CN112387262B - Preparation method of chiral stationary phase based on photocatalytic cross-linked protein, chiral stationary phase and application - Google Patents
Preparation method of chiral stationary phase based on photocatalytic cross-linked protein, chiral stationary phase and application Download PDFInfo
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- CN112387262B CN112387262B CN202011244164.0A CN202011244164A CN112387262B CN 112387262 B CN112387262 B CN 112387262B CN 202011244164 A CN202011244164 A CN 202011244164A CN 112387262 B CN112387262 B CN 112387262B
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Abstract
The invention discloses a preparation method of a chiral stationary phase based on photocatalytic cross-linked protein, the chiral stationary phase and application, wherein the preparation method of the chiral stationary phase based on photocatalytic cross-linked protein comprises the steps of photocatalytic cross-linking, sequentially adding tris (2,2' -bipyridyl) dichlororuthenium (II) hexahydrate and ammonium persulfate into a protein solution, and carrying out photocatalytic cross-linking reaction under illumination; and (3) carrying, namely adding the modified and coupled silica gel into a protein solution subjected to photocatalytic crosslinking, and fully stirring, filtering and drying to obtain the chiral stationary phase. The BSA-CSP is a chiral separation stationary phase with a brand new structure, and can be suitable for chiral separation of chemicals including amino acids and having certain binding capacity with protein sequences.
Description
Technical Field
The invention belongs to the technical field of chiral separation, and particularly relates to a preparation method of a chiral stationary phase based on photocatalytic cross-linked protein, the chiral stationary phase and application.
Background
Chirality, expressed as the difference of an object from its mirror image, is a special form of symmetry that exists widely in nature. Chiral objects and their mirror images are called enantiomers, and the spatial arrangement order of the groups of the enantiomers is different, which often produces differences in their physiological activities, and enantiomers with different configurations also show great differences in pharmacological activities, toxic and side effects, and the like.
Single enantiomers are limited in nature and often need to be synthesized artificially when the material is needed in large quantities in life. However, asymmetric synthesis of a single enantiomer is difficult, and the racemate, i.e. a mixture of two enantiomers in the same ratio, is often obtained industrially first. This results in, on the one hand, the simultaneous synthesis of the non-physiologically active enantiomers, which is of no value, which leads to a waste of raw materials and an increase in the production costs of the whole product; on the other hand, the physiological activities and even toxicity of the two enantiomers are obviously different, and the indiscriminate distinction of the enantiomers often brings about devastating results, for example, the Thalidomide (Thalidomide) event in 1969 results in the birth of about 1.3 ten thousand sea leopards, and tens of thousands of pregnant women are deeply harmed. Therefore, chiral separation is very important in the chemical industry, especially in the pharmaceutical industry.
Chiral resolution can be performed by various methods, such as Thin Layer Chromatography (TLC), Gas Chromatography (GC), Supercritical Fluid Chromatography (SFC), Capillary Electrophoresis (CE), Capillary Electrochromatography (CEC), simulated moving bed chromatography (SMB), and High Performance Liquid Chromatography (HPLC). The HPLC method is the most widely used method in chiral separation at present, because of its advantages of high speed of measuring enantiomer, high column efficiency, strong separation ability and wide application range.
The chiral stationary phase method is the most commonly used method for high performance liquid chromatography, and the development and preparation of the chiral stationary phase are the core of the method. The synthesis of the chiral stationary phase can be mainly divided into two modes of bonding and coating according to the synthesis thought. According to the separation mechanism, the separation type can be divided into an independent type stationary phase and a synergistic type stationary phase, and the more specific classification is mainly divided into a brush type, a cyclodextrin type, a macrocyclic antibiotic type, a crown ether type, a ligand exchange type, a polysaccharide type, a protein type and the like.
The protein chiral stationary phase refers to a chiral chromatographic stationary phase packed column or a chiral monolithic column obtained by fixing protein substances on a suitable carrier. Proteins are important constituent substances in organisms, and are natural biopolymers which are composed of amino acids or amino acids and sugars, and all of them have chirality, so that all proteins have chiral recognition ability. However, since most of proteins have poor stability and many reactive groups, it is difficult to uniformly bind them to an appropriate carrier, and only a small portion of proteins, such as Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), Glycoproteins (glycoprotens), Ovomucoid (Ovomucoid), Avidin (Avidin), etc., are currently used for chiral analysis. Immobilization methods for the preparation of protein-based CSP include physical adsorption, covalent immobilization, and encapsulation. The main advantage of physical adsorption is its simplicity, which is usually achieved by pumping the protein solution through a column pump at the appropriate pH and solution conditions. This technique can be used to rapidly evaluate new candidate proteins for use in CSPs. However, there is a disadvantage in that the CSP prepared by physical adsorption may have stability problems due to weak interaction between the adsorbed protein and the carrier.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.
