein, A is CH or N；B is NH, O or S；D is NH2, COOH, NCS or SH；X is selected from following any structure：
Description
Technical field
The invention belongs to medical domains, particularly belong to pharmaceutical carrier field.The present invention relates to a kind of non-small point of peptides polyguanidine
Son can be coupled as carrier with protein the protein transport of coupling entering cell interior by cell membrane.The present invention
Further relate to the protein import technology based on non-peptides polyguanidine small molecule-protein matter couplet.
Background technique
With the development of biotechnology, many biologically active large biological molecules such as albumen, polypeptide, nucleic acid drug
Clinically using increasingly extensive.But these current biopharmaceutical macromolecular drugs are primarily directed to extracellular and cell surface target
Point, due to the barrier action of cell membrane, these large biological molecules are difficult to cross-film and reach its action target spot in the cell.How will
Biologically active large biological molecule safely, effectively is delivered to intracellular target spot position and plays its effect, is drug
One of delivery system main problem to be solved.
The technology of a variety of transdermal delivery large biological molecules has been developed in people, for example, electroporation and microinjection etc., though
These right methods are widely used in biological study field, but there are still significant limitations, are such as transferred to low efficiency, to thin
The damage of born of the same parents is big, and cytotoxicity is big etc., is not suitable for using on clinical medicine.In recent years, people have developed liposome, macromolecule
The new technologies such as polymer, cell-penetrating peptide and inorganic nano-particle.
Liposome is that phosphatide is dispersed in water the vesica with single or double layer membrane structure to be formed, the rouge that it is wrapped to form
Liposome medicament has good targeting, can be improved and extend curative effect of medication, mitigates drug toxicity.However, due to liposome
Thermodynamic phase prevent it from long-term preservation, and the partial size of liposome is uneven, and partial size is larger, so that it is not easy
Tissue is penetrated by capillary wall, so that almost all is absorbed by mononuclear phagocyte system, therefore liposome is as medicine
The application of object carrier has biggish limitation.
Currently, the high molecular polymer with special topological structure is due to biggish cavity and a large amount of function end
Base has become the comparatively ideal carrier of drug and is concerned.Polymer drug carrier has the water solubility of enhancing drug, extends
Circulation time reduces the features such as human body rejection, has obtained extensive research.However high molecular polymer still has as pharmaceutical carrier
Many urgent problems to be solved, if carrying medicament amount is compared with small, toxic side effect is big, conveying drug does not have targeting etc.
Cell-penetrating peptide (CPPs) is the small peptide that a major class is made of 10~30 amino acid, drug-carrying small molecule
Enter cell with large biological molecule, while the bioactivity of large biological molecule will not be had an impact.However most CPPs
Inorganizable specificity and cell type specificity in vivo, are easy to be degraded by enzymes in vivo, and positive charge density is high, therefore
It is big to the toxicity of cell.In addition, most CPPs complex synthetic route, high production cost.
Recently, scientist by simulation CPPs structure, design synthesized cell membrane will not be caused at low concentrations it is bright
The oligomerization arginine (RPPs) of aobvious damage, RPPs generally contains 6~12 positive charges, so the efficiency of penetrating cell film is low compared with CPP,
And stability is poor in serum.
In order to overcome the problems, such as the efficiency and stability of penetrating cell film, people also developed a kind of trephocyte cell-penetrating peptide
(cCPPs), the efficiency and stability of its penetrating cell film are all enhanced.However, due to trephocyte cell-penetrating peptide
(cCPPs) it is merged by cell-penetrating peptide and Functional Polypeptides two parts, so it, which can only carry lesser peptides molecule, enters cell, nothing
Method carries large biological molecule and enters cell, and there are significant limitations.In addition, the synthesis cost of cCPPs is high, be not suitable for extensive
Using.
It is specific thin that the acceptor interaction on ligand and cell membrane that nanoparticle passes through surface can enter drug
In born of the same parents, a completely new thinking is opened for the design of intellectual drug carrier.Nanoparticle carries medicine and shows good water solubility, targeting
Property high, numerous advantage such as the body-internal-circulation period is long.But when nanoparticle is as pharmaceutical carrier, biggish cell is often shown
Toxicity.Also, the poor reproducibility of nanoparticle preparation, particle distribution is uneven, and shape and size will affect it and wear membrane efficiency.Cause
This, nanoparticle carries medicine and is difficult to obtain the approval of food and medicine Surveillance Authority applied to clinical practice.
