CN116573992B - Non-classical solid phase synthesis carrier and preparation method and application thereof - Google Patents

Non-classical solid phase synthesis carrier and preparation method and application thereof Download PDF

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CN116573992B
CN116573992B CN202310863391.9A CN202310863391A CN116573992B CN 116573992 B CN116573992 B CN 116573992B CN 202310863391 A CN202310863391 A CN 202310863391A CN 116573992 B CN116573992 B CN 116573992B
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reaction
carrier
phase synthesis
solid phase
polypeptide
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CN116573992A (en
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徐峰
魏席
李原强
邓德雄
胡美玲
董事
姚炜炜
杨涵冰
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Ruibo Suzhou Pharmaceutical Co Ltd
Ruibo Hangzhou Pharmaceutical Technology Co Ltd
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Ruibo Hangzhou Pharmaceutical Technology 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
    • C07K1/04—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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/18—Preparation of ethers by reactions not forming ether-oxygen bonds
    • C07C41/22—Preparation of ethers by reactions not forming ether-oxygen bonds by introduction of halogens; by substitution of halogen atoms by other halogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/18—Preparation of ethers by reactions not forming ether-oxygen bonds
    • C07C41/26—Preparation of ethers by reactions not forming ether-oxygen bonds by introduction of hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/225—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/64—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by introduction of functional groups containing oxygen only in singly bound form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups
    • 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
    • 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

Abstract

The invention discloses a non-classical solid phase synthesis carrier, a preparation method and application thereof, wherein the prepared non-classical solid phase synthesis carrier not only realizes homogeneous coupling of the carrier or peptide carrier and amino acid, but also can complete cracking in a cracking solution, and simultaneously retains side chain protecting groups of amino acid in a peptide chain, thereby realizing synthesis of polypeptide products and full-protection cracking of the amino acid side chain; under the same production scale, the volume of the reactor can be reduced by 30% -50%, the consumption of raw material amino acid and condensing agent can be reduced by 50% -60%, the consumption of organic solvent for reaction and washing can be reduced by 60% -80%, and finally the production cost can be reduced by 50% -70%. The carrier prepared by the invention can realize the synthesis of polypeptide products and the full-protection cleavage of amino acid side chains, realize the mass production of the polypeptide products, effectively reduce the production cost, improve the production efficiency, reduce the environmental pollution and realize environmental protection.

Description

Non-classical solid phase synthesis carrier and preparation method and application thereof
Technical Field
The invention relates to the technical field of compound synthesis, in particular to a non-classical solid phase synthesis carrier and a preparation method and application thereof.
Background
The chemical synthesis method of polypeptide can be mainly divided into liquid phase synthesis method and solid phase synthesis method, and the synthesis research of human beings on polypeptide is started from liquid phase synthesis. However, the liquid phase synthesis method of the polypeptide has a small synthesis range, is generally only suitable for synthesizing less than 10 polypeptides, and also needs to purify intermediates in the synthesis process, and has long purification operation time and large workload. In the solid phase synthesis method of polypeptide, the purification purpose can be achieved by simply washing the carrier resin after each step of reaction, the difficulty that the product of each step needs to be purified in the classical liquid phase synthesis method is overcome, and the foundation is laid for the rapid development of the polypeptide synthesis technology. With the development of polypeptide synthesis technology and amino protection technology, the solid phase synthesis method rapidly replaces the liquid phase synthesis method, and becomes the first choice method for polypeptide synthesis.
However, the above-mentioned existing Fmoc solid phase synthesis method still has a lot of problems to be solved: (1) After the solid-phase synthesis carrier of the polypeptide swells by a reaction medium, the volume can be increased by 5-10 times, and compared with the traditional liquid-phase synthesis method, the solid-phase synthesis method of the same reaction scale generally needs a reactor with larger volume, so that the large-scale production of the polypeptide is limited; (2) The solid phase synthesis of the polypeptide is not suitable for being carried out in a common reaction kettle, and a specially designed solid phase reactor such as a screen filter plate and the like is arranged at the bottom of the kettle, so that the equipment cost is increased; (3) In solid phase synthesis of polypeptide, the coupling reaction is carried out in heterogeneous phase, so that excessive amino acid and condensation reagent are often required to be added in a reaction system to ensure the reaction is complete, and the production cost is increased; (4) The greatest disadvantage of polypeptide solid-phase synthesis is the huge consumption of organic solvents for washing, which not only increases the production cost, but also is contrary to the existing green chemistry concept; (5) In the solid-phase synthesis method of polypeptide, because peptide chain is not cracked with resin in the process of repeatedly coupling amino acid, and an effective central control means is lacked, it is difficult to fully research the difficulty of coupling each amino acid and various side reactions possibly occurring in the reaction process.
