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CN112159456A - Green synthesis method of polypeptide - Google Patents

Green synthesis method of polypeptide Download PDF

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CN112159456A
CN112159456A CN202011091435.3A CN202011091435A CN112159456A CN 112159456 A CN112159456 A CN 112159456A CN 202011091435 A CN202011091435 A CN 202011091435A CN 112159456 A CN112159456 A CN 112159456A
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polypeptide
carbon dioxide
supercritical carbon
resin
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向双春
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Dalian Alaning Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract

The invention discloses a method for using supercritical carbon dioxide fluid for continuous flow solid phase synthesis of polypeptide, which comprises the steps of performing polypeptide coupling or deprotection by using an organic solvent, and washing polypeptide resin by using supercritical carbon dioxide or modified supercritical carbon dioxide, thereby saving a large amount of organic solvent.

Description

Green synthesis method of polypeptide
Technical Field
The invention belongs to the field of polypeptide drug synthesis, and particularly relates to a polypeptide synthesis process.
Background
There are three main methods for producing and manufacturing polypeptide, including liquid phase synthesis, solid phase synthesis and recombinant fermentation. The solid phase synthesis method is fast and flexible, is suitable for modifying medium-length and long-length peptides, and has the main defect of higher cost in amplification production. The liquid phase synthesis is suitable for some short peptides and is not suitable for preparing medium and long peptides with medicinal value. The gene recombination method for preparing the polypeptide has long research and development period and high production cost, is suitable for producing large varieties such as insulin, and has no competitive advantage for synthesizing most of medicinal peptides. Because the polypeptide medicament has high activity, definite target and smaller actual clinical dose, the solid-phase synthesis method can meet the production and preparation of most polypeptide medicament varieties at present. The solid phase peptide synthesis methods currently prevalent in the industry have the following problems:
the incomplete formation of the amido bond of the polypeptide in each step causes a large amount of fragment impurities, and the longer the chain is, the lower the content of the product is; the polypeptide formed on the resin is not dissolved, so that the reaction cannot be carried out; the polypeptide formed on the resin is folded, so that the reaction center is shielded, and the reaction cannot be carried out; the side reactions are increased due to the slow reaction speed; the amino acid is greatly used in excess, and the cost is high; the difficulty of long-chain polypeptide synthesis is particularly high; the generated mixed solvent is not easy to be recovered and has great influence on the environment.
The utilization of the microwave solid-phase polypeptide synthesizer improves the synthesis efficiency of the polypeptide, but the problems of low yield, serious racemization of individual amino acid and large solvent consumption are still solved (org. Lett.2014,16, 940-943).
Continuous-Flow polypeptide Synthesis processes in recent years have greatly improved the efficiency of Solid-Phase polypeptide Synthesis (A recovery Reduction of the Amino Acid Excess, ChemSus Chem 2014,7, 3172-3176). The continuous flow polypeptide synthesis is that reaction liquid circulates through a reaction column, unreacted raw materials and impurities are washed away by organic solvent after the reaction is finished, and then the next operation is carried out. The consumption of amino acid and solvent in the continuous flow polypeptide synthesis process is lower than that of other existing processes. But it still does not achieve the problem of achieving a significant reduction in solvent consumption.
1kg of 6 peptides with a molecular weight of about 700Da, the material consumption statistics using different solid phase polypeptide synthesis processes are given in the following table:
TABLE 1 statistics of material consumption for different solid phase polypeptide synthesis processes
Figure BDA0002722207240000011
As can be seen from Table 1, if the number of amino acids contained in the polypeptide is increased, the amount of the produced polypeptide is increased, and the solvent consumption is very large. The large solvent consumption is one of the main disadvantages of the current solid phase polypeptide synthesis method. Therefore, it is necessary to develop a process for producing a polypeptide which can greatly reduce the consumption of a solvent, rather than to solve the problem of how to recover the solvent.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a green synthesis method of polypeptide, which aims to solve the problems in the prior art.
The coupling or deprotection of the polypeptide is completed through a continuous flow solid phase polypeptide synthesis process, and then the original organic solvent is replaced by the supercritical carbon dioxide fluid or the supercritical carbon dioxide mixed fluid containing the modified solvent to wash the polypeptide resin.
