CN112110981B - Preparation method of polypeptide containing long-chain fatty diacid side chain - Google Patents
Preparation method of polypeptide containing long-chain fatty diacid side chain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- 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
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- 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
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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
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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
- C07K14/435—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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- 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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- 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 relates to a preparation method of polypeptide containing a long-chain fatty diacid side chain, wherein the polypeptide containing the long-chain fatty diacid has a structure shown in a formula I;
the method comprises the following steps: 1) coupling a long-chain diacid compound containing 6-32 carbon atoms onto a solid-phase synthetic resin; obtaining long-chain diacid-solid phase synthetic resin conjugate; 2) coupling H-Glu (OAll) -OtBu on a long-chain diacid-solid phase synthetic resin conjugate; 3) removing OAll protecting groups, and then sequentially coupling side chain groups; 4) then coupled one by one for forming A2OAll of the polypeptide segment protects amino acid of alpha carboxyl; 5) fmoc protecting groups were removed and coupled one by one for A formation1Fmoc of the polypeptide segment protects amino acid of alpha amino; 6) cleaving all protecting groups while cleaving the resin to obtain a crude peptide; 7) purifying to obtain polypeptide containing long-chain fatty diacid. The preparation method has high efficiency and simple synthesis method.
Description
Technical Field
The present invention relates to the field of polypeptide synthesis, and in particular to a method for preparing a polypeptide containing long-chain fatty diacid.
Technical Field
At present, the research and development of polypeptide drugs are hot spots in the field of drugs, and the long-acting of polypeptide drugs is the development trend of the current polypeptide drugs.
The long-acting of polypeptide drugs is mainly completed by the structural modification of polypeptides, and the side chain modification of short-chain PEG combined with long-chain fatty diacid is a hot point in the research of heating points of the current polypeptide drugs. Such as semaglutide, currently marketed by norand nordnode, Tirzepatide, currently in clinical phase iii, and BPI-3016, etc., which is currently in clinical study in the berda pharmaceutical industry.
Semetreuptade is a long-acting GLP-1 analogue developed by Novonide, and used for treating type 2 diabetes, and the injection form of the Semetreuptade is firstly approved by FDA to be marketed in 2017, and only needs to be injected and administered once per week subcutaneously. In 2019, oral semaglutide approved by FDA is sold on the market and used as the first oral GLP-1 analogue diabetes drug, and the market prospect of the drug is widely seen.
Tirzepatide is a heavily weighted research product in the field of pharmacy, currently undergoing a three-phase clinical study, and is expected to be approved for marketing in 2022. Is considered to be the GLP-1 analogue hypoglycemic drug which most possibly brings market challenges to the scagliola.
Structurally, the semaglutide backbone has 31 amino acid residues with side chains Lys at position 20 to which two AEEA, gamma-glutamic acid and octadecanedioic acid fatty chains are attached. The sequence structure of semaglutide is as follows:
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH
the structure of telaprepide is substantially similar to semaglutide, but the backbone is longer with 39 amino acids, and the side chain is also substantially similar to semaglutide, with two aea, gamma-glutamic acid and eicosanedioic acid fatty chains attached to Lys at position 20. The sequence structure of telapremide is as follows:
H-Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-Lys20(AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2
at present, the synthesis method of semaglutide and telaprepide is mainly completed by combining biological fermentation with chemical synthesis, and the other method is realized by a simple chemical synthesis method.
The method of combining biological fermentation with chemical synthesis is adopted, firstly, the main chain partial fragment is prepared by the biological fermentation method, then, the chemical synthesis method is adopted, the main chain fragment directly reacts with OSu ester of the whole side chain, and then, other fragments of the main chain are coupled by the chemical synthesis method. The synthesis method has the advantages that the amino acid side chain of the main chain segment does not have any protecting group, so that multi-site reaction is easily caused, a large amount of unexpected impurities are formed, the later separation and purification are not facilitated, and the yield is low.