Therefore, it is imperative to find a kind of carrier small molecule efficient, that toxicity is low and stability is good.
Summary of the invention
The object of the present invention is to provide a kind of carrier small molecule efficient, toxicity is low and stability is good, small point of the carriers
Bioactive protein molecule can be delivered to cell interior by son.
To achieve the above object, the present invention provides a kind of non-peptides polyguanidine small molecule, lactate oxidase can be carried
(LAO), the biologically active protein such as horseradish peroxidase, cromoci (CytC) enters cell across cell membrane
And it functions.By taking lactate oxidase as an example：Due to that can generate a large amount of lactic acid in cancer cell, lactic acid concn is much higher by just
Lactic acid concn in normal cell.After lactate oxidase is sent into cancer cell, lactate oxidase can generate pyruvic acid with Oxidation of Lactic,
Hydrogen peroxide (H is generated simultaneously2O2), it can efficiently kill cancer cell.But in normal cell, lactate oxidase will not play work
With, will not to normal cell generate toxicity.
The non-peptides polyguanidine small molecule is high with penetrating cell membrane efficiency, serum stability is good, cytotoxicity is low, water-soluble
Property and the advantages such as stability is good, and cell membrane will not be damaged, be mentioned for the design of novel transmembrane carrier molecule with synthesis
A kind of new thinking is supplied.The synthetic route of the organic micromolecule is short, and preparation cost is low, and synthesis condition is easily controllable, as
Pharmaceutical carrier has good researching value and development prospect.
In a first aspect, it and bioactive substance pass through covalently the present invention provides a kind of non-peptides polyguanidine small molecule
Biomolecule can be efficiently transported to intracellular by key connection.The general formula structure of the non-peptides polyguanidine small molecule is following formula
I：
Non- peptides polyguanidine small molecule of the invention is tree-shaped it can be seen from Formulas I.
The non-peptides polyguanidine that the non-peptides polyguanidine small molecule is formed after being covalently keyed with bioactive protein molecule is small
Molecule-protein couplet has the structure of Formula Il：
Wherein, in Formulas I and II,
A is CH or N；
B is NH, O or S；
D is NH2, COOH, NCS or SH；
X is optionally from following any structure：
In preferred embodiments, the present invention provides small point of non-peptides polyguanidine with any structure in following formula 11~34
Son：
It should be appreciated by those skilled in the art that in formula 11~34 the non-peptides polyguanidine small molecule of any structure pharmaceutical salts
It is included within the scope of the present invention.
In second aspect, the present invention provides a kind of method of non-peptides polyguanidine small molecule for preparing first aspect.The side
The reaction equation of method is as follows：
Wherein, compound 9 is general formula I compound represented of the present invention,
A is CH or N；
B is NH, O or S；
D is NH2, COOH, NCS or SH；
X is selected from following any structure：
The method of the non-peptides polyguanidine small molecule of the preparation first aspect includes the following steps：
(a) make compound 1 and (BOC)2O is in CH2Cl2It is reacted in system, obtains compound 2；
(b) compound 2 is dissolved in solvent, under the action of highly basic, with acrylonitrile reactor, obtains compound 3；
(c) compound 3 and nickel chloride are dissolved in solvent, and NaBH is added4Reaction；Solvent is added after reaction product separation and takes off
Protective agent trifluoroacetic acid obtains compound 4 after deprotection agent BOC；
(d) it reacts compound 4 in aqueous systems with guanidinated reagent O- methyl-isourea Hemisulphate, obtains compound 5；
(e) compound 6 is dissolved in solvent, in the presence of alkali, is reacted with compound 5,
Obtain compound 7；
(f) it reacts compound 7 and 8 in the mixed solvent in the presence of base, obtains compound 9.
Wherein, compound 9 is non-peptides polyguanidine small molecule shown in first aspect present invention Formulas I.
In step (a)~(c), reaction temperature is preferably 10~40 DEG C, and more preferably 25 DEG C.
In the step (b), the present invention is to the highly basic added in reaction and is not particularly limited, and can be those skilled in the art
Alkali compounds known to member, present invention preferably uses sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxides etc., more preferably use
Potassium hydroxide.The preferred dioxane of solvent used in step (b).