Aiming at the problems of the Fmoc solid-phase synthesis method, chinese patent CN 115626867A discloses the preparation of a non-classical solid-phase synthesis carrier and the application of the carrier in the synthesis of thymopentin. The dissolubility of the non-classical solid phase synthesis carrier prepared by the method is greatly different in different solvents, and the carrier or peptide carrier can be coupled with amino acid in a homogeneous phase in benign solvents of the carrier or peptide carrier, so that the problems existing in the existing Fmoc solid phase synthesis method can be solved to a certain extent. However, in the non-classical solid phase synthesis carrier, a cleavage solution with 85% trifluoroacetic acid content is required in the cleavage stage of the peptide carrier, and the acid content is too high, so that some acid-sensitive amino acid side chain protecting groups are also cleaved off while the peptide is cleaved off from the resin, and therefore, the non-classical solid phase synthesis carrier is not suitable for the synthesis of the side chain active group full-protecting polypeptide.
However, the side chain active group full-protection polypeptide has important significance in the chemical synthesis of polypeptide medicaments. For example, with the development of polypeptide synthesis technology and process, in the process of synthesizing semaglutin (temporary name: cable Ma Lutai; english name: semaglutine) by adopting a method of fragment peptide access at a specific site, obvious advantages are exhibited, for example, chinese patent CN111217901 a discloses a method for synthesizing semaglutin, which adopts Fmoc solid-phase polypeptide synthesis to synthesize a specific protected amino acid fragment, fmoc-Lys [ AEEa-gamma-Glu (α -OtBu) -Octadecanedioic Acid-18 (tert-butoxy) ] -OH in advance, and applies the same to the synthesis of semaglutin, so that the problem of lower product purity in the large-scale preparation method of semaglutin is solved, but the method also has the disadvantages of the solid-phase synthesis listed above.
Therefore, developing a full-protection peptide solid-phase synthesis technology which can solve the problems existing in the existing Fmoc solid-phase method and protect side chain active groups in peptide chains has important significance.
Disclosure of Invention
The invention solves the problems in the prior art by providing a non-classical solid phase synthesis carrier, a preparation method and application thereof.
In order to solve the technical problems, the invention provides a non-classical solid phase synthesis carrier, which at least comprises a compound with the following structural formula:
wherein R is 1 Is C 10 ～C 22 Straight or branched alkyl of (a); r is R 2 Is C 1 ～C 3 Is a hydrocarbon group.
In order to solve the technical problems, the invention also provides a preparation method of the non-classical solid phase synthesis carrier, which comprises the following steps:
s1: reacting trihydroxybenzaldehyde with halogenated alkane in the presence of alkali and a first catalyst to obtain trialkyl ether benzaldehyde, wherein the reaction formula is as follows:
；
s2: reducing the trialkyl ether benzaldehyde in the presence of a reducing agent to obtain trialkyl ether benzyl alcohol, wherein the reaction formula is as follows:
；
s3: reacting the trialkyl ether benzyl alcohol with a halogenated succinimide in the presence of a second catalyst to obtain a trialkyl ether benzyl halide, wherein the reaction formula is as follows:
；
s4: combining the trialkyl ether benzyl halide with 2-hydroxy-4-R 2 The oxybenzaldehyde reacts in the presence of alkali and a first catalyst to obtain a non-classical solid-phase synthesis carrier precursor, and the reaction formula is as follows:
；
s5: reducing the non-classical solid phase synthesis carrier precursor in the presence of a reducing agent to obtain the non-classical solid phase synthesis carrier, wherein the reaction formula is as follows:
；
in each of the above reaction formulae, R 1 Is C 10 ～C 22 Is a linear or branched alkyl group, X is Cl, br or I, R 2 Is C 1 ～C 3 Is a hydrocarbon group.
In order to solve the technical problems, the invention also provides application of the non-classical solid-phase synthesis carrier, which is used as a solid-phase carrier for synthesizing polypeptide substances including side chain active group full-protection polypeptides.
In order to solve the technical problems, the invention also provides a method for synthesizing polypeptide substances by using the non-classical solid phase synthesis carrier, which comprises the following steps:
(1) Amino acid coupling reaction: in a reaction medium, the non-classical solid phase synthesis carrier and Fmoc-amino acid are subjected to coupling reaction under the action of a condensation reagent, and the reaction solution is concentrated, precipitated and filtered to obtain the Fmoc-peptide carrier;
(2) Fmoc protecting group removal reaction: adding the Fmoc-peptide carrier obtained in the step (1) into a deprotection solution for deprotection reaction to obtain an extended peptide carrier;
(3) Repeating the steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier;
(4) And (3) adding cutting fluid into the polypeptide substance carrier obtained in the step (3) for cracking, and washing, drying and concentrating the cracking fluid under reduced pressure to obtain the polypeptide substance.
The english abbreviations and corresponding compound names according to the invention are shown in table 1 below.