The process is realized by the following steps:
step 1, deprotection before polypeptide coupling: deprotection of polypeptide resins
According to the general method for deprotecting polypeptide, circularly inputting a deprotected solution into a reaction column filled with Boc or Fmoc protected amino acid-polypeptide synthetic resin, draining the reaction solution in the polypeptide synthesis column by using a pump after the deprotection is finished, and then introducing supercritical carbon dioxide fluid or supercritical carbon dioxide mixed fluid containing a modified solvent to flush the removed protecting group, protecting group removal reagent and impurities until the detection shows that the removed protecting group, protecting group removal reagent and impurities are completely flushed away.
The modified supercritical carbon dioxide is a mixed fluid of a modified solvent and supercritical carbon dioxide, and the volume ratio of the modified solvent to the supercritical carbon dioxide mixed fluid is 1-50: 50-99.
The modified solvent is selected from one or a mixture of more than two of N, N '-dimethylformamide, N' -dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane, acetone, methanol, ethanol, propanol, isopropanol, dichloromethane, chloroform, carbon tetrachloride, benzene, toluene, N-methylpyrrolidone and carbon disulfide in any proportion.
When the supercritical carbon dioxide fluid or the modified supercritical carbon dioxide is used for flushing the polypeptide resin, the reaction temperature is 3-60 ℃. And 2, performing polypeptide coupling after deprotection: boc or Fmoc protected amino acids were coupled to the polypeptide resin by common Boc or Fmoc solid phase polypeptide coupling methods.
Respectively dissolving Boc or Fmoc protected amino acid, a polypeptide condensation reagent and organic base in an organic solution, circularly inputting the three solvents into the reaction column obtained in the step 1 through a pump, draining the reaction solution in the polypeptide synthesis column by using the pump after the coupling is finished, and then introducing supercritical carbon dioxide fluid or supercritical carbon dioxide mixed fluid containing a modified solvent to wash unreacted raw materials and impurities until the detection shows that the raw materials and the impurities are completely washed away.
The modified supercritical carbon dioxide is a mixed fluid of a modified solvent and supercritical carbon dioxide; the volume ratio of the modified solvent to the supercritical carbon dioxide is 1-50: 50-99.
The modified solvent comprises one or a mixture of more than two of N, N '-dimethylformamide, N' -dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane, acetone, methanol, ethanol, propanol, isopropanol, dichloromethane, chloroform, carbon tetrachloride, benzene, toluene, N-methylpyrrolidone and carbon disulfide in any proportion.
When the supercritical carbon dioxide fluid or the modified supercritical carbon dioxide is used for flushing the polypeptide resin, the temperature is 3-60 ℃.
And the carbon dioxide in the reaction system is decompressed, collected and condensed into liquid carbon dioxide, so that the carbon dioxide can be recycled.
Abbreviations used in the present invention and meanings corresponding to English
TFA Trifluoroacetic acid
DMF N, N-dimethylformamide
Fmoc 9-fluorenylmethoxycarbonyl group
DBU
1, 8-diazabicycloodec-7-enes
HOBt 1-hydroxybenzotriazoles
HBTU benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
DCM Methylene dichloride
T3P 1-Propylphosphoric acid anhydride
DIEA N, N-diisopropylethylamine
Boc Tert-butyloxycarbonyl radical
PS resin Polystyrene resin
Pbf 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
Compared with the prior art, the beneficial effects of the invention are that:
1. the use of organic solvents in polypeptide synthesis is greatly reduced. Continuous flow solid phase polypeptide synthesis in the cyclic reaction phase, the organic solvent usage is fixed, the major consumption occurs in the washing phase, usually the amount of washing solvent is 8-10 times the amount of solvent required for synthesis. It is clear that the present invention reduces the amount of solvent required for the reaction by around 80%.
2. The carbon dioxide has low cost, is extracted from the atmosphere and is recycled to the atmosphere, and the problem of environmental pollution is completely solved.