In the step (c), the solvent of first step reaction is preferably methanol, ethyl alcohol, isopropanol, acetone etc., more preferably
Methanol；The solvent of second step reaction is preferably CH2Cl2, acetone, dioxane etc., more preferably CH2Cl2。
In the step (d), reaction temperature is preferably 30~70 DEG C, and more preferably 50 DEG C.
In the step (e), reaction temperature is preferably 0~50 DEG C, for example, can react prior to 0 DEG C, is then heated to 40
DEG C reaction.The present invention is to the alkali added in step (e) reaction and is not particularly limited, and can be alkali well known to those skilled in the art
Property compound, present invention preferably uses sodium carbonate, sodium bicarbonate, saleratus, triethylamine, n,N-diisopropylethylamine etc..Step
Suddenly the solvent of (e) reaction is preferably water/acetonitrile, water/acetone, water/tetrahydrofuran etc..Wherein water/acetonitrile indicates that water and acetonitrile are mixed
Object is closed, water/acetone indicates water and acetone mixture, and water/tetrahydrofuran indicates water and tetrahydrofuran compound.Those skilled in the art
For member it should be understood that the volume proportion of ingredient each for mixed solvent does not require specifically, they can be according to real reaction
It needs to determine suitable volume proportion with specific solvent property.Preferably, in step (e), in water/acetonitrile solvent used
The volume ratio of water and acetonitrile is 0.5-3: 1, preferably 1-2: 1, more preferably 2: 1；Water and acetone in water/acetone solvent used
Volume ratio be 0.5-3: 1, preferably 1-2: 1, more preferably 2: 1；Water and tetrahydrofuran in water/tetrahydrofuran used
Volume ratio is 0.5-3: 1, preferably 1-2: 1, more preferably 2: 1.
In step (e), A can be CH or N in 6 structural formula of compound.
In the step (f), reaction temperature is preferably 70~100 DEG C, and more preferably 85 DEG C.The present invention is anti-to step (f)
It answers the alkali of middle addition and is not particularly limited, can be alkali compounds well known to those skilled in the art, the present invention is preferably carbon
Sour sodium, sodium bicarbonate, saleratus, triethylamine etc..Step (f) reaction mixed solvent be preferably water/acetonitrile, water/acetone,
Water/tetrahydrofuran etc..Preferably, in step (f), the volume ratio of water and acetonitrile is 0.5-3 in water/acetonitrile solvent used:
1, preferably 1-2: 1, more preferably 2: 1；The volume ratio of water and acetone is 0.5-3: 1 in water/acetone solvent used, preferably
1-2: 1, more preferably 2: 1；The volume ratio of water and tetrahydrofuran is 0.5-3: 1 in water/tetrahydrofuran used, preferably 1-2:
1, more preferably 2: 1.
In step (f), B, X and D above for Formulas I as defined in 8 structural formula of compound.
In the third aspect, the present invention provide make non-peptides polyguanidine small molecule or its pharmaceutically acceptable salt of the invention with
The method that active biomolecule is covalently attached.Wherein the active biomolecule is not particularly limited, as long as itself cannot be by
Be delivered to cell interior, but with non-peptides polyguanidine small molecule or derivatives thereof shown in general formula I of the present invention or theirs is medicinal
Salt can be delivered to cell interior after covalent coupling.The present inventor has found under study for action, due to of the invention non-
Peptides polyguanidine small molecule or derivatives thereof or their pharmaceutically acceptable salts itself carry a large amount of positive charges, these positive charges have
Help it through cell membrane, therefore, non-peptides polyguanidine small molecule of the invention or derivatives thereof or they are pharmaceutically acceptable
Salt is not suitable for being used for drug delivery with bioactive molecule (for example, nucleic acid) covalent coupling with a large amount of negative electrical charges.The present invention
Non- peptides polyguanidine small molecule or derivatives thereof or their pharmaceutically acceptable salts be more suitable for it is total with bioactive protein molecule
Valence coupling, can be delivered to cell interior for the protein molecular of coupling.That is, the active biomolecule is preferably albumen
Matter.For example, the protein can be selected from, but it is not limited to, lactate oxidase (LAO), horseradish peroxidase, cromoci
Deng.Non- peptides polyguanidine small molecule-protein matter couplet is obtained by being covalently attached, which can be by the biology of covalent linkage
Bioactive molecule is delivered to cell interior, ties shown in Formula II wherein the non-peptide class polyguanidine small molecule-protein matter couplet has
Structure.
