CN115490759A - Solid phase synthesis method of polypeptide - Google Patents

Solid phase synthesis method of polypeptide Download PDF

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CN115490759A
CN115490759A CN202111404435.9A CN202111404435A CN115490759A CN 115490759 A CN115490759 A CN 115490759A CN 202111404435 A CN202111404435 A CN 202111404435A CN 115490759 A CN115490759 A CN 115490759A
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polypeptide
fmoc
leu
resin
fragment
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董龙辉
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Shanghai Qilong Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention discloses a solid phase synthesis method of polypeptide, which comprises the following steps: solid-phase synthesis of Fmoc series resin; coupling amino acid on the resin to obtain three sections of polypeptide fragments; then the three sections of polypeptide fragments are deprotected and coupled to obtain the polypeptide sequence; separating and purifying by high performance liquid chromatography, and lyophilizing to obtain pure polypeptide, wherein the deprotection reagent is a mixed reagent of DMA and DMAC with a volume ratio of 1 (1-2). The invention can effectively solve the problems of low yield, more byproducts and the like frequently occurring in the process of synthesizing the polypeptide; the invention adopts a method for synthesizing polypeptide sequences in a sectional mode, reduces the times of condensation circulation and can effectively improve the yield of the reaction; meanwhile, gly and/or Leu with Hmb and/or Dmb are coupled into the polypeptide sequence, so that the steric hindrance of amino acid can be increased, and amide hydrogen of the amino acid can be substituted, thereby preventing the formation of a secondary structure, greatly reducing the generation of byproducts and improving the yield.

Description

Solid phase synthesis method of polypeptide
Technical Field
The invention relates to the field of pharmacy, in particular to a solid-phase synthesis method of polypeptide, and mainly relates to the field of preparation methods of peptide.
Background
The polypeptide is a compound formed by connecting a plurality of amino acids through peptide bonds, and generally consists of 10-100 amino acid molecules, the connection mode of the polypeptide is the same as that of protein, and the relative molecular mass of the polypeptide is less than 10000. Polypeptides are ubiquitous in organisms, and several tens of thousands of polypeptides have been found in organisms so far, and they are widely involved in and regulate functional activities of various systems, organs, tissues and cells in the organism, and play an important role in life activities. With the maturation of biotechnology and polypeptide synthesis technology, more and more polypeptide drugs are developed and applied clinically. Because of wide adaptation, high safety and obvious curative effect, the polypeptide medicament is widely applied to the prevention, diagnosis and treatment of diseases such as tumor, hepatitis, diabetes, AIDS and the like at present, and has wide development prospect.
The polypeptide synthesis method mainly adopts a solid phase synthesis method and a liquid phase synthesis method. The solid phase synthesis method has the advantages of more protecting group selections, low cost, easy amplification of synthesis scale and the like. The major drawbacks of the liquid phase synthesis compared to the solid phase synthesis are: the synthesis range is small, the synthesis of the polypeptide is generally concentrated within 10 amino acids, and in addition, the purification of intermediates is required in the synthesis, so the time is long, and the workload is large. However, there are still some problems in the synthesis of polypeptide compounds due to the long peptide sequence. Such as: (1) In the process of polypeptide synthesis, because the peptide sequence is long, certain loss exists in each step during synthesis, and the final yield is low. (2) Due to the long peptide sequence, secondary structures may be formed when the peptide chain is subsequently further extended. These secondary structures are mainly α -helices and β -sheets, both of which are largely due to the formation of hydrogen bonds between amide hydrogens and carbonyl hydrogens, resulting in more by-products. In patent CN200510040036.3, a polypeptide microwave solid phase synthesis method is adopted, and a microwave technology is adopted to provide energy for the reaction, which can improve the reaction efficiency, but a secondary structure is easily formed in the reaction process, resulting in more byproducts. Patent CN202010161174.1 is made by fixing fragments on resin continuously for several cycles of condensation, with large losses in the repeated cycling steps, resulting in low final yields.