Therefore, an object of the present invention is to provide a method for preparing a chiral stationary phase based on a photocatalytic cross-linked protein, a chiral stationary phase and an application thereof, wherein a novel technique of photo-induced unmodified protein (PICUP) cross-linking is used to obtain BSA aggregates rich in β -sheet structure, the aggregates have a relatively single structure, and after cross-linking with a carrier silica gel, the problem of non-uniform active center structure caused by direct cross-linking of BSA can be improved, and simultaneously, the hydrophobic surface and the contact area with a compound can be increased, and it is expected that the chiral stationary phase having a strong separation performance for various chemicals having interaction with protein, including amino acids, can be prepared.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a chiral stationary phase based on photocatalytic cross-linked protein comprises the following steps,
carrying out photocatalytic crosslinking, namely sequentially adding tris (2,2' -bipyridyl) dichlororuthenium (II) hexahydrate and ammonium persulfate into a protein solution, and carrying out photocatalytic crosslinking reaction under illumination;
and (3) carrying, namely adding the modified and coupled silica gel into a protein solution subjected to photocatalytic crosslinking, and fully stirring, filtering and drying to obtain the chiral stationary phase.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: the protein comprises one of bovine serum albumin, lysozyme and insulin.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: the concentration of the protein solution is 0.5-15 mg/ml.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: the molar ratio of the protein solution to the tris (2,2' -bipyridine) ruthenium (II) dichloride hexahydrate to the ammonium persulfate is 1: 3-5: 60-100.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: and the illumination is carried out for 5-60 s under a 200W incandescent lamp.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: the mass-to-volume ratio of the modified and coupled silica gel to the protein solution subjected to photocatalytic crosslinking is 1: 1-10.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: the modified and coupled silica gel is silica gel which is modified by sulfhydrylation and coupled by 4-maleimidobutyric acid-N-succinimide ester.
As a preferable embodiment of the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, the method comprises the following steps: and (4) performing suction filtration, and after the suction filtration, washing and performing suction filtration for multiple times by using a PBS buffer solution.
The invention also provides the following technical scheme: the chiral stationary phase based on the photocatalytic cross-linked protein is prepared by the preparation method of the chiral stationary phase based on the photocatalytic cross-linked protein, and comprises a chiral resolving agent bonded on a solid carrier, wherein the solid carrier is silica gel subjected to modification coupling, the chiral resolving agent is protein subjected to photocatalytic cross-linking, and the protein comprises one of bovine serum albumin, lysozyme and insulin.
The invention also provides the following technical scheme: the application of a chiral stationary phase based on photocatalytic cross-linked protein, which is used for a stationary phase of chromatography and is used for separating compounds containing amino acid, in particular the resolution of chiral amino acid.
The invention has the beneficial effects that:
the invention utilizes the reaction coupling of the gamma-mercaptopropyl trimethoxy silane activated and modified silica gel and 4-maleimide butyric acid-N-succinimide ester, and adds the bovine serum albumin with photocatalysis crosslinking to obtain the bovine serum albumin stationary phase (BSA-CSP) rich in beta folded structure. The aggregate has a relatively single structure, and can solve the problem of nonuniform active center structure caused by direct crosslinking of BSA (bovine serum albumin) after being crosslinked with carrier silica gel, and simultaneously can increase the hydrophobic surface and the contact area with a compound; because the protein composed of amino acids is used as a hydrophobic phase, the protein has better interaction with amino acids, polypeptides, proteins and the like; the beta-folded structure formed is single in structure and has better uniformity and stability; the protein is composed of L-amino acid, so that different acting forces are exerted on chiral amino acid, and the protein is suitable for separation of the chiral amino acid, so that the protein has an excellent effect on chiral resolution of the amino acid. Therefore, the BSA-CSP is a chiral separation stationary phase with a brand-new structure, and can be applied to chiral separation of chemicals which contain amino acids and have certain binding capacity with protein sequences.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a synthesis scheme of BSA-CSP according to the present invention;
FIG. 2 is a comparison of BSA-CSP of the present invention and silica gel;
FIG. 3 is a graph comparing time conditions for the reaction of photocatalytic cross-linked proteins according to the present invention;
FIG. 4 is an electrophoretic image of an SDS-PAGE experiment according to the present invention;
FIG. 5 is a comparison graph of the FTIR measurement of silica gel, modified silica gel, and modified silica gel-supported BSA of the present invention;
FIG. 6 is a comparison graph of BSA and photo-crosslinked BSA measured by FTIR of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
In the photocatalytic crosslinking bovine serum albumin test, a 50mL centrifuge tube is taken, 10mg/mL bovine serum albumin solution is added, 0.0125M of tris (2,2' -bipyridyl) ruthenium (II) dichloride hexahydrate and 0.25M of Ammonium Persulfate (APS) are sequentially added, and then the centrifuge tube is placed under a 200W incandescent lamp for photocatalytic crosslinking reaction. And the following tests were carried out.
