CN108376608A - A kind of magnetic nano-particle and its purposes for preparing Magnetic solid phases carrier - Google Patents
A kind of magnetic nano-particle and its purposes for preparing Magnetic solid phases carrier Download PDFInfo
- Publication number
- CN108376608A CN108376608A CN201810138399.8A CN201810138399A CN108376608A CN 108376608 A CN108376608 A CN 108376608A CN 201810138399 A CN201810138399 A CN 201810138399A CN 108376608 A CN108376608 A CN 108376608A
- Authority
- CN
- China
- Prior art keywords
- magnetic
- particle
- solid
- magnetic nano
- nano
- 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
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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 Solid phase peptide synthssis technical fields, and in particular to a kind of magnetic nano-particle for Solid phase peptide synthssis, and further disclose its synthetic method, and its in preparing Solid phase peptide synthssis Magnetic solid phases carrier application.Magnetic nano-particle of the present invention is with super-paramagnetic ferriferrous oxide particle (Fe3O4) it is magnetic core, polystyrene (PS) is coated by oleic acid modification and on its surface, to form hud typed PS@Fe3O4The magnetic nano-particle of structure, and coat Fe in styrene3O4While introduce 4 (4 vinyl benzyloxybenzyl alcohols), to obtain the nano-magnetic ball that a kind of surface polymer is modified.Obtained magnetic nano-particle have it is suitably sized, and with can further functionalization surface chemistry adaptability, and have in the medium well dispersibility and compatibility, the Magnetic solid phases carrier that can be used as in Solid-phase synthesis peptides be used.
Description
Technical field
The invention belongs to Solid phase peptide synthssis technical fields, and in particular to a kind of magnetic Nano for Solid phase peptide synthssis
Particle, and its synthetic method is further disclosed, and its it is used to prepare the purposes of Magnetic solid phases carrier in Solid phase peptide synthssis.
Background technology
Polypeptide as a kind of organic polymer, because its with several functions and due to good biocompatibility by more next
More concern so that the chemical synthesis process of polypeptide also becomes a big important area of organic synthesis field.Currently, polypeptide
The principle of synthesis under the effect of the catalyst, passes through carboxyl between amino acid and ammonia mainly using amino acid as basic unit
The reaction of base forms amido bond and will be attached, and realizes increasing for polypeptide chain with this.But in traditional polypeptide organic synthesis
In method, the connection of carboxyl and amino between the amino acid being connected mostly just relies on freely combining between amino acid,
The polypeptide obtained with this is usually all the polypeptide structure of the random connection of amino acid.As it can be seen that existing polypeptide synthesis method is difficult to
To particular sequence or the polypeptide of specific length amino acid sequence, obtained polypeptide product is also difficult to apply.
1963, Merrifield founded the solid phase polypeptide synthesis (Solid using Boc as amino acid protecting groups
Phase Peptide Synthesis, SPPS), it is distinctive excellent with its by the solid-phase synthesis of raw material of protected amino acid since then
Gesture attracts attention.And between decades thereafter, Solid-phase synthesis peptides technology passes through constantly improve, from it is small-scale,
Short peptide chain synthesizes the synthesis for gradually developing to extensive, long peptide chain.This method passes through firstly the need of suitable solid phase carrier is selected
Linker is coupled to realize the growth and cutting of polypeptide in carrier surface, and then the amino acid of one end radical protection is connect one by one
Branch forms polypeptide chain.In Solid phase synthesis, when reaction carries out on solid phase carrier, it is required to avoid liquid phase method synthesis institute
Cumbersome time-consuming intermediate purification procedures, operating procedure is simply controllable, and the reaction time is rapid, in the synthesis of polypeptide
Occupy increasingly consequence.
Solid phase peptide synthssis technology is a key technology of modern protein chemistry research, and to obtain bioactivity more
A kind of important method of peptide further includes Fmoc chemistry in addition to the Boc chemistry of Merrifield exploitations.It is chemical relative to Boc,
Fmoc chemistry is more convenient other than meeting the requirement of rapid automatized preparation, in biochemical, immune, molecular microbiology
And the more field of the demands polypeptide such as pharmacy is widely used.In the prior art, it is typically all for the solid phase carrier of Peptide systhesis
Micrograde polymer needs that the complex steps such as centrifugation are filtered for multiple times in the synthesis process, and the polypeptide synthesized can not be direct
It uses in organism, next step application can be carried out after need to cutting, has seriously affected the development of Solid phase peptide synthssis technology.
