CN102718844A - Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments - Google Patents
Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments Download PDFInfo
- Publication number
- CN102718844A CN102718844A CN2012102210804A CN201210221080A CN102718844A CN 102718844 A CN102718844 A CN 102718844A CN 2012102210804 A CN2012102210804 A CN 2012102210804A CN 201210221080 A CN201210221080 A CN 201210221080A CN 102718844 A CN102718844 A CN 102718844A
- Authority
- CN
- China
- Prior art keywords
- mitp
- peptide
- cys
- antimicrobial peptide
- antibacterial peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an efficient and low-toxicity acid activated antimicrobial peptide with anti-tumor activity. Antimicrobial peptide MitP is used as a template, lysine (K) in the sequence of the antimicrobial peptide MitP is substituted into glutamic acid (E) and histidine (H), and anionic polypeptide MitPE carrying negative charges under normal physiological conditions is synthesized; and the anionic polypeptide is connected with the antimicrobial peptide MitP through a disulfide bond to form a living antimicrobial peptide compound with acid activation property. The synthesized antimicrobial peptide has obvious acid dependency activity; and the antimicrobial peptide has excellent cell killing activity under acidic conditions, and the cell killing activity of the antimicrobial peptide under the normal physiological conditions is remarkably reduced. The selectivity of the antimicrobial peptide can be improved, injury to normal tissue cells due to the antimicrobial peptide is weakened, the toxic or side effect of the antimicrobial peptide is reduced, and a broad prospect is provided for developing antimicrobial peptide medicaments.
Description
Technical field
The invention belongs to the biochemical drug field, relate to a kind of new antibacterial peptide compound and synthetic, relate in particular to a kind of antibacterial peptide AMitP and compound method thereof with acid active characteristic; The present invention also relates to the application of this antibacterial peptide in the preparation antitumor drug simultaneously.
Background technology
Tumour is one of important diseases that threatens human health.Chemotherapy is a kind of effective ordinary method of treating tumour or slowing down tumor growth.But most traditional antitumor drug poor selectivity, toxic side effect are strong, and the reasons such as multidrug resistance of tumour have had a strong impact on clinical efficacy (Accounts Chem. Res., 2007,41,98).Improve selectivity and become the key issue that the development antitumor drug need solve.Therefore press for development one type of wide spectrum, highly selective now, have the new action target spot and the medicine of mechanism of action.
Antibacterial peptide (antimicrobial peptide) generally is by being less than the amphiphilic cationic polypeptide that 50 amino acid constitute, and it is the part (Nature, 2002,415,389) of the important molecule defense mechanism that remains in the organic evolution process.The molecular weight of antibacterial peptide is little, thermostability and good water solubility, and general microbiotic and antitumor drug do not have immunogenicity and resistance, thereby are with a wide range of applications.Except having anti-microbial effect, antibacterial peptide also has good antitumor activity (Biochim. Biophys. Acta., 2008,1778,357).The main mode of action of antibacterial peptide is the destruction of cell membrane: at first positively charged antibacterial peptide is adsorbed onto on the electronegative cytolemma through electrostatic interaction; After quantity reaches threshold value; Antibacterial peptide just can form the duct and destroy cytolemma on film, finally cause outflow (Pharmacol. Rev., 2003 of entocyte; 55,27).Although the mode of action of this uniqueness is given antibacterial peptide certain selectivity and good antitumor activity, also make antibacterial peptide have certain toxic side effect simultaneously, like damage to erythrocyte and other normal tissue cell.In addition, antibacterial peptide itself with the multivalence positive charge also can primosome in system toxicity.These toxic side effect have seriously hindered antibacterial peptide and have developed into clinical medicine.
Mitoparan (MitP) is to be template with antibacterial peptide Mastoparan, the positive changes and the amphiphilic antibacterial peptide compound that all improves that obtain through the amino acid replacement.Experimental result shows that the cytocidal activity of MitP obviously is superior to Mastoparan (Biochim. Biophys. Acta., 2008,1783,849).But the same with other many antibacterial peptides, toxic side effect has limited it and has been used for clinical as outstanding medicine.