SDS-PAGE assay
According to the molecular cloning experimental guidance, an electrophoresis reagent, separation gel and lamination gel are prepared. Firstly, assembling a glass plate, determining to manufacture 10-hole comb gel with the thickness of 1mm, and sequentially adding reagents according to the formula of the separation gel in the table 1 and uniformly mixing. Pouring the uniformly mixed acrylamide solution into the gap of a glass plate of electrophoresis equipment, carefully adding deionized water to cover the acrylamide solution, and waiting for the completion of polymerization. After polymerization, unpolymerized acrylamide was rinsed with deionized water and the remaining deionized water was aspirated off with filter paper. And then preparing a lamination glue solution, pouring the lamination glue mixed solution above the separation glue, immediately inserting a comb with 10 holes, and waiting for the lamination glue to polymerize while taking care to avoid introducing air bubbles. And (3) taking down the comb after polymerization is finished, washing off residual acrylamide in the holes by using deionized water, putting the prepared gel into electrophoresis equipment, and adding Tris-glycine electrophoresis buffer solution into an upper groove and a lower groove of an electrophoresis tank.
Table 1 formula table of electrophoretic gel
Preparing samples from 7 centrifuge tubes (10mL) according to the table 2, fully and uniformly mixing the samples by using a vortex mixer, performing half-hour denaturation in warm water bath at 50 ℃, and sequentially adding the samples to the bottoms of the gel holes by using a microsyringe according to the sample amount in the table after cooling to room temperature; an additional 3 centrifuge tubes (10mL) were added running buffer as a blank control. The electrophoresis apparatus was connected to the power supply of the electrophoresis apparatus, set at 80V, and electrophoresed until bromophenol blue reached the bottom of the separation gel. After the electrophoresis, the gel was carefully removed from the apparatus, immersed in a 5-fold volume of Coomassie Brilliant blue staining solution, and placed on a low speed shaker for staining at room temperature for at least 4 hours. And after dyeing is finished, discarding the dyeing solution, simply washing the gel with water, and placing the gel in the decoloring solution for decoloring for 4-8 hours. The results of the examination of the decolorization by photographing after the end of the decolorization are shown in FIGS. 3 and 4.
TABLE 2 comparison table of electrophoresis conditions of photocatalytic cross-linked protein
FIG. 3 is a comparison of the photocatalysis conditions and time, wherein A to E are sequentially bovine serum albumin solution and light cross-linking substances added for 0s, 5s, 10s, 30s, 60s and 90 s. The experiment observation shows that the color of the solution in the D, E, F tube is changed from yellow to light green when the illumination of the tube exceeds 30 s; the tube A is not illuminated with light and has no protein floccule, the rest tubes are illuminated to generate protein floccule, and the illumination time is within 10s to generate more protein floccule.
FIG. 4 is an electrophoresis chart, wherein the samples added in 1-7 wells correspond to the samples 1-7 in Table 2, respectively, and the samples fed in 8-10 wells are SDS gel sample adding buffer solution. As can be seen from FIG. 4, no protein polymer remained in the wells of blank control 1 and cross-linked non-illuminated control 2, and bovine serum albumin showed a clear protein band on the electrophoretogram, and the protein bands of well 1 and well 2 were substantially equal. The 3 ~ 7 holes are PICUP protein time group, and it is obvious that the albumen polymer is obviously produced to the visible bovine serum albumin after the photocatalysis cross-linking, and 3 holes (5s), 4 holes (10s) in the illumination time 10s produce more albumen polymer. Therefore, the time for carrying out the photocatalytic crosslinking is controlled to be 10s, so as to obtain a better photocatalytic crosslinked protein chiral stationary phase.
Example 2
Preparation of a chiral stationary phase based on photocatalytic cross-linked protein:
(1) preparation of activated silica gel
20g of silica gel are weighed, dissolved in 60ml of HCl (6.0M), stirred and refluxed for 8h at 150 ℃, and the activated silica gel is filtered by suction and washed to neutrality by distilled water. Finally, the product is dried for 8h at 70 ℃ and filled into a reagent bottle for standby.