Table 1 reagents and apparatus
English abbreviations Chinese meaning
Fmoc 9-fluorenylmethoxycarbonyl
NBS N-bromosuccinimide
NCS N-chlorosuccinimide
NIS N-iodosuccinimide
Dde 5-dimethyl-1, 3-cyclohexanedione
Wang Resin Wang resin
2-CTC Resin 2-chlorotrityl chloride resin
DIC Diisopropylcarbodiimide
DCC N, N' -dicyclohexyl-carboimide
EDCI 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
HOOBT 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one
HOBT 1-hydroxy-benzotriazoles
HOAT N-hydroxy-7-azabenzotriazoles
DIPEA Triisopropylethylamine
NMM N-methylmorpholine
DMAP 4-dimethylaminopyridine
HBTU O-benzotriazol-tetramethylurea hexafluorophosphate
HATU 2- (7-Oxybenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
TBTU 2- (1H-Benzotrisazo L-1-yl) -1, 3-tetramethylurea hexafluorophosphate
PyBOP 1H-benzotriazol-1-yl-oxy-tripyrrolidinyl hexafluorophosphoric acid
Glu Glutamic acid
Lys Lysine
AEEA 2- (2- (2-aminoethoxy) ethoxy) acetic acid
Octadecanedioic Acid Octadecanedioic acid
tert-butoxy Tert-butoxy group
OtBu Oxy-t-butyl
DMF N, N' -dimethylformamide
DCM Dichloromethane (dichloromethane)
THF Tetrahydrofuran (THF)
MTBE Methyl tert-butyl ether
TEA Trifluoroethanol
DEA Diethylamine
TLC Thin layer analytical chromatography
HPLC High performance liquid chromatography
The invention discloses a non-classical solid phase synthesis carrier which has better solubility in benign solvents such as N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide and N-methylpyrrolidone and poorer solubility in poor solvents such as water, methanol, ethanol, N-hexane, N-heptane and acetonitrile.
The invention utilizes the great solubility difference of the non-classical solid phase synthesis carrier in benign solvent and poor solvent to realize homogeneous coupling of the carrier or peptide carrier in benign solvent and separation and purification in poor solvent, thereby effectively reducing the consumption of raw materials such as reaction medium, amino acid, condensing agent and the like, and the reaction can be carried out in a container with a conventional volume, thereby effectively reducing the production cost of polypeptide products, reducing environmental pollution and having the characteristics of green and environment protection.
Specifically, the structural formula of the non-classical solid phase synthesis carrier is as follows:
wherein R is 1 Is C 10 ～C 22 Is specifically any one of n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane or phytane; r is R 2 Is C 1 ～C 3 Specifically, any one of methyl, ethyl, propyl or isopropyl.
The non-classical solid phase synthesis carrier with the structural formula can be used as a carrier of Fmoc solid phase synthesis and used for synthesizing polypeptide substances and performing full-protection cleavage on amino acid side chains.
The preparation method of the non-classical solid phase synthesis carrier comprises the following reaction processes:
wherein R is 1 Is C 10 ～C 22 Is a linear or branched alkyl group, X is Cl, br or I, R 2 Is C 1 ～C 3 Is a hydrocarbon group.
The preparation method comprises the following specific steps:
s1: in a first medium, the trihydroxybenzaldehyde 1 is reacted with a haloalkane R at a temperature of 50 to 100℃and preferably 70 to 90 DEG C 1 X reacts in the presence of alkali (at least one of potassium carbonate, sodium hydroxide or potassium hydroxide) and catalyst (at least one of potassium iodide or sodium iodide), the reaction solution is added into poor solvent, and the trialkyl ether benzaldehyde 2 is obtained through precipitation and filtration;
the first medium comprises at least one of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone;
the poor solvent comprises at least one of water, methanol, ethanol, n-hexane, n-heptane or acetonitrile.
S2: reducing the trialkyl ether benzaldehyde 2 prepared in the step S1 under the action of a reducing agent (at least one of sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride) in a second medium at 30-70 ℃, preferably 50-60 ℃, adding the reducing solution into the poor solvent, and precipitating and filtering to obtain trialkyl ether benzyl alcohol 3;
the second medium comprises at least one of methanol, ethanol, or tetrahydrofuran.
S3: in a third medium, using thiourea and derivatives thereof as a catalyst, reacting the trialkyl ether benzyl alcohol 3 obtained in the step S2 with halogenated succinimide to obtain trialkyl ether benzyl halide 4:
wherein the third medium comprises at least one of dichloromethane, chloroform, tetrahydrofuran or toluene.
S4: in the first medium, the trialkane ether benzyl halide 4 obtained in the step S3 and 2-hydroxy-4-R are mixed at 50 to 100 DEG C 2 Reacting oxybenzaldehyde in the presence of alkali (at least one of potassium carbonate, sodium hydroxide or potassium hydroxide) and a catalyst (at least one of potassium iodide or sodium iodide), adding the reaction solution into the poor solvent, and obtaining a non-classical solid phase synthesis carrier precursor 5 through precipitation and filtration;
s5: and in the second medium, reducing the non-classical solid-phase synthesis carrier precursor obtained in the step S4 in the presence of a reducing agent (at least one of sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride), adding a reducing solution into the poor solvent, precipitating and filtering to obtain the non-classical solid-phase synthesis carrier 6.
