WO2017161619A1 - Method for separating polypeptide from imidazolium ionic liquid - Google Patents

Abstract

The present invention provides a method for separating a polypeptide from an imidazolium ionic liquid and relates to the technical field of purification and separation of a biomolecule. The method for separating a polypeptide from an imidazolium ionic liquid comprises: removing a free imidazolium ionic liquid by means of back-extraction adopting a salting-out method, and then using a silver ion-containing reagent to thoroughly detach an imidazolium ion chemically bound to a polypeptide molecule. The method for separating a polypeptide from an imidazolium ionic liquid is simple and effective, has a low cost and energy consumption, obtains a high product purity, and greatly increases the usability and the scope of application of an imidazolium ionic liquid in polypeptide chemistry.

Description

Method for separating polypeptide from imidazole ionic liquid Technical field:

The invention relates to the technical field of purification and separation of biomolecules, in particular to a method for separating polypeptides from imidazole ionic liquids.

Background technique:

Since polypeptides can contain different combinations and amounts of hydrophilic and hydrophobic amino acid residues, they can be folded to form complex secondary structures, resulting in diversity and variability in their solubility properties. Therefore, in the chemistry of peptides, the choice of solvent is often a challenge. Currently, commonly used inorganic and organic solvents are not compatible with the dissolution of all kinds of polypeptides.

Room temperature ionic liquids, often referred to as ionic liquids, are a class of ionic compounds that remain liquid at room temperature. Ionic liquids are widely used in materials, energy and chemical industries and are well-known green industrial reagents. In recent years, room temperature ionic liquids, especially imidazolium ionic liquids, have begun to be used as novel solvents in the field of peptide chemistry. In the research so far, imidazolium ionic liquids have excellent solvency for various types of peptides, have better biocompatibility and catalytic properties of carbene precursors, and are widely used in the ligation of polypeptide fragments [1], Amino acid coupling reaction [2] and peptide carrier synthesis [3]. However, due to the strong solvency of the imidazole ionic liquid to the polypeptide, it is also very difficult to separate the polypeptide dissolved in the ionic liquid; there are literatures from the ion cycle extraction method [2] or liquid chromatography [4] from the ion The polypeptide is separated in the liquid, but the ionic liquid contained in the polypeptide sample is often not completely removed. This is mainly because the anion or cation of the imidazole ionic liquid has strong chemical binding ability and interaction with amino acid residues in the polypeptide molecule, such as cysteine and lysine [4]. Because the binding of the imidazole ionic liquid on the polypeptide easily affects the conformation of the polypeptide itself and the ion channel of the biofilm, it may cause denaturation or inactivation of the polypeptide drug, which may cause obstacles to the application of the polypeptide in the medical field [4]. In most cases, there is a chemical bond between the peptide and the ionic liquid molecule, containing polar residues, often in the millimolar range, and is not suitable for ion exchange resins and antisolvent crystallization techniques [5]. Therefore, this phenomenon greatly restricts the application of ionic liquids in peptide chemistry.

In response to this problem, the present invention provides a method for separating a polypeptide from an imidazole-based ionic liquid, which is capable of completely and easily removing the ionic liquid by a physical method of salting out effect and a chemical method containing silver ion removal; Maximize the configuration and activity of the polypeptide without being affected.

Summary of the invention:

It is an object of the present invention to provide a method for isolating a polypeptide from an imidazole-based ionic liquid to solve the problem that the polypeptide is difficult to separate and purify from the ionic liquid.

The technical scheme adopted by the present invention combines a physical back extraction separation and a chemical elution separation method: Step 1, first mixing a preferred polypeptide organic solvent with a polypeptide solution containing an imidazole ionic liquid in a certain ratio; Step 2, dropping Preferably, the stripping agent is separated into a solution to form a solvent layer and a stripping agent layer, and the solvent phase and the stripping agent phase are separated; in step 3, step 2 is repeated three or more times for the solvent phase obtained by separating; Step 4 The ionic liquid remover containing silver ions is added to the solvent phase after separation and purification for several times, and the reaction releases heat to rapidly form a white flocculent precipitate of a heterocyclic carbene silver complex [6] or an insoluble silver salt, and is removed by centrifugal filtration. Precipitation; Step 5, after purification of the obtained solution, the solvent is removed by lyophilization to obtain a pure polypeptide free of ionic liquid.

