CN108376608B - Magnetic nano particle and application thereof in preparing magnetic solid phase carrier - Google Patents
Magnetic nano particle and application thereof in preparing magnetic solid phase carrier Download PDFInfo
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
- H01F41/02—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for manufacturing cores, coils, or magnets
- H01F41/0253—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for manufacturing cores, coils, or magnets for manufacturing permanent magnets
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F212/02—Monomers containing only one unsaturated aliphatic radical
- C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F212/06—Hydrocarbons
- C08F212/08—Styrene
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
- C08K3/20—Oxides; Hydroxides
- C08K3/22—Oxides; Hydroxides of metals
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
- C08K3/20—Oxides; Hydroxides
- C08K3/22—Oxides; Hydroxides of metals
- C08K2003/2265—Oxides; Hydroxides of metals of iron
- C08K2003/2275—Ferroso-ferric oxide (Fe3O4)
Abstract
The invention belongs to the technical field of solid-phase polypeptide synthesis, and particularly relates to a magnetic nanoparticle for solid-phase polypeptide synthesis, and further discloses a synthesis method and application of the magnetic nanoparticle in preparation of a magnetic solid-phase carrier for solid-phase polypeptide synthesis. The magnetic nano-particles are superparamagnetic ferroferric oxide particles (Fe)3O4) Is a magnetic core, is modified by oleic acid and is coated with Polystyrene (PS) on the surface to form a core-shell type PS @ Fe3O4Magnetic nanoparticles of structure and coating of styrene with Fe3O4And simultaneously introducing 4- (4-vinylbenzyloxy benzyl alcohol) to obtain the surface polymer modified nano magnetic ball. The obtained magnetic nano-particles have proper size, surface chemical adaptability capable of further functionalization, good dispersibility and compatibility in a medium, and can be used as a magnetic solid phase carrier in polypeptide solid phase synthesis.
Description
Technical Field
The invention belongs to the technical field of solid-phase polypeptide synthesis, and particularly relates to a magnetic nanoparticle for solid-phase polypeptide synthesis, and further discloses a synthesis method and application thereof in preparing a magnetic solid-phase carrier for solid-phase polypeptide synthesis.
Background
As an organic polymer, the polypeptide has been receiving more and more attention due to its multiple functionalities and good biocompatibility, so that the chemical synthesis method of the polypeptide also becomes a large important area in the field of organic synthesis. At present, the principle of polypeptide synthesis mainly uses amino acids as basic units, and under the action of a catalyst, the amino acids are connected through the formation of an amide bond through the reaction of carboxyl groups and amino groups between the amino acids, so that the growth of polypeptide chains is realized. However, in the conventional organic synthesis method of polypeptides, the connection of carboxyl and amino groups between the connected amino acids usually depends only on the free combination between the amino acids, and the obtained polypeptides usually have a polypeptide structure with randomly connected amino acids. Therefore, the polypeptide with a specific sequence or an amino acid sequence with a specific length is difficult to obtain by the existing polypeptide synthesis method, and the obtained polypeptide product is difficult to apply.
In 1963, Solid Phase Peptide Synthesis (SPPS) using Boc as an amino acid protecting group was created by Merrifield, and thus solid phase Synthesis using protected amino acids as a raw material has attracted attention due to its unique advantages. In the next decades, the solid phase peptide synthesis technology has been perfected and gradually developed from small-scale short peptide chain synthesis to large-scale long peptide chain synthesis. According to the method, firstly, a proper solid-phase carrier is selected, polypeptide growth and cutting are realized by coupling a Linker on the surface of the carrier, and then amino acids protected by a terminal group are grafted one by one to form polypeptide chains. In the solid-phase synthesis, when the reaction is carried out on a solid-phase carrier, the complex and time-consuming intermediate separation and purification steps required by the liquid-phase synthesis are avoided, the operation steps are simple and controllable, the reaction time is rapid, and the method plays an increasingly important role in the synthesis of the polypeptide.