The method for synthesizing the polypeptide substance by using the non-classical solid phase synthesis carrier comprises the following specific steps:
(1) Amino acid coupling reaction: selecting at least one of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide, N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide as a reaction medium, carrying out coupling reaction on the non-classical solid phase synthesis carrier and Fmoc-amino acid under the action of a condensation reagent, determining a reaction end point by adopting an HPLC, TLC or NT detection method, concentrating, precipitating and filtering the obtained reaction liquid to obtain the Fmoc-peptide carrier, wherein the reaction temperature is 20-50 ℃, preferably 30-35 ℃;
wherein, the feeding mole ratio of the non-classical solid phase synthesis carrier, fmoc-amino acid and condensation reagent is 1:1:1 to 1:3:3, a step of;
the condensing agent is at least two of A, B, C or D; wherein, A is at least one of DIC, DCC or EDCI; the B is at least one of HOOBT, HOBT or HOAT; the C is at least one of DIPEA or NMM; the D is at least one of DMAP, HBTU, HATU, TBTU or PyBOP.
(2) Fmoc protecting group removal reaction: adding the Fmoc-peptide carrier obtained in the step (1) into a dichloromethane solution of diethylamine with the volume concentration of 20%, and carrying out deprotection reaction at 20-50 ℃, preferably 30-35 ℃ to obtain an extended peptide carrier;
(3) Repeating the steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier;
(4) And (3) adding a lysate into the polypeptide substance carrier obtained in the step (3) for pyrolysis, and washing, drying and concentrating the lysate under reduced pressure to obtain the polypeptide substance.
The cracking solution is a mixed solution of the reaction medium, acid and alcohol in a volume ratio of 85-95:14.5-4:0.5-1; wherein the alcohol is at least one of trifluoroethanol or hexafluoroisopropanol; the acid is at least one of acetic acid, trifluoroacetic acid, methanesulfonic acid or trifluoromethanesulfonic acid.
The following will specifically describe embodiments of the present invention by taking the preparation of a non-classical solid phase synthesis support HZ-WSP-W as an example.
Specifically, the non-classical solid phase synthesis carrier HZ-WSP-W has the structural formula:
the synthesis route of HZ-WSP-W is as follows:
the beneficial effects of the invention are as follows: according to the non-classical solid-phase synthesis carrier, the preparation method and the application thereof, the prepared non-classical solid-phase synthesis carrier realizes homogeneous coupling of the carrier or the peptide carrier and the amino acid, and the peptide carrier synthesized by the carrier can be used for finishing cleavage in a cleavage solution and simultaneously retaining a side chain protecting group of the amino acid in a peptide chain, so that the non-classical solid-phase synthesis carrier is suitable for synthesis of full-protection peptides including full-protection fragments of the side chain of the semaglutin, namely, the synthesis of polypeptide products and the full-protection cleavage of the side chain of the amino acid are realized, and the application range is widened; by using the non-classical solid phase synthesis carrier, the volume of a reactor is reduced by 30% -50%, the consumption of raw material amino acid and condensing agent is reduced by 50% -60%, the consumption of organic solvent for reaction and washing is reduced by 60% -80%, the production cost is reduced by 50% -70%, the production cost is effectively reduced, the production efficiency is improved, the environmental pollution is reduced, and the environment is protected; the carrier prepared by the invention has wide market prospect.
Drawings
FIG. 1 shows the H of a non-classical solid phase synthesis carrier HZ-WSP-W prepared by the invention 1 An NMR spectrum;
FIG. 2 is a fragment of a protective amino acid of semaglutin according to example 6 of the present invention: HPLC profile of Fmoc-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy) ] -OH fine-quality;
FIG. 3 is a fragment of a protective amino acid of semaglutin according to example 6 of the present invention: LC-MS spectra of Fmoc-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy) ] -OH fine.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
Example 1 Synthesis of HZ-WSP-W intermediate HZ-WSP-W10 (2)
In a 2000mL three-necked flask1000mL of DMF,15.4g (100.0 mmol) of 3,4, 5-trihydroxybenzaldehyde and 110.0g (330.0 mmol) of bromooctadecane are added, stirred and dissolved, and the reaction solution is heated to 40-50 ℃ in an oil bath. 48.5g (350.0 mmol) of potassium carbonate and 5.0g (30.0 mmol) of potassium iodide were continuously added to the reaction flask, and the mixture was heated to T in an oil bath Inner part The reaction was started at 70 ℃, and TLC detection monitored for reaction endpoint. After the reaction, the heating was stopped, the reaction mixture was cooled to room temperature, and then the mixture was poured into cold water to precipitate a solid, and the solid was filtered, washed with water, dried and purified to give 81.2g (89.1 mmol) of the intermediate HZ-WSP-W10 (2) in a yield of 89.1%.
