CN117964681A - Desalination method of full R series polypeptide trifluoroacetate by solid phase synthesis - Google Patents
Desalination method of full R series polypeptide trifluoroacetate by solid phase synthesis Download PDFInfo
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 120
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 120
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 120
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 title claims abstract description 41
- 238000010612 desalination reaction Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 72
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000007788 liquid Substances 0.000 claims abstract description 57
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 48
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 39
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000007787 solid Substances 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000011347 resin Substances 0.000 claims abstract description 21
- 229920005989 resin Polymers 0.000 claims abstract description 21
- 239000007790 solid phase Substances 0.000 claims abstract description 19
- 150000001450 anions Chemical class 0.000 claims abstract description 15
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 15
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 239000000337 buffer salt Substances 0.000 claims abstract description 9
- 238000001291 vacuum drying Methods 0.000 claims abstract description 9
- 238000010790 dilution Methods 0.000 claims abstract description 6
- 239000012895 dilution Substances 0.000 claims abstract description 6
- 239000004408 titanium dioxide Substances 0.000 claims abstract 5
- 238000002386 leaching Methods 0.000 claims description 48
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 37
- 150000002500 ions Chemical class 0.000 claims description 18
- 238000011033 desalting Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000012467 final product Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 12
- VFRSADQPWYCXDG-LEUCUCNGSA-N ethyl (2s,5s)-5-methylpyrrolidine-2-carboxylate;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.CCOC(=O)[C@@H]1CC[C@H](C)N1 VFRSADQPWYCXDG-LEUCUCNGSA-N 0.000 claims description 9
- 238000004007 reversed phase HPLC Methods 0.000 claims description 9
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 125000000129 anionic group Chemical group 0.000 claims description 4
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- 239000012043 crude product Substances 0.000 claims description 2
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- 239000004475 Arginine Substances 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
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- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
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- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
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- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 1
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 1
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Abstract
The invention discloses a desalination method of full R series polypeptide trifluoroacetate by solid phase synthesis, which mainly solves the technical problems of high desalination difficulty and poor efficiency of the existing desalination method. The invention comprises the following steps: 1) Purifying the total R series polypeptide synthesized by solid phase by liquid chromatography to obtain a preparation solution; 2) Passing the preparation solution through a titanium dioxide packing chromatographic column to remove free trifluoroacetate; 3) Freezing and vacuum drying the preparation liquid to obtain a solid product, and detecting the content of trifluoroacetate; 4) Dissolving the solid product in DMSO, placing the solid product on a stirrer for dissolution, adding triethylamine, and removing the trifluoroacetate combined on the R guanidyl; 5) Then adding a chromatographic column in which the intermediate solution is mixed with the strong alkaline anion resin and the TiO2 into a column incubator, and removing newly generated trifluoroacetic acid buffer salt; 6) Adding methanol into the intermediate liquid for dilution, performing rotary evaporation in water bath, and removing DMSO; 7) And then pure water is used for dilution, freezing and vacuum drying, so that a solid powder end product is obtained, and the detection of trifluoroacetate is less than 1%.
Description
Technical Field
The invention belongs to the technical field of polypeptide de-trifluoroacetate salt desalination, and particularly relates to a desalination method capable of obtaining full R series polypeptides with high activity and high stability.
Background
Arginine (R) has the following structural formula:
，
arginine is an amino acid compound, and is a basic amino acid. White diamond crystals or monoclinic flaky crystals (no crystal water), odorless and bitter; is easily soluble in water, slightly soluble in ethanol, and insoluble in diethyl ether.