The method includes following reactions：
The method that the non-peptides polyguanidine small molecule is connect with active biomolecule includes the following steps：(g) make compound
9 (that is, non-peptides polyguanidine small molecules shown in first aspect Formulas I) are carried out with the corresponding biological activity protein to covalent coupling
Coupling reaction.Wherein, A, B, D and X above for Formulas I as defined, and reaction temperature can be 20~37 DEG C.
In the step (g), the activator of coupling reaction can voluntarily be selected according to the difference of group D in Formulas I, at this
In the specific embodiment of invention：
When in Formulas I D be COOH when, be coupled with the amino at bioactive substance end, can be used DCC, DIEA, HATU,
HBTU, NHS etc. make activator；
When D is NH in Formulas I2When, it is coupled with the carboxyl at bioactive substance end；Can be used DCC, DIEA, HATU,
HBTU, NHS etc. make activator；
When D is NCS in Formulas I, directly it is coupled with the amino or sulfydryl at bioactive substance end, does not need to activate
Agent；
When D is SH in Formulas I, it is coupled with the sulfydryl or disulfide bond at bioactive substance end, does not need activator.
In fourth aspect, the present invention provides through the non-peptides polyguanidine small molecule derivatives that modification obtains, the knot after modification
The following formula III of structure or formula IV：
Wherein, A is CH or N；
B is NH, O or S；
E is NH or S；
Y is Cl, Br or I；
X is selected from following any structure：
At the 5th aspect, the present invention relates to the applications of non-peptides polyguanidine small molecule drugs with function carrier shown in Formulas I.This hair
Non- peptides polyguanidine small molecule shown in Ming Dynasty style I and biological activity protein class drug covalent coupling, can be by the protein of coupling
Class drug transport passes through cell membrane and enters cell interior.
At the 6th aspect, the present invention provides a kind of method transported biological activity protein class drug and enter cell interior,
The method includes making non-peptides polyguanidine small molecule or its medicine shown in the biological activity protein class drug and formula I
With salt or with derivative covalent coupling shown in formula III or formula IV, the couplet after coupling is applied to subject, formula
Derivative shown in non-peptides polyguanidine small molecule or formula III or formula IV shown in I can be by the protein-based of covalent coupling therewith
Drug transport passes through cell membrane and enters cell interior.In specific embodiments, it is used for biological activity protein class drug
Non- peptides polyguanidine small molecule shown in Formulas I of the present invention inside transporte to cells can be appointing in compound 11-34
It is a kind of.
It can be using derivative shown in non-peptides polyguanidine small molecule or formula III shown in I through the invention or formula IV as load
The biological activity protein class drug of body transhipment includes, but are not limited to lactate oxidase (LAO), horseradish peroxidase, thin
Cytochrome C etc..
It should be noted that in the embodiment of the present invention part, the present inventor is with lactate oxidase and cromoci
Example, it was demonstrated that both protein moleculars can be with non-peptides polyguanidine small molecule covalent coupling of the invention and by favorable presentation to carefully
Portion intracellular.However, being based on these experimental datas, those skilled in the art can be according to the bioactive protein molecule to be delivered
Feature selects non-peptides polyguanidine small molecule appropriate and coupling method to make the bioactive protein molecule and peptides of the invention
Polyguanidine small molecule covalent coupling, to realize the purpose for being delivered to cell interior.
Detailed description of the invention
From detailed description with reference to the accompanying drawing, features described above of the invention and advantage be will be apparent from, wherein：
Fig. 1 shows the compound 11 and lactate oxidase (LAO) covalent coupling compound (G that embodiment 3 is prepared4A-
LAO MALDI-TOF (Bruker) chromatography (A) and circular dichroism spectra (B)).
Fig. 2 shows individual lactate oxidase (LAO) (■) and G4Inhibition of the A-LAO compound (●) to Hela cell
Curve.
Fig. 3 shows G4MALDI-TOF (Bruker) mass spectrogram of A-CytC coupled complex, wherein using CytC and
CytC-SH is as a control group.
Fig. 4 shows G4A-CytC coupled complex is to the suppression curve of Hela cell, wherein using CytC and CytC-SH
As a control group.
Specific embodiment
The present invention is further described referring to specific embodiment, it will be appreciated by those skilled in the art that this hair
It is bright to be not limited to these specific embodiments.