The solid phase polypeptide synthesis technology is a key technology of modern protein chemistry research and is an important method for obtaining bioactive polypeptide, and besides Boc chemistry developed by Merrifield, Fmoc chemistry is also included. Compared with Boc chemistry, Fmoc chemistry not only meets the requirement of rapid automatic preparation, but also is more convenient and rapid, and is widely applied in the fields of biochemistry, immunity, molecular microbiology, pharmacy and the like which require more polypeptides. In the prior art, solid phase carriers for polypeptide synthesis are generally micron-sized polymers, complex steps such as multiple filtration and centrifugation are required in the synthesis process, the synthesized polypeptide cannot be directly used in organisms, the synthesized polypeptide can be applied in the next step after being cut, and the development of the solid phase polypeptide synthesis technology is seriously influenced.
Superparamagnetic (superparamagnnetism) refers to a ferromagnetic substance with a single domain structure when the particle size is smaller than a critical size, and the ferromagnetic substance has a paramagnetic characteristic when the Temperature is lower than the curie Temperature and higher than the transition Temperature (Block Temperature), but the paramagnetic susceptibility of the ferromagnetic substance is far higher than that of a common paramagnetic material under the action of an external magnetic field, and the ferromagnetic substance is called superparamagnetic. The superparamagnetic nano particle has wide application prospect in biomedical fields of targeted drug carriers, cell separation, nuclear magnetic resonance, immunodetection, biomolecule purification and the like, and is often used as a solid phase carrier in a polypeptide solid phase synthesis technology. However, the existing magnetic nanoparticles are easy to agglomerate due to the effects of size effect, magnetic dipole attraction and the like, have low chemical stability, are easy to be oxidized, have insufficient surface hydroxyl groups, are difficult to be directly applied and seriously affect the performance of the synthesized polypeptide.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to provide a magnetic nanoparticle, which has a suitable size, surface chemical adaptability for further functionalization, and good dispersibility and compatibility in a medium, and can be used for preparing a magnetic solid phase carrier in solid phase polypeptide synthesis.
In order to solve the technical problems, the synthesis method of the magnetic nanoparticles comprises the following steps:
(1) superparamagnetic Fe3O4The particles are modified by oleic acid to obtain oleic acid modified superparamagnetic Fe3O4Particles;
(2) taking the oleic acid modified superparamagnetic Fe3O4Dispersing the particles in an organic solvent to form magnetic fluid, and adding styrene, 4- (4-vinylbenzyloxy benzyl alcohol) and divinylbenzene into the magnetic fluid to form a magnetic fluid oil phase;
(3) dropwise adding the obtained magnetofluid oil phase into a Sodium Dodecyl Sulfate (SDS) aqueous solution to be uniformly dispersed to prepare a suspension;
(4) adding a potassium persulfate initiator into the obtained suspension, and reacting at 60-80 ℃ in a nitrogen atmosphere; and after the reaction is finished, carrying out magnetic separation on the solution, discarding the solution and collecting precipitates to obtain the required magnetic nanoparticles.
In the step (1), the superparamagnetic Fe3O4The step of modifying the particles with oleic acid specifically comprises: taking FeCl3·6H2O and FeCl2·4H2Dissolving O in deionized water, and adding ammonia water at 70-80 ℃ in a nitrogen atmosphere to react; after the reaction is finished, adding oleic acid into the mixture, and carrying out modification reaction at 70-80 ℃; washing the magnetic nano particles obtained by the reaction to be neutral, and drying in vacuum to obtain the needed oleic acid modified superparamagnetic Fe3O4Particles.
In the step (2), the organic solvent is n-hexane.
In the step (2), the molar ratio of the styrene to the 4- (4-vinylbenzyloxy-benzyl alcohol) to the divinylbenzene is 3-5: 0.4-0.6: 0.4-0.6, and preferably 4: 0.5: 0.5.
in the step (3), the mass concentration of the Sodium Dodecyl Sulfate (SDS) aqueous solution is 0.5-2 g/L.
The invention also discloses the magnetic nano-particles synthesized by the method.
The invention also discloses application of the magnetic nanoparticles to preparation of a magnetic solid phase carrier in solid phase polypeptide synthesis.