Trifluoroacetic acid (TFA, molecular formula: CF3 COOH) having the following structural formula:
，
The alias trifluoroacetic acid is colorless volatile fuming liquid, and has hygroscopicity and pungent odor. Is miscible with water, fluorocarbon, methanol, ethanol, diethyl ether, acetone, benzene, carbon tetrachloride and hexane, and is an excellent solvent for proteins and polyesters. Trifluoroacetic acid (TFA) is a strong carboxylic acid with slight toxicity. Trifluoroacetic acid (TFA) is often used to cleave polypeptides from solid phase resins during solid phase synthesis of polypeptides. Trifluoroacetic acid or acetic acid may also be used in the purification of polypeptides by reverse phase HPLC. However, in the case of polypeptides used in preclinical and clinical studies, residual trifluoroacetic acid or fluoride cannot be used because of its toxicity.
The polypeptide consisting of solid phase synthesized total arginine (R) is cleaved from the resin by a high concentration TFA cleavage reagent, and the guanidino group on the arginine is easily reacted with TFA to bind. Arginine is a hydrophilic amino acid, and the full arginine series polypeptide has strong hydrophilicity and great polarity. When the liquid phase preparation is carried out, after elution and separation by a mobile phase containing TFA as an ion pair reagent, the total arginine series polypeptide is easy to enrich trifluoroacetic acid, so that the content of the trifluoroacetic acid salt in the total R series polypeptide pure product is extremely high and even reaches as high as 60 percent. Too high trifluoroacetate salt results in low effective content of the full R series polypeptide and greatly reduced activity of the polypeptide. The high trifluoroacetate has great influence on a plurality of subsequent biological experiments, animal experiments and even drug researches. In this case, it is important to reduce or eliminate the trifluoroacetate content of the full R-series polypeptide.
The existing desalination technology adopts reverse phase chromatography, water to wash TFA, or organic alkali is added to neutralize trifluoroacetate, and the organic salt is washed away, and the two methods have poor effects on removing trifluoroacetate from all R-series polypeptides. The first reverse phase chromatography method is difficult to completely elute due to the strong polarity and hydrophilicity of the full R series polypeptide, and has a high loss rate. The second organic base neutralization method not only has no good control of the pH value, but also the organic salt is extremely soluble in water, and the acid-base neutralized organic salt is difficult to easily elute. The invention is that
The method adopts a two-step desalination method, and has the advantages that a novel titanium dioxide (TiO 2) material is introduced to adsorb trifluoroacetic acid to achieve the purpose of removing the trifluoroacetic acid salt, then the trifluoroacetic acid salt in a combined form is changed into a free form, and the combined type and the free type trifluoroacetic acid salt in the full R series polypeptide are reduced or even completely removed by adopting the mixed use of anionic resin and the titanium dioxide (TiO 2) material. Finally, the full R series polypeptide products with higher activity and stability are obtained.
Disclosure of Invention
The invention aims to provide a desalting method for full R series polypeptide trifluoroacetate by solid phase synthesis, which mainly solves the technical problems of high desalting difficulty and poor efficiency of the existing desalting method.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
A desalination method of full R series polypeptide trifluoroacetate by solid phase synthesis comprises the following steps:
1) Purifying and separating the solid-phase synthesized crude product of the all-R series polypeptide by using a reversed-phase high performance liquid chromatography to obtain a preparation solution of the all-R series polypeptide product with required purity, wherein the preparation solution contains not only free trifluoroacetate but also combined trifluoroacetate;
2) Filling titanium dioxide (TiO 2) into a chromatographic column according to the volume ratio of the prepared liquid, and eluting with ethanol serving as an eluting agent for later use;
3) Removing free trifluoroacetate in the first step, and passing the preparation solution obtained after purification through a titanium dioxide (TiO 2) chromatographic column after washing, so as to remove the trifluoroacetate in the preparation solution;
4) Carrying out water bath 40 ℃ rotary steaming, freezing and vacuum drying on the preparation liquid after the first step of desalination to obtain a full R series polypeptide solid intermediate;