Drawings
FIG. 1 is a schematic diagram showing the structure of a production apparatus for preparing polypeptide by combining continuous flow solid phase polypeptide synthesis and supercritical carbon dioxide;
FIG. 2 is a high performance liquid chromatogram of the crude Somalutide prepared in example 1;
FIG. 3 is a mass spectrum of crude somaglutide prepared in example 1;
FIG. 4 is a high performance liquid chromatogram of the crude Somaloutide prepared in comparative example 1;
FIG. 5 is a mass spectrum of crude somaglutide prepared in comparative example 1;
FIG. 6 is a high performance liquid chromatogram of the crude product of Ac-Ser-Val-Val-Val-Arg-Thr-OH prepared in example 2;
FIG. 7 is a mass spectrum of the crude product of Ac-Ser-Val-Val-Val-Arg-Thr-OH prepared in example 2;
FIG. 8 is a high performance liquid chromatogram of Ser-Tyr-Leu-Glu-Gly obtained in example 3;
FIG. 9 is a mass spectrum of the crude product of Ser-Tyr-Leu-Glu-Gly prepared in example 3;
in the figure: 1. the device comprises a pump, 2, a solvent bottle, 3, a reagent bottle, 4, a mixer, 5, an ultraviolet detector, 6, a three-way valve I, 7, a modified solvent container, 8, a supercritical carbon dioxide production device, 9, a carbon dioxide steel bottle, 10, a polypeptide synthesis column, 11 and a three-way valve II.
Detailed Description
The invention discloses a method for preparing polypeptide, which is only a part of the embodiments of the invention, but not all embodiments. The embodiments are only for illustrating the technical concept and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
The invention is further illustrated by the following examples.
To facilitate the description of the process for preparing polypeptides by continuous flow solid phase polypeptide synthesis in conjunction with supercritical carbon dioxide, the example was performed according to fig. 1.
Example 1: preparation of Somalutide
The sequence of the somaglutide polypeptide:
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEAc-AEEAc-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
step 1, removing Fmoc from Fmoc-Gly-HMPA-PEG-AM-PS resin
Figure BDA0002722207240000041
Fmoc-Gly-HMPA-PEG-AM-PS resin structure diagram
80g of Fmoc-Gly-HMPA-PEG-AM-PS resin having a degree of substitution of 0.36mmol/g were weighed into the polypeptide synthesis column 10, the column was filled with the resin just enough, and then the lid was tightened.
DMF was pumped through the mixer 4 and three-way valve one 6 into the polypeptide synthesis column 10 at a rate of 90mL/min, flowed back to the mixer 4 from the top of the polypeptide synthesis column 10, circulated for 5 minutes to swell the resin, and the solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated. The reagent bottle 3 is switched, 200mL of 1% DBU/2% 1-octanethiol DMF solution is pumped into the polypeptide synthesis column 10 at 90mL/min through the mixer 4 and the three-way valve I6, and the polypeptide synthesis column 10 flows back to the mixer 4 from the top three-way valve, and the circulation is carried out for 8 minutes. The solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated, and supercritical carbon dioxide fluid at 31.5 ℃ was pumped into the polypeptide synthesis column 10 at a rate of 40mL/min, through the two-way valve 11, UV12 into the waste liquid collection chamber until the on-line UV monitor indicated that the Fmoc was flushed clean.
Step 2:
synthesis of H-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (AEEA-AEEA- (gamma-Glu- (OtBu)) -monoButyl octanate) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-HMPB-PEG-AM-PS resin
42.6g (65.6mmol) of Fmoc-Arg (Pbf) -OH and 32.6mL (196.8mmol) of DIEA in 120mL of DMF, 24.9(65.6mmol) of HBTU and 8.9g (65.6mmol) of HOBt in 120mL of DMF, and pumping the 2 solutions into the mixer 4 at a rate of 30mL/min, the polypeptide synthesis column 10 through the mixer 4 and the three-way valve one 6, and the polypeptide synthesis column 10 is top three-way flowed back into the mixer 4, and circulated for 10 minutes. The solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated, and supercritical carbon dioxide fluid containing 5% methanol at 31.5 ℃ was pumped into the polypeptide synthesis column 10 at a rate of 40mL/min, through the three-way valve II 11, UV12 into the waste liquid collection chamber until the on-line UV monitor indicated that unreacted Fmoc-Arg (Pbf) -OH was washed clean.
