CN103965285A - Method for taking super-paramagnetism nanometer particles as solid phase to perform peptide synthesis and synchronously construct polypeptide magnetic nanometer probe - Google Patents

Method for taking super-paramagnetism nanometer particles as solid phase to perform peptide synthesis and synchronously construct polypeptide magnetic nanometer probe Download PDF

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CN103965285A
CN103965285A CN201310049011.4A CN201310049011A CN103965285A CN 103965285 A CN103965285 A CN 103965285A CN 201310049011 A CN201310049011 A CN 201310049011A CN 103965285 A CN103965285 A CN 103965285A
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polypeptide
solution
nano particle
involucrum
dioxide
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CN103965285B (en
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沙印林
罗聃
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Peking University
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Abstract

The invention discloses a method for taking super-paramagnetism nanometer particles as a solid phase to perform peptide synthesis and synchronously construct a polypeptide magnetic nanometer probe. The method comprises: preparing silica-cladded super-paramagnetism nanometer particles and rink-amide-linker-coupled silica-cladded super-paramagnetism nanometer particles; respectively taking the two kinds of particles as solid phases to perform peptide synthesis; and adding a K reagent into two groups of the solid phases. Aiming at the silica-cladded super-paramagnetism nanometer particles, the K reagent is capable of removing a side-chain protection group of the synthetic polypeptide but not cutting off the polypeptide, so that the polypeptide magnetic nanometer probe is obtained. Aiming at the rink-amide-linker-coupled silica-cladded super-paramagnetism nanometer particles, the K reagent is capable of removing the side-chain protection group of the synthetic polypeptide and cutting off the polypeptide from the solid phase, so that the synthetic polypeptide is obtained. According to the method, the super-paramagnetism nanometer particles are capable of being rapidly divorced from a reaction system under the effect of an external magnetic field , so that synthesis is greatly facilitated, also polypeptide can be simply precisely synthesized, also the polypeptide magnetic nanometer probe is synchronously constructed for confirming the biological functions of the synthetic peptide in vitro and in vivo, and also the method is applicable to establishment of large-scale polypeptide library and applicable to rapidly screening out functional peptides.

Description

A kind of polypeptide that carries out taking superparamagnetic nano particle as solid phase is synthetic and synchronously build the method for polypeptide magnetic nano-probe
Technical field
The present invention relates to a kind of synthetic method of Solid-phase Polypeptide, be particularly related to a kind of polypeptide synthesis method taking superparamagnetic nano particle as solid phase, and synchronously build synthetic polypeptide nano magnetic probe in synthetic polypeptide, can be used for the detection of its biological function, the invention belongs to Solid-phase Polypeptide synthesis technical field.
Background technology
1, solid-phase polypeptide synthetic technology:
Solid-phase polypeptide synthetic technology is the important technology of current biochemical field, and the pioneer of this technology is RobertBruce Merrifield.Although there is now the method (as utilized bacterial expression etc.) of multiple acquisition polypeptide, but still there are many peptide sequences to be difficult to obtain (sequence as beyond expression of words in bacterium).Utilize peptide synthesis technology can help the peptide of our easy this sequence of acquisition in laboratory.In synthetic process, can in sequence, add arbitrarily alpha-non-natural amino acid or micromolecular compound, this is all that other method hardly matches simultaneously.In peptide synthesis technology, can be divided into liquid phase synthetic technology and solid phase synthesis technique.Wherein solid-phase polypeptide synthetic technology has developed the perfect main flow that becomes this technology.Its principle is to be beneficial to small bead as solid phase, modifies functional group and can form covalent linkage with amino acid monomer on pearl, reacts in order and both can obtain peptide sequence respectively according to implementation sequence with amino acid monomer.In synthetic process, polypeptide is stably covalently bonded in solid phase bead surface, after reaction finishes, utilizes special chemical reagent peptide sequence can be cut from solid phase pearl.Than liquid phase synthetic technology, solid-phase synthesis carries out on pearl, the extraction of more easily carrying out intermediates with separate, give synthetic bringing great convenience.The solid phase of appropriate design is the key of this technology, its should meet following some:
1) solid phase can be easy, quickly with liquid phase separation
2) Chemical properties of solid phas is stable, can stable existence in the solvent of reaction, unreactiveness is good
3) in solid phase, there is functional group, can with amino acid coupling
The solid phase that meets above-mentioned condition can be divided into again following three kinds from material:
1) gel-type solid phase (refer to contain functional group superpolymer) is comprising vinylbenzene solid phase, polyacrylamide solid phase, polyoxyethylene glycol solid phase etc.
2) surface type solid phase: comprise Bio-Glas, cellulosic fibre etc.