The simple chemical synthesis method mainly utilizes Fmoc solid phase peptide synthesis and adopts a direct coupling or fragment condensation method to complete the coupling of sequences. In these synthetic methods, the sequence structure and characteristics of the long-sequence polypeptide itself are not considered, such as the influence of the secondary structure of the polypeptide on the solid-phase synthesis of the polypeptide, the influence of the amino acid side chain protecting group on the solid-phase coupling of the polypeptide, the influence of the hydrophobicity of the fatty chain with a longer side chain on the solid-phase synthesis of the polypeptide, the influence of coupling side reactions on the product yield, the solubility and the ease of condensation of the fragment condensation fragment, etc. If the solid phase carrier resin is loaded on the first residue at the C-terminal of the sequence, the coupling of the subsequent terminal residue is very little constrained by the rigidity of the solid phase carrier resin, and the coupling efficiency of the subsequent residue is seriously influenced in the solid phase polypeptide synthesis; the coupling of the pendant long chain diacids is very difficult due to their hydrophobic nature.
If the synthesis is carried out by adopting a fragment condensation method, not only is the dissolution of the fully protected long-sequence large fragment very difficult, but also the coupling efficiency is very low.
There is an urgent need for a method of synthesizing polypeptides that can be adapted to contain side chains for long-cycle use.
Disclosure of Invention
In view of the above, the invention provides a brand new preparation method of polypeptide with a side chain containing symmetrical dicarboxyl, which adopts the steps that one carboxyl in dicarboxyl monomers at the tail end of the side chain is directly connected to solid-phase carrier resin, and then different protected amino acid monomers are gradually adopted from the side chain to the two ends of a main chain, so that the coupling of the whole peptide sequence is gradually completed; the method for synthesizing the polypeptide has the advantages that the solid phase carrier is closer to the N-terminal and the C-terminal, the rigid structure of the solid phase carrier can restrict the formation of a beta-folding secondary structure, and a good synthesis effect is easy to obtain.
In order to achieve the above object, the present invention provides the following technical solutions:
one aspect of the invention provides a solid phase synthesis method of a polypeptide comprising a long chain fatty diacid side chain, wherein the polypeptide comprising a long chain fatty diacid has a structure shown in formula I;
wherein n is 4-30;
A1and A2Respectively polypeptide segments, wherein the A1 polypeptide segment comprises 5-50 amino acid residues;
A2the polypeptide segment comprises 5-50 amino acid residues;
the method is characterized by comprising the following steps:
1) coupling a long-chain diacid compound containing 6-32 carbon atoms onto a solid-phase synthetic resin; obtaining long-chain diacid-solid phase synthetic resin conjugate;
2) coupling H-Glu (OAll) -OtBu on a long-chain diacid-solid phase synthetic resin conjugate;
3) removing OAll protecting groups, and then sequentially coupling H-AEEA-OAll, H-AEEA-OAll and Fmoc-Lys-OAll;
4) then coupled one by one for forming A2Protecting alpha-carboxyl amino acid or amido amino acid amide protected by carboxyl protecting group of the polypeptide segment;
5) fmoc protecting groups were removed and coupled one by one for A formation1Fmoc of the polypeptide segment protects amino acid of alpha amino;
6) cleaving all protecting groups while cleaving the resin to obtain a crude peptide;
7) purifying to obtain polypeptide containing long-chain fatty diacid.
In an embodiment of the invention, the solid phase synthetic resin is selected from 2CTC resins. Preferably, the degree of substitution is in the range of 0.2 to 1.2mmol/g, more preferably 0.3 to 1.0mmol/g, most preferably 0.6 to 0.8 mmol/g;
in an embodiment of the invention, the long chain diacid compound in step 1) is selected from diacid compounds containing 16 to 26 carbons, preferably linear diacid compounds such as hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid, docosanedioic acid and the like.
In an embodiment of the invention, step 1) one of the carboxyl groups of the long chain diacid compound is coupled to a solid phase synthetic resin to form a conjugate.
In an embodiment of the invention, the coupling conditions in step 1) are a reaction under basic conditions obtained by adding N, N-diisopropylethylamine, triethylamine.
In an embodiment of the present invention, 0.2 to 1.2mmol, preferably 0.3 to 1.0mmol, and more preferably 0.3 to 0.5mmol of long-chain diacid compound is added per gram of solid-phase synthetic resin in step 1).
In an embodiment of the present invention, the amino group of H-glu (oall) -OtBu in step 2) is coupled with the carboxyl group of the long-chain diacid-solid phase synthetic resin conjugate to form an amide bond.