By using the polypeptide preparation method of step 1 and step 2, the remaining amino acids and polypeptide fragments Fmoc-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-OH and Fmoc-Lys (AEEA-AEEA- (gamma-Glu- (OtBu)) -monoButyl octanate) -OH fragments are coupled in sequence according to the amino acid sequence.
The molar ratio of the polypeptide fragment, the condensation reagent, the organic base and the polypeptide synthetic resin (HMPB-PEG-AM-PS resin) is 2: 2: 6: 1; the molar ratio of the polypeptide fragment, the condensation reagent, the organic base and the polypeptide synthetic resin (HMPB-PEG-AM-PS resin) is 2: 2: 6: 1; the mol ratio of the protected amino acid to the condensation reagent to the organic base to the polypeptide synthetic resin (HMPB-PEG-AM-PS resin) is 1.5: 2: 6: 1;
the polypeptide synthesis solvent is DMF, and because the protected amino acids are different, the mass ratio of the polypeptide synthesis solvent to the polypeptide synthesis resin is 10-20: 1; the organic base is DIEA, and the condensing agent is HBTU/HOBt. The flow rate of the liquid for protecting the amino acid is controlled between 50mL/min and 100 mL/min. The temperature of the supercritical carbon dioxide fluid washing is controlled to be about 33 ℃, and the flow rate range is 40mL/min to 60 mL/min. Wherein Fmoc-Trp (Boc) -OH and Fmoc-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-OH were coupled, the resin was washed with supercritical carbon dioxide fluid containing 7% 2-methyltetrahydrofuran, and the other amino acids or fragments were coupled and the resin was washed with supercritical carbon dioxide fluid containing 9% methanol.
Fmoc removal conditions: each 200mL of 1% DBU/2% 1-octanethiol DMF solution has a flow rate of 90mL/min, and the temperature of the supercritical carbon dioxide fluid for washing the resin after Fmoc removal is controlled to be about 33 ℃, and the flow rate ranges from 40mL/min to 60 mL/min. The resin was washed with supercritical carbon dioxide fluid for coupling of other protected amino acids and fragments except for coupling of Fmoc-Gln (Trt) -OH using 9% acetonitrile as the modifying solvent.
And step 3:
Boc-His (Boc) -Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (Psi (me) Pro) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (AEEA-AEEA- (gamma-Glu- (OtBu)) -monoButylOctadecanoate) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-HMPB-PEG-AM-PS resin fragment
Refer to the data and operation method of step 2. 24.9g (65.6mmol) HBTU, 8.9g (65.6mmol) HOBt in 120mL DMMF, DIEA21.6mL (131.2mmol) and 82.4g (65.6mmol) Fmoc-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (Psi (me.me) Pro) -OH in 240mL DMF, 2 solutions were pumped into the mixer 4 at 50mL/min and 100mL/min, respectively, into the polypeptide synthesis column 10 via the mixer 4 and the three-way valve one 6, and back into the mixer 4 from the top of the polypeptide synthesis column 10, and the cycle was 10 minutes. The solution inside the mixer 4 and the polypeptide synthesis column 10 was evacuated, and a supercritical carbon dioxide fluid containing 9% dichloromethane at 32 ℃ was introduced into the waste liquid collection chamber from UV12 at a rate of 40mL/min via the polypeptide synthesis column 10 and the second three-way valve 11 until the on-line UV monitor showed that unreacted Fmoc-thr (tbu) -Phe-thr (tbu) -Ser (tbu) -asp (otbu) -Val-Ser (Psi (me. me) Pro) -OH was washed clean. Fmoc removal conditions were performed with reference to step 1.
Coupling Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH to a polypeptide resin by the steps of: 24.9g (65.6mmol) HBTU, 8.9g (65.6mmol) HOBt in 120mL DMMF, Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH 46g (65.6mmol) and DIEA21.6mL (131.2mmol) in 240mL DMF, and 2 solutions were pumped into mixer 4 at 50mL/min and 100mL/min, respectively, into polypeptide synthesis column 10 via mixer 4 and three-way valve one 6, and from the top of polypeptide synthesis column 10 three-way flow back into mixer 4, cycling for 10 minutes. The solution in the mixer 4 and the peptide synthesizing column 10 was emptied, and a supercritical carbon dioxide fluid containing 10% acetonitrile at 31.5 ℃ was introduced into the waste liquid collecting chamber from UV12 at a rate of 40mL/min via the peptide synthesizing column 10 and the three-way valve II 11 until the on-line UV monitor indicated that unreacted Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH was washed clean. After washing and vacuum drying, 256g of polypeptide resin is obtained.