3) compound solid phase: refer to the gel-type superpolymer that supported by rigid matrix
2, superparamagnetic nano particle:
In the time that Ferrite Material is decreased to some scale, at a certain temperature, iron anisotropic crystalline energy barrier can be offset with the hot kinetic energy of particle, and macro manifestations is disorderly and unsystematic the cancelling each other of each magnetocrystalline magnetic moment, thereby does not show magnetic.When under the effect at externally-applied magnetic field, magnetocrystalline magnetic moment tends to arrange along outer magnetic field direction, thus the magnetic of showing.The magnetic nanoparticle that is rich in this character is called as superparamagnetic nano particle.Under the effect of superparamagnetic nano particle outside magnetic field, be convenient to handlingly, can be used for Magnetic resonance imaging, drug conveying etc. simultaneously, there is huge using value.
Summary of the invention
The object of this invention is to provide a kind of method that polypeptide synthesizes and synchronously build polypeptide magnetic nano-probe of carrying out taking superparamagnetic nano particle as solid phase.
In order to reach above object, the technical solution adopted in the present invention is:
A kind of method that polypeptide synthesizes and synchronously build polypeptide magnetic nano-probe of carrying out taking superparamagnetic nano particle as solid phase of the present invention, is characterized in that, comprises the steps:
(1) prepare the superparamagnetic nano particle of silicon-dioxide involucrum, surface exposes amino;
(2) the superparamagnetic nano particle of silicon-dioxide involucrum, coupling strategy by HBTU/HOBt/DIEA is at its surperficial coupling rink amide linker(4-[(2,4-Dimethoxyphenyl) (Fmoc-amino) methyl] phenoxy acetic acid);
(3) respectively taking the superparamagnetic nano particle of the superparamagnetic nano particle of silicon-dioxide involucrum and the silicon-dioxide involucrum of coupling rink amide linker as solid phase, the coupling strategy that utilizes HBTU/HOBt/DIEA is taking the amino acid of Fmoc protection as monomer, synchronously connect one by one amino acid, carry out polypeptide and synthesize;
(4) in two groups of solid phases, add K reagent respectively, described K reagent is by trifluoracetic acid, thioanisole, 1,2-dimercaptoethane, phenol and water are formulated, preferred trifluoracetic acid, thioanisole, 1,2-dimercaptoethane, the volume ratio of phenol and water is 82.5:5:2.5:5:5; K reagent needles plays a part different to two kinds of different solid phases:
A is for the solid phase of the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker, for synthetic polypeptide is taken off to side chain protected and cuts from this solid phase;
B, for the solid phase of the superparamagnetic nano particle of silicon-dioxide involucrum, for synthesizing the de-side chain protected of polypeptide but polypeptide not being cut, obtains the magnetic nano-probe of conjugated polypeptide;
(5) by the polypeptide that in above-mentioned steps (4) a, the superparamagnetic nano particle solid phase from the silicon-dioxide involucrum of coupling rink amide linker cuts with ether sedimentation, collect product and with MALDI-TOF-MS(ground substance assistant laser desorption ionization flight time mass spectrum, English name Matrix-Assisted Laser Desorption/IonizationTime of Flight Mass Spectrometry) chemical structure of the synthetic polypeptide of checking;
(6) the magnetic nano-probe of the conjugated polypeptide being obtained by the superparamagnetic nano particle solid phase of silicon-dioxide involucrum in above-mentioned steps (4) b is dissolved in to PBS(phosphoric acid buffer) in, test confirmation synthetic peptide by vitro and in vivo and whether there is biological function.
Described experiment in vitro comprises cell in vitro magnetic separation, Electronic Speculum.In described body, experiment comprises small animal living body Magnetic resonance imaging.
In the present invention, preferred, described superparamagnetic nano particle be in following three kinds any one: superparamagnetic Fe 3o 4nano particle, superparamagnetic Fe 2o 3nano particle, superparamagnetic FePt nano particle.
Wherein, superparamagnetic nano particle can be according to being prepared with the disclosed method of Publication about Document: MonodisperseMFe 2o 4(M) Fe, Co, Mn) Nanoparticles, Shouheng Sun, et.al, J.AM.CHEM.SOC.2004,126,273-279.