In the above technical solution, the polypeptide organic solvent may be isopropanol, isobutanol, propylene glycol, butanediol, acetonitrile, acetone, methyl acetate, ethyl acetate, dimethylformamide, dimethyl sulfoxide, Tetrahydrofuran, dichloromethane, chloroform; wherein the solvent is preferably based on a solubility in the isolated target polypeptide that is higher than that of the ionic liquid used.

In the above technical solution, the imidazole-based ionic liquid may be an ionic liquid containing an imidazole-based cation or an imidazole-based anion. The ionic liquid structural formula containing an imidazolium cation may be:

The ionic liquid structural formula containing an imidazolium anion can be:

Wherein the structural formula _{R 1, R 2, R 3} , R 4, R 5 can be hydrogen, a hydrocarbon group, a halogen-containing group, oxygen-containing functional groups, nitrogen-containing functional groups, phosphorus-containing functional group, a sulfur-containing functional group; X ^- may be an inorganic An acid anion of an acid or an organic acid; Y ⁺ may be a choline, an amine, a mononuclear imidazole, or a dinuclear imidazole cation.

In the above technical solution, the polypeptide may be a biomolecule containing a natural or unnatural amino acid residue linked by a peptide bond.

In the above technical solution, the stripping agent may be water (including a buffered aqueous solution), methanol, ethanol or n-propanol; the preferred standard is: the solvating ability to the ionic liquid is higher than the solvency of the isolated target polypeptide. And the salting-out effect of the solution system can be formed at a certain mixing ratio to form a liquid-liquid phase separation.

In the above technical solution, the silver ion-containing ionic liquid removing agent is a uniform dispersion system containing silver ions or a uniform dispersion system capable of generating silver ions by physical and chemical means, and may be, but not limited to, a silver salt solution or a silver salt. A suspension, a solution of silver oxide, a suspension of silver oxide or a mixture thereof; preferred standards are: do not affect the stability of the polypeptide, react rapidly with the ionic liquid, and form a precipitate that is easily separated.

The method for separating polypeptides from imidazole-based ionic liquids provided by the present invention has the following innovations and advantages compared with the prior art:

1. The method for separating a polypeptide from an imidazole ionic liquid provided by the invention utilizes the salting out effect of an ionic liquid in a miscible azeotrope to easily remove most of the free ionic liquid, thereby avoiding the polypeptide product in the separation process. Loss

2. The method for separating a polypeptide from an imidazole ionic liquid provided by the present invention utilizes the chemical property of an imidazole ionic liquid to rapidly remove the imidazolium ion chemically bonded to the polypeptide residue by adding a silver ion remover. In addition, the separation cost is low;

3. The method for separating a polypeptide from an imidazole-based ionic liquid provided by the present invention is compatible with a polypeptide containing different kinds of residues, and simultaneously removes an imidazolium cation and an imidazole anion, and the activity of the polypeptide itself is not affected.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 is a high performance liquid chromatogram of the polypeptide LYRAGCRANK in the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Figure 2 is a high performance liquid color separation of the polypeptide LYRAGCRANK from the ionic liquid 1-ethyl-3-methylimidazolium acetate Spectrum.

Figure 3 is an ESI mass spectrum of the polypeptide LYRAGCRANK isolated from the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Figure 4 is a high performance liquid chromatogram of leucine enkephalin in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate.

Figure 5 is a high performance liquid chromatogram of the separation of leucine enkephalin from the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate.

Figure 6 is an ESI mass spectrum of leucine enkephalin isolated from the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate.

Figure 7 is a high performance liquid chromatogram of μ-conotoxin SIIIA in ionic liquid 1-ethyl-3-methylimidazolium acetate.

Figure 8 is a high performance liquid chromatogram of the separation of μ-conotoxin SIIIA from the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Figure 9 is an ESI mass spectrum of the μ-conotoxin SIIIA isolated from the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Figure 10 is a high performance liquid chromatogram of μ-conotoxin SIIIA in the ionic liquid choline imidazolium salt.

Figure 11 is a high performance liquid chromatogram of the separation of μ-conotoxin SIIIA from the ionic liquid choline imidazole salt.