Superparamagnetism (Superparamagnetism) refers to when particle is less than critical dimension with the ferromagnetic of one-domain structure
Substance shows as paramagnetism feature when temperature is less than Curie temperature and is higher than transition temperature (Block Temperature), but
Its paramagnetic susceptibility is far above the magnetic susceptibility of general paramagnetic material, referred to as superparamagnetism under external magnetic field.Superparamagnetism
Nano-particle is in biomedical sectors such as target medicine carrier, cell separation, nuclear magnetic resonance, immune detection and purifying biomolecules
It has broad application prospects, and in Solid-phase synthesis peptides technology, is used frequently as solid phase carrier.However, existing
The effects that magnetic nano-particle is due to dimensional effect and magnetic dipole gravitation so that magnetic particle is easy to reunite, and chemical stabilization
Property it is not high, and be vulnerable to oxidation, cause its surface hydroxyl insufficient, so that being difficult to directly apply, seriously affected the more of synthesis
The performance of peptide.
Invention content
For this purpose, technical problem to be solved by the present invention lies in provide a kind of magnetic nano-particle, the magnetic nano particle
Son have it is suitably sized, and with can further functionalization surface chemistry adaptability, and have in the medium well
Dispersibility and compatibility, can be used in preparing the Magnetic solid phases carrier in Solid phase peptide synthssis.
In order to solve the above technical problems, a kind of synthetic method of magnetic nano-particle of the present invention, including walk as follows
Suddenly：
(1) by superparamagnetic Fe3O4Particle carries out oleic acid modification, obtains the superparamagnetic Fe that oleic acid is modified3O4Particle；
(2) the superparamagnetic Fe for taking the oleic acid to be modified3O4Particle, which is scattered in organic solvent, forms magnetic fluid, and to above-mentioned
Styrene, 4- (4- vinyl benzyloxybenzyl alcohol) and divinylbenzene are added in magnetic fluid, forms magnetic fluid oil phase；
(3) magnetic fluid oil phase obtained above is added drop-wise in lauryl sodium sulfate (SDS) aqueous solution and is uniformly dispersed, made
Obtain suspension；
(4) potassium peroxydisulfate initiator is added into suspension obtained above, in a nitrogen atmosphere, in 60-80 DEG C of progress
Reaction；After reaction, solution is subjected to Magnetic Isolation, discards solution and collects sediment to get required magnetic nano particle
Son.
In the step (1), the superparamagnetic Fe3O4The step that particle carries out oleic acid modification specifically includes：Take FeCl3·
6H2O and FeCl2·4H2O is dissolved in deionized water, and in a nitrogen atmosphere, addition ammonium hydroxide is reacted under the conditions of 70-80 DEG C；
After reaction, oleic acid is added into said mixture, reaction is modified at 70-80 DEG C；It will reaction gained magnetic Nano
Particle washs and obtains the superparamagnetic Fe that required oleic acid is modified after vacuum drying to neutrality3O4Particle.
In the step (2), the organic solvent is n-hexane.
In the step (2), the styrene, 4- (4- vinyl benzyloxybenzyl alcohol) and divinylbenzene dosage molar ratio
For 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 lauryl sodium sulfate (SDS) aqueous solution is 0.5-2g/L.
The invention also discloses the magnetic nano-particles synthesized by the method.
The invention also discloses the magnetic nano-particles to be used to prepare Magnetic solid phases carrier in Solid phase peptide synthssis
Purposes.
The invention also discloses a kind of solid-phase peptide synthesis, include the following steps：
S1, the magnetic nano-particle is synthesized according to the method, and as Magnetic solid phases carrier；
S2, by the active ester coupling systems of HOBt/DIC/DMAP first is connected on the surface of the Magnetic solid phases carrier
Fmoc- amino acid；
S3, fmoc-protected amino acid monomer is individually connected to gained solid phase load using HOBt/HBTU/DIEA coupling systems
On body, the synthesis of particular sequence polypeptide is carried out；
S4, K reagents are added into the above-mentioned system for connecting particular polypeptide, by the deprotection of the peptide side chain of synthesis and from magnetic
It is cut in property ball solid phase；
S5, the above-mentioned polypeptide cut is rotated and is settled with ice ether, collect sediment to get required polypeptide.
In the step S2, the Magnetic solid phases carrier, Fmoc- amino acid, HOBt, DIC, DMAP amount ratio be 1：3：
6：4：0.1；
In the step S3, the Magnetic solid phases carrier, Fmoc- amino acid, HBTU, HOBt, DIEA amount ratio be 1：
3：3：3：6.
The invention also discloses the polypeptides or peptide library that are synthesized by the method.