(2) Sulfhydrylation modification:
first, 1.2mL of gamma-mercaptopropyltrimethoxysilane was added to a 20mL beaker containing 10mL of 95% ethanol, mixed thoroughly using a vortex mixer, the pH of the system was adjusted to 5 with concentrated hydrochloric acid, and poured into a conical flask. 15g of activated silica gel was added, and the flask was then placed in a constant temperature shaker at 37 ℃ and 150rpm for 4 hours. After being taken out, the mixture is cleaned by repeated ultrasonic vibration of ethanol and then is fully cleaned by deionized water until the ethanol on the surface is cleaned.
(3) Coupling of 4-maleimidobutanoic acid-N-succinimidyl ester
20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution is placed in a 50mL conical flask, then thiolated modified silica gel is added into the conical flask, then the conical flask is placed in a constant temperature shaking table, the temperature is set to 37 ℃, the rotating speed is 150rpm, the conical flask is taken out after reaction is carried out for 1h, and the conical flask is placed in a cool and ventilated place to be air-dried.
(4) Photocatalytic cross-linked bovine serum albumin
A50 mL centrifuge tube (covered with tinfoil and protected from light) was charged with 30mL of 10mg/mL bovine serum albumin solution, followed by 1mL of 0.0125M tris (2,2' -bipyridyl) ruthenium (II) dichloride hexahydrate and 1mL of 0.25M Ammonium Persulfate (APS), and placed under a 200W incandescent lamp for 10 seconds.
(5) Activated and modified silica gel immobilized bovine serum albumin
The modified and coupled silica gel 10g was placed in a 20mL beaker, the photocatalytic cross-linked bovine serum albumin solution was added, the mixture was stirred sufficiently with a glass rod to attach the cross-linked bovine serum albumin to the modified silica gel as much as possible, filtered, washed with PBS buffer (PH 7.4) several times, and filtered. Finally drying at 37 ℃ to obtain a bovine serum albumin chiral stationary phase (BSA-CSP).
FIG. 1 is a synthesis scheme of BSA-CSP according to the present invention; FIG. 2 shows a macroscopic comparison of silica gel with BSA-CSP, and the samples were subjected to the following tests.
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) can carry out qualitative analysis to the characteristic group that bovine serum albumin contains, and this experiment carries out infrared characterization to bovine serum albumin chiral stationary phase, modified silica gel, do not do processing silica gel, bovine serum albumin solution, the BSA solution through PICUP processing. BSA-CSP, modified silica gel and untreated silica gel are taken as solid groups, and the experimental steps are as follows: an appropriate amount of potassium bromide solid was taken and added to the sample to be characterized (mixed in a ratio of sample: potassium bromide-1: 100). Grinding the materials into powder, pouring the powder into a die, pressing the powder into a transparent sheet which can smoothly penetrate through a light path by using a tablet press, taking out the die, putting the die into an FTIR-650 Fourier transform infrared spectrometer for scanning (measuring an air background before scanning), setting the scanning frequency to be 4 times, and setting the scanning range to be 4500cm-1～400cm-1. Bovine serum albumin solution and PICUP treated BSA were used as liquid groups, and the experimental steps were: directly preparing pure potassium bromide into a slice, and dripping the solution to be detected to the center of the sliceThe scan was performed rapidly, with the same scan conditions as for the solid group. The results are shown in FIGS. 5 and 6.
FIG. 5 shows that FTIR measures silica gel, modified silica gel, and modified silica gel-supported BSA, respectively. As can be seen from FIG. 5, silica gel is present at 3500cm-1And (3) if the stretching vibration occurs, judging that the stretching vibration of the hydroxyl of the silica gel is absorbed, and obviously shifting the silica gel to the right after modification treatment and the stretching vibration of the hydroxyl of BSA immobilized on the modified silica gel, which shows that the gamma-mercaptopropyl trimethoxysilane and the GMBS are successfully bonded on the silica gel.
FIG. 6 shows FTIR measurements of BSA and photo-crosslinked BSA, respectively. From FIG. 6, it can be seen that the distance is 1700cm-1～1600cm-1Stretching vibration of C ═ O occurs between amide bands, and protein secondary structure increase occurs in the BSA subjected to photocatalytic cross-linking, indicating that the application of the photocatalytic cross-linking technology enables the BSA to obtain a secondary structure rich in beta sheet.
Example 3
Preparation of a chiral stationary phase based on photocatalytic cross-linked protein:
(1) preparation of activated silica gel
20g of silica gel are weighed, dissolved in 60ml of HCl (6.0M), stirred and refluxed for 8h at 150 ℃, and the activated silica gel is filtered by suction and washed to neutrality by distilled water. Finally, the product is dried for 8h at 70 ℃ and filled into a reagent bottle for standby.