EXAMPLE 2 Synthesis of HZ-WSP-W intermediate HZ-WSP-W20 (3)
In a 3000mL three-necked flask, 800mL THF and 80.0g (87.7 mmol) of HZ-WSP-W10 were added, and after stirring and dissolving, 1.7g (43.9 mmol) of sodium borohydride was added, and the mixture was heated to T in an oil bath Inner part The reaction was started at =50 ℃, and TLC detection monitored for reaction endpoint. After the reaction, solid is precipitated in the reaction solution, heating is stopped, after the temperature is returned to room temperature, 160 mL of 0.5mol/L HCl aqueous solution is dripped into the reaction flask, a great amount of solid is precipitated after the reaction solution is firstly dissolved out in the dripping process, the solution is filtered, a filter cake is washed and dried, and 80.2g (87.7 mmol) of HZ-WSP-W20 (3) is obtained, and the yield is 100%.
EXAMPLE 3 Synthesis of HZ-WSP-W intermediate HZ-WSP-W30 (4)
In a 2000mL three-necked flask, 760mL of DCM,80.0g (87.6 mmol) of HZ-WSP-W20 and 18.7g (105.1 mmol) of NBS were added, dissolved with stirring, and heated to T in an oil bath Inner part =30 ℃; another 4.1g (39.4 mmol) of dimethylthiourea was taken and dissolved in 40mL of DCM, and the reaction flask was added dropwise to start the reaction, followed by TLC detection and monitoring of the reaction end point. After the reaction was completed, the reaction mixture was treated with 5% NaHCO 3 The aqueous solution was washed, separated, dried, concentrated under reduced pressure, and the crude product was purified to give 83.7g (85.7 mmol) of HZ-WSP-W30 (4) in a yield of 97.8%.
Example 4 Synthesis of HZ-WSP-W intermediate HZ-WSP-W40 (5)
In a 2000mL three-necked flask, 850mL of DMF,83.0g (85.0 mmol) of HZ-WSP-W30 and 15.5g (102.0 mmol) of 2-hydroxy-4-methoxybenzaldehyde were added and stirredDissolving, heating in oil bath to T Inner part Reaction solution was cleared at 40-50 ℃. To the reaction flask, 17.6g (127.5 mmol) of potassium carbonate and 1.4g (8.5 mmol) of potassium iodide were further added, and the temperature was raised to T in an oil bath Inner part The reaction was started at 80 ℃, and TLC detection monitored for reaction endpoint. After the reaction, the heating was stopped, the reaction solution was cooled to room temperature, and then was poured into cold water, whereupon solid was precipitated, filtered, and the cake was washed with water, dried and purified to obtain 84.8g (80.9 mmol) of intermediate HZ-WSP-W40 (5) in a yield of 95.2%.
EXAMPLE 5 Synthesis of non-classical solid phase Synthesis Carrier HZ-WSP-W (6)
In a 3000mL three-necked flask, 800mL THF and 80.0g (76.4 mmol) of HZ-WSP-W40 were added, and after stirring and dissolving, 1.5g (38.2 mmol) of sodium borohydride was added, and the mixture was heated to T in an oil bath Inner part The reaction was started at =50 ℃, and TLC detection monitored for reaction endpoint. After the reaction, solid is precipitated in the reaction solution, heating is stopped, after the temperature is returned to room temperature, 160 mL of 0.5mol/L HCl aqueous solution is dripped into the reaction flask, a great amount of solid is precipitated after the reaction solution is firstly dissolved out in the dripping process, the solution is filtered, a filter cake is washed and dried, and 80.0g (76.2 mmol) of HZ-WSP-W (6) is obtained, and the yield is 100%.
The total synthesis yield of the non-classical solid phase synthesis carrier HZ-WSP-W is 82.9%.
The following is the application of the non-classical solid phase synthesis support HZ-WSP-W prepared in the above example.
Example 6
The non-classical solid phase synthesis carrier HZ-WSP-W prepared in the embodiment is taken as a carrier to synthesize the semaglutin protected amino acid fragment: fmoc-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy) ] -OH.
1-1, loading the first amino acid Lys: into a 250mL three-necked flask, 100mL of DCM and 10.5g (10.0 mmol; molecular weight: 1049.75, degree of substitution: 0.95 mmol/g) of HZ-WSP-W,10.7g (20.0 mmol) of Dde-Lys (Fmoc) -OH and 0.2g (1.5 mmol) of DMAP were charged, and the solution was stirred and heated to T in an oil bath Inner part =30-35 ℃. 3.1mL (2.5 g,20.0 mmol) of DIC was added dropwise, the reaction was started while maintaining the temperature, and the TLC detection was monitored for the end of the reaction.
1-2, loading a first amino acid Lys coupling reaction post-treatment: after the coupling reaction, the reaction solution was washed with water and saturated brine in this order, separated, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, the concentrated residue was slurried with n-hexane, filtered, and dried to give Dde-Lys (Fmoc) -HZ-WSP-W crude product as a white solid.