Magnetic nano-particle of the present invention is with super-paramagnetic ferriferrous oxide particle (Fe3O4) it is magnetic core, changed by oleic acid
Property and its surface cladding polystyrene (PS), to form hud typed PS@Fe3O4The magnetic nano-particle of structure, and in benzene second
Alkene coats Fe3O4While introduce 4- (4- vinyl benzyloxybenzyl alcohol), to obtain the nanometer that a kind of surface polymer is modified
Magnetic ball.Characterization result proves, superparamagnetism Fe prepared by the present invention3O4Particle has suitable size and good monodisperse
Property.Using microemulsion method to Fe3O4Surface has carried out function of surface modification while coating PS shells to magnetic ball, obtained magnetism
Nano-particle have it is suitably sized, and with can further functionalization surface group, and and have in the medium well
Dispersibility and compatibility.
Magnetic nano-particle of the present invention can be evenly dispersed in Peptide systhesis reagent, and in the work of externally-applied magnetic field
It can realize separation rapidly under, prodigious facility is provided for Peptide systhesis, and being capable of easy, accurately synthesis particular sequence
Polypeptide can be used for the solid phase carrier of Solid-phase synthesis peptides.Meanwhile the magnetic nanoparticle synthesized through the method for the present invention can be used directly
Into organism, the targeting application for polypeptide in vivo can be met after the cleaved reagent cutting of polypeptide of synthesis, it is also possible to
Large-scale peptide library is synthesized in establishing, to filter out functional polypeptide rapidly, and carries out targeting screening and the biological function of polypeptide
Certification.
Description of the drawings
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the superparamagnetic Fe that oleic acid is made in embodiment 1 and is modified3O4The transmission electron microscope picture (TEM) of particle；
Fig. 2 is the transmission electron microscope picture (TEM) that the magnetic nano-particle that surface polymer is modified is made in embodiment 1；
Fig. 3 is the ESI-MS figures of the hexapeptide synthesized in the embodiment of the present invention 4.
Specific implementation mode
Transmission electron microscope photo is measured by JEOL JEM-1200 type transmission electron microscopes in following embodiments；Fourier-transform infrared
Spectrum analysis picture is measured by 6700 type infrared spectrometers of Nicolet.
Embodiment 1
The synthetic method of magnetic nano-particle described in the present embodiment, includes the following steps：
(1) by 12g FeCl3·6H2O and 4.9g FeCl2·4H2O is dissolved in 50mL deionized waters, then in 80 DEG C,
N2In atmosphere, 50ml ammonium hydroxide is quickly adding into above-mentioned solution with vigorous stirring, mixing；
It stirs and 2g oleic acid is added in 30 minutes backward said mixtures, suspension is kept to reaction 1 hour at 80 DEG C, instead
Magnetic nanoparticle is washed with deionized to the superparamagnetic Fe for obtaining oleic acid modification after vacuum drying to neutrality after answering3O4
Particle；
The superparamagnetic Fe that obtained oleic acid is modified using transmission electron microscope3O4Particle carries out Morphology analysis, knot
Fruit is as shown in Figure 1, it can be seen from the figure that the superparamagnetic Fe that oleic acid is modified3O4Particle monodispersity is good, uniform particle diameter, and grain size is about
For 10nm；
(2) obtained oleic acid is modified superparamagnetic Fe3O4Particle is distributed to formation magnetic fluid in n-hexane and (controls the magnetic
The solid-to-liquid ratio of fluid and solvent is 0.125g/mL), styrene, 4- (4- is added into above-mentioned magnetic fluid under usual ultrasound condition
Vinyl benzyloxybenzyl alcohol) and divinylbenzene (DVB) (molar ratio 4：0.5：0.5) magnetic fluid oil phase is formed, with the magnetic current
The gauge of body, every milliliter of magnetic fluid add 400 μ L styrene；
(3) above-mentioned magnetic fluid oil phase is added drop-wise in lauryl sodium sulfate (SDS) aqueous solution of 1g/L, in ice-water bath item
It is 10 minutes ultrasonic in cell disruptor under part, obtain suspension；
(4) potassium peroxydisulfate (KPS) initiator is added in the above-mentioned suspension crushed and (0.6g is added with every milliliter of magnetic fluid
KPS), reacted 3 hours under 70 DEG C, nitrogen atmosphere vigorous mechanical agitation；After reaction, acquired solution is positioned over magnet
On, placement discards solution after a certain period of time, collects precipitation and is washed with water three times, obtains surface cladding polystyrene (PS) and forms nucleocapsid
Type PS@Fe3O4The magnetic nano-particle of structure, you can be used as the Magnetic solid phases carrier in Solid phase peptide synthssis.
The magnetic nano-particle being modified to the surface polymer obtained in the present embodiment using transmission electron microscope is carried out
Morphology analysis, the results are shown in Figure 2, it can be seen from the figure that the magnetic nano-particle of gained changes through surface polymer
After property, monodispersity is good, and shell is apparent, uniform particle diameter, and grain size is about 100nm.