5) Detecting the content of trifluoroacetate at the moment of the solid intermediate of the full R series polypeptide by utilizing an ion chromatograph;
6) Dissolving the solid intermediates of the full R series polypeptides in dimethyl sulfoxide (DMSO), placing the solid intermediates on a stirrer, fully dissolving, gradually adding triethylamine, stirring for reaction, and removing the trifluoroacetate bound on R guanidyl, so that the trifluoroacetate in a bound form is converted into free trifluoroacetate buffer salt;
7) Filling a chromatographic column with an upper layer of strong alkaline anion resin and a lower layer of titanium dioxide (TiO 2) mixed, and eluting with ethanol for later use;
8) The newly generated free trifluoroacetic acid buffer salt polypeptide intermediate solution is washed clean by a mixed chromatographic column, a column temperature box is increased, the temperature is set at 45 ℃, and the newly generated trifluoroacetic acid buffer salt is removed to obtain full R series polypeptide solution;
9) Adding methanol into the full R-series polypeptide liquid subjected to the second step of desalination for dilution, performing rotary evaporation at the temperature of 42 ℃ in water bath, and removing DMSO reagent in the full R-series polypeptide liquid to obtain a full R-series polypeptide intermediate liquid;
10 Diluting the full R series polypeptide intermediate liquid with pure water, freezing and drying in vacuum to obtain solid powdery full R series polypeptide final product;
11 Ion chromatograph to detect TFA root content of less than 1% for all R series polypeptide end products.
The preferable scheme is as follows: the ethanol leaching speed in the step 2) is 450-600 drops/min.
The preferable scheme is as follows: the leaching speed of the preparation liquid in the step 3) is 240-300 drops/min.
The preferable scheme is as follows: the temperature of the stirrer in the step 6) is 45 ℃.
The preferable scheme is as follows: the adding amount of the triethylamine in the step 6) is 8-10 per mill of the weight of the DMSO.
The preferable scheme is as follows: the reaction time of the step 6) is 3-7h.
The preferable scheme is as follows: the mixing weight ratio of the strong alkaline anionic resin to the titanium dioxide (TiO 2) in the step 7) is 7:5.
The preferable scheme is as follows: the volume ratio of methanol to DMSO added in the step 9) is 4:1.
The preferable scheme is as follows: the dilution ratio of the pure water in the step 10) is 7-9 times of that of the liquid after rotary evaporation.
The beneficial effects of the invention are as follows: the method provided by the invention can be used for more effectively removing the trifluoroacetate in the full R-series polypeptides. Desalting is carried out in steps and methods according to the existence form of trifluoroacetate in the polypeptide product. In the first step, a chromatographic column with titanium dioxide (TiO 2) as a filler is introduced, the titanium dioxide (TiO 2) can adsorb trifluoroacetic acid, meanwhile, the titanium dioxide (TiO 2) is insoluble in water, and the preparation liquid of the full R series polypeptide obtained by reverse phase high performance liquid chromatography can be completely used as a eluent and can be adsorbed and eluted at the speed of 240-300 drops/min. And then dissolving the polypeptide in DMSO, changing the combined trifluoroacetic acid into a free type by utilizing triethylamine reaction, and setting the temperature to 45 ℃ can not only increase the solubility of the polypeptide in DMSO and reduce the viscosity of DMSO, but also ensure the stability of the polypeptide without denaturing the polypeptide. In the second desalting step, the strongly basic anion resin can release a large amount of OH -, and when the trifluoroacetic acid salt is desorbed by using DMSO and triethylamine, the trifluoroacetic acid buffer salt is newly formed. At this time, H + in the free salt is more active, interacts with OH - released by the strongly alkaline anion resin, and then a certain amount of titanium dioxide (TiO 2) is proportioned at proper time, so that the purpose of removing the trifluoroacetic acid buffer salt is achieved. So that the full R series polypeptide can obtain the effect of completely removing the trifluoroacetate. In the present invention, the whole desalting process involves introducing new titanium dioxide (TiO 2), and using a strongly basic anionic resin in combination with titanium dioxide (TiO 2) to adsorb and remove trifluoroacetate. In addition, the reagent and the instrument used in the invention are simple and easy to obtain, the operation cost is low, the purpose of completely removing the full R-type polypeptide high trifluoroacetate can be achieved, and a new thought and a new method can be provided for the polypeptide desalination technology.