Therefore, a solid-phase synthesis method of a polypeptide capable of solving the above problems is highly desired. Based on the above-mentioned findings, the present invention provides a solid-phase synthesis method for a polypeptide, which is capable of effectively solving the problems of low yield and a large amount of by-products that are likely to occur in the synthesis of a polypeptide, in view of the above-mentioned problems.
Disclosure of Invention
In order to solve the above problems, the first aspect of the present invention provides a solid phase synthesis method for a polypeptide, comprising the steps of:
(1) Solid-phase synthesis of Fmoc-Cys-HMBA-AM resin, fmoc-Lys-HMBA-AM resin and Fmoc-Tyr-HMBA-AM resin;
(2) Coupling amino acids on the Fmoc-Cys-HMBA-AM resin, the Fmoc-Lys-HMBA-AM resin and the Fmoc-Tyr-HMBA-AM resin according to the peptide sequence of the polypeptide to obtain three polypeptide fragments;
(3) Then the three polypeptide fragments are deprotected and coupled to obtain the polypeptide sequence;
(4) The polypeptide sequence is a crude product, and a pure product of the polypeptide is obtained by separation, purification and freeze-drying through a high performance liquid chromatography.
The deprotection reagent used for deprotection is a mixed reagent of DMA and DMAC with the volume ratio of 1 (1-2).
As a preferable technical scheme, the step (1) comprises the steps of soaking HMBA-AM resin in DCM, fully swelling, performing suction filtration and drying, adding Fmoc-Cys-OH, fmoc-Lys-OH and Fmoc-Tyr-OH, adding a catalyst and a solvent, reacting for 4-12h, performing suction filtration and drying, and washing to obtain the Fmoc-Cys-HMBA-AM resin, the Fmoc-Lys-HMBA-AM resin and the Fmoc-Tyr-HMBA-AM resin.
The catalyst was a combination of EDCI, HOBT and DIPEA.
As a preferred technical scheme, the synthesis method of the first segment of polypeptide fragment in the step (2) comprises the following steps: adding a deprotection reagent into the obtained Fmoc-Cys-HMBA-AM resin, reacting for 1-2 hours, carrying out suction filtration and drying, adding Fmoc-Ser-OH, adding a catalyst and a solvent, reacting for 4-12 hours, detecting a reaction end point by a ninhydrin reaction method, carrying out suction filtration and drying, and washing to obtain Fmoc-Ser-Cys-HMBA-AM resin; adding deprotection reagent, sequentially adding amino acids according to the sequence of the first section of polypeptide fragment, performing condensation reaction, reacting continuously until the last fragment Gly is connected to obtain the first polypeptide resin fragment, adding peptide cutting reagent, reacting for 3-5 hr, vacuum filtering, adding diethyl ether into the filtrate to crystallize, washing with solvent for three times, drying, and making into solid phase I 2 Cyclizing the amino acids at the first position and the seventh position to form a disulfide bond to obtain a first polypeptide segment.
The solvent is a mixed solvent of DCM and DMAC with the volume ratio of 1:1.
As a preferred technical scheme, the synthesis method of the second segment of polypeptide fragment in the step (2) is as follows: adding a deprotection reagent into the obtained Fmoc-Lys-HMBA-AM resin, reacting for 1-2 hours, carrying out suction filtration and drying, adding Fmoc-Leu-OH, adding a catalyst and a solvent, reacting for 4-12 hours, detecting the reaction end point by a ninhydrin reaction method, carrying out suction filtration and drying, washing to obtain Fmoc-Leu-Lys-HMBA-AM resin, then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the second-stage polypeptide fragment, carrying out condensation reaction until the last fragment Thr is connected to obtain a second polypeptide resin fragment, then adding a peptide cutting reagent, reacting for 3-5 hours, carrying out suction filtration, adding diethyl ether into filtrate for crystallization and precipitation, washing the crystallization solvent for three times, and drying to obtain the second-stage polypeptide fragment.