In the present invention, preferred, prepare by following steps as the superparamagnetic nano particle of silicon-dioxide involucrum of solid phase and the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rinkamide linker:
The preparation of the superparamagnetic nano particle solid phase of silicon-dioxide involucrum:
1) the chloroformic solution A of the superparamagnetic nano particle that configuration 100ml concentration is 0.05mg/ml;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml 15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours; After reaction finishes, solution is positioned on magnet, places and discard solution after 10 hours; Collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of silicon-dioxide involucrum;
The preparation of the superparamagnetic nano particle solid phase of the silicon-dioxide involucrum of coupling rink amide linker:
1) the chloroformic solution A of the superparamagnetic nano particle that configuration 100ml concentration is 0.05mg/ml;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours; After reaction finishes, solution is positioned on magnet, places and discard solution after 10 hours, collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of silicon-dioxide involucrum;
5) 0.38mmol rink amide linker is dissolved in dimethyl formamide (DMF) solution of the HBTU/HOBt of the 0.4mmol/L of 1ml; add 69 microlitre N; N-diisopropylethylamine (DIEA) obtains solution C; and the superparamagnetic nano particle of the silicon-dioxide involucrum obtaining is added to solution C, be placed on shaking table room temperature reaction 24 hours;
6) solution that reaction after finishing obtains step 5) is placed on magnet, places and discards solution after 10 hours, and collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker.
In the present invention, preferred, the described superparamagnetic nano particle of silicon-dioxide involucrum taking coupling rink amide linker or the superparamagnetic nano particle of silicon-dioxide involucrum are synthetic as the polypeptide of solid phase, comprise the following steps:
1) pre-treatment of solid phase: by after the superparamagnetic nano particle freeze-drying of the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker or silicon-dioxide involucrum, with dimethyl formamide (DMF) washing;
2) in two kinds of solid phases that obtain to step 1), add respectively the active ester solution of the amino acid monomer of 1ml Fmoc protection, described active ester solution is that the amino acid monomer of 0.38mmol Fmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, then add 69 microlitre N, N-diisopropylethylamine (DIEA) activation obtains for two minutes, reaction is placed in shaking table, room temperature reaction 3 hours;
3) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively;
4) contain 20%(v/v to adding in two groups of solid phases respectively) the DMF solution of piperidines, react 30 minutes deaminize acid Fmoc protection, reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF;
5) respectively to the active ester solution of amino acid monomer that adds as stated above next Fmoc protection in two groups of solid phases.
Further, the present invention also provides the polypeptide and the polypeptide magnetic nano-probe that prepare according to the method described in above any one.And
Described magnetic nano-probe is the application in cell magnetic separation, external electron microscopic observation and confirmation synthetic peptide biological function in vivo in vitro.
Present method has following advantage:
(1) taking superparamagnetic nano particle as solid phase, can be dispersed in polypeptide synthetic agent, and can hightail reaction system under the effect of outside magnetic field, be the synthetic convenience of bringing;
(2) taking magnetic nanoparticle as basis, builds two kinds of solid phases, easy, accurately synthesize when polypeptide and synchronously built the nano-probe of puting together this polypeptide, and can whether there is biological function to it and judge rapidly.This technology can be used for the foundation for peptide library on a large scale, and can filter out rapidly functional peptides.
Using superparamagnetic nano particle is raw material.This material as solid phase not only reached requirement to solid phase (1, solid phase can be easy, quickly with liquid phase separation; 2, Chemical properties of solid phas is stable, can stable existence in the solvent of reaction, unreactiveness is good; 3, in solid phase, there is functional group, can with amino acid coupling.), have more following advantage than traditional solid phase:
1, the centrifuging that separates solid phase than tradition, can directly separate under the effect of superparamagnetic nano particle solid phase outside magnetic field, convenient and swift.
2, by superparamagnetic nano grain surface being carried out to the different solid phase of chemically modified availability matter, for the synthetic polypeptide magnetic nano particle probe of subsequent synchronisation provides the foundation.Described superparamagnetic nano particle be in following three kinds any one: superparamagnetic Fe 3o 4nano particle, superparamagnetic Fe 2o 3nano particle, superparamagnetic FePt nano particle.
The probe of synchronously synthetic conjugated polypeptide when synthetic polypeptide, reason is to add K reagent to produce different-effect to two kinds of solid phases.For the superparamagnetic nano particle solid phase of the silicon-dioxide involucrum of coupling rink amide linker, its rink amide linker can rupture under sour environment, and therefore synthetic polypeptide can depart from solid phase, can be used for mass spectrometric detection.And for the superparamagnetic nano particle solid phase of silicon-dioxide involucrum, synthetic polypeptide and nano particle form amido linkage, can under the effect of K reagent, not depart from solid phase, can obtain thus the bioprobe of polypeptide coupling.The biological function of (small animal living body Magnetic resonance imaging) experiment confirmation synthetic peptide in (cell magnetic separation, Electronic Speculum) and body in vitro.
Described cell in vitro magnetic separation experiment comprises the following steps:
1, the polypeptide magnetic probe of the superparamagnetic nano particle solid phase gained by silicon-dioxide involucrum is added in tumour cell suspension, be placed in 4 ° of C of rotation blending instrument and hatch 2 hours.
2, the cell after hatching is placed on magnet, places after 5 minutes, discard solution, and wash three times with PBS.