In order to further illustrate the present invention, below with reference to embodiment to non-peptides polyguanidine small organic molecule provided by the invention
Covalent linkage with bioactive substance of preparation, non-peptides polyguanidine small organic molecule, Flow cytometry experiments and cytotoxicity it is real
It tests and using being described in detail.
Nuclear magnetic resoance spectrum (NMR) is recorded with Bruker Avance-400 type Nuclear Magnetic Resonance, and the unit of chemical shift δ is
ppm.High resolution mass spectrum figure is recorded with Waters Q-Tof Permier type mass spectrograph.Polarity is used compared with the separating-purifying of large compound
Full-automatic preparative chromatograph (ISCO Combiflash RF+).Column silica gel for chromatography (200~300 mesh) is Qingdao Haiyang chemical industry
Subsidiary factory's production, tlc silica gel plate (HSGF254 type) are the production of Yantai Jiang You silica gel development corporation, Ltd..Solvent for use is point
Analyse pure reagent.
Embodiment 1：The preparation of compound 11
Referring to following formulas, prepare compound 11：
Step a：Compound 1 (diethanol amine, 0.45g, 4.0mmol, 1eq) and (BOC)2O (1.79 g, 8.0mmol,
It 2eq) is dissolved in methanol (10mL), reacts at room temperature 12h, end of reaction.Silica gel chromatography separation, obtains compound 2；
Step b：Compound 2 (0.41g, 2.0mmol, 1eq) is dissolved in dioxane (6mL), is acted at KOH (40%)
Under, it is added acrylonitrile (0.55g, 10mmol, 5eq), 12h is stirred at room temperature, the product after separation is dissolved in by silica gel column chromatography separation
Obtain compound 3；
Step c：Compound 3 (0.64g, 2.0mmol, 1eq), nickel chloride (0.94g, 4.0mmol, 2eq) are dissolved in methanol
It in (30mL), is stirred at room temperature, is slowly added to NaBH4(0.74g, 20mmol, 10eq) reacts at room temperature 8h, silica gel column chromatography separation
Purifying, is dissolved in CH for product after purification2Cl2In, trifluoroacetic acid is added and removes BOC, obtains compound 4；
Step d：Compound 4 (0.34g, 1.5mmol, 1eq) and O- Methyl Isourea Sulfate (0.39 g, 3.3mmol,
2.3eq) it is dissolved in H2In O (7mL), 50 DEG C of reaction 6h, end of reaction.4 DEG C of refrigerators are placed in, white solid is precipitated, are filtered, according to
It is secondary to use ice water, ethanol washing, drying to obtain compound 5；
Step e：Cyanuric Chloride (0.19g, 1mmol, 1eq) is dissolved in acetonitrile (5mL), is cooled to 0 DEG C, compound 5
(0.48g, 2.2mmol, 2.2eq) is dissolved in 10mL H2O, then with NaHCO3(0.35g, 4mmol, 4eq) sequentially adds above-mentioned molten
In liquid, then 0 DEG C of reaction 1h is added heat to 35 DEG C of stirring 1.5h, is tracked and reacted with high performance liquid chromatography (HPLC).Reaction terminates
It does not post-process afterwards, directly progress next step reaction；
Step f：Beta-alanine (0.45g, 5mmol, 5eq) is added into above-mentioned reaction solution, is heated to 80 DEG C of stirrings
5h is tracked with high performance liquid chromatography and is reacted, end of reaction.Concentration is separated with full-automatic preparative chromatograph, and decompression rotation removes solvent,
Freeze-dried acquisition compound 11.
The compound that embodiment 1 is prepared carries out nuclear magnetic resoance spectrum and high resolution mass spec characterization, and data are as follows：
1H NMR (400MHz, D2O)：δ 3.61 (t, J=6.5Hz, 2H), 3.47 (br, 6H), 3.31 (br, 2H), 3.07
(m, 8H), 2.64 (t, J=8Hz, 2H), 1.51 (m, 16H), 1.27 (m, 16H).
13C NMR (100MHz, D2O)：δ 175.73,156.71,154.95,152.85,48.01,41.14,36.31,
33.39,27.95,27.31,25.91,25.46.