Reducing the toxic method of antibacterial peptide so far mainly is amino acid whose replacement; Although this method can reduce the toxicity (mainly being to erythrocytic toxicity) of antibacterial peptide to a certain extent; But the antibacterial peptide compound that obtains still has certain lethal effect to normal cell; Particularly during vivo medicine-feeding, tend to normal cell caused damage, thereby produce inevitable toxic side effect because the local antibacterial peptide concentration is too high.It is (Cancer Res., 2009,69,3458) of cost with the cell killing activity of sacrificing antibacterial peptide often that other reduces the toxic method of antibacterial peptide, thereby causes fish and bear's paw not to get both.Thereby the development New Policy is very necessary for improving its clinical application with the toxic side effect that reduces antibacterial peptide.
How reducing the toxic side effect of antibacterial peptide and keeping its rupture of membranes activity is the primary problem that solves of the antibiotic peptide medicament of development.Utilize difference between normal tissue cell and the tumour cell can effectively improve the selectivity of antibacterial peptide, reduce the toxicity of peptide.Research shows reasons such as tumor tissue cell is fast owing to metabolism, oxygen supply deficiency, and cell provides energy to produce a large amount of lactic acid through glycolysis-, but tumor vessel can not in time remove again unusually, finally causes the tumor tissues microenvironment to be acid.In recent years, be that the medicine of target spot has obtained development (J. Control Release, 2008,132,164) faster with this acidic micro-environment.Therefore this acidic micro-environment can become and improve the effective target spot of antibacterial peptide selectivity.
Great majority research shows that it is the active the first step of performance rupture of membranes that positively charged antibacterial peptide is adsorbed onto on the electronegative cytolemma through electrostatic interaction, also is very crucial step (Nature, 2002,415,389; Biochim. Biophys. Acta., 2008,1778,357).Thereby through controlling the cytotoxicity that electrostatic adsorption can significantly reduce antibacterial peptide.
Summary of the invention
The purpose of this invention is to provide a kind of selectivity that can improve antibacterial peptide, reduce the antibacterial peptide compd A MitP with acid active characteristic of the toxic side effect of antibacterial peptide;
Another object of the present invention provides a kind of synthetic method with antibacterial peptide compd A MitP of acid active characteristic;
A further object of the invention, just providing a kind of is activeconstituents with this antibacterial peptide compd A MitP with acid active characteristic, the application in the preparation antitumor drug.
(1) has the antibacterial peptide compd A MitP of acid active characteristic
The present invention has the antibacterial peptide compd A MitP of acid active characteristic; Be to be template with antibacterial peptide MitP; Methionin in its sequence (K) is replaced to L-glutamic acid (E) and Histidine (H), synthetic a series of negatively charged ion polypeptide MitP electronegative under normal physiological conditions
EThe negatively charged ion polypeptide is connected through disulfide linkage with antibacterial peptide MitP again, obtains having the antibacterial peptide compd A MitP alive of acid active characteristic.Its structure is as follows:
The present invention has the structural changes that the antibacterial peptide compound of acid active characteristic takes place under condition of different pH following: under normal physiological conditions, and negatively charged ion polypeptide MitP
EL-glutamic acid in the sequence (E) can attract together through Methionin (K) in electrostatic interaction and the MitP sequence, both can reduce the positive charge quantity among the MitP, also can limit the structural changes of MitP, thereby reduces the activity of MitP; But under condition of acidic pH, negatively charged ion polypeptide MitP
EIn L-glutamic acid (E) side chain carboxyl group be neutralized, lose with MitP in the electrostatic attraction of Methionin (K), thereby discharge MitP, recover its activity.Therefore, through utilizing negatively charged ion polypeptide MitP
EIn following charged variation of condition of different pH, thereby control the electrostatic adsorption of coupled antibacterial peptide and cytolemma, improve optionally purpose (see figure 1) of antibacterial peptide thereby reach.
(2) has the preparation of the antibacterial peptide of acid active characteristic
The present invention has the preparation method of the antibacterial peptide compound of acid active characteristic, comprises following process step:
(1) antibacterial peptide Cys-MitP and corresponding negatively charged ion polypeptide compound Cys-MitP thereof
ESolid phase synthesis:
A. resin pre-treatment: the Rink-Amide-MBHA resin is added methylene dichloride, stir 20 ~ 40 min, make decompressing and extracting solvent after the abundant swelling of resin;
B. take off the F-moc protection: add deprotecting regent in the resin after swelling, drain behind stirring 2 ~ 5min, repeat 4 times, the Fmoc group is removed fully,, obtain removing the resin of Fmoc radical protection at last with DMF washing Ex-all deprotecting regent;
Said deprotecting regent is mixed with the volume ratio of 1:3 ~ 1:5 by piperidines and DMF.