Embodiment 2
The synthetic method of magnetic nano-particle described in the present embodiment, includes the following steps：
(1) by 12g FeCl3·6H2O and 4.9g FeCl2·4H2O is dissolved in 50mL deionized waters, then in 70 DEG C,
N2In atmosphere, 50mL ammonium hydroxide is quickly adding into above-mentioned solution with vigorous stirring, mixing；
2g oleic acid is added in stirring into said mixture after twenty minutes, suspension is kept to reaction 2 hours at 70 DEG C, instead
Magnetic nanoparticle is washed with deionized to the superparamagnetic Fe for obtaining oleic acid modification after vacuum drying to neutrality after answering3O4
Particle；
(2) obtained oleic acid is modified superparamagnetic Fe3O4Particle is distributed to formation magnetic fluid in n-hexane and (controls the magnetic
The solid-to-liquid ratio of fluid and solvent is 0.125g/mL), styrene, 4- (4- is added into above-mentioned magnetic fluid under the conditions of conventional Ultrasound
Vinyl benzyloxybenzyl alcohol) and divinylbenzene (DVB) (molar ratio 3：0.4：0.4) magnetic fluid oil phase, is formed, with the magnetic current
The gauge of body, every milliliter of magnetic fluid add 400 μ L styrene；
(3) above-mentioned magnetic fluid oil phase is added drop-wise in lauryl sodium sulfate (SDS) aqueous solution of 2g/L, in ice-water bath item
It is 20 minutes ultrasonic in cell disruptor under part, obtain suspension；
(4) potassium peroxydisulfate (KPS) initiator is added in the above-mentioned suspension crushed and (0.6g is added with every milliliter of magnetic fluid
KPS), reacted 2 hours under 80 DEG C, nitrogen atmosphere vigorous mechanical agitation；After reaction, acquired solution is positioned over magnet
On, placement discards solution after a certain period of time, collects precipitation and is washed with water three times, obtains surface cladding polystyrene (PS) and forms nucleocapsid
Type PS@Fe3O4The magnetic nano-particle of structure, you can be used as the Magnetic solid phases carrier in Solid phase peptide synthssis.
Embodiment 3
The synthetic method of magnetic nano-particle described in the present embodiment, includes the following steps：
(1) by 12g FeCl3·6H2O and 4.9g FeCl2·4H2O is dissolved in 50mL deionized waters, then in 80 DEG C,
N2In atmosphere, 50mL ammonium hydroxide is quickly adding into above-mentioned solution with vigorous stirring, mixing；
It stirs and 2g oleic acid is added in 30 minutes backward said mixtures, suspension is kept to reaction 2 hours at 80 DEG C, instead
Magnetic nanoparticle is washed with deionized to the superparamagnetic Fe for obtaining oleic acid modification after vacuum drying to neutrality after answering3O4
Particle；
(2) obtained oleic acid is modified superparamagnetic Fe3O4Particle is distributed to formation magnetic fluid in n-hexane and (controls the magnetic
The solid-to-liquid ratio of fluid and solvent is 0.125g/mL), styrene, 4- (4- is added into above-mentioned magnetic fluid under the conditions of conventional Ultrasound
Vinyl benzyloxybenzyl alcohol) and divinylbenzene (DVB) (three's molar ratio be 5：0.6：0.6) magnetic fluid oil phase, is formed, with every milli
It rises magnetic fluid and adds 400 μ L styrene；
(3) above-mentioned magnetic fluid oil phase is added drop-wise in lauryl sodium sulfate (SDS) aqueous solution of 0.5g/L, in ice-water bath
Under the conditions of in cell disruptor ultrasound 20 minutes, obtain suspension；
(4) potassium peroxydisulfate (KPS) initiator is added in the above-mentioned suspension crushed and (0.6g is added with every milliliter of magnetic fluid
KPS), reacted 2 hours under 60 DEG C, nitrogen atmosphere vigorous mechanical agitation；After reaction, acquired solution is positioned over magnet
On, placement discards solution after a certain period of time, collects precipitation and is washed with water three times, obtains surface cladding polystyrene (PS) and forms nucleocapsid
Type PS@Fe3O4The magnetic nano-particle of structure, you can be used as the Magnetic solid phases carrier in Solid phase peptide synthssis.