ESI-HRMS m/z：MF C30H62N8O6MW 770.5100.[M+2H]2+Calculated value：386.2623, measured value：
386.26929。
Embodiment 2：Cytotoxicity experiment
IC50 is the concentration for inhibiting the growth of 50% cell.Hela, A594 and HepG2 cell is chosen to carry out using MTT method
Cytotoxicity experiment, the specific method is as follows：
Hela, A594 and HepG2 are cultivated in the DMEM of the fetal calf serum containing 10%, 1% penicillin/streptomycin respectively
37 DEG C of culture logarithmic phases in base are inoculated in 96 orifice plates (10000 cells/wells).The volume number that compound 11 calculates is added to pair
In the hole answered, cultivated for 24 hours under conditions of 37 DEG C together with cell.Then the cell MTT of 0.5mg/mL is handled into 4h, used
150 μ L DMSO dissolution, and optical density is read at 570nm by microplate reader.Data are soft by carrying out Igor Pro 6
Part obtains the numerical value of IC50 after duplicate sigmoid curve is fitted three times.
Toxicity of 1 compound 11 of table to cell
| Cell line | Tissue | IC50(μM) |
| HeLa | Uterine neck | 368±5 |
| A549 | Lung | 430±6 |
| HepG2 | Liver | 393±8 |
The IC50 of table 1 is statistics indicate that compound 11 goes out very low poison to tri- kinds of cell line expresses of Hela, A594 and HepG2
Property.
Embodiment 3：Compound G4The synthesis and characterization of A- lactate oxidase (LAO) conjugate
Step a：By compound 11, benzotriazole-N, N, N, N '-tetramethylurea hexafluorophosphoric acid ester (HBTU) and N- hydroxyl
Base succimide (NHS) stirs 1h in DMF solution, obtains reaction intermediate.
Step b：Lactate oxidase (LAO) is dissolved in PBS buffer solution, is added into the reaction solution of step a, number is stirred at room temperature
Hour, it is isolated and purified with super filter tube, product is characterized with MALDI-TOF (Bruker).
The conjugate product G that will be prepared4A-LAO is characterized with MALDI-TOF (Bruker) and circular dichroism spectrometer,
As a result as shown in Figure 1.
The mass spectrometric data of Figure 1A shows that average each LAO protein molecular is connected to 2 G4A molecule.The circular dichroism spectra of Figure 1B
Statistics indicate that connection G4Conjugate product G after A molecule4The secondary structure of A-LAO does not change (that is, G4A-LAO's and LAO
Circular dichroism spectra curve is substantially completely overlapped), illustrate G4A modification has not significant impact the protein three-dimensional structure of HRP.
Embodiment 4：The G that embodiment 3 obtains4The cytotoxicity experiment of A-LAO
IC50 is the concentration for inhibiting the growth of 50% cell.It chooses Hela cell and cytotoxicity experiment is carried out using MTT method,
The specific method is as follows：
By Hela the fetal calf serum containing 10%, 1% penicillin/streptomycin DMEM culture medium in 37 DEG C of cultures to right
The number phase, it is inoculated in 96 orifice plates (10000 cells/wells).By G4Concentration adds in corresponding hole A-LAO as shown in Figure 2, and thin
Born of the same parents cultivate for 24 hours under conditions of 37 DEG C together.Then the cell MTT of 0.5mg/mL is handled into 4h, is dissolved with 150 μ L DMSO,
And optical density is read at 570nm by microplate reader.By data, by carrying out 6 software of Igor Pro, duplicate S-shaped is bent three times
The numerical value of IC50 is obtained after line fitting, as shown in Figure 2.In Fig. 2, (■) indicates individual LAO, and (●) indicates G4A-LAO is compound
Object, the IC50 of LAO is 2.5 μM, and G4A-LAO is 0.2 μM to the IC50 of Hela cell, illustrates G4A-LAO enters HeLa cell
Afterwards, 10 times or more itself improves to the killing ability ratio LAO of cell.
Embodiment 5：The synthesis and characterization of compound 20- cromoci (CytC) conjugate
Step a：Compound 20 is modified, compound 20 is reacted with bromoacetic acid, obtains compound G4A-Br。
Step b：By cromoci (CytC) sulfhydrylation, modified outcome CytC-SH is obtained.
Step c：Cromoci (CytC) is dissolved in PBS buffer solution, is added into the reaction solution of step a, is stirred at room temperature
A few hours are isolated and purified with super filter tube, and product is characterized with MALDI-TOF (Bruker).