C. condensation: successively with amino acid, N-hydroxy benzo triazole, the O-benzotriazole-N of the Fmoc radical protection of 3 ~ 4 times of molar weights of Rink-Amide-MBHA resin; N; N'; N'-tetramethyl-urea-hexafluorophosphate is dissolved among the DMF, add the diisopropylethylamine of 6 ~ 8 times of molar weights of Rink-Amide-MBHA resin again after mixing get mixing solutions; Put into the resin of the said Fmoc of removing radical protection then, stirring reaction 40 ~ 60min under argon shield; Through indenes check reagent detection reaction degree, the DMF repeated washing is used in the back that reacts completely;
D. the prolongation of peptide chain: press order repeating step b, the c of CINLKKLAKL (Aib) KKIL, CINLEHLAHL (Aib) EEIL respectively, till connecing the peptide completion;
E. the cutting of peptide chain from the resin: remove fully according to the method for step b amino acid whose Fmoc group respectively with last connection; Replace washing resin with DCM, MeOH; After fully draining solvent; By the cutting reagent of every gram peptide resin adding 10 ~ 25mL, cleavage reaction is 1.5 ~ 3 hours under room temperature; Collect cutting reagent and decompression and revolve driedly, use ether sedimentation, the abundant dissolution precipitation of water and remove the ether phase again, water must thick peptide Cys-MitP of white solid powder and Cys-MitP through lyophilize
E
Said cutting reagent is by trifluoroacetic acid, phenol, thioanisole, 1, and 2-ethylene dithiol ether, water mix with the volume ratio of 82.5:5:5:5:2.5:5.
The desalination and the purifying of f. thick peptide: the acetic acid soln with volumetric concentration 10 ~ 20% is a moving phase, respectively with thick peptide Cys-MitP and Cys-MitP
EThrough the desalination of Sephadex G25 cross-linked dextran gel column, utilize Ultraviolet Detector to collect the main peak postlyophilization, obtain the peptide compounds of desalination; Utilize reverse performance liquid chromatographic column to carry out separation and purification again, collect main peak, obtain the pure peptide pressed powder Cys-MitP and the Cys-MitP of white after the lyophilize
E
(2) activation of cysteine side chain sulfydryl in the antibacterial peptide Cys-MitP sequence: Cys-MitP is dissolved in the mixing solutions of methyl alcohol and water; Two sulphur, two pyridines that add 10 ~ 12 times of Cys-MitP molar weights, reaction 10 ~ 16 h utilize reverse performance liquid chromatographic column to carry out separation and purification, collect main peak, and lyophilize obtains sulfydryl activatory Cys-MitP;
In the mixing solutions of said methyl alcohol and water, the volume ratio of methyl alcohol and water is 1:1 ~ 1:5.
(3) with sulfydryl activatory Cys-MitP and Cys-MitP
EMol ratio with 1:1 ~ 1:2 is dissolved in the mixing solutions of methyl alcohol and water; Reaction 10 ~ 16 h utilize reverse performance liquid chromatographic column to carry out separation and purification, collect main peak, and lyophilize obtains title product AMitP;
In the mixing solutions of said methyl alcohol and water, the volume ratio of methyl alcohol and water is 1:1 ~ 1:5.
Synthetic polypeptide products of the present invention characterizes through electrospray ionization mass spectrometry (ESI-MS), and the result shows that the actual molecular weight of the purified polypeptide that obtains conforms to theoretical molecular.
(3) the antibacterial peptide anti-tumor activity that has the acid active characteristic is tested
1, anti tumor activity in vitro experiment
TP: adopt mtt assay to detect the anti-tumor activity of acid active antibacterial peptide: preceding 24 hours of dosing with the Hela cell according to 1 * 10
4The density of individual cells/well is inoculated in 96 well culture plates, every hole 100 μ L cell suspensions.Nutrient solution during dosing in the sucking-off culture plate and in every hole, add 100 μ L pH values be respectively 7.4,6.0,5.0 contain the different concns peptide nutrient solution.After the drug incubation 4 hours, in every hole, add the MTT solution (5 mg/mL) of 10 μ L, hatched 4 hours for 37 ℃.The DMSO that adds 150 μ L afterwards with the nutrient solution sucking-off and in every hole dissolves the crystallization of hepatic first a ceremonial jade-ladle, used in libation.Behind the mixing ten minutes, detect the absorbance value under the 570nm wavelength and calculate the IC50 value with ELIASA (Bio-reader 680).