In an embodiment of the invention, the method for sequentially coupling H-AEEA-OAll, H-AEEA-OAll and Fmoc-Lys-OAll in step 3) comprises coupling H-AEEA-OAll, removing OAll protecting groups, coupling Fmoc-Lys-OAll, and removing OAll protecting groups.
In an embodiment of the invention, in step 4), the coupling is extended from the N-terminus to the C-terminus on the Lys coupled in step 3), and the amino acids are coupled in sequence by coupling the amino acids with the side chain and the carboxyl group both protected by the protecting group, then removing the protecting group for the carboxyl group, then coupling the amino acids with the side chain and the carboxyl group both protected by the protecting group, then removing the protecting group for the carboxyl group, and continuing the cycle until A is completed2A polypeptide fragment;
wherein the carboxyl protecting groups of the amino acids other than the last amino acid coupled at the C-terminus are selected from OAll protecting groups;
the carboxyl protecting group of the last amino acid coupled at the C-terminus is selected from the group consisting of-OtBu protecting groups;
alternatively, the first and second electrodes may be,
coupling amino acid with side chain and carboxyl protected by protecting group, then removing carboxyl protecting group, continuously circulating to form polypeptide chain, and finally coupling amino acid amide with side chain and amide protected at C terminal end;
wherein the carboxyl protecting groups of the amino acids other than the last amino acid coupled at the C-terminus are selected from OAll protecting groups;
the amide protecting group of the last amino acid amide coupled at the C-terminus is selected from the Trt protecting groups.
In an embodiment of the invention, the amino acid in step 4) is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, 2-aminoisobutyric acid, serine amide (Ser-NH)2) Glycine amide, alanine amide, valine amide, leucine amide, isoleucine amide, methionine amide, phenylalanine amide, proline amide, tryptophan amide, serine amide, tyrosine amide, cysteine amide, phenylalanine amide, asparagine amide, glutamine amide, threonine amide, asparagine amide, glutamic acid amide, lysine amide, arginine amide, histidine amide, 2-aminoisobutyric acid amide. And when the amino acid contains a side chain to be protected or a carboxyl group, the side chain is protected by a protecting group, and the carboxyl group is protected by an OAll protecting group. The amino acid amides having the main chain amide protected by a Trt protecting group, e.g. serine amides (Ser-NH)2) The main chain amide of (A) is protected by a Trt protecting group
In the present invention, the OAll protecting group is a carboxyl protecting group. The carboxyl structure after OAll protection is specifically as
In an embodiment of the invention, H-Glu (OtBu) -OAll, H-Phe-OAll, H-Ile-OAll, H-Ala-OAll, H-Trp (Boc) -OAll, H-Leu-OAll, H-Val-OAll, H-Arg (Pbf) -OAll, H-Gly-OtBu;
or sequentially coupling H-Ala-OAll, H-Phe-OAll, H-Val-OAll, H-Gln (Trt) -OAll, H-Trp (Boc) -OAll, H-Leu-OAll, H-Ile-OAll, H-Ala-OAll, H-Gly-OAll, H-Pro-OAll, H-Ser (tBu) -OAll, H-Gly-OAll, H-Ala-OAll, H-Pro-OAll, H-Ser (tBu) -NH (Trt).
In an embodiment of the invention the coupling conditions in steps 2), 3), 4) and 5) are DIC + a or B + a + C or C + D, wherein a is HOBt or HOAt or Oxyma, B is HBTU, HATU, PyBOP, C is DIPEA or TMP or DMAP and D is T3P.
Preferably, the amount of condensing agent used in the coupling system is 0.8 to 3.0 times the amino acid equivalent. The reaction time is 1-4 hours.
In an embodiment of the invention, the OAll protecting group is removed in steps 3) and 4) by Pd0(Ph3P)4And PhSiH3Removing the OAll protecting group.
Preferably, wherein Pd0(Ph3P)4The molar amount of (a) is 0.01 to 1.0, more preferably 0.01 to 0.5, of the molar amount of OAll protecting groups; PhSiH3The molar amount of (a) is 1 to 10 times, more preferably 1 to 5 times the molar amount of the OAll protecting group. The reaction time for removing the OAll protecting group is 0.5 to 4 hours, preferably 0.5 hour. After removal of the OAll protecting group, the peptide resin was washed with DCM.