The G that will be prepared4A-CytC conjugate product is characterized with MALDI-TOF (Bruker), as a result such as Fig. 3 institute
Show.The mass spectrometric data of Fig. 3 shows that average each CytC molecule is connected to 1 G4A molecule.
Embodiment 6：The G that embodiment 5 is prepared4The cytotoxicity experiment of A-CytC
IC50 is the concentration for inhibiting the growth of 50% cell.It chooses Hela cell and cytotoxicity experiment is carried out using MTT method,
The specific method is as follows：
By Hela the fetal calf serum containing 10%, 1% penicillin/streptomycin DMEM culture medium in 37 DEG C of cultures to right
The number phase, it is inoculated in 96 orifice plates (10000 cells/wells).By G4Concentration adds in corresponding hole A-CytC as shown in Figure 4, and thin
Born of the same parents cultivate for 24 hours under conditions of 37 DEG C together.Then the cell MTT of 0.5mg/mL is handled into 4h, is dissolved with 150 μ L DMSO,
And optical density is read at 570nm by microplate reader.By data, by carrying out 6 software of Igor Pro, duplicate S-shaped is bent three times
The numerical value of IC50 is obtained after line fitting.Fig. 4 shows that CytC and the CytC (CytC-SH) of sulfhydrylation modification cannot be introduced into HeLa
Cell does not show toxicity to cell in the concentration range of measurement, and G4A-CytC is 2.4nM to the IC50 of Hela cell,
Illustrate G4After A-CytC enters HeLa cell, 1000 times are at least improved to the killing ability ratio CytC and CytC-SH of cell
More than.This illustrates couplet G4After A-CytC enters into the cell, efficient it can induce cell apoptosis.
Therefore, present invention design, the non-peptides polyguanidine small molecule of synthesis can carry biological activity protein and efficiently penetrate carefully
After birth enters into the cell, the original activity of albumen is not influenced, so that biological activity protein be made to efficiently perform specific function.
In addition, it should be appreciated by those skilled in the art that although embodiment part is with lactate oxidase and cromoci
Example, it was demonstrated that both protein moleculars can with non-peptides polyguanidine small molecule covalent coupling of the invention and be successfully delivered cell
Inside, however, being based on these experimental datas, those skilled in the art can be according to the spy for the bioactive protein molecule to be delivered
Point selects non-peptides polyguanidine small molecule appropriate and coupling method to keep the bioactive protein molecule and peptides of the invention more
Guanidine small molecule covalent coupling, to realize the purpose for being delivered to cell interior.
It should be understood that although carrying out particularly shown and description to the present invention with reference to its illustrative embodiment,
It should be understood by those skilled in the art that without departing substantially from spirit of the invention as defined in appended claims
Under conditions of range, any of various embodiments can be carried out in the variation for wherein carrying out various forms and details
Combination.
Claims (10)
1. a kind of non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt, the non-peptides polyguanidine small organic molecule tool
There is structure shown in Formulas I：
Wherein, A is CH or N；
B is NH, O or S；
D is NH2, COOH, NCS or SH；
X is selected from following any structure：
2. non-peptides polyguanidine small organic molecule according to claim 1 or its pharmaceutically acceptable salt, wherein described non-
Peptides polyguanidine small organic molecule has following any structure：
3. a kind of method for preparing non-peptides polyguanidine small organic molecule described in claim 1, the described method comprises the following steps：
(a) make compound 1 and (BOC)2O is in CH2Cl2It is reacted in system, obtains compound 2；
(b) compound 2 is dissolved in solvent, under the action of highly basic, with acrylonitrile reactor, obtains compound 3；
(c) compound 3 and nickel chloride are dissolved in solvent, and NaBH is added4Reaction；Solvent and deprotection is added after reaction product separation
Agent trifluoroacetic acid obtains compound 4 after deprotection agent BOC；
(d) it reacts compound 4 in aqueous systems with guanidinated reagent O- methyl-isourea Hemisulphate, obtains compound 5；
(e) compound 6 is dissolved in solvent, in the presence of alkali, reacts with compound 5, obtains compound 7；
(f) it is reacting compound 7 and 8 in the mixed solvent in the presence of base, obtains compound 9；
Wherein compound 9 is non-peptides polyguanidine small organic molecule shown in Formulas I；
Wherein each step reaction formula is as follows：
Wherein, A is CH or N；
B is NH, O or S；
D is NH2, COOH, NCS or SH；
X is selected from following any structure：
4. according to the method described in claim 3, wherein step (a)~(c) reaction temperature be 10~30 DEG C, preferably 25
DEG C, the reaction temperature of step (d) is 30~70 DEG C, and the reaction temperature of step (e) is 20~50 DEG C, the reaction temperature of step (f)
It is 70~100 DEG C.