Experimental result is seen table 1.The result of table 1 shows that the anti-tumor activity of MitP does not show the pH dependency, negatively charged ion polypeptide MitP
E(0.625 mol/L ~ 20 mol/L) do not show activity in the detectable level scope, have only both are linked to each other and synthetic AMitP has very high anti-tumor activity under acidic conditions, and the anti-tumor activity under normal physiological conditions obviously reduces.
Table 1 MitP, AMitP and MitP
EAnti-tumor activity under condition of different pH
ND: fail to detect toxicity
2, the detection of serum lactic dehydrogenase (LDH) release
Through Cytotoxicity Det. Kit PLUS (LDH) (Roche) detection kit detect the influence of acid active antibacterial peptide cell membrane integrity.Preceding 24 hours of dosing with the Hela cell suspension of 100 μ L with 1 * 10
4The density in individual/hole is inoculated in the 96 porocyte culture plates.Nutrient solution during dosing in the sucking-off culture plate and in every hole, add 100 μ L pH values be respectively 7.4,6.0,5.0 contain different concns peptide (0.625 mol/L ~ 20 mol/L) nutrient solution; Hatched 30 minutes; The supernatant solution of sucking-off 40 μ L was transferred in another piece 96 porocyte culture plates after 96 orifice plates were vibrated 5 seconds on the vibration plate device; The reaction solution that adds 40 μ L again, 22 ℃ of lucifuges were hatched 15 minutes, added the stop buffer of 20 μ L afterwards rapidly.Detect the absorbance value under the 490nm wavelength with ELIASA (Bio-reader 680).The light absorption value of dosing cell is not decided to be negative control, is 100% LDH overflow outward and use the light absorption value of the cell of institute's band lysis solution processing in the test kit.
Experimental result is like Fig. 2,3, shown in 4: negatively charged ion polypeptide MitP
EIn the detectable level scope (0.625 mol/L ~ 20 mol/L) not cell membrane cause damage, the damage of MitP cell membrane under condition of different pH and has only the damage of AMitP cell membrane to show tangible pH dependency much at one.
3, hemolytic experiment
Adopt the eyeball blood taking method to collect fresh mouse blood after in centrifuge tube, adding heparin sodium; And with 1; The rotating speed of 000 * g was removed serum in centrifugal 10 minutes to obtain red corpuscle, used the PBS buffer solution for cleaning then 3 times, afterwards red corpuscle was diluted to the cell suspension of 8% (V/V) with PBS.The red cell suspension for preparing is joined in 96 well culture plates, and every hole 100 μ L add the peptide solution that 100 μ L contain different concns again.The cell that this experiment was handled with the Triton X-100 of PBS and 0.1% respectively is as 0 and 100% hemolysis rate.96 orifice plates were hatched 1 hour centrifugal 10 minutes afterwards with the rotating speed of 1,200 * g under 37 ℃.After centrifugal 100 μ L supernatants are transferred in another piece 96 orifice plates, used ELIASA (Bio-reader 680) to detect the absorbance value under 450 nm wavelength then and calculate hemolysis rate.
Experimental result is as shown in Figure 5: compare with MitP, the hemolytic activity of AMitP obviously reduces, and this will help to reduce its toxic side effect.
4, circular dichroism spectrum experiment
Detecting the secondary structure of acid active antibacterial peptide in the membrane simulation environment of different pH values through Olis DSM 1000 CD spectrophotometers (USA) changes: peptide is dissolved in the pH value and is respectively in 7.4 and 5.0 the phosphate buffered saline buffer that contains 50% (V/V) trifluoroethanol, final concentration is 50 μ M.Under the room temperature peptide solution being put into the path then is that the cell of 2 millimeters long detects, and the parameter that experiment is provided with is that speed is 50 nm/min, and step-length is 0.1 nm, 0.5 s answering time, and 1 nm broadband and wavelength are 195 ~ 260 nm.
Experimental result is as shown in Figure 6, MitP, AMitP and MitP
ECan both in the membrane simulation environment, form alpha-helix, but have only the helicity of AMitP under acidic conditions, to increase to some extent, this will help to recover the cell killing activity of MitP.