(2) Sulfhydrylation modification:
first, 1.2mL of gamma-mercaptopropyltrimethoxysilane was added to a 20mL beaker containing 10mL of 95% ethanol, mixed thoroughly using a vortex mixer, the pH of the system was adjusted to 5 with concentrated hydrochloric acid, and poured into a conical flask. 15g of activated silica gel was added, and the flask was then placed in a constant temperature shaker at 37 ℃ and 150rpm for 4 hours. After being taken out, the mixture is cleaned by repeated ultrasonic vibration of ethanol and then is fully cleaned by deionized water until the ethanol on the surface is cleaned.
(3) Coupling of 4-maleimidobutanoic acid-N-succinimidyl ester
20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution is placed in a 50mL conical flask, then thiolated modified silica gel is added into the conical flask, then the conical flask is placed in a constant temperature shaking table, the temperature is set to 37 ℃, the rotating speed is 150rpm, the conical flask is taken out after reaction is carried out for 1h, and the conical flask is placed in a cool and ventilated place to be air-dried.
(4) Photocatalytic cross-linked bovine serum albumin
A50 mL centrifuge tube (covered with tinfoil and protected from light) was charged with 30mL of 10mg/mL bovine serum albumin solution, followed by 1mL of 0.0125M tris (2,2' -bipyridyl) ruthenium (II) dichloride hexahydrate and 1mL of 0.25M Ammonium Persulfate (APS), and placed under a 200W incandescent lamp for 10 seconds.
(5) Activated and modified silica gel immobilized bovine serum albumin
The modified and coupled silica gel 10g was placed in a 20mL beaker, 10mL of a solution of bovine serum albumin after photocatalytic cross-linking was added, the solution was stirred sufficiently with a glass rod to attach the cross-linked bovine serum albumin to the modified silica gel as much as possible, filtered, washed with PBS buffer (PH 7.4) several times, and filtered. Finally drying at 37 ℃ to obtain a bovine serum albumin chiral stationary phase (BSA-CSP).
Resolution test of column chromatography on amino acid
Selecting a small-aperture chromatographic column (the inner diameter is 1cm, the length is 20cm), and respectively filling untreated silica gel and the prepared bovine serum albumin chiral stationary phase, wherein the height of the filled column is 7 cm. D, L-tryptophan, tyrosine and cysteine are respectively prepared into 0.03mol/L sample solution, 1.0mL of mixed exosomal amino acid is added to a chromatographic column, and absolute ethyl alcohol is used as eluent, and the flow rate is controlled at 1 mL/min. Collecting 2mL of chromatographic solution in 10mL centrifuge tubes, diluting the collected chromatographic solution by 5 times with absolute ethanol, and measuring the alpha value of the optical rotation by using an automatic polarimeter and the A value of the ultraviolet absorption by using an ultraviolet spectrophotometer (the ultraviolet absorption wavelengths of tryptophan, tyrosine and cysteine are all set to 280 nm). The results are shown in tables 3 and 4.
TABLE 3 chiral resolution of D, L-tyrosine, phenylalanine, tryptophan by silica gel
The optically active alpha value in Table 3 indicates the resolution of the enantiomer, and the UV absorption A value indicates the concentration of amino acids in the solution. As shown in Table 3, silica gel has no resolving effect on D, L-tyrosine, and has a certain resolving power on the racemate of D, L-cysteine and D, L-tryptophan.
TABLE 4 chiral resolution of the BSA-CSP pair D, L-tyrosine, phenylalanine, tryptophan
The optically active alpha value in Table 4 indicates the resolution of the enantiomer, and the UV absorption A value indicates the concentration of amino acids in the solution. It is evident from Table 4 that tyrosine, cysteine and tryptophan gave better resolution on BSA-CSP.
And (3) respectively eluting 0.03mol/L of L-tyrosine, L-phenylalanine and L-tryptophan by taking 1mL of column chromatography (silica gel column and BSA-CSP column), measuring the corresponding optical rotation value when the concentration of the collected liquid is maximum, and dividing the optical rotation value by the maximum negative value of the optical rotation values measured by eluting the collected liquid on the column chromatography of D, L-tyrosine, cysteine and tryptophan to obtain the resolution ratio of the amino acid. The maximum concentration optical rotation values measured by elution in a BSA-CSP column are-0.028, -0.074 and-0.030 in sequence, and the resolution ratios are 57%, 70% and 53% respectively; the maximum concentration optical rotation values measured by silica gel column elution are respectively 0.000, -0.034-0.031, and the obtained resolution ratios are 0%, 35% and 25%. Therefore, the prepared bovine serum albumin chiral fixation has better resolution effect relative to amino acid.