The somaglutide polypeptide resin is processed by trifluoroacetic acid cutting solution to obtain 81g of crude somaglutide product with purity of 45.43 percent and total yield of the crude product: 69.6 percent. The HPLC spectrum of the prepared crude somaluopeptide is shown in FIG. 2, and the mass spectrum is shown in FIG. 3.
Example 1 deprotection and coupling the amount of co-solvent used was 6.2 liters and the amount of solvent used to wash the resin was 1.6 liters.
Example 2: synthesizing Ac-Ser-Val-Val-Val-Arg-Thr-OH
Step 1, Fmoc-Thr (tBu) -HMPA-PEG-SO2-Carboxyl-PS resin Fmoc removal
Figure BDA0002722207240000071
Structure of Fmoc-Thr (tBu) -HMPA-PEG-SO2-Carboxyl-PS resin
12g of Fmoc-Gly-HMPA-PEG-SO with a degree of substitution of 0.34mmol/g were weighed out2The Carboxyl-PS resin is packed into the polypeptide synthesis column 10, the resin is just filled in the column, and then the lid is screwed down.
DMF was pumped into the polypeptide synthesis column 10 at a rate of 30mL/min through the mixer 4 and the three-way valve one 6, flowed back to the mixer 4 from the top of the polypeptide synthesis column 10, circulated for 5 minutes to swell the resin, and the solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated. The reagent bottle 3 is switched, 40mL of 5% piperidine/DMF solution is pumped into the polypeptide synthesis column 10 at 30mL/min through the mixer 4 and the three-way valve I6, and flows back to the mixer 4 from the top of the polypeptide synthesis column 10 in a three-way manner, and the circulation is carried out for 6 minutes. The solution in the mixer 4 and the polypeptide synthesis column 10 was emptied and a 32 ℃ supercritical carbon dioxide fluid containing 11% 1, 4-dioxane was passed through the polypeptide synthesis column 10 and the two-way valve 11 at a rate of 20mL/min from UV12 into the waste collection chamber until the on-line UV monitor indicated that the Fmoc was flushed clean.
Step 2, synthesizing Fmoc-Arg (pbf) -Thr (tBu) -HMPA-PEG-SO2-Carboxyl-PS resin
5.3g (8.16mmol) of Fmoc-Arg (Pbf) -OH and 2.7mL (16.32mmol) of DIEA in 20mL of DMF, 3.1(8.16mmol) of HBTU and 1.1g (8.16mmol) of HOBt in 20mL of DMF, and the 2 solutions were pumped into the mixer 4 at a rate of 30mL/min, the polypeptide synthesis column 10 through the mixer 4 and the three-way valve one 6, and the polypeptide synthesis column 10 was top three-way flowed back into the mixer 4, and circulated for 10 minutes. The solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated, and a 32 ℃ supercritical carbon dioxide fluid containing 6% acetone was passed through the polypeptide synthesis column 10 and the two-way valve 11 at a rate of 20mL/min from UV12 to the waste liquid collection chamber until the in-line UV monitor indicated that unreacted Fmoc-Arg (Pbf) -OH was washed clean.
The polypeptide preparation method of the steps 1 and 2 is adopted to complete the coupling and deprotection of 3 Fmoc-Val-OH and Fmoc-Ser (tBu) -OH according to the amino acid sequence to obtain H-Ser-Val-Val-Val-Thr (tBu) -HMPA-PEG-SO2the-Carboxyl-PS resin was finally blocked with acetic anhydride. Washing of acetic anhydride polypeptide synthesis column 10 was performed using supercritical carbon dioxide fluid containing 5% dichloromethane, 10% isopropanol at a rate of 20mL/min until UV showed that acetic anhydride was washed clean.