As a preferred technical scheme, the method for synthesizing the third segment of polypeptide fragment in the step (2) is referred to the method for synthesizing the second segment of polypeptide fragment, and the difference lies in that: taking Fmoc-Tyr-HMBA-AM resin as a base, adding Fmoc-Pro-OH for reaction, sequentially adding amino acid according to the sequence of the third section of polypeptide fragment, carrying out condensation reaction until the last fragment Pro is connected to obtain a third polypeptide resin fragment, adding a peptide cutting reagent, reacting for 3-5 hours, carrying out suction filtration, adding diethyl ether into filtrate for crystallization and precipitation, washing the crystals for three times by using a solvent, and drying to obtain the third section of polypeptide fragment.
As a preferred technical solution, the step (3) includes the following specific contents: adding a deprotection reagent into the first section of polypeptide fragment obtained in the step (2), reacting for 1-2 hours, performing suction filtration and drying, adding a second section of polypeptide fragment, adding a catalyst and a solvent, reacting for 4-12 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration and drying, and washing to obtain a fourth section of polypeptide fragment: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-Gly-X-Val-Cys-Thr-Ser-Leu-Asn-Ser-OH; then adding a deprotection reagent, reacting for 1-2 hours, performing suction filtration drying, adding a third section of polypeptide fragment, adding a catalyst and a solvent, reacting for 4-12 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration drying, washing, adding a deprotection reagent, reacting for 1-2 hours, and performing suction filtration drying to obtain the polypeptide sequence;
the deprotection reagent is a mixed reagent of DEA and DMF with the volume ratio of 1:1.
As a preferred technical scheme, the molar ratio of EDCI, HOBT and DIPEA in the solid-phase synthesis method of the polypeptide is 1.5:1.5:2.3.
as a preferred technical scheme, the solid phase synthesis method of the polypeptide comprises Gly in peptide sequence 3 、Gly 5 And Gly 23 With Gly having Hmb and/or Dmb groups, leu 14 、Leu 17 、Leu 21 And Leu 29 Are coupled with Leu of Hmb and/or Dmb groups.
As a preferred technical scheme, the first segment of polypeptide fragment in the step (2) is: fmoc-Gly-Leu-Val-Cys-Thr-Ser-Leu-Asn-Ser-Cys-OH, and the second section of polypeptide fragment is: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-OH, and the third polypeptide segment is as follows: fmoc-Pro-Thr-Gly-Ser-Gly-Thr-Asn-Thr-Arg-Pro-Tyr-OH; the polypeptide sequence obtained after coupling in the step (3) is NH 2 -Pro-Thr-Gly 3 -Ser-Gly 5 -Thr-Asn-Thr-Arg-Pro-Tyr-Thr-Gln-Leu 14 -Lys-His-Leu 17 -Gln-Gln-Ser-Leu 21 -Lys-Gly 23 -Leu-Val-Cys-Thr-Ser-Leu 29 -Asn-Ser-Cys-OH。
As a preferred technical scheme, in the solid phase synthesis method of the polypeptide, the peptide cutting reagent comprises TAS, PDT, TES and H, wherein the volume ratio of the TAS to the PDT to the TES to the H is 91.2 2 Mixed reagent of O.
Some common abbreviations in the present invention have the following specific meanings:
EDCI: (1-Ethyl-3 (3-dimethylpropylamine) carbodiimide)
HOBT: 1-hydroxybenzotriazoles
DIPEA: diisopropylethylamine
DCM: methylene dichloride
DMAC: n, N-dimethyl acetamide
DMA: dimethylamine
TAS: trichloroacetic acid
PDT: propyl mercaptan
TES: triethylsilane
Fmoc: fmoc group
Pro: proline
Thr: threonine
Gly: glycine
Ser: serine
Asn: aspartic acid
Arg: arginine
Tyr: tyrosine
Gln: glutamine
Leu: leucine
Lys: lysine
His: histidine (His)
Val: valine
Cys: cysteine
Has the beneficial effects that: the invention provides a solid-phase synthesis method of polypeptide, which can effectively solve the problems of low yield, more byproducts and the like frequently occurring in the polypeptide synthesis process. The invention adopts a method for synthesizing polypeptide sequences in a sectional mode, reduces the times of condensation circulation and can effectively improve the yield of the reaction. Meanwhile, gly and/or Leu with Hmb and/or Dmb are coupled into the polypeptide sequence, so that the steric hindrance of the amino acid can be increased, the amide hydrogen of the amino acid can be replaced, the formation of a secondary structure of the amino acid can be prevented, the generation of byproducts can be greatly reduced, and the yield of the amino acid can be improved.