5. according to the method described in claim 3, wherein highly basic used in step (b) is selected from sodium hydroxide, potassium hydroxide, first
Sodium alkoxide or sodium ethoxide, the solvent are dioxane.
6. according to the method described in claim 3, wherein in step (c) first step reaction solvent used be selected from methanol, ethyl alcohol,
Isopropanol or acetone, second step reaction solvent used are selected from CH2Cl2, acetone or dioxane.
7. according to the method described in claim 3, wherein solvent used in step (e) be selected from water and acetonitrile mixture, water and
Acetone mixture or water and tetrahydrofuran compound, mixed solvent used in step (f) be selected from water and acetonitrile mixture, water and
Acetone mixture or water and tetrahydrofuran compound.
8. one kind makes non-peptides polyguanidine small organic molecule described in claim 1 or its pharmaceutically acceptable salt and bioactivity
Protein molecular be covalently attached at non-peptides polyguanidine small molecule-protein matter couplet method, obtained in non-peptides polyguanidine it is small
Molecule-protein couplet has structure shown in Formula II：
Wherein A, B and X are as defined in claim 1,
When D is COOH in Formulas I, the non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt and biological active matter
The amino at matter end is crosslinked, and DCC, DIEA, HATU, HBTU or NHS is used to be crosslinked as activator；
When D is NH in Formulas I2When, the non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt and bioactive substance
The carboxyl at end is crosslinked；DCC, DIEA, HATU, HBTU or NHS is used to be crosslinked as activator；
When D is NCS in Formulas I, the non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt and biological active matter
The amino or sulfydryl at matter end are crosslinked, and crosslinking is not needed using activator；Or
When D is SH in Formulas I, the non-peptides polyguanidine small organic molecule or its pharmaceutically acceptable salt and bioactive substance
The sulfydryl or disulfide bond at end are crosslinked, and crosslinking is not needed using activator.
9. the derivative of non-peptides polyguanidine small organic molecule described in claim 1, wherein the derivative passes through the D in Formulas I
Substituent group is modified, and the structure after modification is as shown in formula III or formula IV：
Wherein, A is CH or N；
B is NH, O or S；
E is NH or S；
Y is Cl, Br or I；
X is selected from following any structure：
10. a kind of transhipment biological activity protein class drug enters the method for cell interior, the method includes making the biology
Reactive protein class drug and non-peptides polyguanidine small organic molecule of any of claims 1 or 2 or its pharmaceutically acceptable salt
With the derivative covalent coupling of non-peptides polyguanidine small organic molecule as claimed in claim 9, and obtained couplet is applied
To subject, the couplet can pass through cell membrane and enter cell.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263473A (en) * | 1997-05-21 | 2000-08-16 | 利兰·斯坦福青年大学托管委员会 | Composition and method for enhancing transport across biological membranes |
| WO2009055027A2 (en) * | 2007-10-25 | 2009-04-30 | Li Yong-Fu | Molecular transporter compositions comprising dendrimeric oligoguanidine with a tri-functional core that facilitate delivery into cells in vivo |
-
2018
- 2018-05-18 CN CN201810485318.1A patent/CN108822052A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263473A (en) * | 1997-05-21 | 2000-08-16 | 利兰·斯坦福青年大学托管委员会 | Composition and method for enhancing transport across biological membranes |
| WO2009055027A2 (en) * | 2007-10-25 | 2009-04-30 | Li Yong-Fu | Molecular transporter compositions comprising dendrimeric oligoguanidine with a tri-functional core that facilitate delivery into cells in vivo |
| US20090111775A1 (en) * | 2007-10-25 | 2009-04-30 | Li Yong-Fu | Molecular transporter compositions comprising dendrimeric oligoguanidine with a tri-functional core that facilitates delivery into cells in vivo |
Non-Patent Citations (1)
| Title |
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| NING ZHANG ET AL.: "Efficient Mini-Transporter for Cytosolic Protein Delivery", 《ACS APPLIED MATERIALS & INTERFACES》 * |
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