Figure 12 is an ESI mass spectrum of μ-conotoxin SIIIA isolated from ionic liquid choline imidazolium salt

detailed description:

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The experimental materials, reagents and the like used in the following examples can be obtained by a commercial route or a known experimental method.

Example 1

In the literature [1], the polypeptide LYRAGCRANK was synthesized using the ionic liquid 1-ethyl-3-methylimidazolium acetate as a solvent. In this example, the separation of the polypeptide LYRAGCRANK in the ionic liquid 1-ethyl-3-methylimidazolium acetate was carried out using the method provided by the present invention.

The experimental device is mainly a 50 ml test tube with a graduated tip, a filter, a pipette, a centrifuge, a water bath device, a shaking stirrer and a low temperature vacuum drying device. In step 1, the polypeptide LYRAGCRANK is dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate to form 1 mL of a polypeptide ionic liquid solution having a concentration of 3 nmol/L. In step 2, 10 mL of propylene glycol was added and stirred under stirring. In step 3, 200 μL of pure water was added to cause a mist, and the resulting mixture was centrifuged at the highest speed for 30 minutes to separate the layers. Step 4. Carefully remove the aqueous layer containing the ionic liquid from the bottom of the tube. In step 5, steps 3 and 4 are repeated for the resulting solution until no more fogging occurs when pure water is added dropwise. Step 6. Slowly add a solution of propylene glycol containing 20% silver nitrate under the condition of a water bath; immediately stop the dropwise addition when the white precipitate no longer continues to be produced; the reaction formula of the step 6 is:

In step 7, the mixture is centrifuged for 20 minutes, and the precipitate is removed by filtration through a quartz fiber filter paper; in step 8, the obtained solution is purified by a gel filtration method using a dilute hydrochloric acid eluate, and lyophilized under a low temperature vacuum to obtain a white polypeptide crystalline powder.

The obtained product is the polypeptide LYRAGCRANK, the yield is 95.4% (not including the oxidized polypeptide); the characteristic peak of the ionic liquid is not found in the high performance liquid chromatogram, compared with the peak of the polypeptide product in the ionic liquid (Fig. 1), after separation The peak of the peptide product did not shift or split (Fig. 2); ESI mass spectrometry did not detect ionic liquid residue (Fig. 3).

Example 2

In the literature [3], the polypeptide leucine enkephalin has been synthesized using an imidazole ionic liquid as a carrier. In this example, the separation of leucine enkephalin in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was carried out using the method provided by the present invention.

The experimental device is mainly a 50 ml test tube with a graduated tip, a filter, a pipette, a centrifuge, a water bath device, a shaking stirrer and a low temperature vacuum drying device. Step 1. The leucine enkephalin is dissolved in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate to form 1 mL of a polypeptide ionic liquid solution having a concentration of 3 nmol/L. Step 2, add 10 mL of methyl acetate, shake and stir. Step 3, 150 μL of dichloromethane was added dropwise to cause a mist; the resulting mixture was centrifuged for 30 minutes to separate the layers. Step 4. Carefully remove the ionic liquid-containing methylene chloride layer from the bottom of the tube. In step 5, steps 3 and 4 are repeated for the resulting solution until no more fogging occurs when pure water is added dropwise. Step 6. Slowly add a methanol solution containing saturated silver oxide under the condition of a water bath; immediately stop the dropwise addition when the white precipitate no longer continues to be produced; the reaction formula of the step 6 is:

In step 7, the mixture is centrifuged for 20 minutes, and the precipitate is removed by filtration through a quartz fiber filter paper; in step 8, the obtained solution is purified by a gel filtration method using a dilute hydrochloric acid eluate, and lyophilized under a low temperature vacuum to obtain a white polypeptide crystalline powder.

The obtained product is leucine enkephalin, the yield is 99.2%; the characteristic peak of the ionic liquid is not found in the high performance liquid chromatogram, and the peak of the separated polypeptide product is compared with the peak of the polypeptide product in the ionic liquid (Fig. 4). No displacement or fragmentation occurred (Fig. 5); no ionic liquid residue was detected by ESI mass spectrometry (Fig. 6).