Detailed Description
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
Examples
The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to the examples. Unless otherwise specified, the starting materials in the present invention are all commercially available.
Example 1
Gly in this example 3 、Gly 5 And Gly 23 All adopt Gly couples with Dmb groupsBi, leu 14 、Leu 17 、Leu 21 And Leu 29 All are coupled with Leu of Dmb group.
(1) Preparation of Fmoc-Cys-HMBA-AM resin, fmoc-Lys-HMBA-AM resin and Fmoc-Tyr-HMBA-AM resin
Taking HMBA-AM resin (10 g, the substitution degree of 1.0 mmol/g), soaking with DCM (100 mL) for 1 hour to fully swell the resin, performing suction filtration and drying, adding Fmoc-Cys-OH (4.13g, 12mmol), fmoc-Lys-OH (4.43g, 12mmol) and Fmoc-Tyr-OH (4.10g, 12mmol) respectively, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, performing suction filtration and drying, washing with DCM (50 mL) for three times, and drying to obtain the Fmoc-Cys-AM resin, the Fmoc-HMBA-AM resin and the Fmoc-Tyr-HMBA-AM resin.
(2) Preparation of the first polypeptide fragment
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Cys-HMBA-AM resin obtained in the step (1), reacting for 1 hour, performing suction filtration and drying, adding Fmoc-Ser-OH (3.95g and 12mmol), selecting EDCI (3.55g and 18mmol), HOBT (2.44g and 18mmol) and DIPEA (4.56mL and 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by an ninhydrin reaction method, performing suction filtration and drying, and washing for three times by DCM (50 mL) to obtain Fmoc-Ser-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the first section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Gly is connected to the first polypeptide resin fragment, and obtaining a first polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting a peptide cutting reagent of O (2.05 mL) for 3 hours, performing suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and performing vacuum drying to obtain a first section of polypeptide fragment; through a solid phase I 2 Cyclizing the amino acids at the first and seventh positions to form disulfide bonds.
(3) Preparation of the second polypeptide fragment
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Lys-HMBA-AM resin obtained in the step (1), reacting for 1-2 hours, performing suction filtration and drying, adding Fmoc-Leu-OH (4.25g and 12mmol), selecting EDCI (3.55g and 18mmol), HOBT (2.44g and 18mmol) and DIPEA (4.56mL and 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction endpoint by an ninhydrin reaction method, performing suction filtration and drying, and washing with DCM (50 mL) for three times to obtain the Fmoc-Leu-Lys-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the second section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Thr connected to the second polypeptide resin fragment is obtained, and obtaining a second polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting the peptide cutting reagent O (2.05 mL) for 3 hours, carrying out suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and carrying out vacuum drying to obtain a second section of polypeptide fragment.
(4) Preparation of the third fragment of the polypeptide
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Tyr-HMBA-AM resin obtained in the step (1), reacting for 1 hour, performing suction filtration and drying, adding Fmoc-Pro-OH (4.06g, 12mmol), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by an ninhydrin reaction method, performing suction filtration and drying, and washing Tyr with DCM (50 mL) for three times to obtain the Fmoc-Pro-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the third section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Pro is connected to the third polypeptide resin fragment, and obtaining a third polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.2 mL) at a volume ratio of 911 mL) and H 2 Reacting the peptide cutting reagent O (2.05 mL) for 3 hours, carrying out suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and carrying out vacuum drying to obtain a third section of polypeptide fragment.