3, the suspension of step 2 gained is placed in to light Microscopic observation, can judges by the quantity of cell in the visual field power of the effect of polypeptide to this cell.
Described external electron microscope experiment comprises the following steps:
1, the polypeptide magnetic probe of the superparamagnetic nano particle solid phase gained by silicon-dioxide involucrum is added in tumour cell suspension, be placed in 4 ° of C of rotation blending instrument and hatch 2 hours.
2, the cell after hatching is placed on magnet, places after 5 minutes, discard solution, and wash three times with PBS.
3, step 2 gained PBS suspension is placed in to whizzer 3000rpm centrifugal 10 minutes, supernatant discarded, and carefully add glutaraldehyde.
4, step 3 gained sample is fixed, dehydration, dyeing, after cell section, is positioned over electron microscopic observation, further judges the effect of polypeptide to this cell by observing the distribution of magnetic probe in cell.
In described body, animalcule Magnetic resonance imaging further confirms the biological function of synthetic peptide at live body, comprises the following steps:
1, prepare the mouse (as tumour etc.) of certain disease model.
2, the polypeptide magnetic nano-probe by the superparamagnetic nano particle solid phase gained of silicon-dioxide involucrum by tail vein injection.
3, after 2 hours, will be placed in nuclear magnetic resonance analyser through step 2 mouse after treatment, and whether have signal to reduce to judge the biological effect of synthetic polypeptide to live body by T2 imaging lesions position.
Brief description of the drawings
Fig. 1 is the schematic diagram that the polypeptide of carrying out taking superparamagnetic nano particle as solid phase synthesizes and synchronously build the method for polypeptide probe;
Fig. 2 is the exploded view that polypeptide is synthetic and magnetic separates taking superparamagnetic nano particle as solid phase;
Fig. 3 is the MALDI-TOF-MS(ground substance assistant laser desorption ionization flight time mass spectrum of synthetic pentapeptide (RGDGG), English name Matrix-Assisted Laser Desorption/Ionization Time of Flight MassSpectrometry);
Fig. 4 is that synchronous synthetic RGDGG-magnetic nano-probe carries out cell sorting in vitro, HepG2 cell is α v β 3 high expressing cells, can more efficiently be combined with rgd peptide, therefore by utilizing the HepG2 cell of RGDGG-magnetic nano-probe sorting obviously more than the low expression Hela of α v β 3 cell;
Fig. 5 be to use HepG2 cell section under the sorting of RGDGG-magnetic nano-probe and with electric Microscopic observation, can find the effect by rgd peptide, magnetic probe is attached to cell surface, simultaneously visible receptoe mediated endocytosis;
Fig. 6 is that RGDGG-magnetic nano-probe carries out Magnetic resonance imaging to the nude mice of inoculation HepG2 tumour, further proves that rgd peptide has recognition reaction to HepG2 tumour cell.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 is with superparamagnetic Fe 3o 4nano particle is solid phase synthesis RGDGG pentapeptide and synchronously builds RGDGG-magnetic nano-probe
Schematic diagram as shown in Figure 1.
1, prepare the superparamagnetic Fe of silicon-dioxide involucrum 3o 4nano particle (is prepared according to the disclosed method of document: Monodisperse MFe 2o 4(M) Fe, Co, Mn) Nanoparticles, Shouheng Sun, et.al, J.AM.CHEM.SOC.2004,126,273-279.) and coupling rink amide linker(4-[(2,4-Dimethoxyphenyl) (Fmoc-amino) methyl] phenoxy acetic acid, buy from the biochemical Shanghai of gill company limited.) the superparamagnetic Fe of silicon-dioxide involucrum 3o 4nano particle solid phase:
the superparamagnetic Fe of silicon-dioxide involucrum 3 o 4 the preparation of nano particle solid phase:
1) the superparamagnetic Fe that configuration 100ml concentration is 0.05mg/ml 3o 4the chloroformic solution A of nano particle;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours.
5) after reaction finishes, the solution of step 4) is positioned on magnet, places and discard solution after 10 hours.Collecting precipitation washes with water three times, obtains the superparamagnetic Fe of silicon-dioxide involucrum 3o 4nano particle.
the superparamagnetic Fe of the silicon-dioxide involucrum of coupling rink amide linker 3 o 4 the preparation of nano particle solid phase:
1) the superparamagnetic Fe that configuration 100ml concentration is 0.05mg/ml 3o 4the chloroformic solution A of nano particle;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours.After reaction finishes, solution is positioned on magnet, places and discard solution after 10 hours.Collecting precipitation washes with water three times, obtains the superparamagnetic Fe of silicon-dioxide involucrum 3o 4nano particle.