The invention also discloses a solid phase polypeptide synthesis method, which comprises the following steps:
s1, synthesizing the magnetic nanoparticles according to the method, and using the magnetic nanoparticles as a magnetic solid phase carrier;
s2, connecting a first Fmoc-amino acid on the surface of the magnetic solid phase carrier through a HOBt/DIC/DMAP active ester coupling system;
s3, connecting Fmoc-protected amino acid monomers to the obtained solid phase carrier one by utilizing a HOBt/HBTU/DIEA coupling system, and synthesizing a polypeptide with a specific sequence;
s4, adding a K reagent into the system connected with the specific polypeptide, deprotecting the synthesized polypeptide side chain, and cutting off the polypeptide side chain from the magnetic sphere solid phase;
s5, carrying out rotary evaporation on the cut polypeptide, carrying out sedimentation by using ethyl acetate, and collecting sediment to obtain the required polypeptide.
In the step S2, the dosage ratio of the magnetic solid phase carrier, Fmoc-amino acid, HOBt, DIC, and DMAP is 1: 3: 6: 4: 0.1;
in the step S3, the usage ratio of the magnetic solid phase carrier, Fmoc-amino acid, HBTU, HOBt, DIEA is 1: 3: 3: 3: 6.
the invention also discloses the polypeptide or polypeptide library synthesized by the method.
The magnetic nano-particles are superparamagnetic ferroferric oxide particles (Fe)3O4) Is a magnetic core, is modified by oleic acid and is coated with Polystyrene (PS) on the surface to form a core-shell type PS @ Fe3O4Magnetic nanoparticles of structure and coating of styrene with Fe3O4And simultaneously introducing 4- (4-vinylbenzyloxy benzyl alcohol) to obtain the surface polymer modified nano magnetic ball. The characterization result proves that the superparamagnetism Fe prepared by the invention3O4The particles have a suitable size and good monodispersity. Using micro-emulsion method to treat Fe3O4The surface of the magnetic ball is coated with the PS shell, and simultaneously, the surface function modification is carried out on the magnetic ball, so that the obtained magnetic nano particle has a proper size, surface groups capable of further functionalization and good dispersibility and compatibility in a medium.
The magnetic nano-particles can be uniformly dispersed in a polypeptide synthesis reagent, can be quickly separated under the action of an external magnetic field, provides great convenience for polypeptide synthesis, can simply, conveniently and accurately synthesize the polypeptide with a specific sequence, and can be used as a solid phase carrier for polypeptide solid phase synthesis. Meanwhile, the magnetic nano-particles synthesized by the method can be directly used in organisms, the synthesized polypeptide can meet the target application of the polypeptide in the organisms after being cut by a cutting reagent, and the method can also be used for establishing a large-scale synthesized polypeptide library so as to quickly screen out functional polypeptides and carry out the target screening and biological function authentication of the polypeptides.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 shows oleic acid-modified superparamagnetic Fe prepared in example 13O4Transmission Electron Micrographs (TEMs) of the particles;
FIG. 2 is a Transmission Electron Micrograph (TEM) of the surface polymer-modified magnetic nanoparticles prepared in example 1;
FIG. 3 is an ESI-MS diagram of the hexapeptide synthesized in example 4 of the present invention.
Detailed Description
The transmission electron microscope photographs in the following examples were measured by a JEOL JEM-1200 type transmission electron microscope; the Fourier transform infrared spectrum analysis picture is measured by a Nicolet 6700 type infrared spectrometer.