(5) Preparing the crude peptide sequence of the polypeptide
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the first section of polypeptide fragment obtained in the step (2), reacting for 1 hour, performing suction filtration and drying, adding a second section of polypeptide fragment obtained in the step (2), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6 mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction endpoint by a ninhydrin reaction method, performing suction filtration and drying, and washing with DCM (50 mL) for three times to obtain a fourth section of polypeptide fragment: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-Gly-X-Val-Cys-Thr-Ser-Leu-Asn-Ser-OH; and (2) adding a deprotection reagent, reacting for 1 hour, performing suction filtration and drying, adding a third section of polypeptide fragment obtained in the step (2), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6 mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration and drying, washing with DCM (50 mL) for three times, adding the deprotection reagent, reacting for 1 hour, performing suction filtration and drying, and obtaining the polypeptide sequence.
(6) Preparing the pure peptide sequence product of the polypeptide
And (4) separating and purifying the polypeptide sequence obtained in the step (3) by using a high performance liquid chromatography, and freeze-drying to obtain a pure polypeptide product, wherein the total yield is 23.5w.t.%.
Total yield = mass of actually obtained polypeptide/mass of theoretically obtained polypeptide × 100%
Example 2
Gly in this example 23 Using Gly coupling with Dmb group, leu 29 Leu coupling with Hmb group.
(1) Preparation of Fmoc-Cys-HMBA-AM resin, fmoc-Lys-HMBA-AM resin and Fmoc-Tyr-HMBA-AM resin
HMBA-AM resin (10 g, degree of substitution 1.0 mmol/g) was taken, soaked with DCM (100 mL) for 1 hour to sufficiently swell the resin, suction-filtered and dried, fmoc-Cys-OH (4.13g, 12mmol), fmoc-Lys-OH (4.43g, 12mmol) and Fmoc-Tyr-OH (4.10g, 12mmol) were added, EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6mmol) were used as catalysts, a mixed solvent of DCM (100 mL) and DMAC (100 mL) at a volume ratio of 1:1 was selected as a solvent, reacted for 4 hours, suction-filtered and dried, washed three times with DCM (50 mL), and dried to obtain Fmoc-Cys-AM resin, fmoc-Lys resin and Fmoc-Tyr-HMBA-AM resin.
(2) Preparation of the first polypeptide fragment
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Cys-HMBA-AM resin obtained in the step (1), reacting for 1 hour, performing suction filtration and drying, adding Fmoc-Ser-OH (3.95g, 12mmol), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by an ninhydrin reaction method, performing suction filtration and drying, and washing for three times by DCM (50 mL) to obtain Fmoc-Ser-Cys-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the first section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Gly is connected to the first polypeptide resin fragment, and obtaining a first polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting a peptide cutting reagent of O (2.05 mL) for 3 hours, performing suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and performing vacuum drying to obtain a first section of polypeptide fragment; through a solid phase I 2 Cyclizing the amino acids at the first and seventh positions to form disulfide bonds.
(3) Preparation of the second polypeptide fragment
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with a volume ratio of 1:1 to Fmoc-Lys-HMBA-AM resin obtained in step (1)Reacting for 1-2 hours, performing suction filtration and drying, adding Fmoc-Leu-OH (4.25g, 12mmol), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6 mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration and drying, and washing for three times by DCM (50 mL) to obtain Fmoc-Leu-Lys-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the second section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Thr connected to the second polypeptide resin fragment is obtained, and obtaining a second polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting the peptide cutting reagent O (2.05 mL) for 3 hours, carrying out suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and carrying out vacuum drying to obtain a second section of polypeptide fragment.
(4) Preparation of the third fragment of the polypeptide
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Tyr-HMBA-AM resin obtained in the step (1), reacting for 1 hour, performing suction filtration and drying, adding Fmoc-Pro-OH (4.06g, 12mmol), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by an ninhydrin reaction method, performing suction filtration and drying, and washing Tyr with DCM (50 mL) for three times to obtain the Fmoc-Pro-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the third section of polypeptide fragment after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Pro is connected to the third polypeptide resin fragment, and obtaining a third polypeptide resin fragment; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting O (2.05 mL) peptide cutting reagent for 3 hours, filtering, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and vacuum drying to obtain the final productTo third fragment polypeptide.