5, molecular dynamics simulation experiment
Utilize GROMACS software; Method through molecular dynamics simulation detects the secondary structure of acid active antibacterial peptide in the membrane simulation environment of different pH values and changes and the interaction situation: place the pH value to be respectively the simulated environment (normal temperature 315K, normal pressure 1.0 bar) of POPC film-aqueous solution of 7.4 and 5.0 on the peptide model.At the process balancing charge, energy minimization relief system is carried out the unrestricted motion of 50ns.At last structure, energy, electric charge and the movement locus etc. of peptide are made relevant detection.GROMOS96 is used in the field of force that experiment is provided with, and PME (Particle Mesh Ewald) arithmetic calculation ion ability is used LINCS algorithm limits The interaction distance, and weak point/long-range reactive force Cutoff is 9/12, temperature 315K, pressure 1.0 bar.
Experimental result is as shown in Figure 7: AMitP (Residue 2 ~ 16) and MitP
E(Residue 18 ~ 32) can both form alpha-helix (A-Helix) in the membrane simulation environment, wherein the helicity of AMitP under acidic conditions under normal pH condition (A, B).AMitP and MitP
EUnder normal pH condition is polymeric (C), and issues estrangedly from (D) at acidic conditions, and this will make AMitP inactivation and under acidic conditions, recover activity under normal operation.
In sum, the anti tumor activity in vitro experiment shows that synthetic antibacterial peptide of the present invention has tangible acid and relies on active: under acidic conditions, just have outstanding cell killing activity, and the cell killing activity under normal pH condition reduces significantly.These characteristics can improve the selectivity of antibacterial peptide, weaken antibacterial peptide to normal histiocytic damage, reduce the toxic side effect of antibacterial peptide, finally improve its potential applicability in clinical practice.Show that through circular dichroism spectrum and molecular dynamics simulation experiment synthetic acid active antibacterial peptide of the present invention really structural changes as shown in Figure 1 can take place under condition of different pH.The anti tumor activity in vitro experiment shows that this type antibacterial peptide has shown outstanding cell killing activity under condition of acidic pH; Hemolytic experiment is illustrated in and shows lower toxicity under the normal physiological conditions.Therefore, this type antibacterial peptide has good using value in the preparation antitumor drug.
Description of drawings
Fig. 1 is the synoptic diagram that structural changes takes place under condition of different pH the acid active antibacterial peptide;
Fig. 2 is the influence that MitP discharges LDH under condition of different pH;
Fig. 3 is the influence that AMitP discharges LDH under condition of different pH;
Fig. 4 is MitP
EThe influence that under condition of different pH, LDH is discharged;
Fig. 5 is MitP, AMitP and MitP
ETo erythrocytic hemolytic action;
Fig. 6 is MitP, AMitP and MitP
EStructural changes under condition of different pH;
Fig. 7 is the structural changes of molecular simulation AMitP under condition of different pH.
Embodiment
Below through synthetic being described further of specific embodiment to acid active antibacterial peptide AMitP of the present invention.
(1) Cys-MitP and corresponding negatively charged ion polypeptide compound Cys-MitP thereof
ESolid phase synthesis:
A. resin pre-treatment: with amino molar weight is that the Rink-Amide-MBHA resin of 0.2 mmol joins in the reactor drum, adds the 10mL methylene dichloride and also stirs 30 min, makes decompressing and extracting solvent after the abundant swelling of resin.
B. take off the F-moc protection: 10 ~ 15mL deprotecting regent is joined in the reactor drum drain behind the stirring 2min, repeat 4 times, the Fmoc group is removed fully, at last with DMF washing Ex-all deprotecting regent; Deprotecting regent is piperidines/DMF=1:4 (V/V).
C. condensation: successively with amino acid, N-hydroxy benzo triazole, the O-benzotriazole-N of 0.6 ~ 0.8 mol Fmoc radical protection; N; N', N'-tetramethyl-urea-hexafluorophosphate are dissolved among 3 ~ 5mL DMF, add 1.2 ~ 1.6 mol diisopropylethylamine again after mixing get mixing solutions; Put in the reactor drum stirring reaction 40 ~ 60min then; Entire reaction course is used argon shield, and through indenes check reagent detection reaction degree, unreacted reagent is removed with the DMF repeated washing in the back that reacts completely.
D. the prolongation of peptide chain: press order repeating step b, the c of CINLKKLAKL (Aib) KKIL, CINLEHLAHL (Aib) EEIL respectively, till connecing the peptide completion.