Drawings
FIG. 1 shows the TFA root content of crude total R polypeptide synthesized in solid phase.
FIG. 2 shows TFA root content of the product after the first desalting step.
FIG. 3 shows TFA root content of the product after the second desalting step.
FIG. 4 shows the TFA content of the final product of example 1.
FIG. 5 shows the TFA content of the final product of example 2.
FIG. 6 shows the TFA content of the final product of example 3.
FIG. 7 shows the TFA content of the final product of example 4.
FIG. 8 shows the TFA content of the final product of example 5.
Detailed Description
Example 1
Taking full R sequence RRRRRRRRRRRRRRRR RR (RR 18) as an example, the specific implementation steps are as follows:
1. Firstly, detecting the content of trifluoroacetate in the polypeptide by utilizing ion chromatography on RR18 synthesized by solid phase, and detecting the content of TFA (TFA) in the crude polypeptide by ion chromatography instrument model (Switzerland wan tong 883 ion chromatograph) (figure 1).
2. 200Mg of crude polypeptide is weighed, and purified and separated by a reversed phase high performance liquid chromatography to obtain a preparation solution of the full R-series polypeptide product with required purity for standby.
3. 300Mg of titanium dioxide (TiO 2) is weighed and placed in a glass chromatographic column, 20ml of ethanol is added for leaching, the leaching speed is 450 drops/min, and after leaching cleanly, the glass chromatographic column is used.
4. And (3) adding all the preparation liquid obtained after purification into a glass chromatographic column, setting the leaching speed to 240 drops/min, and waiting for the preparation liquid to be completely leached to obtain the polypeptide intermediate liquid after the first step of desalination.
5. And (3) carrying out water bath 40 ℃ rotary evaporation, freezing and vacuum drying on the polypeptide intermediate liquid after the first step of desalination to obtain a solid full R-series polypeptide intermediate. At this time, TFA root content was detected by ion chromatography, still up to 20% or more (fig. 2).
6. The solid intermediate of the full R series polypeptide is dissolved in 5ml of dimethyl sulfoxide (DMSO), placed on a stirrer with the temperature set at 45 ℃ for complete dissolution, and gradually dripped into triethylamine with the dripping amount of 50ul, stirred for reaction, and the reaction time is set for 3h.
7. At this time, 100mg of titanium dioxide (TiO 2) is weighed and placed on the lower layer of the chromatographic column, 140mg of strongly basic anion resin is placed on the upper layer, 20ml of ethanol is added for leaching, the leaching speed is 400 drops/min, and after leaching, the chromatographic column is used for later use.
8. And (3) diluting the intermediate liquid after the reaction with the anion resin with the strong alkalinity and titanium dioxide (TiO 2), simultaneously adding 20ml of methanol to dilute the DMSO, mixing the solution with a chromatographic column, keeping the chromatographic column at a constant temperature of 45 ℃, setting the leaching speed to be 200 drops/min, and completely leaching the solution to obtain the polypeptide liquid after the second step of desalination. The TFA root content was then measured by ion chromatography and was 1.579% (FIG. 3).
9. And (3) performing rotary evaporation on the polypeptide liquid subjected to the second step of desalination at the water bath temperature of 42 ℃ for 30min, and removing a certain amount of DMSO and methanol.
10. At this time, 100ml of pure water was added and mixed uniformly, and then the mixed solution was frozen and dried in vacuo to obtain a solid powder of the all R-series polypeptide of the trifluoroacetate-removed salt. The final product was tested for TFA root content by ion chromatography at 0.908% (FIG. 4).
Example 2
1. Firstly, detecting the content of trifluoroacetate in the polypeptide by utilizing ion chromatography on RR18 synthesized by solid phase, and the model of an ion chromatograph (Switzerland wan tong 883 ion chromatograph).