(5) Preparing the crude product of the peptide sequence of the polypeptide
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) in a volume ratio of 1:1 into the first-stage polypeptide fragment obtained in the step (2), reacting for 1 hour, performing suction filtration and drying, adding the second-stage polypeptide fragment obtained in the step (2), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) in a volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction endpoint by a ninhydrin reaction method, performing suction filtration and drying, and washing with DCM (50 mL) for three times to obtain a fourth-stage polypeptide fragment: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-Gly-X-Val-Cys-Thr-Ser-Leu-Asn-Ser-OH; and (2) adding a deprotection reagent, reacting for 1 hour, performing suction filtration and drying, adding a third section of polypeptide fragment obtained in the step (2), selecting EDCI (3.55g, 18mmol), HOBT (2.44g, 18mmol) and DIPEA (4.56mL, 27.6 mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration and drying, washing with DCM (50 mL) for three times, adding the deprotection reagent, reacting for 1 hour, performing suction filtration and drying, and obtaining the polypeptide sequence.
(6) Preparing the pure peptide sequence product of the polypeptide
And (4) separating and purifying the polypeptide sequence obtained in the step (3) by using a high performance liquid chromatography, and freeze-drying to obtain a pure polypeptide product, wherein the total yield is 19.3w.t.%.
Example 3
The amino acids in this example did not employ coupled amino acids, and the synthetic route also did not employ a stepwise synthetic approach.
(1) Preparation of Fmoc-Cys-HMBA-AM resin
HMBA-AM resin (10 g, substitution degree of 1.0 mmol/g) is taken out, soaked for 1 hour with DCM (100 mL) to fully swell the resin, filtered and dried, fmoc-Cys-OH (4.13g, 12mmol) is respectively added, EDCI (2.96g, 15mmol), HOBT (2.03g, 15mmol) and DIPEA (3.72mL, 22.5mmol) are taken as catalysts, a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 is taken as a solvent, reacted for 4 hours, filtered and dried, washed for three times with DCM (50 mL), and dried to obtain the Fmoc-Cys-HMBA-AM resin.
(2) Preparation of the polypeptide fragment
Adding a deprotection reagent of DMA (50 mL) and DMAC (50 mL) with the volume ratio of 1:1 into the Fmoc-Cys-HMBA-AM resin obtained in the step (1), reacting for 1 hour, performing suction filtration and drying, adding Fmoc-Ser-OH (3.95g and 12mmol), selecting EDCI (3.55g and 18mmol), HOBT (2.44g and 18mmol) and DIPEA (4.56mL and 27.6mmol) as catalysts, selecting a mixed solvent of DCM (100 mL) and DMAC (100 mL) with the volume ratio of 1:1 as a solvent, reacting for 4 hours, detecting a reaction end point by an ninhydrin reaction method, performing suction filtration and drying, and washing for three times by DCM (50 mL) to obtain Fmoc-Ser-HMBA-AM resin; then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the polypeptide fragments after deprotection reaction, carrying out condensation reaction, repeating the steps until the last amino acid Pro is connected, and obtaining the polypeptide resin; then TAS (91 mL), PDT (5.2 mL), TES (3.1 mL) and H at a volume ratio of 91 2 Reacting a peptide cutting reagent of O (2.05 mL) for 3 hours, performing suction filtration, collecting filtrate, adding ether for crystallization, collecting precipitate, washing the precipitate with ether for three times, and performing vacuum drying to obtain the polypeptide fragment; through a solid phase I 2 Cyclizing the amino acids at the first and seventh positions to form disulfide bonds.
(3) Preparing the pure product of the polypeptide sequence
And (3) separating and purifying the polypeptide sequence obtained in the step (3) by high performance liquid chromatography, and freeze-drying to obtain a pure polypeptide product with the total yield of 16.7w.t.%.