Example 4
Preparation of a chiral stationary phase based on photocatalytic cross-linked protein:
(1) preparation of activated silica gel
20g of silica gel are weighed, dissolved in 60ml of HCl (6.0M), stirred and refluxed for 8h at 150 ℃, and the activated silica gel is filtered by suction and washed to neutrality by distilled water. Finally, the product is dried for 8h at 70 ℃ and filled into a reagent bottle for standby.
(2) Sulfhydrylation modification:
first, 1.2mL of gamma-mercaptopropyltrimethoxysilane was added to a 20mL beaker containing 10mL of 95% ethanol, mixed thoroughly using a vortex mixer, the pH of the system was adjusted to 5 with concentrated hydrochloric acid, and poured into a conical flask. 15g of activated silica gel was added, and the flask was then placed in a constant temperature shaker at 37 ℃ and 150rpm for 4 hours. After being taken out, the mixture is cleaned by repeated ultrasonic vibration of ethanol and then is fully cleaned by deionized water until the ethanol on the surface is cleaned.
(3) Coupling of 4-maleimidobutanoic acid-N-succinimidyl ester
20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution is placed in a 50mL conical flask, then thiolated modified silica gel is added into the conical flask, then the conical flask is placed in a constant temperature shaking table, the temperature is set to 37 ℃, the rotating speed is 150rpm, the conical flask is taken out after reaction is carried out for 1h, and the conical flask is placed in a cool and ventilated place to be air-dried.
(4) Photocatalytic cross-linked bovine serum albumin
A500 mL beaker (wrapped with tinfoil and protected from light) was charged with 300mL of a bovine serum albumin solution at a concentration of 0.5mg/mL, and then 500. mu.L of 0.021M tris (2,2' -bipyridine) ruthenium (II) dichloride hexahydrate and 500. mu.L of 0.42M Ammonium Persulfate (APS) were sequentially added thereto at a molar ratio of the above 3 reactants of 1:5:100, followed by placing under a 200W incandescent lamp for 10 seconds.
(5) Activated and modified silica gel immobilized bovine serum albumin
The modified and coupled silica gel 10g was placed in a 200mL beaker, 100mL of the photocatalytic cross-linked bovine serum albumin solution was added, the mixture was stirred sufficiently with a glass rod to attach the cross-linked bovine serum albumin to the modified silica gel as much as possible, filtered, washed with PBS buffer (pH 7.4) several times, and filtered. Finally drying at 37 ℃ to obtain a bovine serum albumin chiral stationary phase (BSA-CSP).
The results of the amino acid resolution test by column chromatography on the samples are shown in table 5.
TABLE 5 chiral resolution of the BSA-CSP pair D, L-cysteine, serine, threonine
The optically active alpha value in Table 5 indicates the resolution of the enantiomer, and the UV absorption A value indicates the concentration of amino acids in the solution. It is evident from Table 5 that cysteine, serine and threonine have a good resolution effect on BSA-CSP.
Respectively eluting 0.03mol/L of L-cysteine, L-serine and L-threonine on a BSA-CSP column by 1mL, measuring the corresponding optical rotation value when the concentration of the collected liquid is maximum, and dividing the optical rotation value by the maximum negative value of the optical rotation measured by eluting D, L-cysteine, serine and threonine on the column to obtain the resolution ratio of the amino acid. The maximum concentration optical rotation values measured by elution on a BSA-CSP column are-0.033, -0.18 and-0.056 in sequence, and the resolution ratios are respectively 17%, 80% and 23%. Therefore, the prepared bovine serum albumin chiral fixation has better resolution effect than the natural amino acid containing hydroxyl.
Example 5
Preparation of a chiral stationary phase based on photocatalytic cross-linked protein:
(1) preparation of activated silica gel
20g of silica gel are weighed, dissolved in 60ml of HCl (6.0M), stirred and refluxed for 8h at 150 ℃, and the activated silica gel is filtered by suction and washed to neutrality by distilled water. Finally, the product is dried for 8h at 70 ℃ and filled into a reagent bottle for standby.
(2) Sulfhydrylation modification:
first, 1.2mL of gamma-mercaptopropyltrimethoxysilane was added to a 20mL beaker containing 10mL of 95% ethanol, mixed thoroughly using a vortex mixer, the pH of the system was adjusted to 5 with concentrated hydrochloric acid, and poured into a conical flask. 15g of activated silica gel was added, and the flask was then placed in a constant temperature shaker at 37 ℃ and 150rpm for 4 hours. After being taken out, the mixture is cleaned by repeated ultrasonic vibration of ethanol and then is fully cleaned by deionized water until the ethanol on the surface is cleaned.