5) by rink amide linker(0.38mmol) be dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, add 69 microlitre DIEA to obtain solution C.And the superparamagnetic nano particle (5mg) of the silicon-dioxide involucrum obtaining is added to solution C, be placed on shaking table room temperature reaction 24 hours.
6) solution that reaction finishes rear mahjong step 5) is placed on magnet, places and discards solution after 10 hours.Collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker
2, respectively with the superparamagnetic Fe of silicon-dioxide involucrum 3o 4the superparamagnetic Fe of the silicon-dioxide involucrum of nano particle and coupling rink amide linker 3o 4nano particle is solid phase, and the coupling strategy that utilizes HBTU/HOBt/DIEA, taking the amino acid of Fmoc protection as monomer, synchronously connects amino acid one by one, carries out polypeptide synthetic:
1) pre-treatment of solid phase: by the superparamagnetic Fe of the silicon-dioxide involucrum of coupling rink amide linker 3o 4the superparamagnetic Fe of nano particle or silicon-dioxide involucrum 3o 4after nano particle freeze-drying, wash with DMF.
2) to above-mentioned two kinds of solid phases (superparamagnetic Fe of the silicon-dioxide involucrum of coupling rink amide linker 3o 4the superparamagnetic Fe of nano particle and silicon-dioxide involucrum 3o 4nano particle) add respectively the active ester solution (Fmoc-Gly of 0.38mmolFmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, adds 69 microlitre DIEA postactivated two minutes) of amino acid glycine (Fmoc-Gly) monomer of first Fmoc of 1ml protection.Reaction is placed in shaking table, room temperature reaction 3 hours.
3) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively.
4), respectively to the DMF solution that adds 20% piperidines in two groups of solid phases, react 30 minutes deaminize acid Fmoc protection.Reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF.
5) discard the DMF of above-mentioned two kinds of solid phases; and add respectively the active ester solution (Fmoc-Gly of 0.38mmolFmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, adds 69 microlitre DIEA postactivated two minutes) of amino acid glycine (Fmoc-Gly) monomer of second Fmoc of 1ml protection.Reaction is placed in shaking table, room temperature reaction 3 hours.
6) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively.
7), respectively to the DMF solution that adds 20% piperidines in two groups of solid phases, react 30 minutes deaminize acid Fmoc protection.Reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF.
8) discard the DMF of above-mentioned two kinds of solid phases; and add respectively the active ester solution (Fmoc-Asp of 0.38mmolFmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, adds 69 microlitre DIEA postactivated two minutes) of amino acid aspartic acid (Fmoc-Asp) monomer of the 3rd Fmoc of 1ml protection.Reaction is placed in shaking table, room temperature reaction 3 hours.
9) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively.
10), respectively to the DMF solution that adds 20% piperidines in two groups of solid phases, react 30 minutes deaminize acid Fmoc protection.Reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF.
11) discard the DMF of above-mentioned two kinds of solid phases; and add respectively the active ester solution (Fmoc-Gly of 0.38mmolFmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, adds 69 microlitre DIEA postactivated two minutes) of amino acid glycine (Fmoc-Gly) monomer of the 4th Fmoc of 1ml protection.Reaction is placed in shaking table, room temperature reaction 3 hours.
12) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively.
13), respectively to the DMF solution that adds 20% piperidines in two groups of solid phases, react 30 minutes deaminize acid Fmoc protection.Reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF.
14) discard the DMF of above-mentioned two kinds of solid phases; and add respectively the active ester solution (Fmoc-Arg of 0.38mmolFmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, adds 69 microlitre DIEA postactivated two minutes) of amino acids Arginine (Fmoc-Arg) monomer of the 5th Fmoc of 1ml protection.Reaction is placed in shaking table, room temperature reaction 3 hours.
15) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively.
16), respectively to the DMF solution that adds 20% piperidines in two groups of solid phases, react 30 minutes deaminize acid Fmoc protection.Reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF.
3, in two groups of solid phases, add K reagent (trifluoracetic acid/thioanisole/1,2-dimercaptoethane/phenol/water: 82.5/5/2.5/5/5) reaction 20 minutes respectively.K reagent needles plays a part different to two kinds of different solid phases:
A is for the superparamagnetic Fe of the silicon-dioxide involucrum of coupling rink amide linker 3o 4the solid phase of nano particle, can and cut from this solid phase de-synthetic polypeptide side chain protected;
B is for the superparamagnetic Fe of silicon-dioxide involucrum 3o 4the solid phase of nano particle, can, by synthesizing the de-side chain protected of polypeptide but polypeptide not cut, obtain the magnetic nano-probe of conjugated polypeptide.
4, by the superparamagnetic Fe from the silicon-dioxide involucrum of coupling rink amide linker in above-mentioned steps 3a 3o 4the polypeptide cutting in nano particle solid phase, with ether sedimentation, is collected product the chemical structure with the synthetic polypeptide of MALDI-TOF-MS checking.