From the above examples 1-3, we can see that: when the solid phase synthesis method of the polypeptide adopts a sectional preparation method, the times of condensation circulation can be reduced, and the reaction yield is effectively improved. Meanwhile, gly and/or Leu with Hmb and/or Dmb are coupled into the polypeptide sequence, so that the steric hindrance of the amino acid can be increased, the amide hydrogen of the amino acid can be replaced, the formation of a secondary structure of the amino acid can be prevented, the generation of byproducts can be greatly reduced, and the yield of the amino acid can be improved.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent replacements within the protection scope of the present invention.

Claims (10)

1. A solid phase synthesis method of polypeptide is characterized by comprising the following steps:
(1) Solid-phase synthesis of Fmoc-Cys-HMBA-AM resin, fmoc-Lys-HMBA-AM resin and Fmoc-Tyr-HMBA-AM resin;
(2) Coupling amino acids on the Fmoc-Cys-HMBA-AM resin, the Fmoc-Lys-HMBA-AM resin and the Fmoc-Tyr-HMBA-AM resin according to the peptide sequence of the polypeptide to obtain a three-segment polypeptide fragment;
(3) Then the three polypeptide fragments are deprotected and coupled to obtain the polypeptide sequence;
(4) The polypeptide sequence is a crude product, and a pure product of the polypeptide is obtained by separation, purification and freeze-drying through a high performance liquid chromatography;
the deprotection reagent used for deprotection is a mixed reagent of DMA and DMAC with the volume ratio of 1 (1-2).
2. The solid-phase synthesis method of polypeptide of claim 1, wherein the step (1) comprises the steps of soaking HMBA-AM resin in DCM, fully swelling, performing suction filtration and drying, adding Fmoc-Cys-OH, fmoc-Lys-OH and Fmoc-Tyr-OH, adding catalyst and solvent, reacting for 4-12h, performing suction filtration and drying, and washing to obtain Fmoc-Cys-HMBA-AM resin, fmoc-Lys-HMBA-AM resin and Fmoc-Tyr
-HMBA-AM resin.
The catalyst was a combination of EDCI, HOBT and DIPEA.
3. The method for solid-phase synthesis of polypeptide according to claim 1, wherein the first segment of polypeptide fragment is synthesized in step (2) by: adding a deprotection reagent into the obtained Fmoc-Cys-HMBA-AM resin, reacting for 1-2 hours, carrying out suction filtration and drying, adding Fmoc-Ser-OH, adding a catalyst and a solvent, reacting for 4-12 hours, detecting the reaction end point by a ninhydrin reaction method, carrying out suction filtration and drying, and washing to obtain Fmoc-Ser
-Cys-HMBA-AM resin; then adding deprotection reagent, adding amino acids in sequence according to the sequence of the first section of polypeptide fragment, carrying out condensation reaction, continuously reacting until the last fragment Gly is connected to obtain the first polypeptide resin fragment, then adding peptide cutting reagent, reacting for 3-5 hours, suction filtering, adding diethyl ether into filtrate, crystallizing, washing crystal with solvent for three times, drying, and passing through solid phase I 2 Cyclizing the amino acids at the first position and the seventh position to form a disulfide bond to obtain a first polypeptide segment.
The solvent is a mixed solvent of DCM and DMAC with the volume ratio of 1:1.
4. The method for solid-phase synthesis of polypeptide according to claim 1, wherein the second polypeptide fragment is synthesized in step (2) by: adding a deprotection reagent into the obtained Fmoc-Lys-HMBA-AM resin, reacting for 1-2 hours, carrying out suction filtration and drying, adding Fmoc-Leu-OH, adding a catalyst and a solvent, reacting for 4-12 hours, detecting the reaction end point by a ninhydrin reaction method, carrying out suction filtration and drying, washing to obtain Fmoc-Leu-Lys-HMBA-AM resin, then adding a deprotection reagent, sequentially adding amino acids according to the sequence of the second-stage polypeptide fragment, carrying out condensation reaction until the last fragment Thr is connected to obtain a second polypeptide resin fragment, then adding a peptide cutting reagent, reacting for 3-5 hours, carrying out suction filtration, adding diethyl ether into filtrate for crystallization and precipitation, washing the crystallization solvent for three times, and drying to obtain the second-stage polypeptide fragment.