E. the cutting of peptide chain: remove fully according to the method for step b amino acid whose Fmoc group with last connection; Replace washing resin with DCM, MeOH; After fully draining solvent; Add the cutting agent (trifluoroacetic acid: phenol: thioanisole: 1,2-ethylene dithiol ether: water=82.5:5:5:5:2.5:5 (V/V)) of 10 ~ 25ml by every gram peptide resin, cleavage reaction is 1.5 ~ 3 hours under room temperature; Collection cutting reagent and decompression are revolved dried; (50 ~ 80mL) join the concussion of exerting oneself in the flask precipitates the refrigerative ether in advance; Leave standstill the back and remove the ether of supernatant earlier; Remove the ether phase with separating funnel after water fully dissolves again, water gets thick peptide Cys-MitP of white solid powder and Cys-MitP through lyophilize
E
The desalination and the purifying of f. thick peptide: the acetic acid soln with volumetric concentration 10 ~ 20% is a moving phase, with thick peptide Cys-MitP and Cys-MitP
EThrough the desalination of Sephadex G25 cross-linked dextran gel column, utilize Ultraviolet Detector to collect the main peak postlyophilization, obtain the peptide compounds of desalination; Utilize the reverse performance liquid chromatographic column of C18 to carry out separation and purification again, collect main peak, obtain the pure peptide pressed powder Cys-MitP and the Cys-MitP of white after the lyophilize
E
(2) activation of cysteine side chain sulfydryl in the antibacterial peptide Cys-MitP sequence: 0.4 mmol, two sulphur, two pyridines are dissolved in the 2mL methanol solution; Then 0.04 mmol Cys-MitP is dissolved in 1mL methanol (V/V=1:1 ~ 1:10), and be added dropwise in the methanol solution of above-mentioned two sulphur, two pyridines reaction 10 ~ 16 h; Utilize the reverse performance liquid chromatographic column of C18 to carry out separation and purification; Collect main peak, lyophilize obtains sulfydryl activatory Cys-MitP.
(3) with 0.02 mmol sulfydryl activatory Cys-MitP and 0.02 mmol Cys-MitP
E(among the V/V=1:1 ~ 1:10), reaction 10 ~ 16 h utilize the reverse performance liquid chromatographic column of C18 to carry out separation and purification, collect main peak, and lyophilize obtains title product AMitP to be dissolved in 2 mL methanol.Productive rate is 30 ~ 42%.
Synthetic polypeptide products of the present invention characterizes as follows through electrospray ionization mass spectrometry (ESI-MS):
Table 2 MitP, AMitP and MitP
EMass spectrometric detection result
The result of table 2 shows that the actual molecular weight of the polypeptide that the present invention obtains conforms to theoretical molecular.
Claims (6)
1. have the antibacterial peptide compd A MitP of acid active characteristic, its structural formula is following:
2. like the said preparation method of claim 1, comprise following process step with antibacterial peptide compd A MitP of acid active characteristic:
(1) antibacterial peptide Cys-MitP and corresponding negatively charged ion polypeptide compound Cys-MitP thereof
ESolid phase synthesis:
A. resin pre-treatment: the Rink-Amide-MBHA resin is added methylene dichloride, stir 20 ~ 40 min, make decompressing and extracting solvent after the abundant swelling of resin;
B. take off the F-moc protection: add deprotecting regent in the resin after swelling, drain behind stirring 2 ~ 5min, repeat 4 times, the Fmoc group is removed fully,, obtain removing the resin of Fmoc radical protection at last with DMF washing Ex-all deprotecting regent;
C. condensation: successively with amino acid, N-hydroxy benzo triazole, the O-benzotriazole-N of the Fmoc radical protection of 3 ~ 4 times of molar weights of Rink-Amide-MBHA resin; N; N'; N'-tetramethyl-urea-hexafluorophosphate is dissolved among the DMF, add the diisopropylethylamine of 6 ~ 8 times of molar weights of Rink-Amide-MBHA resin again after mixing get mixing solutions; Put into the resin of the said Fmoc of removing radical protection then, stirring reaction 40 ~ 60min under argon shield; Through indenes check reagent detection reaction degree, the DMF repeated washing is used in the back that reacts completely;
D. the prolongation of peptide chain: press order repeating step b, the c of CINLKKLAKL (Aib) KKIL, CINLEHLAHL (Aib) EEIL respectively, till connecing the peptide completion;