With superparamagnetic Fe 3o 4nano particle be the polypeptide of solid phase is synthetic and magnetic separates exploded view as shown in Figure 2; The MALDI-TOF-MS(ground substance assistant laser desorption ionization flight time mass spectrum of synthetic pentapeptide (RGDGG), English name Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detected result is as shown in Figure 3.
Embodiment 2 is with superparamagnetic Fe 2o 3nano particle is solid phase synthesis RGDGG pentapeptide and synchronously builds RGDGG-magnetic nano-probe
Prepare the superparamagnetic Fe of silicon-dioxide involucrum according to the method for embodiment 1 2o 3nano particle and coupling rinkamide linker(4-[(2,4-Dimethoxyphenyl) (Fmoc-amino) methyl] phenoxy acetic acid, buy from the biochemical Shanghai of gill company limited.) the superparamagnetic Fe of silicon-dioxide involucrum 2o 3nano particle.Taking the two as solid phase synthesis RGDGG pentapeptide and synchronously build RGDGG-magnetic nano-probe, method is with embodiment 1 respectively.
Embodiment 3 cell in vitro sorting experiments
The magnetic nano-probe of the conjugated polypeptide being obtained by the superparamagnetic nano particle solid phase of silicon-dioxide involucrum in embodiment 1 step 3b is dissolved in PBS to the effect by cell in vitro sorting experiment confirmation synthetic peptide to cell.
Method:
1) the polypeptide magnetic probe (0.15mg) of the superparamagnetic nano particle solid phase gained by silicon-dioxide involucrum is added respectively to (cell concn is 200000/ml) in 1ml Hela cell and HepG2 cell suspension, be placed in 4 ° of C of rotation blending instrument and hatch 2 hours;
2) two groups of cells after hatching are placed on magnet, place after 5 minutes, discard solution, and wash three times with PBS;
3) by through step 2) process after the suspension of gained be placed in light Microscopic observation, the quantity that can pass through cell in the visual field judge the power of the effect of polypeptide to this cell.
Result: Fig. 4 is that synchronous synthetic RGDGG-magnetic nano-probe carries out cell sorting in vitro, HepG2 cell is α v β 3 high expressing cells, can more efficiently be combined with rgd peptide, therefore by utilizing the HepG2 cell of RGDGG-magnetic nano-probe sorting obviously more than the low expression Hela of α v β 3 cell.
The external electron microscope experiment of embodiment 4
Comprise the following steps:
1) the polypeptide magnetic probe (0.15mg) of the superparamagnetic nano particle solid phase gained by silicon-dioxide involucrum embodiment 1 being prepared adds (cell concn is 200000/ml) in 1ml HepG2 cell suspension, is placed in 4 ° of C of rotation blending instrument and hatches 2 hours;
2) cell after hatching is placed on magnet, places after 5 minutes, discard solution, and wash three times with PBS;
3) by step 2) gained PBS suspension is placed in whizzer 3000rpm centrifugal 10 minutes, supernatant discarded, and carefully add glutaraldehyde;
4) step 3) gained sample is fixed, dehydration, dyeing, after cell section, is positioned over electron microscopic observation, further judges the effect of polypeptide to this cell by observing the distribution of magnetic probe in cell.
Result: Fig. 5 be to use HepG2 cell section under the sorting of RGDGG-magnetic nano-probe and with electric Microscopic observation, can find the effect by rgd peptide, magnetic probe is attached to cell surface, simultaneously visible receptoe mediated endocytosis.
Embodiment 5 pentapeptide RGDGG biological function verification in vivo
Adopt animalcule Magnetic resonance imaging in body further to confirm the synthetic pentapeptide RGDGG of embodiment 1 biological function in vivo:
Method:
1) select BABL/c nude mice, raise two weeks in 25 ° of C constant temperature, and with shoulder kind HepG2 tumour;
2) the RGDGG-magnetic nano-probe of the superparamagnetic nano particle solid phase gained by silicon-dioxide involucrum by tail vein injection 0.3mg.Experiment with the superparamagnetic nano particle of dioxide injection silicon involucrum (not participating in polypeptide synthetic) in contrast;
3) will be through step 2 after 2 hours) whether two mouse of processing are placed in nuclear magnetic resonance analyser, have signal to reduce by T2 imaging lesions position.
Result: Fig. 6 is that RGDGG-magnetic nano-probe carries out Magnetic resonance imaging to the nude mice of inoculation HepG2 tumour, further proves that rgd peptide has recognition reaction to HepG2 tumour cell.