5. The method for solid-phase synthesis of a polypeptide according to claim 1, wherein the method for synthesizing the third fragment of the polypeptide in step (2) is different from the method for synthesizing the second fragment of the polypeptide in that: taking Fmoc-Tyr-HMBA-AM resin as a base, adding Fmoc-Pro-OH for reaction, sequentially adding amino acid according to the sequence of the third section of polypeptide fragment, carrying out condensation reaction until the last fragment Pro is connected to obtain a third polypeptide resin fragment, adding a peptide cutting reagent, reacting for 3-5 hours, carrying out suction filtration, adding diethyl ether into filtrate for crystallization and precipitation, washing the crystals for three times by using a solvent, and drying to obtain the third section of polypeptide fragment.
6. The method for solid-phase synthesis of a polypeptide according to claim 1, wherein the step (3) comprises the following steps: adding a deprotection reagent into the first section of polypeptide fragment obtained in the step (2), reacting for 1-2 hours, performing suction filtration and drying, adding a second section of polypeptide fragment, adding a catalyst and a solvent, reacting for 4-12 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration and drying, and washing to obtain a fourth section of polypeptide fragment: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-Gly-X-Val-Cys-Thr-Ser-Leu-Asn-Ser-OH; then adding a deprotection reagent, reacting for 1-2 hours, performing suction filtration drying, adding a third section of polypeptide fragment, adding a catalyst and a solvent, reacting for 4-12 hours, detecting a reaction end point by a ninhydrin reaction method, performing suction filtration drying, washing, adding a deprotection reagent, reacting for 1-2 hours, and performing suction filtration drying to obtain the polypeptide sequence;
the deprotection reagent is a mixed reagent of DEA and DMF with the volume ratio of 1:1.
7. The solid phase synthesis of a polypeptide according to any one of claims 2-6, wherein the catalyst is a combination of EDCI, HOBT and DIPEA in a molar ratio of 1.5:1.5:2.3.
8. the solid-phase synthesis method for polypeptide according to claim 1, wherein Gly is contained in the peptide sequence 3 、Gly 5 And Gly 23 With Gly having Hmb and/or Dmb groups, leu 14 、Leu 17 、Leu 21 And Leu 29 Are coupled with Leu of Hmb and/or Dmb groups.
9. The solid-phase synthesis method for polypeptide according to claim 1, wherein the first segment of polypeptide fragment in step (2) is: fmoc-Gly-Leu-Val-Cys-Thr-Ser-Leu-Asn-Ser-Cys-OH, and the second polypeptide segment is as follows: fmoc-Thr-Gln-Leu-Lys-His-Leu-Gln-Gln-Ser-Leu-Lys-OH, and the third polypeptide segment is: fmoc-Pro-Thr-Gly-Ser-Gly-Thr-Asn-Thr-Arg-Pro-Tyr-OH; the polypeptide sequence obtained after coupling in the step (3) is NH 2 -Pro-Thr-Gly 3 -Ser-Gly 5 -Thr-Asn-Thr-Arg-Pro-Tyr-Thr-Gln-Leu 14 -Lys-His-Leu 17 -Gln-Gln-Ser-Leu 21 -Lys-Gly 23 -Leu-Val-Cys-Thr-Ser-Leu 29 -Asn-Ser-Cys-OH。
10. The solid-phase synthesis method for polypeptide according to any one of claims 3-5, wherein the peptide-cleaving agent is TAS, PDT, TES and H at a volume ratio of 91.2 2 Mixed reagent of O.
CN202111404435.9A 2021-11-24 2021-11-24 Solid phase synthesis method of polypeptide Withdrawn CN115490759A (en)

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Application publication date: 20221220