(3) Coupling of 4-maleimidobutanoic acid-N-succinimidyl ester
20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution is placed in a 50mL conical flask, then thiolated modified silica gel is added into the conical flask, then the conical flask is placed in a constant temperature shaking table, the temperature is set to 37 ℃, the rotating speed is 150rpm, the conical flask is taken out after reaction is carried out for 1h, and the conical flask is placed in a cool and ventilated place to be air-dried.
(4) Photocatalytic cross-linked porcine insulin
A50 mL centrifuge tube (wrapped with tinfoil and protected from light) was charged with 30mL of a 5mg/mL porcine insulin solution, followed by 500. mu.L of 0.0125M ruthenium (II) tris (2,2' -bipyridine) dichloride hexahydrate and 500. mu.L of 0.25M Ammonium Persulfate (APS), and placed under a 200W incandescent lamp for 10 seconds.
(5) Activated and modified silica gel immobilized porcine insulin
And (2) putting 10g of modified and coupled silica gel into a small 20mL beaker, adding a pig insulin solution subjected to photocatalytic crosslinking, fully stirring with a glass rod to ensure that the crosslinked pig insulin is attached to the modified silica gel as much as possible, performing suction filtration, washing with PBS (phosphate buffered saline) (pH 7.4) for multiple times, and performing suction filtration. Finally drying at 37 ℃ to prepare the porcine insulin chiral stationary phase (INS-CSP).
The results of the amino acid resolution test by column chromatography on the samples are shown in table 6.
TABLE 6 chiral resolution of the INS-CSP pair D, L-tyrosine, cysteine, tryptophan
The optically active alpha value in Table 6 indicates the resolution ratio of the enantiomer, and the ultraviolet absorption A value indicates the concentration of amino acid in the solution. It is apparent from Table 6 that glutamic acid, aspartic acid and alanine gave better resolution on INS-CSP.
And (3) respectively eluting 0.03mol/L of L-tyrosine, L-cysteine and L-tryptophan on 1mL of upper columns (a silica gel column and an INS-CSP column), measuring the corresponding optical rotation value when the concentration of the collected liquid is maximum, and dividing the optical rotation value by the maximum negative value of the optical rotation values measured by eluting on the upper columns of D, L-tyrosine, cysteine and tryptophan to obtain the resolution ratio of the amino acid. The maximum concentration optical rotation values measured by elution on the INS-CSP column are-0.046, -0.094 and-0.086 in sequence, and the resolution ratios are respectively 29%, 43% and 33%. Therefore, the prepared porcine insulin has better resolution effect relative to amino acid chiral fixation.
Example 6
Preparation of a chiral stationary phase based on photocatalytic cross-linked protein:
(1) preparation of activated silica gel
20g of silica gel are weighed, dissolved in 60ml of HCl (6.0M), stirred and refluxed for 8h at 150 ℃, and the activated silica gel is filtered by suction and washed to neutrality by distilled water. Finally, the product is dried for 8h at 70 ℃ and filled into a reagent bottle for standby.
(2) Sulfhydrylation modification:
first, 1.2mL of gamma-mercaptopropyltrimethoxysilane was added to a 20mL beaker containing 10mL of 95% ethanol, mixed thoroughly using a vortex mixer, the pH of the system was adjusted to 5 with concentrated hydrochloric acid, and poured into a conical flask. 15g of activated silica gel was added, and the flask was then placed in a constant temperature shaker at 37 ℃ and 150rpm for 4 hours. After being taken out, the mixture is cleaned by repeated ultrasonic vibration of ethanol and then is fully cleaned by deionized water until the ethanol on the surface is cleaned.
(3) Coupling of 4-maleimidobutanoic acid-N-succinimidyl ester
20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution is placed in a 50mL conical flask, then thiolated modified silica gel is added into the conical flask, then the conical flask is placed in a constant temperature shaking table, the temperature is set to 37 ℃, the rotating speed is 150rpm, the conical flask is taken out after reaction is carried out for 1h, and the conical flask is placed in a cool and ventilated place to be air-dried.
(4) Photocatalytic cross-linked lysozyme protein
A50 mL centrifuge tube was charged with 30mL of a 5mg/mL lysozyme solution, followed by 500. mu.L of 0.0125M ruthenium (II) dichloride hexahydrate and 500. mu.L of 0.25M Ammonium Persulfate (APS), and placed under a 200W incandescent lamp for 10 seconds.