2. Weighing 500mg of crude polypeptide, and purifying and separating by reverse phase high performance liquid chromatography to obtain the preparation solution of the full R-series polypeptide product with required purity.
3. 600Mg of titanium dioxide (TiO 2) is weighed and placed in a glass chromatographic column, 40ml of ethanol is added for leaching, the leaching speed is 450 drops/min, and after leaching cleanly, the glass chromatographic column is used.
4. And (3) adding all the preparation liquid obtained after purification into a glass chromatographic column, setting the leaching speed to 240 drops/min, and waiting for the preparation liquid to be completely leached to obtain the polypeptide intermediate liquid after the first step of desalination.
5. And (3) carrying out water bath 40 ℃ rotary evaporation, freezing and vacuum drying on the polypeptide intermediate liquid after the first step of desalination to obtain a solid full R-series polypeptide intermediate. At this time, the TFA root content was detected by ion chromatography.
6. The solid intermediate of the full R series polypeptide is dissolved in 8ml of dimethyl sulfoxide (DMSO), placed on a stirrer with the temperature set at 45 ℃ for complete dissolution, and gradually dripped into triethylamine with the dripping amount of 75ul, stirred for reaction, and the reaction time is set for 4 hours.
7. At this time, 250mg of titanium dioxide (TiO 2) is weighed and placed on the lower layer of the chromatographic column, 350mg of strongly basic anion resin is placed on the upper layer, 40ml of ethanol is added for leaching, the leaching speed is 450 drops/min, and after leaching, the chromatographic column is used for standby.
8. And (3) diluting the intermediate liquid after the reaction with the anion resin with the strong alkalinity and titanium dioxide (TiO 2), simultaneously adding 32ml of methanol to dilute the DMSO, mixing the solution with a chromatographic column, keeping the chromatographic column at a constant temperature of 45 ℃, setting the leaching speed to 240 drops/min, and completely leaching the solution to obtain the polypeptide liquid after the second step of desalination. And detecting the TFA root content by using an ion chromatograph.
9. And (3) performing rotary evaporation on the polypeptide liquid subjected to the second step of desalination at the water bath temperature of 42 ℃ for 50min, and removing a certain amount of DMSO and methanol.
10. At this time, 150ml of pure water was added and mixed uniformly, and then the mixed solution was frozen and dried in vacuo to obtain a solid powder of the all R-series polypeptide of the trifluoroacetate-removed salt. The final product was tested for TFA root content by ion chromatography at 0.62% (fig. 5).
Example 3
1. Firstly, detecting the content of trifluoroacetate in the polypeptide by utilizing ion chromatography on RR18 synthesized by solid phase, and the model of an ion chromatograph (Switzerland wan tong 883 ion chromatograph).
2.1 G of crude polypeptide is weighed, and purified and separated by a reversed phase high performance liquid chromatography to obtain a preparation solution of the full R-series polypeptide product with required purity for standby.
3. 1.5G of titanium dioxide (TiO 2) is weighed and placed in a glass chromatographic column, and is added with 100ml of ethanol for leaching, wherein the leaching speed is 500 drops/min, and after leaching cleanly, the glass chromatographic column is used.
4. And (3) adding all the preparation liquid obtained after purification into a glass chromatographic column, setting the leaching speed to 280 drops/min, and waiting for the preparation liquid to be completely leached to obtain the polypeptide intermediate liquid after the first step of desalination.
5. And (3) carrying out water bath 40 ℃ rotary evaporation, freezing and vacuum drying on the polypeptide intermediate liquid after the first step of desalination to obtain a solid full R-series polypeptide intermediate. At this time, the TFA root content was detected by ion chromatography.
6. The solid intermediate of the full R series polypeptide is dissolved in 15ml of dimethyl sulfoxide (DMSO), placed on a stirrer with the temperature set at 45 ℃ for complete dissolution, and gradually dripped into triethylamine with the dripping amount of 135ul, stirred for reaction, and the reaction time is set for 4.5h.