Example 1
The synthesis method of the magnetic nanoparticles comprises the following steps:
(1) 12g of FeCl3·6H2O and 4.9g FeCl2·4H2O was dissolved in 50mL of deionized water and then N at 80 deg.C2In the atmosphere, 50ml of ammonia water is rapidly added into the solution under the condition of violent stirring, and the mixture is uniformly mixed;
stirring for 30 minutes, adding 2g of oleic acid into the mixture, keeping the suspension at 80 ℃ for reacting for 1 hour, washing the magnetic nanoparticles to be neutral by using deionized water after the reaction is finished, and drying in vacuum to obtain oleic acid modified superparamagnetic Fe3O4Particles;
transmission electron microscope is adopted to carry out superparamagnetic Fe modified by oleic acid3O4The microscopic morphology analysis of the particles is shown in FIG. 1, from which it can be seen that oleic acid modified superparamagnetic Fe3O4The monodispersity of the particles is good, the particle size is uniform, and the particle size is about 10 nm;
(2) the obtained oleic acid modified superparamagnetic Fe3O4Dispersing the particles into n-hexane to form magnetic fluid (the solid-to-liquid ratio of the magnetic fluid to a solvent is controlled to be 0.125g/mL), adding styrene, 4- (4-vinylbenzyloxy benzyl alcohol) and Divinylbenzene (DVB) (the molar ratio is 4: 0.5: 0.5) into the magnetic fluid under the normal ultrasonic condition to form a magnetic fluid oil phase, and adding 400 mu L of styrene into each milliliter of the magnetic fluid according to the amount of the magnetic fluid;
(3) dropwise adding the magnetofluid oil phase into 1g/L Sodium Dodecyl Sulfate (SDS) aqueous solution, and carrying out ultrasonic treatment in a cell crusher for 10 minutes under the condition of ice-water bath to obtain suspension;
(4) adding potassium persulfate (KPS) initiator (0.6 g KPS per ml of magnetofluid) into the crushed suspension, and reacting for 3 hours at 70 ℃ under the condition of vigorous mechanical stirring in nitrogen atmosphere; after the reaction is finished, placing the obtained solution on a magnet, placing for a certain time, removing the solution, collecting the precipitate, washing with water for three times to obtain the surface-coated Polystyrene (PS) to form a core-shell type PS @ Fe3O4The magnetic nano-particle with the structure can be used as a magnetic solid phase carrier in solid phase polypeptide synthesis.
The microscopic morphology analysis of the surface polymer-modified magnetic nanoparticles obtained in this example was performed by using a transmission electron microscope, and the results are shown in fig. 2, which shows that the obtained magnetic nanoparticles, after being modified by the surface polymer, have good monodispersity, an obvious shell layer, a uniform particle size of about 100 nm.
Example 2
The synthesis method of the magnetic nanoparticles comprises the following steps:
(1) 12g of FeCl3·6H2O and 4.9g FeCl2·4H2O was dissolved in 50mL deionized water and then N at 70 deg.C2In the atmosphere, 50mL of ammonia water is rapidly added into the solution under vigorous stirring, and the mixture is uniformly mixed;
stirring for 20min, adding 2g oleic acid into the mixture, maintaining the suspension at 70 deg.c for 2 hr, washing the magnetic nanometer particle with deionized water to neutralityVacuum drying to obtain oleic acid modified superparamagnetic Fe3O4Particles;
(2) the obtained oleic acid modified superparamagnetic Fe3O4Dispersing the particles into n-hexane to form magnetic fluid (the solid-to-liquid ratio of the magnetic fluid to a solvent is controlled to be 0.125g/mL), adding styrene, 4- (4-vinylbenzyloxy benzyl alcohol) and Divinylbenzene (DVB) (the molar ratio is 3: 0.4: 0.4) into the magnetic fluid under the conventional ultrasonic condition to form magnetic fluid oil phase, and adding 400 mu L of styrene into each milliliter of magnetic fluid according to the amount of the magnetic fluid;
(3) dropwise adding the magnetofluid oil phase into 2g/L Sodium Dodecyl Sulfate (SDS) aqueous solution, and carrying out ultrasonic treatment in a cell crusher for 20 minutes under the condition of ice-water bath to obtain suspension;
(4) adding potassium persulfate (KPS) initiator (0.6 g KPS per ml of magnetofluid) into the crushed suspension, and reacting for 2 hours at 80 ℃ under the action of vigorous mechanical stirring in nitrogen atmosphere; after the reaction is finished, placing the obtained solution on a magnet, placing for a certain time, removing the solution, collecting the precipitate, washing with water for three times to obtain the surface-coated Polystyrene (PS) to form a core-shell type PS @ Fe3O4The magnetic nano-particle with the structure can be used as a magnetic solid phase carrier in solid phase polypeptide synthesis.