In an embodiment of the invention, in step 5), the coupling is extended from the C-terminus to the N-terminus on the Lys coupled in step 2), the amino acids being coupled in sequence by coupling the amino acids with side chains and amino groups protected by protecting groups, then removing the protecting groups for the amino groups, and coupling is carried out until A is completed1A polypeptide fragment; wherein the amino protecting group is selected from the group consisting of Fmoc protecting groups.
In an embodiment of the invention, the amino acid in step 5) is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, 2-aminoisobutyric acid. And the amino acid coupling side chain and the amino group are protected by protecting groups.
In embodiments of the invention, side chain protecting groups may be selected from protecting groups conventional in the art, for example: tBu, OtBu, Trt, Boc, Pbf, etc.
In an embodiment of the present invention, in step 5), Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH;
or coupling Fmoc-Gln (Trt) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr tBu- (OH), Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-Tyr (tBu) -OH in this order.
In an embodiment of the present invention, the Fmoc protecting group is removed in step 5) by a mixed solution of piperidine and DMF, preferably in a volume ratio of 1: 4.
In an embodiment of the invention, the cleavage agent used for cleavage in step 6) is TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2O＝85-95:2-5:0-3:0-2:1-5。
Preferably, the lysis time is 1.5-3.5 hours.
In the embodiment of the present invention, the purification method in step 7) refers to a conventional purification method in the art, such as HPLC, supercritical fluid chromatography, and the like.
In an embodiment of the invention, in the compounds of formula I, n is 12 to 26, for example n is 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, preferably 16 to 18.
In an embodiment of the invention, said A is1The polypeptide fragment comprises 15-30 amino acid residues, such as 15,16,17,18,19,20,21,22,23,24,25,26, 27, 28, 29, 30.
In an embodiment of the invention, said A is2The polypeptide segment comprises 6-25 amino acid residues, such as 6, 7, 8,9、10、11、12、13、14、15、16、17、18、19、20、22、23、24、25。
In an embodiment of the invention, said A is1The polypeptide segment contains 15-30 amino acid residues.
In an embodiment of the invention, said A is2The polypeptide segment comprises 11 amino acid residues or 19 amino acid residues.
In an embodiment of the invention, said A is2The C-terminal of the polypeptide fragment has NH2Modified or not modified.
In an embodiment of the invention, said A is1The polypeptide segment is H-AA1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-AA10-Ser11-AA12-AA13-Leu14-AA15-AA16-AA17-Ala18-AA19-, wherein AA1Selected from His1Or Tyr1、AA10Selected from Val10Or Tyr10、AA12Is selected from Ser12Or Ile12、AA13Is selected from Tyr13Or Aib13、AA15Selected from Glu15Or Asp15、AA16Is selected from Gly16Or Lys16、AA17Selected from Gln17Or Ile17、AA19Is selected from Ala19Or Gln19；
Preferably, A1The polypeptide segment is H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-, or is H-Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-。
In an embodiment of the invention, said A is2The polypeptide segment is-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH or is-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2。
In a preferred embodiment of the invention, in the compounds of the formula I, n is 16 and A1The polypeptide segment is H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-；A2The polypeptide segment is-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH。
In a preferred embodiment of the invention, in the compounds of the formula I, n is 18 and A1The polypeptide segment is H-Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-，A2The polypeptide fragment is-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2。
In a preferred embodiment of the invention, the polypeptide comprising a long chain fatty diacid is selected from Semaglutide (Semaglutide) or tipepide (Tirzepatide).
In a preferred embodiment of the invention, the polypeptide comprising a long chain fatty diacid side chain is semaglutide, a compound of formula I wherein n is 16 and a1The polypeptide segment is H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-；A2The polypeptide segment is-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH；
Which comprises the following steps:
1) coupling octadecanedioic acid to the solid-phase synthetic resin; obtaining an octadecanedioic acid-solid phase synthetic resin conjugate;
2) coupling the carboxyl group of the octadecanedioic acid-solid phase synthesis resin conjugate with the amino group of H-Glu (OAll) -OtBu;
3) removing OAll protecting groups, and then sequentially coupling H-AEEA-OAll, H-AEEA-OAll and Fmoc-Lys-OAll;
4) then coupling-OAll to protect amino acid of alpha carboxyl one by one to form A2The polypeptide segment is-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH；
5) Removing Fmoc protecting group, coupling Fmoc protecting alpha amino acid one by one to form A1The polypeptide segment is H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-；
6) Cracking all protecting groups and simultaneously cracking the solid-phase synthetic resin to obtain crude peptide;
7) purifying to obtain the polypeptide containing the long-chain fatty diacid side chain.