7. At this time, 650mg of titanium dioxide (TiO 2) is weighed and placed on the lower layer of the chromatographic column, 910mg of strongly basic anion resin is placed on the upper layer, 100ml of ethanol is added for leaching, the leaching speed is 500 drops/min, and after leaching, the chromatographic column is used for standby.
8. And (3) diluting the intermediate liquid after the reaction with the anion resin with the strong alkalinity and titanium dioxide (TiO 2), simultaneously adding 60ml of methanol to dilute the DMSO, mixing the solution with a chromatographic column, keeping the chromatographic column at a constant temperature of 45 ℃, setting the leaching speed to 280 drops/min, and completely leaching the solution to obtain the polypeptide liquid after the second step of desalination. And detecting the TFA root content by using an ion chromatograph.
9. And (3) performing rotary evaporation on the polypeptide liquid subjected to the second step of desalination at the water bath temperature of 42 ℃ for 1.5h, and removing a certain amount of DMSO and methanol.
10. At this time, 600ml of pure water was added and mixed uniformly, and then the mixed solution was frozen and dried in vacuo to obtain a solid powder of the all R-series polypeptide of the trifluoroacetate-removed salt. The final product was tested for TFA root content by ion chromatography at 0.469% (FIG. 6).
Example 4
1. Firstly, detecting the content of trifluoroacetate in the polypeptide by utilizing ion chromatography on RR18 synthesized by solid phase, and the model of an ion chromatograph (Switzerland wan tong 883 ion chromatograph).
2. Weighing 5g of crude polypeptide, and purifying and separating by reverse phase high performance liquid chromatography to obtain the preparation liquid of the full R-series polypeptide product with required purity.
3.6 G of titanium dioxide (TiO 2) is weighed and placed in a glass chromatographic column, and is added with 200ml of ethanol for leaching, wherein the leaching speed is 600 drops/min, and after leaching cleanly, the glass chromatographic column is used.
4. And (3) adding all the preparation liquid obtained after purification into a glass chromatographic column, setting the leaching speed to 300 drops/min, and waiting for the preparation liquid to be completely leached to obtain the polypeptide intermediate liquid after the first step of desalination.
5. And (3) carrying out water bath 40 ℃ rotary evaporation, freezing and vacuum drying on the polypeptide intermediate liquid after the first step of desalination to obtain a solid full R-series polypeptide intermediate. At this time, the TFA root content was detected by ion chromatography.
6. The solid intermediate of the full R series polypeptide is dissolved in 50ml of dimethyl sulfoxide (DMSO), placed on a stirrer with the temperature set at 45 ℃ for complete dissolution, and gradually dripped into triethylamine with the dripping amount of 400ul, stirred for reaction, and the reaction time is set for 6h.
7. At this time, 2.5g of titanium dioxide (TiO 2) is weighed and placed on the lower layer of the chromatographic column, 3.5g of strongly basic anion resin is placed on the upper layer, 200ml of ethanol is added for leaching, the leaching speed is 600 drops/min, and after leaching, the membrane is completely leached for later use.
8. And (3) diluting the intermediate liquid after the reaction with the anion resin with the strong alkalinity and titanium dioxide (TiO 2), adding 200ml of methanol to dilute DMSO, mixing the mixture with a chromatographic column, keeping the chromatographic column at a constant temperature of 45 ℃, setting the leaching speed to 300 drops/min, and completely leaching to obtain the polypeptide liquid after the second step of desalination. And detecting the TFA root content by using an ion chromatograph.
9. And (3) performing rotary evaporation on the polypeptide liquid subjected to the second step of desalination at the water bath temperature of 42 ℃ for 2.5 hours, and removing a certain amount of DMSO and methanol.