Embodiment 4
The magnetic nanoparticle synthesized using in above-described embodiment 1 in the present embodiment carries out 6 peptides as Magnetic solid phases carrier
(VMIMIV) synthesis in solid state, specifically includes following step：
S1, the magnetic nano-particle is made according to method in embodiment 1, and as Magnetic solid phases carrier；It is carried out at the same time
The processing of reactor：By reaction vessel chromic acid lotion soaked overnight, with 10% dichloro-dimethyl silicon after distilled water clean dry
Alkane-toluene solution impregnates 20min, confirms that instrument inner surface is all contacted with reagent and bubble-free is adhered to；Then absolute methanol is used
Drying for standby after immersion 20min；
S2, by gained magnetic nano-particle NN, it is solid to obtained magnetism after-dimethylformamide (DMF) washing by soaking
The active ester coupling liquid of fmoc-protected amino acid is added in phase carrier, the active ester solution is by fmoc-protected amino acid
Monomer, which is dissolved in activating 15 minutes in the solution of HOBt/DIC/DMAP/DMF, to be obtained, and the molar ratio of dosage is：Magnetic solid phases carry
Body：Fmoc-Val：HOBt：DIC：DMAP=1：3：6：4：0.1, reaction is placed in 25 DEG C of shaking table and is reacted 3 hours；
After reaction, reaction is placed on magnet and collects magnetic particle and discards solution, washed Magnetic solid phases carrier with DMF
It washs 4 times and obtains the solid phase carrier of one Fmoc-Val of connection；
20wt% diethylamine/DMF solution is added into above-mentioned solid phase, reacts the Fmoc sloughed on amino acid for 20 minutes and protects
Reaction is placed on magnet and collects magnetic particle and discard solution, washed solid phase 4 times with DMF, obtains one amino acid of connection by shield
Solid phase carrier；
S3, fmoc-protected amino acid monomer (Met) is added to above-mentioned be connected in the solid phase of first amino acid (Val)
Active ester solution, the active ester solution is the DMF solution containing fmoc-protected amino acid/HBTU/HOBt/DIEA,
The molar ratio of its dosage is：Solid phase carrier：Fmoc-Met：HBTU：HOBt：DIEA=1：3：3：3：6, reaction is placed in shaking table 25
DEG C reaction 3 hours；
After reaction, reaction is placed on magnet and collects magnetic particle and discards solution, washed 4 times with DMF；
20wt% diethylamine/DMF solution is added into above-mentioned solid phase, reacts the Fmoc sloughed on amino acid for 20 minutes and protects
Reaction is placed on magnet and collects magnetic particle and discard solution, washed solid phase 4 times with DMF by shield；
S4, continue the work that the amino acid monomer of next protection is added as stated above into solid phase carrier obtained above
Ester solution is sprinkled, until synthesis target peptide；
K reagents (10-20mL) are added into the system for connecting particular polypeptide of synthesis, by the peptide side chain remove-insurance of synthesis
It protects and is cut from magnetic ball solid phase；The K reagents press 82.5 by trifluoroacetic acid, thioanisole, dithioglycol, phenol and water：
5：2.5：5：5 mass ratio is formulated；
S5, the above-mentioned polypeptide cut is rotated and is settled with ice ether, collect sediment to get required hexapeptide
(VMIMIV)。
Hexapeptide obtained above (VMIMIV) is verified to the chemical constitution of synthesis polypeptide, testing result such as Fig. 3 institutes with ESI
Show, it is seen then that the method for the present invention can synthesize the polypeptide structure of particular sequence, and product structure is correct.