E. the cutting of peptide chain from the resin: remove fully according to the method for step b amino acid whose Fmoc group respectively with last connection; Replace washing resin with DCM, MeOH; After fully draining solvent; By the cutting reagent of every gram peptide resin adding 10 ~ 25mL, cleavage reaction is 1.5 ~ 3 hours under room temperature; Collect cutting reagent and decompression and revolve driedly, use ether sedimentation, the abundant dissolution precipitation of water and remove the ether phase again, water must thick peptide Cys-MitP of white solid powder and Cys-MitP through lyophilize
E
The desalination and the purifying of f. thick peptide: the acetic acid soln with volumetric concentration 10 ~ 20% is a moving phase, respectively with thick peptide Cys-MitP and Cys-MitP
EThrough the desalination of Sephadex G25 cross-linked dextran gel column, utilize Ultraviolet Detector to collect the main peak postlyophilization, obtain the peptide compounds of desalination; Utilize reverse performance liquid chromatographic column to carry out separation and purification again, collect main peak, obtain the pure peptide pressed powder Cys-MitP and the Cys-MitP of white after the lyophilize
E
(2) activation of cysteine side chain sulfydryl in the antibacterial peptide Cys-MitP sequence: Cys-MitP is dissolved in the mixing solutions of methyl alcohol and water; Two sulphur, two pyridines that add 10 ~ 12 times of Cys-MitP molar weights, reaction 10 ~ 16 h utilize reverse performance liquid chromatographic column to carry out separation and purification, collect main peak, and lyophilize obtains sulfydryl activatory Cys-MitP;
(3) with sulfydryl activatory Cys-MitP and Cys-MitP
EMol ratio with 1:1 ~ 1:2 is dissolved in the mixing solutions of methyl alcohol and water; Reaction 10 ~ 16 h utilize reverse performance liquid chromatographic column to carry out separation and purification, collect main peak, and lyophilize obtains title product AMitP.
3. like the said preparation method with antibacterial peptide compd A MitP of acid active characteristic of claim 2, it is characterized in that: among step (1) b, said deprotecting regent is mixed with the volume ratio of 1:3 ~ 1:5 by piperidines and DMF.
4. like the said preparation method of claim 2 with antibacterial peptide compd A MitP of acid active characteristic; It is characterized in that: among step (1) e; Said cutting reagent is by trifluoroacetic acid, phenol, thioanisole, 1, and 2-ethylene dithiol ether, water mix with the volume ratio of 82.5:5:5:5:2.5:5.
5. like the said preparation method with antibacterial peptide compd A MitP of acid active characteristic of claim 2, it is characterized in that: in step (2), (3), in the mixing solutions of said methyl alcohol and water, the volume ratio of methyl alcohol and water is 1:1 ~ 1:5.
6. the application of antibacterial peptide compd A MitP in the preparation antitumor drug that has the acid active characteristic according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210221080.4A CN102718844B (en) | 2012-06-29 | 2012-06-29 | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210221080.4A CN102718844B (en) | 2012-06-29 | 2012-06-29 | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102718844A true CN102718844A (en) | 2012-10-10 |
| CN102718844B CN102718844B (en) | 2014-04-02 |
Family
ID=46944735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210221080.4A Active CN102718844B (en) | 2012-06-29 | 2012-06-29 | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102718844B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109232719A (en) * | 2018-09-21 | 2019-01-18 | 中国科学院理化技术研究所 | A kind of antibacterial peptide and its preparation method and application of pH response |
| CN110218760A (en) * | 2019-06-18 | 2019-09-10 | 青岛大学 | It is a kind of antitumor with antibacterial peptide Activity determination experimental provision |
| CN110862458A (en) * | 2019-11-13 | 2020-03-06 | 倪京满 | Combined peptide with acid-activated antitumor activity and clinical application thereof |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
| CN115925802A (en) * | 2023-02-22 | 2023-04-07 | 西南交通大学 | Antibacterial peptide with enhanced antibacterial activity and serum stability and application thereof |
-
2012
- 2012-06-29 CN CN201210221080.4A patent/CN102718844B/en active Active
Non-Patent Citations (6)
| Title |
|---|
| KAIRONG WANG等: "Membrane-Active Action Mode of Polybia-CP, a Novel Antimicrobial Peptide Isolated from the Venom of Polybia paulista", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| SARAH JONES 等: "Mitoparan and target-selective chimeric analogues: Membrane translocation and intracellular redistribution induces mitochondrial apoptosis", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| SARAH JONES,等: "Charge delocalisation and the design of novel mastoparan analogues:enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP", 《REGULATORY PEPTIDES》 * |