In a preferred embodiment of the invention, the polypeptide comprising a long chain fatty diacid side chain is a telapremide, a compound of formula I wherein n is 18, a1The polypeptide segment is H-Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-，A2The polypeptide fragment is-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2；
Which comprises the following steps:
1) coupling the eicosanedioic acid to the solid phase synthetic resin; to obtain the didecyl diacid-solid phase synthetic resin conjugate;
2) coupling the carboxyl group of the didecanedioic acid-solid phase synthetic resin conjugate with the amino group of H-Glu (OAll) -OtBu;
3) removing OAll protecting groups, and then sequentially coupling H-AEEA-OAll, H-AEEA-OAll and Fmoc-Lys-OAll;
4) then coupling-OAll to protect amino acid of alpha carboxyl one by one to form A2The polypeptide fragment is-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2；
5) Removing Fmoc protecting group, coupling Fmoc protecting alpha amino acid one by one to form A1The polypeptide segment is H-Tyr1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-Aib13-Leu14-Asp15-Lys16-Ile17-Ala18-Gln19-；
6) Cracking all protecting groups and simultaneously cracking the solid-phase synthetic resin to obtain crude peptide;
7) purifying to obtain the polypeptide analogue containing the long-chain fatty diacid side chain.
Advantageous effects
1) The invention provides a solid-phase synthesis method, which does not adopt a conventional method of starting coupling from the C terminal, but starts coupling from a side chain containing a long fatty chain one by one, and avoids the problems of low residue coupling efficiency caused by small rigid constraint of solid-phase synthetic resin in the subsequent residue coupling process of the conventional solid-phase synthesis long-chain polypeptide.
2) The method directly couples the long-chain fatty diacid to the solid-phase resin in a first residue mode, and avoids the problem that the long-chain fatty diacid is low in subsequent coupling efficiency due to high hydrophobicity.
3) The method utilizes the symmetrical characteristic of two carboxyl groups of the long-chain fatty diacid to directly couple one of the carboxyl groups to the solid phase carrier, does not need to carry out the protection of the mono-tert-butyl ester on the long-chain fatty diacid, and can reduce the synthesis cost (the price of the octadecanedioic acid of the mono-tert-butyl ester is more than 5 times of the price of the unprotected octadecanedioic acid at present).
4) The method does not adopt the method of synthesizing polypeptide chain first and then coupling the long-chain diacid side chain with strong hydrophobicity in the prior art. The problem of difficult synthesis caused by overlarge difference between the hydrophilicity and the hydrophobicity of polypeptide chains and side chains is avoided.
5) The fragments coupled one by the method of the invention are all very short, are single amino acids, and are not polypeptide fragments. The method of the invention avoids the problem of difficult dissolution of the fully-protected polypeptide fragment and improves the coupling efficiency.
Drawings
FIG. 1: HPLC profile of crude semaglutide prepared in example 7.
FIG. 2: MS spectrum of crude semaglutide prepared in example 7.
FIG. 3: HPLC profile of the semaglutide protamine prepared in example 7.
FIG. 4: HPLC profile of crude semaglutide prepared in example 9.
Detailed Description
The invention discloses a preparation method of polypeptide, which can be realized by appropriately improving process parameters by the technical personnel in the field with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Abbreviations used in the specification and claims have the following specific meanings:
the polypeptide provided by the invention, the preparation method and the raw materials, auxiliary materials and reagents used in the application can be purchased from the market or produced by the inventor.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of Octadecanedioic acid-2CTC resin
Weighing 200 grams of 2CTC Resin with the substitution degree of 0.8mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling and swelling for 60 minutes by nitrogen; 18.9 g (60mmol) of octadecanedioic acid was weighed, 22.1mL of DIPEA (120mmol) was added, and after 1.0 hour of reaction, 30mL of methanol and 11mL of DIPEA were added and the reaction was continued for 30 minutes. Washing with DMF for three times, shrinking methanol, and draining the resin to obtain 213.9 g of octadecanedioic acid-2CTC resin, wherein the resin substitution is calculated to be 0.22mmol/g through the weight gain of the resin.
EXAMPLE 2 preparation of Dicranodianedioic acid-2CTC resin
Weighing 200 grams of 2CTC Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling and swelling for 60 minutes by nitrogen; 26.2 g (80mmol) of eicosanedioic acid was weighed, 30mL of DIPEA (160mmol) was added, and after 0.5 hour of reaction, 30mL of methanol and 11mL of DIPEA were added and the reaction was continued for 30 minutes. DMF was washed three times, and after methanol shrinkage, the Resin was drained to obtain 219.5 g total of didecyl diacid-2 CTC Resin, and the Resin substitution was calculated as 0.31mmol/g by Resin weight gain.
Example 3 solid phase Synthesis of Semetreuptatide side chain to C-terminus
45.5 g (10mmol) of octaneedioic acid-2CTC resin obtained in example 1 (substitution: 0.22mmol/g) was placed in a reaction column, washed 3 times with DMF, and swollen with DMF for 30 minutes. Then 6.87 g (30mmol) of H-Glu (OAll) -OtBu and 4.86 g (36mmol) of HOAt were weighed out, dissolved in DMF, 6.1mL DIC (36mmol) was added, the reaction column was added, the reaction was allowed to react for 2 hours, then the resin was washed 3 times with DMF, and 1.6 g of phenylsilane and 0.1 g of Pd were weighed out0(Ph3P)4800mL of DCM was measured out and added to the reaction column to conduct reaction for 30 minutes. The resin was then washed 3 times with DCM and 4 times with DMF. The above operation is repeated, and H-AEEA-OAll, Fmoc-Lys-OAll, H-Glu (OtBu) -OAll, H-Phe-OAll, H-Ile-OAll, H-Ala-OAll, H-Trp (Boc) -OAll, H-Leu-OAll, H-Val-OAll, H-Arg (Pbf) -OAll, H-Gly-OtBu are coupled according to the sequence in sequence. After the reaction was completed, the peptide resin was washed with DMF.
Example 4 solid phase Synthesis of Teteprepid side chain to C-terminus
The didecanedioic acid-2CTC resin obtained in example 2 (Sub ═ 0.31mmol/g)32.3 g (10mmol) was placed in a reaction column, washed 3 times with DMF and swollen with DMF for 30 min. Then 6.87 g (30mmol) of H-Glu (OAll) -OtBu and 6.87 g (36mmol) of T3P 11.5.5 g (36mmol) were weighed, dissolved in DMF, 0.4 g DMAP (3mmol) was added, the reaction column was added, the reaction was allowed to react for 2 hours, then the resin was washed 3 times with DMF, 1.6 g phenylsilane and 0.1 g Pd were weighed0(Ph3P)4800mL of DCM was measured out and added to the reaction column to conduct reaction for 30 minutes. The resin was then washed 3 times with DCM and 4 times with DMF. The above operation is repeated, and H-AEEA-OAll, Fmoc-Lys-OAll, H-Ala-OAll, H-Phe-OAll, H-Val-OAll, H-Gln (Trt) -OAll, H-Trp (Boc) -OAll, H-Leu-OAll, H-Ile-OAll, H-Ala-OAll, H-Gly-OAll, H-Pro-OAll, H-Ser (tBu) -OAll, H-Gly-OAll, H-Ala-OAll, H-Pro-OAll, H-Ser (tBu) -NH (Trt) are coupled in sequence. After the reaction was completed, the peptide resin was washed with DMF.
Example 5 Synthesis of Semetreuptatide peptide resin
The peptide resin obtained in example 3 was subjected to removal of the Fmoc protecting group with DBLK and then washed 6 times with DMF. Fmoc-Ala 9.35 g (30mmol), HOBt 4.86 g (36mmol) was weighed, dissolved in DMF, 6.1mL DIC (36mmol) was added in an ice water bath at 0 deg.C, activated for 5 min, added to the reaction column, reacted for 2 h, and then the Fmoc protecting group was removed with DBLK. The above-described procedure was repeated by coupling Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH according to the sequence, and after the reaction was completed, the peptide resin was washed with DMF 3 times. Washing with DCM again 3 times, followed by addition of methanol for 3 × 10 min, and vacuum drying gave 132.6 g of semaglutide peptide resin.
Example 6 Synthesis of telopeptide peptide resins
The peptide resin obtained in example 4 was subjected to removal of the Fmoc protecting group with DBLK and then washed 6 times with DMF. Fmoc-Gln (Trt) -OH 18.35 g (30mmol), HOAt 4.86 g (36mmol) were weighed, dissolved in DMF, 6.1mL DIC (36mmol) were added in an ice water bath at 0 deg.C, activated for 5 min, charged to the reaction column, reacted for 2 h, and then the Fmoc protecting group was removed with DBLK. The above procedure was repeated by coupling Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-Lys (tBu) -OH in the order. After the reaction was completed, the peptide resin was washed 3 times with DMF. Washing with DCM was again carried out 3 times, then methanol was added for 3X 10 minutes, and vacuum drying was carried out to obtain 136.2 g of telaprepin peptide resin.
Example 7 preparation of semaglutide
132.6 g of the peptide resin obtained in example 5 was added to a 2000mL single-neck flask, and lysate 1400mL of TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2Adding lysate into a flask, reacting at room temperature for 2.5 hours, filtering off resin, washing the resin with 50mL of TFA, combining filtrates, adding 14000mL of anhydrous ether to precipitate white solid, centrifuging, washing the solid with the anhydrous ether, and drying in vacuum to obtain 42.03 g of crude semaglutide peptide as the white solid, wherein the yield is 102.18%. HPLC purity 70.41%.
The crude peptide was prepared by HPLC to yield 14.5 g of semaglutide protamine with a purity of 99.07% and a total yield of 35.25%.
EXAMPLE 8 preparation of telaprepin
136.2 g of the peptide resin obtained in example 6 was added to a 2000mL single-neck flask to prepare a lysate of 1400mL TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2Adding lysate into flask, reacting at room temperature for 2.5 hr, filtering to remove resin, washing resin with 50mL of THF, mixing filtrates, adding 14000mL of anhydrous ether to precipitate white solid, centrifuging, washing solid with anhydrous ether, and vacuum dryingCrude telporopeptide peptide 40.18 g to a white solid in 97.69% yield. HPLC purity 71.33%.
The crude peptide was prepared by HPLC to obtain 13.8 g of telapraxin refined peptide with a purity of 98.89% and a total yield of 33.55%.
Example 9 preparation of Semetreuptade by Standard Fmoc solid phase peptide Synthesis method
Solid phase synthesis was performed using Wang Resin starting from C-terminal Gly in a conventional polypeptide solid phase reaction column using standard Fmoc solid phase peptide synthesis protocol, followed by coupling Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH; after the reaction is finished, removing Alloc protecting groups by using Pd, then sequentially coupling each residue of a side chain, and then cracking to obtain crude semaglutide peptide with the HPLC purity of 36.7% (figure 4).
The crude peptide is prepared by HPLC, so that the semaglutide refined peptide with the purity of 99.01 percent is obtained, and the total yield of the product is 10.32 percent.
From the above crude peptide purity and refined peptide yield obtained in examples 9 and 7, the synthesis by the method of the present invention, both crude peptide purity and refined peptide yield, was much better than standard Fmoc solid phase polypeptide synthesis.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, numerous process modifications and reagent substitutions can be made without departing from the principles of the present invention, and such modifications and substitutions should be considered to be within the scope of the present invention.
Claims (10)
1. A solid phase synthesis method of polypeptide containing long-chain fatty diacid side chain, wherein the polypeptide containing long-chain fatty diacid has a structure shown in a formula I;
wherein n is 4-30;
A1and A2Respectively is a polypeptide segment A1The polypeptide segment comprises 5-50 amino acid residues;
A2the polypeptide segment comprises 5-50 amino acid residues;
the method is characterized by comprising the following steps:
1) coupling a long-chain diacid compound containing 6-32 carbon atoms onto a solid-phase synthetic resin; obtaining long-chain diacid-solid phase synthetic resin conjugate;
2) coupling H-Glu (OAll) -OtBu on a long-chain diacid-solid phase synthetic resin conjugate;
3) removing OAll protecting groups, and then sequentially coupling H-AEEA-OAll, H-AEEA-OAll and Fmoc-Lys-OAll;
4) then coupled one by one for forming A2Protecting alpha-carboxyl amino acid or amido amino acid amide protected by carboxyl protecting group of the polypeptide segment;
5) fmoc protecting groups were removed and coupled one by one for A formation1Fmoc of the polypeptide segment protects amino acid of alpha amino;
6) cleaving all protecting groups while cleaving the resin to obtain a crude peptide;
7) purifying to obtain polypeptide containing long-chain fatty diacid;
wherein the polypeptide comprising a long chain fatty diacid is selected from Semaglutide (Semaglutide) or tipepide (Tirzepatide).
2. The solid-phase synthesis method according to claim 1, wherein the H-AEEA-OAll, and Fmoc-Lys-OAll are sequentially coupled in step 3) by coupling H-AEEA-OAll, removing OAll protecting groups, coupling Fmoc-Lys-OAll, and removing OAll protecting groups.
3. The solid-phase synthesis process according to claim 1, wherein in step 4), the coupling is extended from the N-terminus to the C-terminus on Lys coupled in step 3), and the amino acids are coupled in sequence by coupling the amino acid having both the side chain and the carboxyl group protected by the protecting group, then removing the protecting group for the carboxyl group, then coupling the amino acid having both the side chain and the carboxyl group protected by the protecting group, then removing the protecting group for the carboxyl group, and continuing the cycle until A is completed2A polypeptide fragment; wherein the carboxyl protecting groups of the amino acids other than the last amino acid coupled at the C-terminus are selected from OAll protecting groups;
the last amino acid carboxyl protecting group coupled to the C-terminus is selected from the group consisting of-OtBu protecting groups;
alternatively, the first and second electrodes may be,
coupling amino acid with side chain and carboxyl protected by protecting group, then removing carboxyl protecting group, continuously circulating to form polypeptide chain, and finally coupling amino acid amide with side chain and amide protected at C terminal end;
wherein the carboxyl protecting groups of the amino acids other than the last amino acid coupled at the C-terminus are selected from OAll protecting groups;
the amide protecting group of the last amino acid amide coupled at the C-terminus is selected from the Trt protecting groups.
4. The solid phase synthesis process of claim 1, wherein the coupling conditions in steps 2), 3), 4) and 5) are DIC in combination with A, or B, A in combination with C, or C in combination with D, wherein A is HOBt or HOAt or Oxyma, B is HBTU, HATU, PyBOP, C is DIPEA or TMP or DMAP, and D is T3P.
5. The solid-phase synthesis method of claim 1, wherein the OAll protecting group is removed in steps 3) and 4) by Pd0(Ph3P)4And PhSiH3Removing the OAll protecting group.
6. The solid phase synthesis method of claim 1, wherein Pd is0(Ph3P)4PhSiH in a molar amount of 0.01 to 1.0 molar amount of OAll protecting group3The molar amount of (a) is 1-10 times of the molar amount of the OAll protecting group.
7. The solid-phase synthesis method according to claim 1, wherein in step 5), the coupling is performed by extending from the C-terminus to the N-terminus on the Lys coupled in step 2), and the amino acids are sequentially coupled by coupling the amino acids having the side chains and the amino groups protected with the protecting groups, removing the protecting groups from the amino groups, and then coupling is performed until A is completed1A polypeptide fragment; wherein the amino protecting group is selected from the group consisting of Fmoc protecting groups.
8. The solid phase synthesis method according to claim 1, wherein the Fmoc protecting group is removed in step 5) by using a mixed solution of piperidine and DMF.
9. The solid phase synthesis method according to claim 1, wherein the cleavage agent used in the cleavage in step 6) is TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2O＝85-95:2-5:0-3:0-2:1-5。
10. The solid phase synthesis method of claim 1, wherein A is1The polypeptide segment is H-AA1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-AA10-Ser11-AA12-AA13-Leu14-AA15-AA16-AA17-Ala18-AA19-, wherein AA1Selected from His1Or Tyr1、AA10Selected from Val10Or Tyr10、AA12Is selected from Ser12Or Ile12、AA13Is selected from Tyr13Or Aib13、AA15Selected from Glu15Or Asp15、AA16Is selected from Gly16Or Lys16、AA17Selected from Gln17Or Ile17、AA19Is selected from Ala19Or Gln19(ii) a A is described2The polypeptide segment is-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH or is-Ala21-Phe22-Val23-Gln24-Trp25-Leu26-Ile27-Ala28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2。
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