1-3, removing Fmoc protecting group from the first amino acid and post-treatment: dde-Lys (Fmoc) -HZ-WSP-W was added to a 20% diethyl amine in dichloromethane deprotected solution, and the solution was stirred and heated to T in an oil bath Inner part The reaction was started at 30-35 ℃, and TLC detection monitored for reaction endpoint. After the deprotection reaction is completed, the mixture is concentrated under reduced pressure, the concentrated residue is pulped by methyl tertiary butyl ether, filtered and dried, and 11.9g of a crude product of the extended peptide carrier Dde-Lys-HZ-WSP-W is obtained as a white solid.
2-1, fmoc-AEEA coupled: into a 250mL three-necked flask, 100mL DCM and 11.9g Dde-Lys-HZ-WSP-W crude product were added, the solution was stirred and heated to T in an oil bath Inner part =30-35 ℃. Into an activation flask, 10mL of N, N-dimethylformamide, 4.6g (12.0 mmol) of Fmoc-AEEA and 1.6g (12.0 mmol) of HOBT were added, dissolved with stirring, and pre-cooled to T in a low temperature tank Inner part =0-5 ℃; to the activation flask was added 1.9mL (1.5 g,12.0 mmol) DIC, and the temperature was maintained for 5min of pre-activation. After the activation is completed, the activated solution is added into the reaction flask, the reaction is started, and TLC or NT detection monitors the end point of the reaction.
2-2, fmoc-AEEA coupled post-treatment: after the coupling reaction is finished, the reaction solution is washed by water and saturated saline in sequence, the solution is separated, the organic phase is dried by anhydrous sodium sulfate, the filtrate is filtered, the filtrate is concentrated under reduced pressure, concentrated residues are combined and pulped by n-hexane, methyl tertiary butyl ether, methanol and acetonitrile, the filtrate is filtered and dried, and Dde-Lys (AEEA-Fmoc) -HZ-WSP-W crude product is obtained as white solid.
2-3, fmoc protecting group removal of peptide chain and post treatment: dde-Lys (AEEA-Fmoc) -HZ-WSP-W was added to a 20% diethyl amine in dichloromethane deprotected solution, and the solution was stirred and heated to T in an oil bath Inner part The reaction was started at 30-35 ℃, and TLC detection monitored for reaction endpoint. After the deprotection reaction is completed, the mixture is concentrated under reduced pressure, the concentrated residue is beaten by the combination of normal hexane and acetonitrile,filtration and drying gave 13.8g of crude extended peptide carrier Dde-Lys (AEEA) -HZ-WSP-W as a white solid.
3-1, fmoc-AEEA coupled: into a 250mL three-necked flask, 100mL DCM and 13.8g Dde-Lys (AEEA) -HZ-WSP-W crude product were added, and the solution was stirred and heated to T in an oil bath Inner part =30-35 ℃. Into an activation flask, 10mL of N, N-dimethylformamide, 4.6g (12.0 mmol) of Fmoc-AEEA and 1.6g (12.0 mmol) of HOBT were added, dissolved with stirring, and pre-cooled to T in a low temperature tank Inner part =0-5 ℃; to the activation flask was added 1.9mL (1.5 g,12.0 mmol) DIC, and the temperature was maintained for 5min of pre-activation. After the activation is completed, the activated solution is added into the reaction flask, the reaction is started, and TLC or NT detection monitors the end point of the reaction.
3-2, fmoc-AEEA coupled post-treatment: after the coupling reaction, the reaction solution was washed with water and saturated brine in this order, separated, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the concentrated residue was slurried with n-hexane, methyl t-butyl ether, methanol and acetonitrile in combination, filtered, and dried to give Dde-Lys (AEEA-AEEA-Fmoc) -HZ-WSP-W crude product as a white solid.
3-3, fmoc protecting group removal of peptide chain and post treatment: dde-Lys (AEEA-AEEA-Fmoc) -HZ-WSP-W was added to a dichloromethane deprotected solution of 20% diethylamine, and the solution was stirred and heated to T in an oil bath Inner part The reaction was started at 30-35 ℃, and TLC detection monitored for reaction endpoint. After the deprotection reaction was completed, the mixture was concentrated under reduced pressure, and the concentrated residue was slurried with a combination of n-hexane and acetonitrile, filtered, and dried to give 15.0g of a crude peptide carrier Dde-Lys (AEEA-AEEA) -HZ-WSP-W as a white solid after extension.
4-1, fmoc-Glu-OtBu coupled: into a 250mL three-necked flask, 100mL DCM and 15.0g Dde-Lys (AEEA-AEEA) -HZ-WSP-W crude product were added, the solution was stirred and heated to T in an oil bath Inner part =30-35 ℃. Into an activation flask, 10mL of N, N-dimethylformamide, 5.1g (12.0 mmol) of Fmoc-Glu-OtBu and 1.6g (12.0 mmol) of HOBT were added, dissolved with stirring, and pre-cooled to T in a low temperature tank Inner part =0-5 ℃; to the activation flask was added 1.9mL (1.5 g,12.0 mmol) DIC, and the temperature was maintained for 5min of pre-activation. After activation is completed, the activated solution is added into a reaction flask to start the reaction, TLC orNT detection monitors the end point of the reaction.