Claims (9)

1. carry out polypeptide taking superparamagnetic nano particle as solid phase synthetic and synchronously build the method for polypeptide magnetic nano-probe, it is characterized in that, comprise the steps:
(1) prepare the superparamagnetic nano particle of silicon-dioxide involucrum, surface exposes amino;
(2) the superparamagnetic nano particle of silicon-dioxide involucrum, the coupling strategy by HBTU/HOBt/DIEA is at its surperficial coupling rink amide linker;
(3) respectively taking the superparamagnetic nano particle of the superparamagnetic nano particle of silicon-dioxide involucrum and the silicon-dioxide involucrum of coupling rink amide linker as solid phase, the coupling strategy that utilizes HBTU/HOBt/DIEA is taking the amino acid of Fmoc protection as monomer, synchronously connect one by one amino acid, carry out polypeptide and synthesize;
(4) in two groups of solid phases, add K reagent respectively, described K reagent is by trifluoracetic acid, thioanisole, 1,2-dimercaptoethane, phenol and water are formulated, preferred trifluoracetic acid, thioanisole, 1,2-dimercaptoethane, the volume ratio of phenol and water is 82.5:5:2.5:5:5; K reagent needles plays a part different to two kinds of different solid phases:
A is for the solid phase of the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker, for synthetic polypeptide is taken off to side chain protected and cuts from this solid phase;
B, for the solid phase of the superparamagnetic nano particle of silicon-dioxide involucrum, for synthesizing the de-side chain protected of polypeptide but polypeptide not being cut, obtains the magnetic nano-probe of conjugated polypeptide;
(5) by the polypeptide that in above-mentioned steps (4) a, the superparamagnetic nano particle solid phase from the silicon-dioxide involucrum of coupling rink amide linker cuts with ether sedimentation, collect product also by the chemical structure of the synthetic polypeptide of MALDI-TOF-MS checking;
(6) the magnetic nano-probe of the conjugated polypeptide being obtained by the superparamagnetic nano particle solid phase of silicon-dioxide involucrum in above-mentioned steps (4) b is dissolved in PBS, tests confirmation synthetic peptide by vitro and in vivo and whether there is biological function.
2. method according to claim 1, it is characterized in that described superparamagnetic nano particle be in following three kinds any one: superparamagnetic Fe 3o 4nano particle, superparamagnetic Fe 2o 3nano particle, superparamagnetic FePt nano particle.
3. method according to claim 1, is characterized in that preparing by following steps as the superparamagnetic nano particle of silicon-dioxide involucrum of solid phase and the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker:
The preparation of the superparamagnetic nano particle solid phase of silicon-dioxide involucrum:
1) the chloroformic solution A of the superparamagnetic nano particle that configuration 100ml concentration is 0.05mg/ml;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours; After reaction finishes, solution is positioned on magnet, places and discard solution after 10 hours; Collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of silicon-dioxide involucrum;
The preparation of the superparamagnetic nano particle solid phase of the silicon-dioxide involucrum of coupling rink amide linker:
1) the chloroformic solution A of the superparamagnetic nano particle that configuration 100ml concentration is 0.05mg/ml;
2) solution A is placed under 40 ° of C, with the rotating speed underpressure distillation of 150rpm, solution is concentrated into 10 microlitres;
3) to the sodium dodecyl sulfate aqueous solution that adds 100ml15mM in the solution A after concentrated, the ultrasonic 10min of ultrasonic power with 60W in 40 ° of C water-baths obtains solution B;
4) solution B is placed in to shaking table, adds 50 microlitre n-octyl Trimethoxy silanes (OCTMO), shake up after 30 minutes; Continue to drip 50 microlitre APTESs (APS), continue reaction 48 hours; After reaction finishes, solution is positioned on magnet, places and discard solution after 10 hours, collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of silicon-dioxide involucrum;
5) 0.38mmol rink amide linker is dissolved in dimethyl formamide (DMF) solution of the HBTU/HOBt of the 0.4mmol/L of 1ml; add 69 microlitre N; N-diisopropylethylamine (DIEA) obtains solution C; and the superparamagnetic nano particle of the silicon-dioxide involucrum obtaining is added to solution C, be placed on shaking table room temperature reaction 24 hours;
6) solution that reaction after finishing obtains step 5) is placed on magnet, places and discards solution after 10 hours, and collecting precipitation washes with water three times, obtains the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker.