Embodiment 5
The magnetic nanoparticle synthesized using in above-described embodiment 2 in the present embodiment carries out 6 peptides as Magnetic solid phases carrier
(VMIMIV) synthesis in solid state, specifically includes following step：
S1, the magnetic nano-particle is made according to method in embodiment 2, and as Magnetic solid phases carrier；It is carried out at the same time
The processing of reactor：By reaction vessel chromic acid lotion soaked overnight, with 10% dichloro-dimethyl silicon after distilled water clean dry
Alkane-toluene solution impregnates 25min, confirms that instrument inner surface is all contacted with reagent and bubble-free is adhered to；Then absolute methanol is used
Drying for standby after immersion 25min；
S2, by gained magnetic nano-particle NN, it is solid to obtained magnetism after-dimethylformamide (DMF) washing by soaking
The active ester coupling liquid of fmoc-protected amino acid is added in phase carrier, the active ester solution is by fmoc-protected amino acid
Monomer, which is dissolved in activating 20 minutes in the solution of HOBt/DIC/DMAP/DMF, to be obtained, and the molar ratio of dosage is：Magnetic solid phases carry
Body：Fmoc-Val：HOBt：DIC：DMAP=1：3：6：4：0.1, reaction is placed in 25 DEG C of shaking table and is reacted 4 hours；
After reaction, reaction is placed on magnet and collects magnetic particle and discards solution, solid phase carrier is washed 6 with DMF
All over the solid phase carrier for obtaining connecting a Fmoc-Val amino acid；
20wt% diethylamine/DMF solution is added into above-mentioned solid phase, reacts the Fmoc sloughed on amino acid for 30 minutes and protects
Reaction is placed on magnet and collects magnetic particle and discard solution, washed solid phase 6 times with DMF, obtains one amino acid of connection by shield
Solid phase carrier；
S3, fmoc-protected amino acid monomer (Met) is added to above-mentioned be connected in the solid phase of first amino acid (Val)
Active ester solution, the active ester solution is the DMF solution containing fmoc-protected amino acid/HBTU/HOBt/DIEA,
The molar ratio of its dosage is：Solid phase carrier：Fmoc-Met：HBTU：HOBt：DIEA=1：3：3：3：6, reaction is placed in shaking table 25
DEG C reaction 4 hours；
After reaction, reaction is placed on magnet and collects magnetic particle and discards solution, washed 4 times with DMF；
20wt% diethylamine/DMF solution is added into above-mentioned solid phase, reacts the Fmoc sloughed on amino acid for 30 minutes and protects
Reaction is placed on magnet and collects magnetic particle and discard solution, washed solid phase 6 times with DMF by shield；
S4, continue the work that the amino acid monomer of next protection is added as stated above into solid phase carrier obtained above
Ester solution is sprinkled, until synthesis target peptide；
K reagent (trifluoroacetic acids are added into the system for connecting particular polypeptide of synthesis：Thioanisole：Dithioglycol：Benzene
Phenol：Water=82.5：5：2.5：5：5), the peptide side chain of synthesis is deprotected and is cut from magnetic ball solid phase；
S5, the polypeptide cut from nano particle solid phase in above-mentioned steps is settled with ether, collects product up to required
Hexapeptide (VMIMIV).After testing, gained polypeptide product structure is correct.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of synthetic method of magnetic nano-particle, which is characterized in that include the following steps：
(1) by superparamagnetic Fe3O4Particle carries out oleic acid modification, obtains the superparamagnetic Fe that oleic acid is modified3O4Particle；
(2) the superparamagnetic Fe for taking the oleic acid to be modified3O4Particle, which is scattered in organic solvent, forms magnetic fluid, and to above-mentioned magnetic current
Styrene, 4- (4- vinyl benzyloxybenzyl alcohol) and divinylbenzene are added in body, forms magnetic fluid oil phase；
(3) magnetic fluid oil phase obtained above is added drop-wise in lauryl sodium sulfate (SDS) aqueous solution and is uniformly dispersed, be made outstanding
Supernatant liquid；
(4) potassium peroxydisulfate initiator is added into suspension obtained above, in a nitrogen atmosphere, is reacted in 60-80 DEG C；
After reaction, solution is subjected to Magnetic Isolation, discards solution and collects sediment to get required magnetic nano-particle.
2. the synthetic method of magnetic nano-particle according to claim 1, which is characterized in that described in the step (1)
Superparamagnetic Fe3O4The step that particle carries out oleic acid modification specifically includes：Take FeCl3·6H2O and FeCl2·4H2O is dissolved in deionization
In water, in a nitrogen atmosphere, addition ammonium hydroxide is reacted under the conditions of 70-80 DEG C；After reaction, add into said mixture
Enter oleic acid, reaction is modified at 70-80 DEG C；Reaction gained magnetic nanoparticle is washed to neutrality, after vacuum drying
The superparamagnetic Fe being modified to required oleic acid3O4Particle.
3. the synthetic method of magnetic nano-particle according to claim 1 or 2, which is characterized in that in the step (2),
The organic solvent is n-hexane.
4. according to the synthetic method of claim 1-3 any one of them magnetic nano-particles, which is characterized in that the step
(2) in, the dosage molar ratio of the styrene, 4- (4- vinyl benzyloxybenzyl alcohol) and divinylbenzene is 3-5：0.4-0.6：
0.4-0.6。
5. according to the synthetic method of claim 1-4 any one of them magnetic nano-particles, which is characterized in that the step
(3) in, the mass concentration of lauryl sodium sulfate (SDS) aqueous solution is 0.5-2g/L.
6. the magnetic nano-particle synthesized by any one of claim 1-5 the methods.
7. the magnetic nano-particle described in claim 6 is used to prepare the purposes of Magnetic solid phases carrier in Solid phase peptide synthssis.