4-2, fmoc-Glu-OtBu coupled post-treatment: after the coupling reaction is finished, the reaction solution is washed with water and saturated saline in sequence, the solution is separated, the organic phase is dried with anhydrous sodium sulfate, filtered, the filtrate is concentrated under reduced pressure, concentrated residues are pulped by using n-hexane, methyl tertiary butyl ether, methanol and acetonitrile, filtered and dried, and Dde-Lys [ AEEA-AEEA-gamma-Glu (alpha-OtBu) -Fmoc ] -HZ-WSP-W crude product is obtained as white solid.
4-3, fmoc protecting group removal of peptide chain and post treatment: to a dichloromethane deprotected solution of 20% diethylamine was added Dde-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Fmoc]-HZ-WSP-W, stirring to dissolve, heating in oil bath to T Inner part The reaction was started at 30-35 ℃, and TLC detection monitored for reaction endpoint. After the deprotection reaction is completed, concentrating under reduced pressure, pulping the concentrated residue by using n-hexane and acetonitrile combination, filtering and drying to obtain the extended peptide carrier Dde-Lys [ AEEA-AEEA-gamma-Glu (alpha-OtBu)]16.7g of crude product of HZ-WSP-W is white solid.
5-1, coupled HOOC-C 16 COOtBu: into a 250mL three-necked flask, 100mL DCM and 16.7g Dde-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) were added]Stirring and dissolving the crude product of HZ-WSP-W, and heating to T in an oil bath Inner part =30-35 ℃. Into an activation flask, 10mL of N, N-dimethylformamide, 4.5g (12.0 mmol) of HOOC-C were added 16 COOtBu and 1.6g (12.0 mmol) HOBT, stirring to dissolve, pre-cooling in a low temperature tank to T Inner part =0-5 ℃; to the activation flask was added 1.9mL (1.5 g,12.0 mmol) DIC, and the temperature was maintained for 5min of pre-activation. After the activation is completed, the activated solution is added into the reaction flask, the reaction is started, and TLC or NT detection monitors the end point of the reaction.
5-2, coupled HOOC-C 16 COOtBu post-treatment: after the coupling reaction, the reaction solution was washed with water and saturated brine in this order, separated, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the concentrated residue was slurried with n-hexane, methyl t-butyl ether, methanol and acetonitrile, filtered, and dried to give Dde-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy)]-HZ-WSP-W crude 21.3g as white solid.
6-1, lys backbone amino removalDde protecting group: to 200mL of a 3% strength by volume solution of hydrazine hydrate in tetrahydrofuran was added 21.3g of Dde-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy)]Stirring and dissolving the crude product of HZ-WSP-W, and heating to T in an oil bath Inner part The reaction was started at 30-35 ℃, and TLC detection monitored for reaction endpoint. After the deprotection reaction is completed, the reaction solution is paved into 2000mL of cold water, and the solid is separated out, filtered, washed, dried and purified to obtain H-Lys [ AEEA-AEEA-gamma-Glu (alpha-OtBu) -Octadecanedioic Acid-18 (tert-butoxy)]-HZ-WSP-W crude 19.1g.
Fmoc protecting group on amino group of 6-2, lys backbone: to 200mL of tetrahydrofuran solution, 19.1-g H-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy) was added]Crude HZ-WSP-W and 1.6g (12.4 mmol) DIPEA were stirred for clearing. Heating in oil bath to T Inner part 3.8g (11.4 mmol) Fmoc-OSU was added in multiple portions at 30-35℃to start the reaction, and TLC detection monitored the end of the reaction. After the reaction, the reaction solution was poured into 2000mL of cold water to precipitate a solid, and the solid was filtered, washed, dried and purified to give Fmoc-Lys [ AEEA-AEEA-gamma-Glu (. Alpha. -OtBu) -Octadecanedioic Acid-18 (tert-butoxy)]-HZ-WSP-W21.0 g (9.5 mmol), overall yield 95%.
7. Cleavage reaction:
the lysate was prepared in a volume ratio of DCM: TEA: TFA=90:9:1, and precooled to 5 ℃. 200mL of the precooled lysate and 21.0g (9.5 mmol) of the semaglutin protected amino acid fragment prepared in the step 3 are added into a 1000mL pyrolysis bottle, and the mixture is heated to T in an oil bath Inner part The reaction was started at 20-25 ℃, and TLC detection monitored for reaction endpoint. After the completion of the cleavage, the reaction solution was cooled to T in a low-temperature tank Inner part Adding 100mL of 5% sodium carbonate aqueous solution at the temperature of between-5 and 0 ℃, stirring and washing, separating, washing the organic phase by water and saturated saline water successively, drying, concentrating under reduced pressure to obtain a crude product, and purifying the crude product to obtain Fmoc-Lys [ AEEA-AEEA-gamma-Glu (alpha-OtBu) -Octadecanedioic Acid-18 (tert-butoxy)]9.8g (8.7 mmol) of OH fine product with a yield of 87.5% and a HPLC purity of 98.38%.