(5) Activated modified silica gel immobilized lysozyme protein
And (2) putting 10g of modified and coupled silica gel into a small 20mL beaker, adding a photocatalytic cross-linked lysozyme protein solution, fully stirring with a glass rod to ensure that the cross-linked lysozyme protein is attached to the modified silica gel as much as possible, performing suction filtration, washing with PBS (phosphate buffered saline) (pH 7.4) for multiple times, and performing suction filtration. Finally drying at 37 ℃ to prepare lysozyme chiral stationary phase (Lys-CSP).
The results of the amino acid resolution test by column chromatography on the samples are shown in Table 7.
TABLE 7 chiral resolution of Lys-CSP to D, L-lysine, arginine, leucine
The optically active alpha value in Table 7 indicates the resolution ratio of the enantiomer, and the UV absorption A value indicates the concentration of amino acids in the solution. It is evident from Table 7 that tyrosine, cysteine and tryptophan gave better resolution on Lys-CSP.
Respectively eluting 0.03mol/L of L-lysine, L-arginine and L-leucine on 1mL of upper column (silica gel column and Lys-CSP column), measuring the corresponding optical rotation value when the concentration of the collected liquid is maximum, and dividing the optical rotation value by the maximum negative value of the optical rotation measured by eluting on D, L-lysine, arginine and leucine on the upper column to obtain the resolution ratio of the amino acid. The maximum concentration optical rotation values measured by eluting in a Lys-CSP column are-0.026, -0.072 and-0.032 in sequence, and the resolution ratios are respectively 37%, 50% and 25%. Therefore, the prepared lysozyme chirally fixed has a better resolution effect compared with amino acid.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (1)
1. The application of the chiral stationary phase based on the photocatalytic cross-linked protein is characterized in that: for separating mixed exosomal amino acids by:
selecting a chromatographic column with the inner diameter of 1cm and the length of 20cm, filling the prepared bovine serum albumin chiral stationary phase, and filling the column with the height of 7 cm; preparing the mixed exosomal amino acid into a sample solution of 0.03mol/L, adding 1.0mL of the mixed exosomal amino acid to a chromatographic column, using absolute ethyl alcohol as eluent, controlling the flow rate at 1mL/min, collecting 2mL of chromatographic solution in 10mL centrifuge tubes, and diluting the collected chromatographic solution by 5 times by using the absolute ethyl alcohol;
the preparation method of the bovine serum albumin chiral stationary phase comprises the following steps:
weighing 20g of silica gel, dissolving in 60mL6.0M HCl, stirring and refluxing for 8h at 150 ℃, carrying out suction filtration on the activated silica gel, washing with distilled water to be neutral, drying the product for 8h at 70 ℃, and filling the product into a reagent bottle for later use to obtain activated silica gel;
adding 1.2mL of gamma-mercaptopropyl trimethoxysilane into a 20mL beaker containing 10mL of 95% ethanol, fully mixing by using a vortex mixer, adjusting the pH value of the system to 5 by using concentrated hydrochloric acid, pouring into a conical flask, adding 15g of activated silica gel, then placing the conical flask in a constant-temperature shaking table, reacting for 4 hours at 37 ℃ and 150rpm, taking out, repeatedly washing with ethanol by ultrasonic vibration, and then fully washing with deionized water until the ethanol on the surface is cleaned, thus obtaining the silica gel modified by sulfhydrylation;
placing 20mL of prepared 1mM 4-maleimidobutyrate-N-succinimidyl ester solution in a 50mL conical flask, adding the sulfhydrylation modified silica gel into the conical flask, placing the conical flask in a constant-temperature shaking table, setting the temperature to be 37 ℃, setting the rotating speed to be 150rpm, reacting for 1h, taking out, placing in a cool and ventilated place, and air-drying to obtain modified and coupled silica gel;
taking a 50mL centrifuge tube, wrapping the centrifuge tube with tinfoil to avoid light, adding 30mL bovine serum albumin solution with the concentration of 10mg/mL, sequentially adding 1mL tris (2,2' -bipyridyl) dichlororuthenium (II) hexahydrate with the concentration of 0.0125M and 1mL ammonium persulfate with the concentration of 0.25M, then placing under a 200W incandescent lamp, and controlling the illumination time to be 10s to obtain the photocatalytic cross-linked bovine serum albumin;
putting 10g of modified and coupled silica gel into a 20mL small beaker, adding 10mL of a bovine serum albumin solution after photocatalytic crosslinking, fully stirring by using a glass rod to ensure that the crosslinked bovine serum albumin is attached to the modified silica gel as much as possible, performing suction filtration, washing for multiple times by using PBS (phosphate buffered saline) with pH of 7.4, performing suction filtration, and finally drying at 37 ℃ to obtain the bovine serum albumin chiral stationary phase.
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