Example 3
The synthesis method of the magnetic nanoparticles comprises the following steps:
(1) 12g of FeCl3·6H2O and 4.9g FeCl2·4H2O was dissolved in 50mL of deionized water and then N at 80 deg.C2In the atmosphere, 50mL of ammonia water is rapidly added into the solution under vigorous stirring, and the mixture is uniformly mixed;
stirring for 30 minutes, adding 2g of oleic acid into the mixture, keeping the suspension at 80 ℃ for reacting for 2 hours, washing the magnetic nanoparticles to be neutral by using deionized water after the reaction is finished, and drying in vacuum to obtain oleic acid modified superparamagnetic Fe3O4Particles;
(2) the obtained oleic acid modified superparamagnetic Fe3O4The particles are dispersed in n-hexane to formAdding styrene, 4- (4-vinylbenzyloxy benzyl alcohol) and Divinylbenzene (DVB) (the molar ratio of the styrene to the 4- (4-vinylbenzyloxy) benzyl alcohol to the magnetic fluid is 5: 0.6: 0.6) under the conventional ultrasonic condition to form a magnetic fluid oil phase, wherein 400 mu L of styrene is added into each milliliter of magnetic fluid;
(3) dropwise adding the magnetofluid oil phase into 0.5g/L Sodium Dodecyl Sulfate (SDS) aqueous solution, and carrying out ultrasonic treatment in a cell crusher for 20 minutes under the condition of ice-water bath to obtain suspension;
(4) adding potassium persulfate (KPS) initiator (0.6 g KPS per ml of magnetofluid) into the crushed suspension, and reacting for 2 hours at 60 ℃ under the action of vigorous mechanical stirring in nitrogen atmosphere; after the reaction is finished, placing the obtained solution on a magnet, placing for a certain time, removing the solution, collecting the precipitate, washing with water for three times to obtain the surface-coated Polystyrene (PS) to form a core-shell type PS @ Fe3O4The magnetic nano-particle with the structure can be used as a magnetic solid phase carrier in solid phase polypeptide synthesis.
Example 4
In this example, the solid phase synthesis of 6 peptide (VMIMIV) was performed using the nano-magnetic particles synthesized in example 1 as a magnetic solid phase carrier, and specifically includes the following steps:
s1, preparing the magnetic nanoparticles by the method in the embodiment 1, and using the magnetic nanoparticles as a magnetic solid phase carrier; the reactor treatment was carried out simultaneously: soaking the reaction container with chromic acid washing solution overnight, washing with distilled water, drying, soaking with 10% dichlorodimethylsilane-toluene solution for 20min, and confirming that the inner surface of the apparatus is completely contacted with the reagent and no bubbles are attached; soaking in anhydrous methanol for 20min, and drying;
s2, soaking and washing the obtained magnetic nanoparticles with NN-Dimethylformamide (DMF), and adding Fmoc-protected amino acid active ester coupling solution into the obtained magnetic solid phase carrier, wherein the active ester solution is obtained by dissolving Fmoc-protected amino acid monomers in a HOBt/DIC/DMAP/DMF solution and activating for 15 minutes, and the molar ratio of the used amount is as follows: magnetic solid phase carrier: Fmoc-Val: HOBt: DIC: DMAP ═ 1: 3: 6: 4: 0.1, placing the reaction in a shaking table for reaction at 25 ℃ for 3 hours;
after the reaction is finished, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing the magnetic solid-phase carrier for 4 times by using DMF (dimethyl formamide) to obtain a solid-phase carrier connected with Fmoc-Val;
adding 20 wt% of diethylamine/DMF solution into the solid phase, reacting for 20 minutes to remove Fmoc protection on the amino acid, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing the solid phase for 4 times by using DMF to obtain a solid phase carrier connected with an amino acid;
s3, adding an active ester solution of Fmoc protected amino acid monomer (Met) into the solid phase connected with the first amino acid (Val), wherein the active ester solution is a DMF solution containing Fmoc protected amino acid/HBTU/HOBt/DIEA, and the molar ratio of the dosage is as follows: solid phase carrier: Fmoc-Met: HBTU: HOBt: DIEA 1: 3: 3: 3: 6, placing the reaction in a shaking table for reaction at 25 ℃ for 3 hours;