| WEI ZHANG等: "A novel analog of antimicrobial peptide Polybia-MPI, with thioamide bond substitution, exhibits increased therapeutic efficacy against cancer and diminished toxicity in mice", 《PEPTIDES》 * |
| WEI ZHANG等: "Design of Acid-Activated Cell Penetrating Peptide for Delivery of Active Molecules into Cancer Cells", 《BIOCONJUGATE CHEMISTRY》 * |
| 李佳: "具有硫取代酰胺键的抗菌肽活性研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109232719A (en) * | 2018-09-21 | 2019-01-18 | 中国科学院理化技术研究所 | A kind of antibacterial peptide and its preparation method and application of pH response |
| CN109232719B (en) * | 2018-09-21 | 2021-06-29 | 中国科学院理化技术研究所 | PH-responsive antibacterial peptide and preparation method and application thereof |
| CN110218760A (en) * | 2019-06-18 | 2019-09-10 | 青岛大学 | It is a kind of antitumor with antibacterial peptide Activity determination experimental provision |
| CN110218760B (en) * | 2019-06-18 | 2023-01-13 | 青岛大学 | Experimental device for activity detection of antitumor antibacterial peptide |
| CN110862458A (en) * | 2019-11-13 | 2020-03-06 | 倪京满 | Combined peptide with acid-activated antitumor activity and clinical application thereof |
| CN110862458B (en) * | 2019-11-13 | 2023-05-05 | 倪京满 | Combined peptide with acid activated antitumor activity and clinical application thereof |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
| CN115925802A (en) * | 2023-02-22 | 2023-04-07 | 西南交通大学 | Antibacterial peptide with enhanced antibacterial activity and serum stability and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102718844B (en) | 2014-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102718844B (en) | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments | |
| Lu et al. | BODIPY-based macromolecular photosensitizer with cation-enhanced antibacterial activity | |
| CN104961801B (en) | A kind of synthesis and application of antibacterial polypeptide conjugate and its dimer with Mutiple Targets | |
| CN107298710A (en) | A kind of extracting method of spirulina phycocyanin | |
| US9127250B2 (en) | Immunosuppressive cell-capturing material and immunosuppressive cell-capturing column | |
| CN105943520A (en) | Biological responsiveness targeted double-drug slow-release carrier material and preparation method thereof | |
| WO2014180533A2 (en) | Conjugates for protection from nephrotoxic active substances | |
| CN105769926A (en) | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof | |
| CN106749559A (en) | Antibacterial peptide and its synthetic method of the one kind based on cell-penetrating peptide Tat (49 57) | |
| Xu et al. | Antisenescence ZIF-8/Resveratrol Nanoformulation with Potential for Enhancement of Bone Fracture Healing in the Elderly | |
| Sozmen et al. | Synthesis of multifunctional organic nanoparticles combining photodynamic therapy and chemotherapeutic drug release | |
| CN102558569B (en) | Lipopolysaccharide amine cationic polymer and preparation method and application thereof | |
| Cao et al. | Starch and chitosan-based antibacterial dressing for infected wound treatment via self-activated NO release strategy | |
| AU2011362979B2 (en) | Compositions and methods for separating, characterizing and administering soluble selenoglycoproteins | |
| CN108998427B (en) | Preparation method of phenol oxidase active protein | |
| CN111423493A (en) | Palmitic acid anti-enzymolysis antibacterial peptide and preparation method and application thereof | |
| CN116731108A (en) | Straw mushroom antioxidant peptide and application thereof | |
| CN102153746B (en) | Degradable polyamino acid derivative as well as preparation method and application thereof | |
| CN102719477A (en) | Non-viral gene vector constructed on basis of N-terminal octadecane acylated antibacterial peptide | |
| CN105037739A (en) | Reduced sensitive type polymer with effect of arginine membrane penetration as well as preparation method and application of reduced sensitive type polymer | |
| CN103120797A (en) | Nano-drug carrier for reductive response, nano-drug particle, nano-drug particle preparation and preparation methods thereof | |
| CN104478994A (en) | Fullerene single-addition line peptide as well as preparation method and application thereof | |
| CN110878129B (en) | Glucosamine heparin salt and application thereof | |
| CN103554305A (en) | Synthetic method of affinity precipitation medium and application thereof to preparation of enoxaparin sodium | |
| CN106176675B (en) | Application of honokiol nanoparticles targeting hepatic sinus endothelial cells in treatment of hepatic fibrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |