CN102408289B - Separation method of chiral medicament by applying protein functionalized magnetic nano-particles - Google Patents
Separation method of chiral medicament by applying protein functionalized magnetic nano-particles Download PDFInfo
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Abstract
The invention discloses a separation method of a chiral medicament by applying protein functionalized magnetic nano-particles. The method comprises the following steps: (1) modifying the surfaces of superparamagnetic particles by adopting a physical adsorption method; (2) loading protein with chiral identification effect; and (3) mixing protein-modified superparamagnetic nano-particle aqueous solution and a chiral medicament racemic aqueous solution at a volume ratio of 1:1, adsorbing, and performing magnetic separation, wherein the supernatant is liquid containing the separation product of one enantiomer, and the superparamagnetic particle adsorbate is the separation product of the other enantiomer. The method is simple to operate, and has mild condition, short reaction time, and large protein immobilization capacity; the medicament identification capability of the protein is maintained; and the optical purity of the product is remarkably increased.
Description
Technical field
The invention belongs to medical technical field, concrete relate to a kind of method of applying protein function magnetic nanoparticle resolving chiral medicine.
Background technology
The enantiomer of chiral drug shows different physiological behaviors conventionally---they often a kind of steric isomer have drug effect, and its mirror image molecule or there is toxic side effect or there is contrary drug effect or just there is no drug effect [J.Chromat.A at all, 2001,906,3-33].Therefore, research and develop the focus that high efficiency single enantiomer chipal compounds production technology has become scientific research department and industry member, the chipal compounds of preparing single enantiomer will rely on stereoselectivity to synthesize and chiral separation technology.At present, the method splitting for enantiomers of chiral drugs mainly contains crystallization process, film Split Method, chromatography, capillary electrophoresis etc.Chromatogram and capillary electrophoresis are widely used, though separation efficiency is high, operation can not serialization, does not reach preparative-scale, is only applicable to pharmaceutical analysis and detection; And crystallization process can reach preparative-scale, but require medicine racemic modification to exist with aggregate form, thereby limited the kind of separable medicine; Film splits and can carry out operate continuously, but contradiction between the equilibrium separation factor and flux is problem demanding prompt solution [Anal.Chem.2010,82,4712-4722].
Albumen is extensively used as chiral selector because of its unique drug binding site and stereoselectivity, wherein bovine serum albumin, ovoglycoprotein, glycoprotein, ovomucoid, cellobiohydrolase, avidin, Quimotrase etc. have been successfully applied to high performance liquid chromatography and capillary electrophoresis fractionation drug enantiomer, there is good separation effect [J.Chromat.A, 2000,875,235-254; J.Chromat.A, 2001,906,253-273].The immobilized albumen of Chinese patent 200510110689.4 a kind of carbon nanotube of invention as stationary phase be filled in PMMA chip separated logical in, based on micro flow chip, carry out the method for chiral separation analysis, this method compartment analysis speed is fast, be applicable to drug testing and analysis, but because volume containing the sample is little, be unsuitable for the preparative that treatment capacity is large separated.Chinese patent 200810113779.2 has been invented a kind of tandem continuous multilevel chiral disassemble apparatus, first utilize macromole chiral selector (BSA etc.) and medicine to be split to carry out site recognition reaction, then by ripe ultrafiltration or nanofiltration membrane, realize and splitting, this method has overcome the separated flux of chiral film and the lower shortcoming of enantio-selectivity, but device is comparatively complicated, disengaging time 1-4h.
Functional magnetic composite nano-granule, owing to thering is magnetic responsiveness and surface-functional simultaneously, by magnetic stablizing bed technology, separate targets molecule from medium fast and efficiently, at field widespread use [Biotechnol.Bioeng. such as biomedicine, cytology and physiotechnologys, 1997,53,79-87; Angew.Chem.Int.Ed., 2009,48,1620-1624].In recent years, due to the demand of biological detection and enzyme technology, the method for magnetic nanoparticle surface being carried out to protein modification has obtained increasing concern.At present, by supperparamagnetic particles area load functionalization protein, and also do not report by the fractionation that enantiomers of chiral drugs is carried out in magnetic separation.
Summary of the invention
The object of this invention is to provide a kind of method of applying protein function magnetic nanoparticle resolving chiral medicine.
Technical scheme of the present invention is summarized as follows:
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The Ionomer aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 2-10g/L by mass concentration is 0.02-1g/L with mass concentration mixes, and at 10-45 ℃, adsorbs 20-40 minute, obtains the supperparamagnetic particles of surface modification;
(2) load has the protein of chiral recognition effect:
The protein water soln equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2-10g/L by mass concentration is 0.8-10g/L with concentration mixes, and adsorbs 2-5h, obtains the magnetic nanoparticle of protein modification; Described protein is bovine serum albumin, human serum albumin, α 1 acid glycoprotein, ovomucoid, ovoglycoprotein or avidin;
(3) the chiral drug racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1-10g/L by mass concentration is 1-100mg/L with mass concentration mixes, at 15-40 ℃, and absorption 2-4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer.
Described supperparamagnetic particles is that median size is the Fe that 10-80nm, saturation magnetization are 10-80emu/g
3o
4, γ-Fe
2o
3, CoFe
2o
4, NiFe
2o
4or Ni
0.5zn
0.5fe
2o
4.
Described Ionomer is diallyl dimethyl ammoniumchloride, polymine, glycol-chitosan, polypropylene amine, polyacrylic acid or poly (sodium 4-styrenesulfonate).
Described in described step (1), the mass concentration of the Ionomer aqueous solution is 0.1-0.5g/L, and described temperature is 20-30 ℃.
The mass concentration of the chiral drug racemic modification aqueous solution described in described step (3) is 5-20mg/L, and described temperature is 20-30 ℃.
Described chiral drug is Ibuprofen BP/EP, warfarin, Ofloxacine USP 23, Naproxen Base, Ketoprofen, oxazepam, flurbiprofen, citalopram, Proprasylyte or Toldrin.
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the silylating reagent solution that concentration expressed in percentage by volume is 0.3-10%, to make the concentration of supperparamagnetic particles powder be 1-10g/L, at 25-130 ℃, reaction 3-48h, separation, obtains the supperparamagnetic particles of surface modification; The solvent of described silylating reagent solution is: toluene, hexanaphthene or ethanol;
(2) load has the protein of chiral recognition effect:
The protein water soln equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2-10g/L by mass concentration is 0.8-10g/L with mass concentration mixes, add again covalently bound reagent, making covalently bound reagent volumetric molar concentration is 0.005-0.2mol/L, react 2-5h, obtain the magnetic nanoparticle of protein modification; Described protein is bovine serum albumin, human serum albumin, α 1 acid glycoprotein, ovomucoid, ovoglycoprotein, stomach en-or cellulolytic enzyme I;
(3) the chiral drug racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1-10g/L by mass concentration is 1-100mg/L with mass concentration mixes, at 15-40 ℃, and absorption 2-4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer.
Described supperparamagnetic particles is that median size is the Fe that 10-80nm, saturation magnetization are 10-80emu/g
3o
4, γ-Fe
2o
3, CoFe
2o
4, NiFe
2o
4or Ni
0.5zn
0.5fe
2o
4.
Described silylating reagent is aminopropyl trimethoxysilane or mercaptopropyl trimethoxysilane.
Described covalently bound reagent is glutaraldehyde, ethyl-(3-dimethyl propyl) carbodiimide hydrochloride, nitrogen-succinimido-3 (2-pyridine dithio)-propionic ester or bitoscanate.
Described in described step (1), the concentration expressed in percentage by volume of silylating reagent solution is 1-5%, and described temperature is 40-80 ℃, and the described reaction times is 6-12h.
The mass concentration of the chiral drug racemic modification aqueous solution described in described step (3) is 5-20mg/L, and described temperature is 20-30 ℃.
Described chiral drug is Ibuprofen BP/EP, warfarin, Ofloxacine USP 23, Naproxen Base, Ketoprofen, oxazepam, flurbiprofen, citalopram, Proprasylyte or Toldrin.
Advantage of the present invention:
(1) method of the present invention is easy and simple to handle, mild condition, and the reaction times is short, and proteinaceous solid carrying capacity is large and can keep its medicine recognition capability;
(2) magnetic separation can make optical purity of products significantly improve.
Embodiment
The following examples are in order to enable those skilled in the art to understand better the present invention, but the present invention are not imposed any restrictions.
The preparation of supperparamagnetic particles is even with the synthetic particle diameter of the methods such as low-temperature co-precipitation method, overcritical comminution granulation, hydrothermal synthesis method, sol-gel method, micro emulsion method, presoma thermal decomposition method, to have superparamagnetism nano particle.
Embodiment 1
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The diallyl dimethyl ammoniumchloride aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 2g/L by mass concentration is 0.1g/L with mass concentration mixes, and at 25 ℃, adsorbs 25 minutes, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the Fe that 15nm, saturation magnetization are 70emu/g
3o
4;
(2) load has the protein of chiral recognition effect:
The Bovine Serum Albumin in Aqueous Solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 5g/L by mass concentration is 1g/L with concentration mixes, and adsorbs 3h, obtains the magnetic nanoparticle of protein modification;
(3) the chirality Ibuprofen BP/EP racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 2g/L by mass concentration is 5mg/L with mass concentration mixes, at 20 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 77.2%.
The method of the present embodiment also can be used for resolving chiral Ofloxacine USP 23, warfarin or Racemic propranolol.
Embodiment 2
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The polyethyleneimine: amine aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 4g/L by mass concentration is 0.02g/L with mass concentration mixes, and at 30 ℃, adsorbs 20 minutes, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is γ-Fe that 10nm, saturation magnetization are 80emu/g
2o
3;
(2) load has the protein of chiral recognition effect:
The human serum albumin aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2g/L by mass concentration is 0.8g/L with concentration mixes, and adsorbs 2h, obtains the magnetic nanoparticle of protein modification;
(3) the chirality Naproxen Base racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 5g/L by mass concentration is 1mg/L with mass concentration mixes, at 25 ℃, and absorption 4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 80.1%.
The method of the present embodiment also can be used for resolving chiral Ibuprofen BP/EP, Ketoprofen, oxazepam or flurbiprofen racemic modification.
Embodiment 3
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The polypropylene amine aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 6g/L by mass concentration is 1g/L with mass concentration mixes, and at 45 ℃, adsorbs 30 minutes, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the CoFe that 20nm, saturation magnetization are 50emu/g
2o
4;
(2) load has the protein of chiral recognition effect:
The ovomucoid aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 7g/L by mass concentration is 5g/L with concentration mixes, and adsorbs 3h, obtains the magnetic nanoparticle of protein modification;
(3) the chirality Toldrin racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1g/L by mass concentration is 20mg/L with mass concentration mixes, at 30 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 68.1%.
Also can adopt ovoglycoprotein to substitute the ovomucoid in the present embodiment, resolving chiral Toldrin racemic modification.
Embodiment 4
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The poly (sodium 4-styrenesulfonate) aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 8g/L by mass concentration is 0.2g/L with mass concentration mixes, and at 10 ℃, adsorbs 25 minutes, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the NiFe that 30nm, saturation magnetization are 30emu/g
2o
4;
(2) load has the protein of chiral recognition effect:
The avidin aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 5g/L by mass concentration is 2g/L with concentration mixes, and adsorbs 3h, obtains the magnetic nanoparticle of protein modification;
(3) the chirality Ketoprofen racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 10g/L by mass concentration is 100mg/L with mass concentration mixes, at 40 ℃, and absorption 2h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 68.2%.
Also can adopt polyacrylic acid to substitute the poly (sodium 4-styrenesulfonate) in the present embodiment, chirality Ketoprofen is split.
Embodiment 5
A method of applying protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The glycol-chitosan aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 10g/L by mass concentration is 0.5g/L with mass concentration mixes, and at 20 ℃, adsorbs 40 minutes, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the Ni that 80nm, saturation magnetization are 10emu/g
0.5zn
0.5fe
2o
4;
(2) load has the protein of chiral recognition effect:
The α 1 acid glycoprotein aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 10g/L by mass concentration is 10g/L with concentration mixes, and adsorbs 5h, obtains the magnetic nanoparticle of protein modification;
(3) the chirality citalopram racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 5g/L by mass concentration is 20mg/L with mass concentration mixes, at 15 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 63.8%.
Embodiment 6
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the cyclohexane solution of the aminopropyl trimethoxysilane that concentration expressed in percentage by volume is 2%, to make the concentration of supperparamagnetic particles powder be 5g/L, at 80 ℃, reaction 8h, separation, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the Fe that 15nm, saturation magnetization are 70emu/g
3o
4;
(2) load has the protein of chiral recognition effect:
The Bovine Serum Albumin in Aqueous Solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 5g/L by mass concentration is 2g/L with mass concentration mixes, add again covalently bound reagent glutaraldehyde, making covalently bound reagent volumetric molar concentration is 0.1mol/L, react 4h, obtain the magnetic nanoparticle of protein modification;
(3) the chirality Ibuprofen BP/EP racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 2g/L by mass concentration is 5mg/L with mass concentration mixes, at 20 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 70.1%.
The method of the present embodiment also can be used for resolving chiral Ofloxacine USP 23, warfarin or Racemic propranolol.
Embodiment 7
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the cyclohexane solution of the mercaptopropyl trimethoxysilane that concentration expressed in percentage by volume is 0.3%, to make the concentration of supperparamagnetic particles powder be 1g/L, at 40 ℃, reaction 12h, separation, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is γ-Fe that 10nm, saturation magnetization are 80emu/g
2o
3;
(2) load has the protein of chiral recognition effect:
The human serum albumin aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2g/L by mass concentration is 0.8g/L with mass concentration mixes, add again covalently bound reagent nitrogen-succinimido-3 (2-pyridine dithio)-propionic ester, making covalently bound reagent volumetric molar concentration is 0.005mol/L, react 3h, obtain the magnetic nanoparticle of protein modification;
(3) the chirality Naproxen Base racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 5g/L by mass concentration is 1mg/L with mass concentration mixes, at 25 ℃, and absorption 4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 81.0%.
The method of the present embodiment also can be used for resolving chiral Ibuprofen BP/EP, Ketoprofen, oxazepam or flurbiprofen racemic modification.
Embodiment 8
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the ethanolic soln of the aminopropyl trimethoxysilane that concentration expressed in percentage by volume is 5%, to make the concentration of supperparamagnetic particles powder be 5g/L, at 25 ℃, reaction 48h, separation, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the CoFe that 20nm, saturation magnetization are 50emu/g
2o
4;
(2) load has the protein of chiral recognition effect:
The ovomucoid aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 5g/L by mass concentration is 3g/L with mass concentration mixes, add again covalently bound reagent glutaraldehyde, making covalently bound reagent volumetric molar concentration is 0.1mol/L, react 2h, obtain the magnetic nanoparticle of protein modification;
(3) the chirality Toldrin racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1g/L by mass concentration is 20mg/L with mass concentration mixes, at 30 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 63.8%.
Also can adopt ovoglycoprotein to substitute the ovomucoid in the present embodiment, resolving chiral Toldrin racemic modification.
Embodiment 9
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the toluene solution of the mercaptopropyl trimethoxysilane that concentration expressed in percentage by volume is 10%, to make the concentration of supperparamagnetic particles powder be 10g/L, at 130 ℃, reaction 3h, separation, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the NiFe that 30nm, saturation magnetization are 30emu/g
2o
4;
(2) load has the protein of chiral recognition effect:
The cellulolytic enzyme I aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 5g/L by mass concentration is 5g/L with mass concentration mixes, add again covalently bound reagent bitoscanate, making covalently bound reagent volumetric molar concentration is 0.2mol/L, react 4h, obtain the magnetic nanoparticle of protein modification;
(3) the chirality Racemic propranolol aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 10g/L by mass concentration is 100mg/L with mass concentration mixes, at 40 ℃, and absorption 2h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 56.2%.
Also can adopt stomach en-to substitute the cellulolytic enzyme I in the present embodiment, resolving chiral Racemic propranolol.
Embodiment 10
The method of the second application protein function magnetic nanoparticle resolving chiral medicine, comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the toluene solution of the aminopropyl trimethoxysilane that concentration expressed in percentage by volume is 1%, to make the concentration of supperparamagnetic particles powder be 2g/L, at 80 ℃, reaction 6h, separation, obtains the supperparamagnetic particles of surface modification; Described supperparamagnetic particles is that median size is the Ni that 80nm, saturation magnetization are 10emu/g
0.5zn
0.5fe
2o
4;
(2) load has the protein of chiral recognition effect:
The α 1 acid glycoprotein aqueous solution equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 10g/L by mass concentration is 10g/L with mass concentration mixes, add again covalently bound reagent ethyl-(3-dimethyl propyl) carbodiimide hydrochloride, making covalently bound reagent volumetric molar concentration is 0.2mol/L, react 5h, obtain the magnetic nanoparticle of protein modification;
(3) the chirality citalopram racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 5g/L by mass concentration is 20mg/L with mass concentration mixes, at 15 ℃, and absorption 3h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer, and in supernatant liquor, enantiomeric excess value is 77.2%.