after the reaction is finished, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing for 4 times by using DMF (dimethyl formamide);
adding 20 wt% diethylamine/DMF solution into the solid phase, reacting for 20min to remove Fmoc protection on the amino acid, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing the solid phase 4 times with DMF;
s4, continuously adding the active ester solution of the next protected amino acid monomer into the obtained solid phase carrier according to the method until the target peptide is synthesized;
adding a K reagent (10-20mL) into the synthesized system connected with the specific polypeptide, deprotecting the side chain of the synthesized polypeptide, and cutting off the side chain from the solid phase of the magnetic sphere; the K reagent is prepared from trifluoroacetic acid, thioanisole, ethanedithiol, phenol and water according to a weight ratio of 82.5: 5: 2.5: 5: 5, preparing the raw materials according to a mass ratio;
s5, performing rotary evaporation on the cut polypeptide, performing sedimentation by using ethyl acetate, and collecting sediment to obtain the required hexapeptide (VMIMIV).
ESI is used for verifying the chemical structure of the synthesized polypeptide by the hexapeptide (VMIMIV), and the detection result is shown in figure 3.
Example 5
In this example, the solid phase synthesis of 6 peptide (VMIMIV) was performed using the nano-magnetic particles synthesized in the above example 2 as a magnetic solid phase carrier, and specifically includes the following steps:
s1, preparing the magnetic nanoparticles according to the method in the embodiment 2, and using the magnetic nanoparticles as a magnetic solid phase carrier; the reactor treatment was carried out simultaneously: soaking the reaction container with chromic acid washing solution overnight, washing with distilled water, drying, soaking with 10% dichlorodimethylsilane-toluene solution for 25min, and confirming that the inner surface of the apparatus is completely contacted with the reagent and no bubbles are attached; soaking in anhydrous methanol for 25min, and drying;
s2, soaking and washing the obtained magnetic nanoparticles with NN-Dimethylformamide (DMF), and adding Fmoc-protected amino acid active ester coupling solution into the obtained magnetic solid phase carrier, wherein the active ester solution is obtained by dissolving Fmoc-protected amino acid monomers in a HOBt/DIC/DMAP/DMF solution and activating for 20 minutes, and the molar ratio of the used amount is as follows: magnetic solid phase carrier: Fmoc-Val: HOBt: DIC: DMAP ═ 1: 3: 6: 4: 0.1, placing the reaction in a shaking table for reacting for 4 hours at 25 ℃;
after the reaction is finished, placing the reaction on a magnet to collect magnetic particles and discard the solution, and washing the solid phase carrier for 6 times by using DMF to obtain the solid phase carrier connected with Fmoc-Val amino acid;
adding 20 wt% of diethylamine/DMF solution into the solid phase, reacting for 30 minutes to remove Fmoc protection on the amino acid, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing the solid phase for 6 times by using DMF to obtain a solid phase carrier connected with an amino acid;
s3, adding an active ester solution of Fmoc protected amino acid monomer (Met) into the solid phase connected with the first amino acid (Val), wherein the active ester solution is a DMF solution containing Fmoc protected amino acid/HBTU/HOBt/DIEA, and the molar ratio of the dosage is as follows: solid phase carrier: Fmoc-Met: HBTU: HOBt: DIEA 1: 3: 3: 3: 6, placing the reaction in a shaking table for reacting for 4 hours at 25 ℃;
after the reaction is finished, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing for 4 times by using DMF (dimethyl formamide);
adding 20 wt% diethylamine/DMF solution into the solid phase, reacting for 30 min to remove Fmoc protection on the amino acid, placing the reaction on a magnet to collect magnetic particles, discarding the solution, and washing the solid phase 6 times with DMF;
s4, continuously adding the active ester solution of the next protected amino acid monomer into the obtained solid phase carrier according to the method until the target peptide is synthesized;