Example 3

In the literature [4], the polypeptide μ-conotoxin SIIIA was oxidatively folded with the ionic liquid 1-ethyl-3-methylimidazolium acetate as a solvent. In this example, the separation of the polypeptide μ-conotoxin SIIIA in the ionic liquid 1-ethyl-3-methylimidazolium acetate was carried out using the method provided by the present invention.

The experimental device is mainly a 50 ml test tube with a graduated tip, a filter, a pipette, a centrifuge, a water bath device, a shaking stirrer and a low temperature vacuum drying device. Step 1. The μ-conotoxin SIIIA was dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate to form 1 mL of a polypeptide ionic liquid solution having a concentration of 3 nmol/L. In step 2, 10 mL of isopropanol was added and stirred under stirring. In step 3, 100 μL of pure water was added dropwise to cause a mist phenomenon; the resulting mixture was centrifuged for 30 minutes to separate the layers. Step 4. Carefully remove the aqueous layer containing the ionic liquid from the bottom of the tube. In step 5, steps 3 and 4 are repeated for the resulting solution until no more fogging occurs when pure water is added dropwise. In step 6, a solution of 2.4 mg/mL silver acetate in isopropanol is slowly added dropwise under a water bath; the dropping is stopped immediately when the white precipitate is no longer produced; the reaction equation of step 6 is:

Step 7, centrifuge for 20 minutes, and remove the precipitate by filtration with quartz fiber filter paper; Step 8, pass the obtained solution The gel filtration method of the dilute hydrochloric acid eluate was purified, and lyophilized under low temperature vacuum to obtain a white crystalline powder of the polypeptide.

The obtained product is μ-conotoxin SIIIA, the yield is 97.5%; the characteristic peak of the ionic liquid is not found in the high performance liquid chromatogram, and the peak of the separated polypeptide product is compared with the peak of the polypeptide product in the ionic liquid (Fig. 7). No displacement or fragmentation occurred (Fig. 8); no ionic liquid residue was detected by ESI mass spectrometry (Fig. 9).

Example 4

In this example, the method provided by the present invention separates the polypeptide μ-conotoxin SIIIA in an ionic liquid choline imidazolium salt.

The experimental device is mainly a 50 ml test tube with a graduated tip, a filter, a pipette, a centrifuge, a shaking stirrer and a low temperature vacuum drying device. Step 1. The μ-conotoxin SIIIA is dissolved in the ionic liquid choline imidazolium salt to form 1 mL of a polypeptide ionic liquid solution having a concentration of 3 nmol/L. In step 2, 15 mL of dichloromethane was added and stirred under stirring. In step 3, 100 μL of water was added dropwise to cause a mist phenomenon; the resulting mixture was centrifuged for 30 minutes to separate the layers. Step 4. Carefully remove the aqueous layer containing the ionic liquid from the bottom of the tube. In step 5, steps 3 and 4 are repeated for the resulting solution until no more fogging occurs when pure water is added dropwise. In step 6, a mixed solution of water and dichloromethane containing 1.5 mg/mL of silver oxide is added dropwise (volume ratio 1:1); when the white precipitate is no longer produced, the dropwise addition is stopped immediately; the reaction equation of step 6 is:

In step 7, the mixture is centrifuged for 20 minutes, and the precipitate is removed by filtration through a quartz fiber filter paper; in step 8, the obtained solution is purified by a gel filtration method using a dilute hydrochloric acid eluate, and lyophilized under a low temperature vacuum to obtain a white polypeptide crystalline powder.

The obtained product is μ-conotoxin SIIIA, the yield is 98.1%; the characteristic peak of the ionic liquid is not found in the high performance liquid chromatogram, and the peak of the separated polypeptide product is compared with the peak of the polypeptide product in the ionic liquid (Fig. 10). No displacement or fragmentation occurred (Fig. 11); no ionic liquid residue was detected by ESI mass spectrometry (Fig. 12).