10. At this time, 1L of pure water was added and mixed uniformly, and then the mixed solution was frozen and dried in vacuo to obtain a solid powder of the all R-series polypeptide of the trifluoroacetate-removed salt. The final product was tested for TFA root content by ion chromatography to 0.385% (fig. 7).
Example 5
1. Firstly, detecting the content of trifluoroacetate in the polypeptide by utilizing ion chromatography on RR18 synthesized by solid phase, and the model of an ion chromatograph (Switzerland wan tong 883 ion chromatograph).
2. Weighing 10g of crude polypeptide, and purifying and separating by reverse phase high performance liquid chromatography to obtain the preparation liquid of the full R-series polypeptide product with required purity.
3. 12G of titanium dioxide (TiO 2) is weighed and placed in a glass chromatographic column, 300ml of ethanol is added for leaching, the leaching speed is 600 drops/min, and after leaching cleanly, the glass chromatographic column is used.
4. And (3) adding all the preparation liquid obtained after purification into a glass chromatographic column, setting the leaching speed to 300 drops/min, and waiting for the preparation liquid to be completely leached to obtain the polypeptide intermediate liquid after the first step of desalination.
5. And (3) carrying out water bath 40 ℃ rotary evaporation, freezing and vacuum drying on the polypeptide intermediate liquid after the first step of desalination to obtain a solid full R-series polypeptide intermediate. At this time, the TFA root content was detected by ion chromatography.
6. The solid intermediate of the full R series polypeptide is dissolved in 80ml of dimethyl sulfoxide (DMSO), placed on a stirrer with the temperature set at 45 ℃ for complete dissolution, and gradually dripped into triethylamine with the dripping amount of 700ul, stirred for reaction, and the reaction time is set at 7h.
7. At this time, 5g of titanium dioxide (TiO 2) is weighed and placed on the lower layer of the chromatographic column, 7g of strongly alkaline anion resin is placed on the upper layer, 300ml of ethanol is added for leaching, the leaching speed is 700 drops/min, and after leaching, the membrane is used for standby.
8. And (3) diluting the intermediate liquid after the reaction with the anion resin with the strong alkalinity and titanium dioxide (TiO 2), adding 320ml of methanol to dilute DMSO, mixing the mixture with a chromatographic column, keeping the chromatographic column at a constant temperature of 45 ℃, setting the leaching speed to 350 drops/min, and completely leaching to obtain the polypeptide liquid after the second step of desalination. And detecting the TFA root content by using an ion chromatograph.
9. And (3) performing rotary evaporation on the polypeptide liquid subjected to the second step of desalination at the water bath temperature of 42 ℃ for 3 hours, and removing a certain amount of DMSO and methanol.
10. At this time, 2L of pure water was added and mixed uniformly, and then the mixed solution was frozen and dried in vacuo to obtain a solid powder of the all R-series polypeptide of the trifluoroacetate-removed salt. The final product was tested for TFA root content by ion chromatography at 0.897% (fig. 8).