8. a kind of solid-phase peptide synthesis, which is characterized in that include the following steps：
S1, the magnetic nano-particle is synthesized according to any one of claim 1-5 the methods, and as Magnetic solid phases carrier；
S2, first Fmoc- is connected on the surface of the Magnetic solid phases carrier by the active ester coupling systems of HOBt/DIC/DMAP
Amino acid；
S3, fmoc-protected amino acid monomer is individually connected to gained solid phase carrier using HOBt/HBTU/DIEA coupling systems
On, carry out the synthesis of particular sequence polypeptide；
S4, K reagents are added into the above-mentioned system for connecting particular polypeptide, by the deprotection of the peptide side chain of synthesis and from magnetic ball
It is cut in solid phase；
S5, the above-mentioned polypeptide cut is rotated and is settled with ice ether, collect sediment to get required polypeptide.
9. solid-phase peptide synthesis according to claim 8, it is characterised in that：
In the step S2, the Magnetic solid phases carrier, Fmoc- amino acid, HOBt, DIC, DMAP amount ratio be 1：3：6：4：
0.1；
In the step S3, the Magnetic solid phases carrier, Fmoc- amino acid, HBTU, HOBt, DIEA amount ratio be 1：3：3：
3：6.
10. the polypeptide or peptide library that are synthesized by the method described in claim 8 or 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810138399.8A CN108376608B (en) | 2018-02-10 | 2018-02-10 | Magnetic nano particle and application thereof in preparing magnetic solid phase carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810138399.8A CN108376608B (en) | 2018-02-10 | 2018-02-10 | Magnetic nano particle and application thereof in preparing magnetic solid phase carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108376608A true CN108376608A (en) | 2018-08-07 |
| CN108376608B CN108376608B (en) | 2020-11-06 |
Family
ID=63017678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810138399.8A Active CN108376608B (en) | 2018-02-10 | 2018-02-10 | Magnetic nano particle and application thereof in preparing magnetic solid phase carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108376608B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110655117A (en) * | 2019-09-30 | 2020-01-07 | 天津大学 | Method for preparing ferroferric oxide with different sizes and good dispersibility |
| CN112868668A (en) * | 2021-03-19 | 2021-06-01 | 常州英诺升康生物医药科技有限公司 | Fe3O4-DA-AMP nano composite antibacterial material and preparation method and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1206001A (en) * | 1997-05-09 | 1999-01-27 | 赫彻斯特股份公司 | Substituted diaminocarboxylic acids |
| CN1283228A (en) * | 1997-12-24 | 2001-02-07 | 武田药品工业株式会社 | Polypeptide, their production method and use |
| US20040219361A1 (en) * | 2003-04-30 | 2004-11-04 | Shanxi Lifegen Co., Ltd. | Super-paramagnetic composite particle with core/shell structure, preparation method and use thereof |
| CN1944471A (en) * | 2006-09-28 | 2007-04-11 | 上海交通大学 | Process for preparing functional high molecule composite micro ball with quick magnetic field responsiveness |
| CN102744420A (en) * | 2012-06-20 | 2012-10-24 | 中国科学院宁波材料技术与工程研究所 | Preparation method of magnetic nanometer particles with adjustable and controllable particle diameter |
| CN103333295A (en) * | 2013-06-18 | 2013-10-02 | 西北工业大学 | Preparation method of thymopentin molecularly-imprinted magnetic microspheres |
| CN103965910A (en) * | 2013-02-06 | 2014-08-06 | 北京大学 | Core-shell type magnetic quantum-dot nanometer particle and preparation method thereof |
| CN103965285A (en) * | 2013-02-06 | 2014-08-06 | 北京大学 | Method for taking super-paramagnetism nanometer particles as solid phase to perform peptide synthesis and synchronously construct polypeptide magnetic nanometer probe |
| CN104368390A (en) * | 2014-10-09 | 2015-02-25 | 浙江工业大学 | Surface grafted multipoint bridged supported 2, 2, 6, 6-tetramethylpiperidinooxy (TEMPO) catalyst and preparation method thereof |
| US20150232616A1 (en) * | 2007-04-30 | 2015-08-20 | Intezyne Technologies, Inc. | Hybrid block copolymer micelles with mixed stereochemistry for encapsulation of hydrophobic agents |
-
2018
- 2018-02-10 CN CN201810138399.8A patent/CN108376608B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1206001A (en) * | 1997-05-09 | 1999-01-27 | 赫彻斯特股份公司 | Substituted diaminocarboxylic acids |
| CN1283228A (en) * | 1997-12-24 | 2001-02-07 | 武田药品工业株式会社 | Polypeptide, their production method and use |
| US20040219361A1 (en) * | 2003-04-30 | 2004-11-04 | Shanxi Lifegen Co., Ltd. | Super-paramagnetic composite particle with core/shell structure, preparation method and use thereof |