adding a K reagent (trifluoroacetic acid: thioanisole: dithioglycol: phenol: water: 82.5: 5: 2.5: 5: 5) into the synthesized system connected with the specific polypeptide, deprotecting the side chain of the synthesized polypeptide, and cutting the polypeptide from the solid phase of the magnetic sphere;
s5, precipitating the polypeptide cut from the solid phase of the nano particles in the step by using ethyl ether, and collecting the product to obtain the required hexapeptide (VMIMIV). The obtained polypeptide product has correct structure by detection.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (7)
1. A synthetic method of magnetic nanoparticles is characterized by comprising the following steps:
(1) superparamagnetic Fe3O4The particles are modified by oleic acid to obtain oleic acid modified superparamagnetic Fe3O4Particles;
the superparamagnetic Fe3O4The step of modifying the particles with oleic acid specifically comprises: taking FeCl3•6H2O and FeCl2•4H2Dissolving O in deionized water, and adding ammonia water at 70-80 ℃ in a nitrogen atmosphere to react; after the reaction is finished, adding oleic acid into the mixture, and carrying out modification reaction at 70-80 ℃; will react withWashing the obtained magnetic nano particles to be neutral, and drying the magnetic nano particles in vacuum to obtain the needed oleic acid modified superparamagnetic Fe3O4Particles;
(2) taking the oleic acid modified superparamagnetic Fe3O4Dispersing the particles in an organic solvent to form a magnetic fluid, and adding styrene, 4- (4-vinylbenzyloxy benzyl alcohol) and divinylbenzene into the magnetic fluid to form a magnetic fluid oil phase;
the molar ratio of the styrene to the 4- (4-vinylbenzyloxy benzyl alcohol) to the divinylbenzene is 3-5: 0.4-0.6: 0.4-0.6;
(3) dropwise adding the obtained magnetofluid oil phase into a sodium dodecyl sulfate aqueous solution to be uniformly dispersed to prepare a suspension; the mass concentration of the sodium dodecyl sulfate aqueous solution is 0.5-2 g/L;
(4) adding a potassium persulfate initiator into the obtained suspension, and reacting at 60-80 ℃ in a nitrogen atmosphere; and after the reaction is finished, carrying out magnetic separation on the solution, discarding the solution and collecting precipitates to obtain the required magnetic nanoparticles.
2. The method for synthesizing magnetic nanoparticles according to claim 1, wherein in the step (2), the organic solvent is n-hexane.
3. Magnetic nanoparticles synthesized by the method of claim 1 or 2.
4. Use of the magnetic nanoparticles of claim 3 for the preparation of magnetic solid phase carriers in solid phase polypeptide synthesis.
5. A solid phase polypeptide synthesis method is characterized by comprising the following steps:
s1, synthesizing the magnetic nano-particles according to the method of claim 1 or 2, and using the magnetic nano-particles as a magnetic solid phase carrier;
s2, connecting a first Fmoc-amino acid on the surface of the magnetic solid phase carrier through a HOBt/DIC/DMAP active ester coupling system;
s3, connecting Fmoc-protected amino acid monomers to the obtained solid phase carrier one by utilizing a HOBt/HBTU/DIEA coupling system, and synthesizing a polypeptide with a specific sequence;
s4, adding a K reagent into a system connected with specific polypeptide, deprotecting the synthesized polypeptide side chain, and cutting off the polypeptide side chain from the magnetic ball solid phase;
s5, carrying out rotary evaporation on the cut polypeptide, carrying out sedimentation by using ethyl acetate, and collecting sediment to obtain the required polypeptide.
6. The solid phase polypeptide synthesis method of claim 5, wherein:
in the step S2, the molar ratio of the magnetic solid phase carrier, Fmoc-amino acid, HOBt, DIC, and DMAP is 1: 3: 6: 4: 0.1;
in the step S3, the molar ratio of the magnetic solid phase carrier to the Fmoc-amino acid to the HBTU to the HOBt to the DIEA is 1: 3: 3: 3: 6.
7. a polypeptide or library of polypeptides synthesized by the method of claim 5 or 6.
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