Claims (9)
1. a method of applying protein function magnetic nanoparticle resolving chiral medicine, its feature comprises the steps:
(1) adopt physisorphtion to supperparamagnetic particles modifying surface:
The Ionomer aqueous solution equal-volume that the supperparamagnetic particles aqueous solution that is 2-10g/L by mass concentration is 0.02-1g/L with mass concentration mixes, and at 10-45 ℃, adsorbs 20-40 minute, obtains the supperparamagnetic particles of surface modification;
(2) load has the protein of chiral recognition effect:
The protein water soln equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2-10g/L by mass concentration is 0.8-10g/L with concentration mixes, and adsorbs 2-5h, obtains the magnetic nanoparticle of protein modification; Described protein is bovine serum albumin, human serum albumin, α 1 acid glycoprotein, ovomucoid, ovoglycoprotein or avidin;
(3) the chiral drug racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1-10g/L by mass concentration is 1-100mg/L with mass concentration mixes, at 15-40 ℃, and absorption 2-4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, supperparamagnetic particles adsorptive is the separated product of another kind of enantiomer, and described Ionomer is diallyl dimethyl ammoniumchloride, polymine, glycol-chitosan, polypropylene amine or poly (sodium 4-styrenesulfonate).
2. a method of applying protein function magnetic nanoparticle resolving chiral medicine, its feature comprises the steps:
(1) adopt chemical bonding to supperparamagnetic particles modifying surface:
Supperparamagnetic particles powder is immersed in the silylating reagent solution that concentration expressed in percentage by volume is 0.3-10%, to make the concentration of supperparamagnetic particles powder be 1-10g/L, at 25-130 ℃, reaction 3-48h, separation, obtains the supperparamagnetic particles of surface modification; The solvent of described silylating reagent solution is: toluene, hexanaphthene or ethanol;
(2) load has the protein of chiral recognition effect:
The protein water soln equal-volume that the supperparamagnetic particles aqueous solution of the described surface modification that is 2-10g/L by mass concentration is 0.8-10g/L with mass concentration mixes, add again covalently bound reagent, making covalently bound reagent volumetric molar concentration is 0.005-0.2mol/L, react 2-5h, obtain the magnetic nanoparticle of protein modification; Described protein is bovine serum albumin, human serum albumin, α 1 acid glycoprotein, ovomucoid, ovoglycoprotein, stomach en-or cellulolytic enzyme I;
(3) the chiral drug racemic modification aqueous solution equal-volume that the superparamagnetic nano particle aqueous solution of the described protein modification that is 1-10g/L by mass concentration is 1-100mg/L with mass concentration mixes, at 15-40 ℃, and absorption 2-4h; Carry out magnetic resolution, supernatant liquor is the liquid of the separated product that contains a kind of enantiomer, the separated product that supperparamagnetic particles adsorptive is another kind of enantiomer.
3. method according to claim 1 and 2, is characterized in that described supperparamagnetic particles is that median size is the Fe that 10-80nm, saturation magnetization are 10-80emu/g
3o
4, γ-Fe
2o
3, CoFe
2o
4, NiFe
2o
4or Ni
0.5zn
0.5fe
2o
4.
4. method according to claim 2, is characterized in that described silylating reagent is aminopropyl trimethoxysilane or mercaptopropyl trimethoxysilane.
5. method according to claim 2, is characterized in that described covalently bound reagent is glutaraldehyde, ethyl-(3-dimethyl propyl) carbodiimide hydrochloride, nitrogen-succinimido-3 (2-pyridine dithio)-propionic ester or bitoscanate.
6. method according to claim 1, the mass concentration that it is characterized in that the Ionomer aqueous solution described in described step (1) is 0.1-0.5g/L, described temperature is 20-30 ℃.
7. method according to claim 2, the concentration expressed in percentage by volume that it is characterized in that silylating reagent solution described in described step (1) is 1-5%, and described temperature is 40-80 ℃, and the described reaction times is 6-12h.
8. method according to claim 1 and 2, is characterized in that the mass concentration of the chiral drug racemic modification aqueous solution described in described step (3) is 5-20mg/L, and described temperature is 20-30 ℃.
9. method according to claim 1 and 2, is characterized in that described chiral drug is Ibuprofen BP/EP, warfarin, Ofloxacine USP 23, Naproxen Base, Ketoprofen, oxazepam, flurbiprofen, citalopram, Proprasylyte or Toldrin.
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