Example 5

Refer to the electrobiological method (sodium ion channel blockade) in [4] to evaluate the activity of the following polypeptides: μ separated from the ionic liquid 1-ethyl-3-methylimidazolium acetate by high performance liquid chromatography. - Conotoxin SIIIA, μ-conotoxin SIIIA isolated from ionic liquid choline imidazolium salt by high performance liquid chromatography, using the method provided by the invention from ionic liquid 1-ethyl-3-methylimidazoliumacetic acid The μ-conotoxin SIIIA isolated from the salt, and the μ-conotoxin SIIIA isolated from the ionic liquid choline imidazolium salt by the method provided by the present invention. The results are shown in Table 1:

Table I

The results showed that the μ-conotoxin SIIIA isolated by high performance liquid chromatography using the literature [4] lost activity, while the μ-conotoxin SIIIA isolated by the method provided by the present invention maintained high activity.

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all included in the scope of protection and disclosure of the present invention.

[1] Kühl, T.; Chen, M.; Teichmann, K.; Stark, A.; Imhof, D. Ionic Liquid 1-Ethyl-3-Methylimidazolium Acetate: An Attractive Solvent for Native Chemical Ligation of Peptides. Tetrahedron Lett .2014, 55 (27), 3658–3662.

[2]Vallette, H.Room Temperature Ionic Liquids (RTIL’s) Are Convenient Solvents for Peptide Synthesis! Arkivoc 2006, 2006 (4), 200.

[3] Miao, W.; Chan, T.H. Ionic-Liquid-Supported Synthesis: A Novel Liquid-Phase Strategy for Organic Synthesis. Acc. Chem. Res. 2006, 39 (12), 897–908.

[4] Heimer, P.; Tietze, AA;

M.; Giernoth, R.; Kuchenbuch, A.; Stark, A.; Leipold, E.; Heinemann, SH; Kandt, C.; Imhof, D. Application of Room-Temperature Aprotic and Protic Ionic Liquids for Oxidative Folding of Cysteine-Rich Peptides. ChemBioChem 2014, 15 (18), 2754–2765.

[5] CN 102559943B: A process for separating glucose from ionic liquids.

[6] Hindi, K.M.; Panzner, M.J.; Tessier, C.A.; Cannon, C.L.; Youngs, W.J. The Medicinal Applications of Imidazolium Carbene-Metal Complexes. Chem. Rev. 2009, 109 (8), 3859-3884.

Claims (9)

1. The invention relates to a method for separating a polypeptide from an imidazole ionic liquid, characterized in that a physical back extraction separation and a chemical elution separation method are combined, comprising the following steps: (1) firstly using a polypeptide organic solvent and a polypeptide containing an imidazole ionic liquid; The solution is mixed according to a certain ratio; (2) using the principle of salting out effect, adding a stripping agent to back-extract the imidazole ionic liquid in the polypeptide solution; (3) adding ions containing silver ions in the separated polypeptide solution a liquid remover; (4) removing the precipitate formed by filtration; (5) purifying the resulting solution to obtain a polypeptide product.
2. A method for separating a polypeptide from an imidazole-based ionic liquid according to claim 1, wherein the imidazole-based ionic liquid is an ionic liquid containing an imidazole-based cation or an imidazole-based anion.
3. The structural formula of the imidazole cation according to claim 2 may be

   
4. The structural formula of the imidazole-based cation according to claim 3, wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be hydrogen, a hydrocarbon group, a halogen-containing group, an oxygen-containing functional group, a nitrogen-containing functional group, a phosphorus-containing functional group, Sulfur-containing functional group.
5. The structural formula of the imidazole anion according to claim 2 may be

   
6. The structural formula of the imidazole-based cation according to claim 5, wherein R ₁ and R ₂ may be hydrogen, a hydrocarbon group, a halogen-containing group, an oxygen-containing functional group, a nitrogen-containing functional group, a phosphorus-containing functional group, and a sulfur-containing functional group.
7. The method for separating a polypeptide from an imidazole-based ionic liquid according to claim 1, wherein the polypeptide organic solvent is isopropanol, isobutanol, propylene glycol, butanediol, acetonitrile, acetone, Methyl acetate, ethyl acetate, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane or chloroform.
8. A method for separating a polypeptide from an imidazole-based ionic liquid according to claim 1, wherein the stripping agent is water, a buffer solution, methanol, ethanol or n-propanol.
9. The method for separating a polypeptide from an imidazole-based ionic liquid according to claim 1, wherein the silver ion-containing ionic liquid removing agent is a uniform dispersion system containing silver ions or can be physically or chemically A uniform dispersion of silver ions is produced.

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