The mol ratio of the protected amino acid, the condensation reagent, the organic base and the polypeptide synthetic resin (HMPB-PEG-AM-PS resin) is 2: 2: 4: 1; the polypeptide synthesis solvent is DMF, and the mass ratio of the polypeptide synthesis solvent to the polypeptide synthetic resin is 10-20: 1; the organic base is DIEA, and the condensing agent is HBTU/HOBt. The flow rate of the liquid for protecting the amino acid is controlled to be 20 mL/min-30 mL/min. The elution solvent used for coupling other amino acids is supercritical carbon dioxide fluid which adopts modified solvent containing 6% acetone, the temperature of the fluid is controlled to be about 33 ℃, and the flow rate range is 20 mL/min-30 mL/min.
Fmoc removal conditions: 40mL of 5% piperidine/DMF solution is used each time, the circulating flow rate is controlled at 30mL/min, the elution solvent for washing the resin after Fmoc removal adopts supercritical carbon dioxide fluid, the temperature of the fluid is controlled at 32 ℃, and the flow rate ranges from 20mL/min to 30 mL/min.
The polypeptide resin is processed by trifluoroacetic acid cutting solution to obtain 2.37g of crude product of Ac-Ser-Val-Val-Val-Arg-Thr-OH, the purity is 81.9 percent, and the total yield of the crude product is as follows: 82.9 percent. The crude HPLC is shown in FIG. 6 and the crude mass spectrum is shown in FIG. 7.
Example 2 deprotection and coupling the amount of co-solvent used was 560 ml and the total amount of solvent used to wash the resin was 210 ml.
Example 3: synthesis of Ser-Tyr-Leu-Glu-Gly
Step 1, Boc-Gly-Merrifield resin Boc removal
20g of Boc-Gly-Merrifield resin with a degree of substitution of 0.72mmol/g was weighed into the peptide synthesis column 10, the column was filled with resin just enough, and then the lid was screwed down.
DCM was pumped into the polypeptide synthesis column 10 at a rate of 30mL/min via the mixer 4 and the three-way valve one 6, and flowed back to the mixer 4 from the top of the polypeptide synthesis column 10, circulated for 3 minutes to swell the resin, and the solution inside the mixer 4 and the polypeptide synthesis column 10 was evacuated. The reagent bottle 3 was switched, and a 50% TFA/DCM solution was pumped into the polypeptide synthesis column 10 at 30mL/min via the mixer 4 and the three-way valve one 6, and flowed back to the mixer 4 from the top three-way of the polypeptide synthesis column 10, and circulated for 5 minutes. The solution in the mixer 4 and the polypeptide synthesis column 10 was evacuated, and a supercritical carbon dioxide fluid containing 7% carbon disulfide at 32.5 ℃ was introduced into the waste liquid collection chamber from UV12 at a rate of 20mL/min through the polypeptide synthesis column 10 and the two-way valve 11, and flushed for a total of 10 minutes.
Step 2, synthesizing Boc-Glu (OBzl) -Gly-Merrifield resin
9.7g (28.8mmol) Boc-Glu (OBzl) -OH and 9.5mL (57.6mmol) DIEA in 70mL DMF, 18.4mL (28.8mmol) 50% T3The P/DMF solution was mixed with 70mL of DMF, and 2 solutions were pumped into the mixer 4 at a rate of 30mL/min, the polypeptide synthesis column 10 through the mixer 4 and the three-way valve one 6, and the polypeptide synthesis column 10 was circulated for 10 minutes from the top of the three-way valve back to the mixer 4. The solution in the mixer 4 and the polypeptide synthesis column 10 is emptied, and 325 ℃ supercritical carbon dioxide fluid containing 4% tetrahydrofuran was passed through the polypeptide synthesis column 10 and the three-way valve II 11 from UV12 into the waste liquid collection chamber at a rate of 20mL/min and flushed for 10 minutes.
Adopting Boc-Leu-OH, Boc-Tyr (tBu) -OH and Boc-Ser (Bzl) -OH Boc protection amino acid to couple the rest amino acid to polypeptide resin according to amino acid sequence by adopting the polypeptide preparation method of step 1 and step 2.
Boc protection of amino acids, T3P, DIEA, the molar ratio of the polypeptide-Merrifield resin is 2: 2: 4: 1; the polypeptide synthesis solvent is DMF, and the mass ratio of the polypeptide synthesis solvent to the polypeptide-Merrifield resin is 10-20: 1; the circulating flow rate is controlled to be 20 mL/min-30 mL/min. The elution solvent adopts supercritical carbon dioxide fluid containing 4 percent of tetrahydrofuran, the temperature of the fluid is controlled to be about 33 ℃, and the flow rate range is 20 mL/min-30 mL/min.