4. method according to claim 1, is characterized in that, the described superparamagnetic nano particle of silicon-dioxide involucrum taking coupling rink amide linker or the superparamagnetic nano particle of silicon-dioxide involucrum are synthetic as the polypeptide of solid phase, comprise the following steps:
1) pre-treatment of solid phase: by after the superparamagnetic nano particle freeze-drying of the superparamagnetic nano particle of the silicon-dioxide involucrum of coupling rink amide linker or silicon-dioxide involucrum, with dimethyl formamide (DMF) washing;
2) in two kinds of solid phases that obtain to step 1), add respectively the active ester solution of the amino acid monomer of 1ml Fmoc protection, described active ester solution is that the amino acid monomer of 0.38mmol Fmoc protection is dissolved in the DMF solution of HBTU/HOBt of the 0.4mmol/L of 1ml, then add 69 microlitre N, N-diisopropylethylamine (DIEA) activation obtains for two minutes, reaction is placed in shaking table, room temperature reaction 3 hours;
3) after reaction finishes, respectively two group reactions are placed on magnet, collect magnetic particle and discard solution, wash three times with DMF respectively;
4) contain 20%(v/v to adding in two groups of solid phases respectively) the DMF solution of piperidines, react 30 minutes deaminize acid Fmoc protection, reaction finishes respectively two group reactions to be placed on magnet afterwards, collects magnetic particle and also discards solution, washes three times respectively with DMF;
5) respectively to the active ester solution of amino acid monomer that adds as stated above next Fmoc protection in two groups of solid phases.
5. the polypeptide preparing according to the method described in claim 1-4 any one and polypeptide magnetic nano-probe.
6. polypeptide magnetic nano-probe claimed in claim 5 application in cell magnetic separation, external electron microscopic observation and confirmation synthetic peptide biological function in vivo in vitro.
7. utilize polypeptide magnetic nano-probe described in claim 5 to carry out a method for cell in vitro magnetic separation, it is characterized in that comprising the following steps:
1) polypeptide magnetic nano-probe claimed in claim 5 is added in tumour cell suspension, be placed in 4 ° of C of rotation blending instrument and hatch 2 hours;
2) cell after hatching is placed on magnet, places after 5 minutes, discard solution, and wash three times with PBS;
3) by step 2) suspension of gained is placed in light Microscopic observation, can judge by the quantity of cell in the visual field power of the effect of polypeptide to this cell.
8. utilize polypeptide magnetic nano-probe described in claim 5 to carry out a method for external electron microscopic observation, it is characterized in that comprising the following steps:
1) polypeptide magnetic nano-probe claimed in claim 5 is added in tumour cell suspension, be placed in 4 ° of C of rotation blending instrument and hatch 2 hours;
2) cell after hatching is placed on magnet, places after 5 minutes, discard solution, and wash three times with PBS;
3) by step 2) gained PBS suspension is placed in whizzer 3000rpm centrifugal 10 minutes, supernatant discarded, and carefully add glutaraldehyde;
4) step 3) gained sample is fixed, dehydration, dyeing, after cell section, is positioned over electron microscopic observation, further judges the effect of polypeptide to this cell by observing the distribution of magnetic probe in cell.
9. a method of utilizing the polypeptide magnetic nano-probe described in claim 5 further to confirm synthetic peptide biological function in vivo by Magnetic resonance imaging, is characterized in that comprising the following steps:
1) prepare the mouse of certain disease model;
2) by tail vein injection polypeptide magnetic claimed in claim 5 nano-probe;
3) will be through step 2 after 2 hours) whether mouse after treatment is placed in nuclear magnetic resonance analyser, have signal to reduce to judge the biological effect of synthetic polypeptide to live body by T2 imaging lesions position.
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CN108376608A (en) * 2018-02-10 2018-08-07 青岛大学 A kind of magnetic nano-particle and its purposes for preparing Magnetic solid phases carrier
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CN108927078A (en) * 2018-07-13 2018-12-04 珠海中科先进技术研究院有限公司 A kind of Aminosilylation magnetic Nano microsphere and the preparation method and application thereof containing Fmoc- diazanyl
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CN1597692A (en) * 2004-07-19 2005-03-23 西安蓝晶生物科技有限公司 Light cut-out magnetic particle molecale label, its preparation method and aplication in multipeptide synthesis preparation
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US10087221B2 (en) 2013-03-21 2018-10-02 Sanofi-Aventis Deutschland Gmbh Synthesis of hydantoin containing peptide products
US10450343B2 (en) 2013-03-21 2019-10-22 Sanofi-Aventis Deutschland Gmbh Synthesis of cyclic imide containing peptide products
CN108376608A (en) * 2018-02-10 2018-08-07 青岛大学 A kind of magnetic nano-particle and its purposes for preparing Magnetic solid phases carrier
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CN108314700B (en) * 2018-02-27 2021-08-03 青岛大学 Method for solid-phase synthesis of polypeptide, synthesized magnetic nano probe and application thereof
CN108927078A (en) * 2018-07-13 2018-12-04 珠海中科先进技术研究院有限公司 A kind of Aminosilylation magnetic Nano microsphere and the preparation method and application thereof containing Fmoc- diazanyl

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