Claims (9)
1. A desalination method of full R series polypeptide trifluoroacetate by solid phase synthesis is characterized by comprising the following steps:
1) Purifying and separating the solid-phase synthesized crude product of the all-R series polypeptide by using a reversed-phase high performance liquid chromatography to obtain a preparation solution of the all-R series polypeptide product with required purity, wherein the preparation solution contains not only free trifluoroacetate but also combined trifluoroacetate;
2) Filling titanium dioxide into a chromatographic column according to the volume ratio of the prepared liquid, and using ethanol as a eluting agent, and eluting the chromatographic column for later use;
3) Removing free trifluoroacetate in the first step, and passing the preparation solution obtained after purification through a titanium dioxide chromatographic column after rinsing, so as to remove the trifluoroacetate in the preparation solution;
4) Carrying out water bath 40 ℃ rotary steaming, freezing and vacuum drying on the preparation liquid after the first step of desalination to obtain a full R series polypeptide solid intermediate;
5) Detecting the content of trifluoroacetate at the moment of the solid intermediate of the full R series polypeptide by utilizing an ion chromatograph;
6) Dissolving the solid intermediate of the full R series polypeptide in dimethyl sulfoxide, placing the solid intermediate on a stirrer, fully dissolving, gradually adding triethylamine, stirring for reaction, and removing the trifluoroacetate bound on R guanidyl, so that the trifluoroacetate in a bound form is converted into free trifluoroacetate buffer salt;
7) Filling a chromatographic column with a strong alkaline anion resin at the upper layer and titanium dioxide mixed at the lower layer, and washing the chromatographic column with ethanol for later use;
8) The newly generated free trifluoroacetic acid buffer salt polypeptide intermediate solution is washed clean by a mixed chromatographic column, a column temperature box is increased, the temperature is set at 45 ℃, and the newly generated trifluoroacetic acid buffer salt is removed to obtain full R series polypeptide solution;
9) Adding methanol into the full R series polypeptide liquid after the second step of desalination for dilution, performing rotary evaporation at the temperature of 42 ℃ in water bath, and removing dimethyl sulfoxide reagent in the polypeptide liquid to obtain full R series polypeptide intermediate liquid;
10 Diluting the full R series polypeptide intermediate liquid with pure water, freezing and drying in vacuum to obtain solid powdery full R series polypeptide final product;
11 Detecting the content of trifluoroacetate by ion chromatograph for the final product of all R series polypeptide, which is less than 1%.
2. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the ethanol leaching speed in the step 2) is 450-600 drops/min.
3. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the elution speed of the preparation liquid in the step 3) is 240-300 drops/min.
4. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the temperature of the stirrer in the step 6) is set to 45 ℃.
5. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: step 6) is gradually added with triethylamine with the weight of 8-10 per mill of the weight of the dimethyl sulfoxide.
6. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: in the step 6), the reaction time is 3-7h.
7. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the mixing ratio of the strongly basic anionic resin to the titanium dioxide in the step 7) is 7:5.
8. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the volume ratio of the methanol to the dimethyl sulfoxide in the step 9) is 4:1.
9. The method for desalting the trifluoroacetate salt of a solid-phase synthesized full-R-series polypeptide of claim 1, wherein the method comprises the following steps: the dilution ratio of the pure water in the step 10) is 7-9 times of the mixed liquid after rotary evaporation.
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| WO2008148645A1 (en) * | 2007-06-07 | 2008-12-11 | Syddansk Universitet | Separation of mono- from multi-phoshorylated peptides |
| CN101386660A (en) * | 2008-10-23 | 2009-03-18 | 北京交通大学 | Functionalized titanic oxide composite microsphere and preparation method thereof |
| WO2011145077A2 (en) * | 2010-05-20 | 2011-11-24 | Soft Flow Hungary Ltd. | Branched polypeptides and their use in promoting adhesion of cells to solid surfaces |
| CN109666063A (en) * | 2018-12-28 | 2019-04-23 | 陕西师范大学 | Maltose functionalized nano composite material and preparation method and application based on ion complementary type self-assembling peptide |
| CN114487198A (en) * | 2022-01-27 | 2022-05-13 | 江汉大学 | Relative quantitative method of phosphorylated peptide |
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| WO2008148645A1 (en) * | 2007-06-07 | 2008-12-11 | Syddansk Universitet | Separation of mono- from multi-phoshorylated peptides |
| CN101386660A (en) * | 2008-10-23 | 2009-03-18 | 北京交通大学 | Functionalized titanic oxide composite microsphere and preparation method thereof |
| WO2011145077A2 (en) * | 2010-05-20 | 2011-11-24 | Soft Flow Hungary Ltd. | Branched polypeptides and their use in promoting adhesion of cells to solid surfaces |
| CN109666063A (en) * | 2018-12-28 | 2019-04-23 | 陕西师范大学 | Maltose functionalized nano composite material and preparation method and application based on ion complementary type self-assembling peptide |
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