Boc removal conditions: the circulation flow rate is controlled to be 30mL/min by using 50% TFA/DCM solution each time, the elution solvent for washing the resin after removing Boc adopts supercritical carbon dioxide fluid containing 7% carbon disulfide, the temperature of the fluid is controlled to be about 33 ℃, and the flow rate ranges from 20mL/min to 30 mL/min.
The polypeptide resin is processed by hydrogen fluoride cutting solution to obtain 5.76g of crude Ser-Tyr-Leu-Glu-Gly with the purity of 84.6 percent and the total yield of the crude product: 70.5 percent. The crude HPLC is shown in FIG. 8 and the crude mass spectrum is shown in FIG. 9.
Example 3 deprotection and coupling the amount of solvent used together was 400 ml and the total amount of solvent used to wash the resin was 110 ml.
Comparative example 1:
the difference between this example and example 1 is that each washing part is washed with organic solvent, i.e. 3 times with DMF, 3 times with methanol, and 3 times with dichloromethane, and the other steps are the same as example 1, and 80.7g of crude thaumautide with purity 46.13%, total yield of crude product is finally obtained: 69.5%, the HPLC spectrum of the prepared crude somalutide is shown in FIG. 4, and the mass spectrum is shown in FIG. 5. The amount of organic solvent used for synthetic deprotection and coupling was 6.2 liters and the amount of organic solvent used for resin washing was 93 liters.
Comparative example 2:
the difference between the present example and example 2 is that each washing part is washed with organic solvent, i.e. 3 times with DMF, 3 times with methanol, 3 times with dichloromethane, and the other steps are the same as example 2, finally obtaining crude Ac-Ser-Val-Val-Val-Arg-Thr-OH 2.28g with purity 46.17%, total yield of crude: 66.4 percent. The total amount of organic solvent used for deprotection and coupling was 560 ml and the total amount of organic solvent used for washing the resin was 7570 ml.
Comparative example 3:
the difference between the present example and example 3 is that each washing part is washed with an organic solvent, i.e. 3 times with DMF, 3 times with methanol, 3 times with dichloromethane, and the other steps are the same as example 2, to finally obtain 5.37g of crude Ac-Ser-Val-Arg-Thr-OH, wherein the purity is 46.58%, and the total yield of the crude product is as follows: 64.1 percent. The total amount of organic solvent used for deprotection and coupling was 400 ml and the total amount of organic solvent used for washing the resin was 6860 ml.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Claims (5)

1. A method of using supercritical carbon dioxide fluid for continuous flow solid phase synthesis of polypeptides, comprising: in the process of continuous flow solid phase synthesis of polypeptide, after the coupling or deprotection step is finished, supercritical carbon dioxide or modified supercritical carbon dioxide is adopted to wash polypeptide resin.
2. The method of claim 1, wherein the supercritical carbon dioxide fluid is used for continuous flow solid phase synthesis of polypeptides, wherein: the modified supercritical carbon dioxide is a mixed fluid of a modified solvent and supercritical carbon dioxide.
3. The method of claim 2, wherein the supercritical carbon dioxide fluid is used for continuous flow solid phase synthesis of polypeptides, wherein: the volume ratio of the modified solvent to the supercritical carbon dioxide is 1-50: 50-99.
4. The method of claim 1, wherein the supercritical carbon dioxide fluid is used for continuous flow solid phase synthesis of polypeptides, wherein: the modified solvent is selected from one or a mixture of more than two of N, N '-dimethylformamide, N' -dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane, acetone, methanol, ethanol, propanol, isopropanol, dichloromethane, chloroform, carbon tetrachloride, benzene, toluene, N-methylpyrrolidone and carbon disulfide in any proportion.
5. The method of claim 1, wherein the supercritical carbon dioxide fluid is used for continuous flow solid phase synthesis of polypeptides, wherein: when the supercritical carbon dioxide fluid or the modified supercritical carbon dioxide is used for flushing the polypeptide resin, the temperature is 3-60 ℃.
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