| CN1944471A (en) * | 2006-09-28 | 2007-04-11 | 上海交通大学 | Process for preparing functional high molecule composite micro ball with quick magnetic field responsiveness |
| US20150232616A1 (en) * | 2007-04-30 | 2015-08-20 | Intezyne Technologies, Inc. | Hybrid block copolymer micelles with mixed stereochemistry for encapsulation of hydrophobic agents |
| CN102744420A (en) * | 2012-06-20 | 2012-10-24 | 中国科学院宁波材料技术与工程研究所 | Preparation method of magnetic nanometer particles with adjustable and controllable particle diameter |
| CN103965910A (en) * | 2013-02-06 | 2014-08-06 | 北京大学 | Core-shell type magnetic quantum-dot nanometer particle and preparation method thereof |
| CN103965285A (en) * | 2013-02-06 | 2014-08-06 | 北京大学 | Method for taking super-paramagnetism nanometer particles as solid phase to perform peptide synthesis and synchronously construct polypeptide magnetic nanometer probe |
| CN103333295A (en) * | 2013-06-18 | 2013-10-02 | 西北工业大学 | Preparation method of thymopentin molecularly-imprinted magnetic microspheres |
| CN104368390A (en) * | 2014-10-09 | 2015-02-25 | 浙江工业大学 | Surface grafted multipoint bridged supported 2, 2, 6, 6-tetramethylpiperidinooxy (TEMPO) catalyst and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110655117A (en) * | 2019-09-30 | 2020-01-07 | 天津大学 | Method for preparing ferroferric oxide with different sizes and good dispersibility |
| CN112868668A (en) * | 2021-03-19 | 2021-06-01 | 常州英诺升康生物医药科技有限公司 | Fe3O4-DA-AMP nano composite antibacterial material and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108376608B (en) | 2020-11-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ladj et al. | Polymer encapsulation of inorganic nanoparticles for biomedical applications | |
| Uchida et al. | Biological containers: protein cages as multifunctional nanoplatforms | |
| US7226636B2 (en) | Gold-coated nanoparticles for use in biotechnology applications | |
| Flenniken et al. | A library of protein cage architectures as nanomaterials | |
| CN104531669B (en) | A kind of magnetic graphene nanocomposite, the preparation method and its usage of cladding hydrophily poly-dopamine | |
| Leal et al. | Effect of the surface treatment on the structural, morphological, magnetic and biological properties of MFe2O4 iron spinels (M= Cu, Ni, Co, Mn and Fe) | |
| CN104762085B (en) | A kind of magnetic fluorescence composite Nano bioprobe and preparation method thereof | |
| CN104174039B (en) | Nanosilicon dioxide/ferroferric oxide shell-core particle surface modifying method and magnetic nano material | |
| CN105936820B (en) | A kind of water-soluble biological compatibility fluorescence magnetic nanocluster and preparation method thereof | |
| CN104387446B (en) | A kind of graphene dispersion agent and the preparation method of graphene dispersing solution | |
| CN108376608A (en) | A kind of magnetic nano-particle and its purposes for preparing Magnetic solid phases carrier | |
| CN104927010B (en) | Core-shell magnetic composite microsphere containing polyelectrolyte and its preparation method and application | |
| CN108440652A (en) | A kind of solid phase synthesis process of Pu Kana peptides | |
| CN110129029A (en) | A kind of single-stranded collagen polypeptide functional probe and preparation method thereof of electrical charge rejection effect induction | |
| You et al. | Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique | |
| KR20040083095A (en) | Organic substance having ferrite bonded thereto and process for producing the same | |
| CN109939624A (en) | A kind of preparation method for the core-shell type magnetic particle that biocompatible coating is controllable | |
| CN105079806B (en) | A kind of carbon nanomaterial and its preparation method and application that polypeptide is directly modified | |
| CN108802015A (en) | A kind of preparation of the nontoxic electrochemical luminescence sensor of zearalenone based on peptide sensor | |
| Magnabosco et al. | Effect of surface chemistry on incorporation of nanoparticles within calcite single crystals | |
| CN105381466B (en) | A kind of optomagnetic temperature-sensitive nano combination drug carrier and preparation method thereof | |
| CN107802845B (en) | Method for phase conversion of hydrophobic nanoparticles by using silk fibroin molecules | |
| CN103965285A (en) | Method for taking super-paramagnetism nanometer particles as solid phase to perform peptide synthesis and synchronously construct polypeptide magnetic nanometer probe | |
| Shao et al. | Characterization of Fe 3 O 4/SiO 2 composite core-shell nanoparticles synthesized in isopropanol medium | |
| Xing et al. | Intracellular uptake of magnetite nanoparticles conjugated with RGDS-peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |