CN112321699B - Synthesis method of semaglutide - Google Patents
Synthesis method of semaglutide Download PDFInfo
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- CN112321699B CN112321699B CN202011221694.3A CN202011221694A CN112321699B CN 112321699 B CN112321699 B CN 112321699B CN 202011221694 A CN202011221694 A CN 202011221694A CN 112321699 B CN112321699 B CN 112321699B
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Abstract
The invention relates to the field of polypeptide synthesis, and in particular relates to a synthetic method of semaglutide. According to the invention, an ionic liquid Linker is introduced on a conventional solid phase carrier, and then the Linker is sequentially connected with corresponding amino acids in a semaglutide sequence through a solid phase synthesis method, and the semaglutide protamine is obtained through cracking and purification. Compared with the conventional chemical synthesis method, the method provided by the invention has the advantages of simple process, low cost and high yield, changes the characteristics of a solid phase carrier and increases the swelling performance of resin in a solvent by increasing the ionic liquid Linker and exchanging anions, thereby improving the coupling efficiency of amino acid residues synthesized by a solid phase, solving the hydrophobic characteristic of PS resin, avoiding the peptide chain aggregation in the process of peptide chain extension and further solving the problems of difficult residue solid phase coupling and low yield of the self sequence of the seraglutide caused by beta-folding.
Description
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a synthetic method of GLP-1 analogue semaglutide.
Technical Field
Semaglutide is a long-acting GLP-1 analog developed by norand incorporated that requires only once weekly subcutaneous administration or once daily oral administration.
Structurally, semaglutide requires the attachment of two AEEA, gamma-glutamic acid and octadecanedioic acid fatty chains at Lys at position 20, with the unnatural amino acid aminoisobutyric acid at position 2. 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 existing synthesis process of the semaglutide comprises a preparation method of a product which is finished by combining a biological fermentation method and a chemical synthesis method selected in the original research (CN 101910193). After GLP-1(11-37) fragments are prepared by the method through synthesis and biological fermentation, the N-terminal amino group and the hydroxyl groups of Ser, Thr and Tyr side chains are not protected, and the fragments directly react with OSu ester of the side chains, so that multi-site reaction is easily caused, a large amount of unexpected impurities are formed, the method is not beneficial to later separation and purification, and the yield is lower.
The existing synthesis process of semaglutide also comprises a plurality of traditional polypeptide solid phase synthesis and fragment condensation synthesis methods (CN 103848910, CN 106928343, CN106478806, WO2016046753 and the like). In all these various methods, it is basically difficult to have a good synthesis effect by using a linear coupling method, mainly the polypeptide sequence structure of semaglutide itself, the N-terminal fragment: H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14Because of strong hydrophobicity, a secondary structure such as beta-sheet is easily formed, coupling is very difficult, and condensation efficiency is very low. Some methods of fragment condensation also aim to solve the problem of difficult linear coupling. However, some of the current methods of fragment condensation, when the synthesis scale is up to production, all of them affect the final synthesis effect, such as the dissolution of fragments, the coupling efficiency of fragments, and the exothermic problem of fragment dissolution. In addition, the fragment condensation is also complicated in operation, and is not favorable for industrial production.
In the current solid-phase synthesis of polypeptides, the commonly used synthesis strategy is Fmoc solid-phase synthesis, which generally employs solid-phase resins of the PS type (e.g. Wang resin, Rink resin, etc.), followed by coupling of the corresponding amino acids one by one. This conventional method, for some long peptides and peptide fragments with strong hydrophobicity, is easy to form beta-sheet secondary structure through intramolecular hydrogen bond during synthesis, thereby causing shrinkage of resin and resulting in coupling difficulty. For example, in solid phase synthesis, the simmerelutide is easy to synthesize poorly due to the formation of beta-sheet secondary structure. The reason for the above phenomenon is not only the peptide sequence itself, but also the strong hydrophobic property of the PS type resin itself used in the synthesis process, for example, the shrinkage of the resin is alleviated to some extent by using the PEG type resin.
Disclosure of Invention
In view of the above-mentioned disadvantages of the methods for synthesizing semaglutide in the prior art, the present invention provides a method for synthesizing semaglutide from Fmoc solid-phase polypeptide: a Linker of ionic liquid is additionally connected to a PS type solid phase carrier, and the hydrophobic property of the PS carrier is solved by utilizing the solubility property of the ionic liquid, so that the problem of poor coupling effect caused by the formation of a beta-folding secondary structure of partial residue of the semaglutide is solved.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for synthesizing semaglutide comprises the steps of coupling ionic liquid serving as a hydrophilic Linker onto solid-phase resin, sequentially connecting corresponding amino acids in a semaglutide sequence through a solid-phase synthesis method, and performing cracking and purification to obtain the semaglutide peptide.
In the technical scheme of the invention, the method for synthesizing the semaglutide comprises the following steps:
(1) coupling an ionic liquid Linker to the solid-phase resin;
(2) coupling Fmoc-Gly-OH to the solid-phase resin containing the ionic liquid Linker according to the first amino acid Gly at the C-terminal of the semaglutide;
(3) removing Fmoc protecting groups, and sequentially coupling by coupling Fmoc or Boc protected alpha amino acid or peptide segments according to the sequence of semaglutide and then removing the Fmoc protecting groups, wherein the coupling sequence of the Fmoc or Boc protected alpha amino acid is as follows: 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-Ser (tBu) -OH (Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-OtBu (Asp), (Asp, Fmoc-Val) -OH, Fmoc-Leu-OH, Fmoc-Leu-Asp (Ser-Leu) -OH, Fmoc-Leu-OH, Fmoc-Ser (Ser-Leu) -OH, Fmoc-Gly-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (Ser-OH, Fmoc-Gly-OH, Fmoc-Gly-Ser-Leu-OH, Fmoc-Ser-OH, Fmoc-Ser-OH, etc, 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;
(4) removing the Alloc protecting group from Lys (Alloc);
(5) coupling Fmoc-AEEA-OH according to the side chain structure of semaglutide, then removing Fmoc protecting groups, and then sequentially coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester;
(6) cracking the resin and the side chain protecting group to obtain crude peptide;
(7) and purifying to obtain the semaglutide protamine.
The ionic liquid Linker in the step (1) comprises 1- (2-hydroxyethyl) imidazole tetrafluoroborate ionic liquid; 1- (2-hydroxyethyl) imidazole hexafluorophosphate ionic liquid; 1- (2-hydroxyethyl) imidazole trifluoromethanesulfonic acid ionic liquid; 1- (2-hydroxyethyl) imidazole bistrifluoromethylsulfonyl imide liquid; 1- (3-hydroxypropyl) -1H-imidazole hexafluorophosphate ionic liquid; 1- (3-hydroxypropyl) -1H-imidazole tetrafluoroborate ionic liquid; 1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid ionic liquid; 1- (3-hydroxypropyl) -1H-imidazole bis (trifluoromethyl) sulfonyl imide ionic liquid and the like.
In the technical scheme of the invention, the coupling of the ionic liquid Linker to the solid-phase resin comprises the following steps: step one, reacting 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole with isobutene, extracting and crystallizing to obtain tert-butyl protected 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole; step two, adding the solid phase resin, reacting for 24 hours at 80 ℃ in N-methylpyrrolidone, carrying out suction filtration, washing the solid phase resin with DCM, adding a DCM solvent of 25% TFA, and reacting for 2 hours at room temperature to obtain the 1- (2-hydroxyethyl) imidazole solid phase resin without tert-butyl protection or the 1- (3-hydroxypropyl) -1H-imidazole solid phase resin without tert-butyl protection; and thirdly, respectively reacting the 1- (2-hydroxyethyl) imidazole solid-phase resin without the tert-butyl protection or the 1- (3-hydroxypropyl) -1H-imidazole solid-phase resin without the tert-butyl protection with sodium tetrafluoroborate, sodium hexafluorophosphate, sodium trifluoromethanesulfonate and lithium bistrifluoromethylsulfonyl imide through anion exchange resin to obtain the corresponding solid-phase resin taking the 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole ionic liquid as a Linker.
In the technical scheme of the invention, the solid phase resin is Merrifield resin, 2CTC resin or Wang resin.
In the technical scheme of the invention, Fmoc-Gly-OH is coupled to solid-phase resin of an ionic liquid Linker in the step (2), and the coupling of amino acid is specifically carried out by adopting Fmoc-Gly-OH and the solid-phase resin containing the ionic liquid Linker.
In the technical scheme of the invention, in the step (2), the substitution degree range of the solid-phase resin is 0.2-1.0mmol/g, and the condensing agent adopted for coupling is a combination of HOBt, DIC and DMAP, a combination of HOBt, DCC and DMAP, a combination of HOAt, DIC and DMAP, a combination of HBTU, HOBt and DIPEA, a combination of TBTU, HOBt and DIPEA, and a combination of PyBOP, HOBt and DIPEA; preferably a combination of HOBt, DIC, DMAP; the dosage of the condensing agent is 0.8 to 3.0 times of the molar weight of the amino acid, and the reaction time is 1 to 4 hours.
In the technical scheme of the invention, the condensing agent adopted in the coupling in the step (3) and the step (5) comprises DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU or PyBOP, and C is DIEA or TMP or DMAP; the dosage of the condensing agent is 0.8 to 3.0 times of the molar weight of the amino acid, and the reaction time is 1 to 4 hours.
In the technical scheme of the invention, in the step (4), the method for removing Alloc is to use Pd0(Ph3P)4And Me2NH·BH3Combination of (2) or Pd0(Ph3P)4And PhSiH3In which Pd is removed0(Ph3P)4The dosage of the compound is 0.05 to 1.0 time of the molar weight of the Alloc protecting group, preferably 0.1 to 0.5 time; me2NH·BH3Or PhSiH3The dosage of the compound is 10 to 100 times, preferably 20 to 60 times of the molar weight of the Alloc protecting group; the reaction time for removing the Alloc protecting group is 0.5 to 4 hours, preferably 1 to 2 hours.
In the technical scheme of the invention, in the step (6), during cracking, the peptide chain and the ionic liquid are cracked, and the ionic liquid and the Linker are cracked together from the resin. The cleavage reagent used for the cleavage is TFA, TIS, EDT, PhOH, H2Mixed solution of O, volume ratio TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2O-85-95: 2-5:0-3:0-2:1-5, with a cleavage time of 1.5-3.5 hours.
In the technical scheme of the present invention, the purification method in step (6) refers to a conventional purification method in the art, such as HPLC purification method.
Compared with the prior art, the invention has the following beneficial effects:
1. the method solves the problems of difficult research on the impurities of the semaglutide, difficult purification and lower yield caused by a biological fermentation method.
2. Compared with the conventional chemical synthesis method, the method provided by the invention has the advantages that the characteristics of the solid phase carrier are changed by adding the ionic liquid Linker and exchanging anions, and the swelling performance of the resin in a solvent is improved, so that the coupling efficiency of the solid phase synthesized amino acid residues is improved.
3. By adopting the method, the self hydrophilic property of the ionic liquid inhibits the semaglutide H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15The formation of hydrogen bonds in the molecule of the coupling difficult fragment avoids the formation of a beta-folding secondary structure, improves the coupling effect of the sequence, and greatly improves the purity and the synthesis yield of the final crude peptide.
4. The method can solve the hydrophobic property of the PS resin and avoid peptide chain aggregation in the peptide chain extension process, thereby solving the problems of difficult residue solid-phase coupling and lower yield of the semaglutide sequence caused by beta-folding, simultaneously solving the problem of difficult coupling of a semaglutide side chain long-chain fatty chain caused by strong hydrophobicity, and greatly improving the coupling effect of octadecanedioic acid.
5. By adopting the method, the synthesis of the semaglutide is completed without adopting a fragment condensation method, and a good effect can be obtained by direct solid-phase sequential coupling.
Drawings
FIG. 1: HPLC profile of crude semaglutide peptide from example 19.
FIG. 2: HPLC profile of crude semaglutide peptide from example 20.
FIG. 3: HPLC profile of crude semaglutide peptide from example 21.
FIG. 4: HPLC profile of crude semaglutide peptide from example 22.
FIG. 5: HPLC profile of crude semaglutide peptide from comparative example 1.
Detailed Description
The invention discloses a preparation method of semaglutide, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the preparation method. 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 Merrifield Resin and 1- (2-hydroxyethyl) imidazole solid phase Supports
Weighing 120mmol of 1- (2-hydroxyethyl) imidazole, dissolving in tetrahydrofuran, dropwise adding a proper amount of concentrated sulfuric acid, adding 180mmol of liquid isobutene under an ice bath condition, stirring overnight at normal temperature, extracting, and crystallizing to obtain the tert-butyl protected hydroxyl 1- (2-hydroxyethyl) imidazole (compound 1).
Merrifield Resin 125 g (100mmol, 0.8mmol/g) is weighed, added to the compound 1 obtained above, reacted in N-methylpyrrolidone at 80 ℃ for 24 hours, filtered, the solid phase Resin is washed with DCM, then added with DCM solvent of 25% TFA and reacted at room temperature for 2 hours to obtain 1- (2-hydroxyethyl) imidazole Merrifield Resin without tert-butyl protection.
Dividing 1- (2-hydroxyethyl) imidazole Merrifield Resin without tert-butyl protection into four parts, and respectively reacting with sodium tetrafluoroborate, sodium hexafluorophosphate, sodium trifluoromethanesulfonate and lithium bistrifluoromethylsulfonyl imide through anion exchange Resin to obtain corresponding Merrifield Resin (SCHEME 1) taking 1- (2-hydroxyethyl) imidazole ionic liquid as a Linker.
EXAMPLE 2 preparation of Merrifield Resin and 1- (3-hydroxypropyl) -1H-imidazole solid phase support
Referring to the synthesis method of example 1, 1- (3-hydroxypropyl) -1H-imidazole reacts with Merrifield Resin to prepare Merrifield Resin with 1- (3-hydroxypropyl) -1H-imidazole ionic liquid as Linker, and the specific structure is as follows:
EXAMPLE 3 preparation of Fmoc-Gly-1- (2-hydroxyethyl) imidazole hexafluorophosphate-Merrifield Resin
Weighing 20 g of 1- (2-hydroxyethyl) imidazole hexafluorophosphate-Merrifield Resin prepared in example 1 into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 4.2 g (14mmol) of Fmoc-Gly-OH, 2.2 g (16.8mmol) of HOBt and 0.2 g (1.4mmol) of DMAP, dissolving with DMF, adding 3.0ml of DIC (20mmol) in ice-water bath at 0 ℃, activating for 5 minutes, adding into a reaction column, reacting for 2 hours, adding 35ml of acetic anhydride and 30ml of pyridine, mixing and sealing for 24 hours, washing three times with DCM, draining the Resin after methanol contraction to obtain 22.4 g of 1- (2-hydroxyethyl) imidazole hexafluorophosphate-Merrifield Resin, wherein the detection substitution degree is 0.28 mmol/g.
EXAMPLE 4 preparation of Fmoc-Gly-1- (2-hydroxyethyl) imidazole tetrafluoroboric acid-Merrifield Resin
Weighing 20 g of 1- (2-hydroxyethyl) imidazole tetrafluoroborate-Merrifield Resin prepared in example 1 into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 4.2 g (14mmol) of Fmoc-Gly-OH, 2.2 g (16.8mmol) of HOBt and 0.2 g (1.4mmol) of DMAP, dissolving with DMF, adding 3.0ml of DIC (20mmol) in an ice-water bath at 0 ℃, activating for 5 minutes, adding the mixture into a reaction column, reacting for 2 hours, adding 35ml of acetic anhydride and 30ml of pyridine, mixing and sealing for 24 hours, washing three times with DCM, draining the Resin after methanol contraction to obtain 23.2 g of 1- (2-hydroxyethyl) imidazole tetrafluoroboric acid-Merrifield Resin, and detecting the substitution degree to be 0.26 mmol/g.
EXAMPLE 5 preparation of Fmoc-Gly-1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid-Merrifield Resin
Weighing 20 g of 1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid-Merrifield Resin prepared in example 2 into a solid-phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 4.2 g (14mmol) of Fmoc-Gly-OH, 2.2 g (16.8mmol) of HOBt and 0.2 g (1.4mmol) of DMAP, dissolving with DMF, adding 3.0ml of DIC (20mmol) in ice-water bath at 0 ℃, activating for 5 minutes, adding into a reaction column, reacting for 2 hours, adding 35ml of acetic anhydride and 30ml of pyridine, mixing and sealing for 24 hours, washing three times with DCM, draining the Resin after methanol contraction to obtain 21.8 g of 1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid-Merrifield Resin in total, wherein the detection substitution degree is 0.30 mmol/g.
EXAMPLE 6 preparation of Fmoc-Gly-1- (3-hydroxypropyl) -1H-imidazole bistrifluoromethylsulfonimide-Merrifield Resin
Weighing 20 g of 1- (3-hydroxypropyl) -1H-imidazole bistrifluoromethylsulfonyl imide-Merrifield Resin prepared in example 2 into a solid phase reaction column, adding DMF, and carrying out bubbling with nitrogen for swelling for 60 minutes; weighing 4.2 g (14mmol) of Fmoc-Gly-OH, 2.2 g (16.8mmol) of HOBt and 0.2 g (1.4mmol) of DMAP, dissolving with DMF, adding 3.0ml of DIC (20mmol) in ice-water bath at 0 ℃, activating for 5 minutes, adding the mixture into a reaction column, reacting for 2 hours, adding 35ml of acetic anhydride and 30ml of pyridine, mixing and sealing for 24 hours, washing three times with DCM, draining the Resin after methanol contraction to obtain 22.8 g of 1- (3-hydroxypropyl) -1H-imidazole bistrifluoromethylsulfonimide-Merrifield Resin, wherein the detection substitution degree is 0.27 mmol/g.
Example 7 preparation of a Semetreuptade backbone peptide resin
17.8 g (5mmol) of Fmoc-Gly-1- (2-hydroxyethyl) imidazole hexafluorophosphate-Merrifield Resin (Sub ═ 0.28mmol/g) obtained in example 3 was weighed into the reaction column, washed 3 times with DCM and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. Fmoc-Arg (Pbf) -OH 9.7 g (15mmol), HOBt 2.4 g (18mmol) were weighed, dissolved in DMF, 3.1ml DIC (18mmol) was added in an ice water bath at 0 ℃ to activate for 5 minutes, the reaction column was charged, reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. Repeating the above procedure by coupling 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-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asp (Ser (tBu) -OH, Fmoc-Val-Leu-Asp (OtBu) -OH, Fmoc-Leu-Asp (TaBu) -OH, Fmoc-Asp (TaOH, Fmoc) and Fmoc-Lys (Boc) -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 was completed, the peptide resin was washed with DMF.
Example 8 preparation of a Semetreuptade backbone peptide resin
19.2 g (5mmol) of Fmoc-Gly-1- (2-hydroxyethyl) imidazole tetrafluoroborate-Merrifield Resin (Sub ═ 0.26mmol/g) obtained in example 4 was weighed in a reaction column, and the same coupling method as in example 7 was followed to obtain the corresponding semaglutide backbone peptide Resin.
Example 9 preparation of a Semetreuptade backbone peptide resin
16.7 g (5mmol) of Fmoc-Gly-1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid-Merrifield Resin (Sub ═ 0.30mmol/g) obtained in example 5 was weighed into a reaction column, and the same coupling procedure as in example 7 was followed to obtain the corresponding semaglutide backbone peptide Resin.
Example 10 preparation of a Semetreuptade backbone peptide resin
18.5 g (5mmol) of Fmoc-Gly-1- (3-hydroxypropyl) -1H-imidazole bistrifluoromethylsulfonimide-Merrifield Resin (Sub ═ 0.27mmol/g) obtained in example 6 was weighed into a reaction column and coupled according to the same coupling method as in example 7 to obtain the corresponding semaglutide backbone peptide Resin.
Example 11 removal of Lys side chain protecting groups
The fully protected peptide resin obtained in example 7 was washed 3 times with DCM. 11.8 g of dimethylamine borane was weighed, 800ml of DCM was weighed, the mixture was put into a reaction column, and after 10 minutes of reaction, 0.58 g of Pd was added0(Ph3P), reaction for 2 hours. The resin was then washed 3 times with DCM, 3 times with DMF and 3 times with DCM to give a selectively Alloc-removed peptide resin for use.
Example 12 removal of Lys side chain protecting groups
The fully protected peptide resin obtained in example 8 was washed 3 times with DCM. 5.4 g of phenylsilane was weighed, 800ml of DCM was added to the reaction column, and after 10 minutes of reaction, 0.58 g of Pd was added0(Ph3P)4And reacting for 2 hours. The resin was then washed 3 times with DCM, 3 times with DMF and 3 times with DCM to give a selectively Alloc-removed peptide resin for use.
Example 13 removal of Lys side chain protecting groups
The fully protected peptide resin obtained in example 9 was subjected to the same procedure as in example 11 to thereby complete the removal of the Lys side chain Alloc protecting group.
Example 14 removal of Lys side chain protecting groups
The fully protected peptide resin obtained in example 10 was subjected to the same procedure as in example 12 to thereby complete the removal of the Lys side chain Alloc protecting group.
Example 15 coupling of the Semetreuptade side chain
6.0 g (10mmol) of Fmoc-AEEA-OH, 2.43 g (18mmol) of HOAt were weighed out, dissolved in DMF, 3.1ml of DIC (18mmol) were added in an ice-water bath at 0 ℃ to activate for 5 minutes, the peptide resin from example 11 was added, reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. Repeating the operation, and coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester according to the sequence; after the reaction was completed, the reaction mixture was washed 6 times with DMF, again 3 times with DCM, then washed 3X 10 minutes with methanol and dried under vacuum to give 59.4 g of a peptide resin of semaglutide.
Example 16 coupling of the Semetreuptade side chain
The peptide resin of example 12 was removed and coupled in exactly the same manner as in example 15 to give 62.6 g of semaglutide peptide resin.
Example 17 conjugation of Semetreuptade side chains
The peptide resin of example 13 was removed and coupled in exactly the same manner as in example 15 to give 58.3 g of semaglutide peptide resin.
Example 18 coupling of the Semetreuptade side chain
The peptide resin of example 14 was removed and coupled in exactly the same manner as in example 15 to give 60.5 g of semaglutide peptide resin.
Example 19 preparation of Semetreuptade
59.4 g of the peptide resin obtained in example 15 was added to a 2000ml single-neck flask, and lysate 600ml of TFA was prepared in advance: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: H2O ═ 90:3:3:2:2 (vol.%), the lysate was added to the flask, the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50ml tfa, the filtrates were combined, 6000ml of anhydrous ether was added to precipitate a white solid, the solid was centrifuged, the solid was washed with anhydrous ether, and 20.2 g of crude semaglutide peptide was dried in vacuo to a white solid, with a yield of 98.24%. HPLC purity 78.48%.
The crude peptide was purified by HPLC to obtain 8.80 g of semaglutide protamine with a purity of 99.25% and a total yield of 42.78%.
Example 20 preparation of Semetreuptade
62.6 g of the peptide resin obtained in example 16 was charged into a 2000ml single-neck flask, and a lysate of 650ml of TFA was prepared: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: H2O ═ 90:3:3:2:2 (vol.%), the lysate was added to the flask, the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50ml tfa, the filtrates were combined, the resulting mixture was added to 6500ml of anhydrous ether to precipitate a white solid, the solid was centrifuged, washed with anhydrous ether, and 21.02 g of crude semaglutide peptide was dried in vacuo to a white solid, with a yield of 102.13%. HPLC purity 85.06%.
The crude peptide was purified by HPLC to obtain 8.94 g of semaglutide refined peptide with a purity of 99.30% and a total yield of 43.46%.
Example 21 preparation of Semetreuptade
58.3 g of the peptide resin obtained in example 17 were charged into a 2000ml single-neck flask, and lysate 600ml of TFA was prepared in advance: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: H2O ═ 90:3:3:2:2 (vol.%), the lysate was added to the flask, the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50ml tfa, the filtrates were combined, 6000ml of anhydrous ether was added to precipitate a white solid, the solid was centrifuged, the solid was washed with anhydrous ether, and 19.98 g of crude semaglutide peptide was dried in vacuo to a white solid, with a yield of 98.41%. HPLC purity 84.66%.
The crude peptide is purified by HPLC, 8.64 g of the semaglutide refined peptide with the purity of 99.09 percent is obtained, and the total yield of the product is 42.00 percent.
Example 22 preparation of Semetreuptade
60.5 g of the peptide resin obtained in example 18 was charged into a 2000ml single-neck flask, and lysate 600ml of TFA was prepared in advance: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2Adding the lysate into a flask, reacting at room temperature for 2.5 hours, filtering off the resin, washing the resin with 50ml of TFA, combining filtrates, adding 6000ml of anhydrous ether to precipitate a white solid, centrifuging, washing the solid with the anhydrous ether, and drying in vacuum to obtain 20.56 g of crude semaglutide peptide as the white solid with the yield of 100.00 percent, wherein O is 90:3:3:2:2 (volume ratio). HPLC purity 84.21%.
The crude peptide was purified by HPLC to obtain 8.73 g of semaglutide protamine with a purity of 99.36% and a total yield of 42.44%.
Comparative example 1 preparation of semaglutide by standard Fmoc solid phase peptide Synthesis procedure
The synthesis of semaglutide was completed using standard Fmoc solid phase peptide synthesis, i.e., using standard Wang Resin, following the same synthesis procedure as described in examples 3-22 above, and the final resulting crude semaglutide peptide had an HPLC purity of 36.7% (fig. 5). After purification by HPLC, the overall yield of product was 10.32%.
From the comparative analysis of the results of comparative example 1 and examples 19, 20, 21 and 22, the synthesis of semaglutide was performed after introducing an ionic liquid Linker on the PS resin solid phase support, and the crude peptide purity and the refined peptide yield were much better than those of the standard Fmoc solid phase polypeptide synthesis method.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that several modifications and partial solvent changes can be made without departing from the principle of the present invention, and these modifications and partial solvent changes should be also considered as the protection scope of the present invention.
Claims (8)
1. A method for synthesizing semaglutide is characterized in that ionic liquid is used as a hydrophilic Linker to be coupled to solid-phase resin, then the ionic liquid is sequentially connected with corresponding amino acids in a semaglutide sequence through a solid-phase synthesis method, and the semaglutide fine peptide is obtained through cracking and purification;
which comprises the following steps:
(1) coupling an ionic liquid Linker to the solid-phase resin;
(2) coupling Fmoc-Gly-OH to the solid-phase resin containing the ionic liquid Linker according to the first amino acid Gly at the C-terminal of the semaglutide;
(3) removing Fmoc protecting groups, and sequentially coupling by coupling Fmoc or Boc protected alpha amino acid or peptide segments according to the sequence of semaglutide and then removing the Fmoc protecting groups, wherein the coupling sequence of the Fmoc or Boc protected alpha amino acid is as follows: 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-Ser (tBu) -OH (Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-OtBu (Asp), (Asp, Fmoc-Val) -OH, Fmoc-Leu-OH, Fmoc-Leu-Asp (Ser-Leu) -OH, Fmoc-Leu-OH, Fmoc-Ser (Ser-Leu) -OH, Fmoc-Gly-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (Ser-OH, Fmoc-Gly-OH, Fmoc-Gly-Ser-Leu-OH, Fmoc-Ser-OH, Fmoc-Ser-OH, etc, 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;
(4) removing the Alloc protecting group from Lys (Alloc);
(5) coupling Fmoc-AEEA-OH according to the side chain structure of the semaglutide, removing an Fmoc protective group, and then sequentially coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester;
(6) cracking the resin and the side chain protecting group to obtain crude peptide;
(7) purifying to obtain the semaglutide protamine;
wherein the ionic liquid is selected from imidazolium ionic liquids; the imidazolium ionic liquid comprises 1- (2-hydroxyethyl) imidazolium tetrafluoroborate ionic liquid, 1- (2-hydroxyethyl) imidazolium hexafluorophosphate ionic liquid, 1- (2-hydroxyethyl) imidazolium trifluoromethanesulfonate ionic liquid and 1- (3-hydroxypropyl) -1H-imidazolium hexafluorophosphate ionic liquid, one of 1- (3-hydroxypropyl) -1H-imidazole tetrafluoroborate ionic liquid, 1- (3-hydroxypropyl) -1H-imidazole trifluoromethanesulfonic acid ionic liquid, 1- (2-hydroxyethyl) imidazole bis-trifluoromethylsulfonyl imide liquid and 1- (3-hydroxypropyl) -1H-imidazole bis-trifluoromethylsulfonyl imide ionic liquid;
the cracking reagent adopted in the cracking in the step (6) is TFA, TIS, EDT, PhOH and H2Mixed solution of O, volume ratio TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2O-90: 3:3:2:2, cleavage time 1.5-3.5 hours.
2. The method for synthesizing semaglutide according to claim 1, wherein coupling an ionic liquid Linker to a solid phase resin comprises the steps of: step one, reacting 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole with isobutene, extracting and crystallizing to obtain tert-butyl protected 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole; step two, adding the solid phase resin, reacting for 24 hours at 80 ℃ in N-methylpyrrolidone, carrying out suction filtration, washing the solid phase resin with DCM, adding a DCM solvent of 25% TFA, and reacting for 2 hours at room temperature to obtain the 1- (2-hydroxyethyl) imidazole solid phase resin without tert-butyl protection or the 1- (3-hydroxypropyl) -1H-imidazole solid phase resin without tert-butyl protection; and thirdly, respectively reacting the 1- (2-hydroxyethyl) imidazole solid-phase resin without the tert-butyl protection or the 1- (3-hydroxypropyl) -1H-imidazole solid-phase resin without the tert-butyl protection with sodium tetrafluoroborate, sodium hexafluorophosphate, sodium trifluoromethanesulfonate and lithium bistrifluoromethylsulfonyl imide through anion exchange resin to obtain the corresponding solid-phase resin taking the 1- (2-hydroxyethyl) imidazole or 1- (3-hydroxypropyl) -1H-imidazole ionic liquid as a Linker.
3. The method of synthesizing semaglutide according to claim 1 or 2, wherein said solid phase resin is selected from the group consisting of Merrifield resin, 2CTC resin or Wang resin.
4. The method for synthesizing semaglutide as claimed in claim 1, wherein said step (2) comprises coupling Fmoc-Gly-OH to a solid-phase resin containing an ionic liquid Linker, specifically coupling amino acids using Fmoc-Gly-OH and the solid-phase resin containing the ionic liquid Linker.
5. The process for the synthesis of semaglutide according to claim 4, wherein in step (2), the degree of substitution of the solid phase resin is in the range of 0.2 to 1.0mmol/g, the condensing agent used for the coupling is a combination of HOBt, DIC, DMAP, a combination of HOBt, DCC, DMAP, a combination of HOAt, DIC, DMAP, a combination of HBTU, HOBt, DIPEA, a combination of TBTU, HOBt, DIPEA, a combination of PyBOP, HOBt, DIPEA; the dosage of the condensing agent is 0.8 to 3.0 times of the molar weight of the amino acid, and the reaction time is 1 to 4 hours.
6. The method of claim 5, wherein the condensing agent used for coupling is a combination of HOBt, DIC, DMAP.
7. The process for the synthesis of semaglutide according to claim 1, wherein said coupling of step (3) and step (5) is carried out using a condensing agent comprising DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU, PyBOP, and C is DIEA or TMP or DMAP; the dosage of the condensing agent is 0.8 to 3.0 times of the molar weight of the amino acid, and the reaction time is 1 to 4 hours.
8. The process for the synthesis of semaglutide as claimed in claim 1, wherein in said step (4), the method for removing Alloc is Pd0(Ph3P)4And Me2NH·BH3Combination of (2) or Pd0(Ph3P)4And PhSiH3In which Pd is removed0(Ph3P)4The dosage of the Alloc protective group is 0.1 to 0.5 time of the molar weight of the Alloc protective group; me2NH·BH3Or PhSiH3The dosage of the Alloc protective group is 20 to 60 times of the molar weight of the Alloc protective group; the reaction time for removing the Alloc protecting group is 1-2 hours.
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