Fmoc-Lys [ AEEA-AEEA-gamma-Glu (alpha-OtBu) -Octadecanedioic Acid-18 (tert-butoxy) ] -OH fine product prepared above has HPLC spectrum shown in figure 2 and LC-MS spectrum shown in figure 3.
The non-classical solid phase synthesis carrier of the invention is adopted to synthesize polypeptide drugs, which comprises the preparation of the semaglutin protected amino acid fragment, the same production scale, the volume of a reactor can be reduced by 30% -50%, the consumption of raw material amino acid and condensing agent can be reduced by 50% -60%, the consumption of organic solvents for reaction and washing can be reduced by 60% -80%, and finally the production cost is reduced by 50% -70%.
The invention designs a novel non-classical solid phase synthesis carrier synthesized by the carrier, and the peptide carrier synthesized by the carrier can complete the cracking in the cracking liquid with lower acid concentration, simultaneously reserves the side chain protecting group of the amino acid in the peptide chain, can realize the large-scale production of polypeptide products, and is also suitable for the synthesis of full-protection peptides including full-protection fragments of the side chain of the semaglutin. The non-classical solid phase synthesis carrier synthesized by the method for producing the polypeptide product has the advantages of reducing production cost, improving production efficiency, reducing environmental pollution, reducing cost, enhancing efficiency, saving energy, reducing emission and protecting environment.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (6)

1. The preparation method of the polypeptide substance is characterized in that the prepared non-classical solid-phase synthesis carrier is used for synthesizing the polypeptide substance, and comprises the following compounds in the structural formula:
，
wherein R is 1 Is C 10 ～C 22 Straight or branched alkyl of (a); r is R 2 Is C 1 ～C 3 Alkyl of (a); the non-classical solid phase synthesis carrier is prepared by adopting the following preparation method：
S1: reacting trihydroxybenzaldehyde with halogenated alkane in the presence of alkali and a first catalyst to obtain trialkyl ether benzaldehyde, wherein the reaction formula is as follows:
；
s2: reducing the trialkyl ether benzaldehyde in the presence of a reducing agent to obtain trialkyl ether benzyl alcohol, wherein the reaction formula is as follows:
；
s3: reacting the trialkyl ether benzyl alcohol with a halogenated succinimide in the presence of a second catalyst to obtain a trialkyl ether benzyl halide, wherein the reaction formula is as follows:
；
s4: combining the trialkyl ether benzyl halide with 2-hydroxy-4-R 2 The oxybenzaldehyde reacts in the presence of alkali and a first catalyst to obtain a non-classical solid-phase synthesis carrier precursor, and the reaction formula is as follows:
；
s5: reducing the non-classical solid phase synthesis carrier precursor in the presence of a reducing agent to obtain the non-classical solid phase synthesis carrier, wherein the reaction formula is as follows:
；
in each of the above reaction formulae, R 1 Is C 10 ～C 22 Is a linear or branched alkyl group, X is Cl, br or I, R 2 Is C 1 ～C 3 Alkyl of (a);
the non-classical solid phase synthesis carrier prepared is used for synthesizing polypeptide substances, and comprises the following steps:
(1) Amino acid coupling reaction: in a reaction medium, the non-classical solid phase synthesis carrier and Fmoc-amino acid are subjected to coupling reaction under the action of a condensation reagent, and the reaction solution is concentrated, precipitated and filtered to obtain the Fmoc-peptide carrier;
(2) Fmoc protecting group removal reaction: adding the Fmoc-peptide carrier obtained in the step (1) into a deprotection solution for deprotection reaction to obtain an extended peptide carrier;
(3) Repeating the steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier;
(4) Adding a lysate into the polypeptide substance carrier obtained in the step (3) for pyrolysis, washing, drying and concentrating under reduced pressure the lysate to obtain polypeptide substances;
wherein the cracking liquid is a mixed solution of a reaction medium, acid and alcohol in a volume ratio of 85-95:14.5-4:0.5-1; wherein the alcohol is at least one of trifluoroethanol or hexafluoroisopropanol; the acid is at least one of acetic acid, trifluoroacetic acid, methanesulfonic acid or trifluoromethanesulfonic acid.
2. The method according to claim 1, wherein in the step (1), the non-classical solid phase synthesis support, fmoc-amino acid and condensation reagent are fed in a molar ratio of 1:1:1 to 1:3:3.
3. the method for producing a polypeptide according to claim 2, wherein the condensing agent is at least two of A, B, C and D; wherein, A is at least one of DIC, DCC or EDCI; the B is at least one of HOOBT, HOBT or HOAT; the C is at least one of DIPEA or NMM; the D is at least one of DMAP, HBTU, HATU, TBTU or PyBOP.
4. The method according to claim 1, wherein in the step (1) or (2), the reaction temperature is 20 to 50 ℃.
5. The method of claim 1, wherein the reaction medium comprises at least one of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide, N-diethylformamide, N-methylpyrrolidone, or dimethylsulfoxide.
6. The method according to claim 1, wherein in the step (2), the deprotecting solution is a dichloromethane solution of diethylamine.
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