CN116023467A - Solid-phase synthesis method of abamectin - Google Patents
Solid-phase synthesis method of abamectin Download PDFInfo
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- CN116023467A CN116023467A CN202211708473.8A CN202211708473A CN116023467A CN 116023467 A CN116023467 A CN 116023467A CN 202211708473 A CN202211708473 A CN 202211708473A CN 116023467 A CN116023467 A CN 116023467A
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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
- 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 provides a solid-phase synthesis method of abamectin, belonging to the field of pharmaceutical chemistry. According to the method, 1-12 fragments, 13-23 fragments and 24-34 fragments are synthesized according to the amino acid sequence from the C end to the N end of the peptide chain of the abamectin, and 3 fragments are coupled to obtain the abamectin. The solid-phase synthesis method of the abamectin has the advantages of low cost, simplicity in operation, high yield and high purity, and is suitable for mass production in factories.
Description
Technical Field
The invention belongs to the field of polypeptide pharmaceutical chemistry, and particularly relates to a solid-phase synthesis method of abamectin.
Background
Abapatadine (abatoparatid) english name: abalopratides; the peptide sequence is: ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NH 2 。
Abalapatide (Abalopatide) was originally developed by Issen pharmaceutical company, france, and was licensed to the United states radio Health biopharmaceutical company for sale and sale in the United states. Abaparib is an artificially synthesized 34-amino acid polypeptide, is an analogue of parathyroid hormone-related protein, and is used for treating postmenopausal women at risk of osteoporosis by selectively activating a signaling pathway of parathyroid hormone type 1 receptor, regulating metabolism and promoting bone formation. The U.S. Food and Drug Administration (FDA) was approved for marketing, under the trade name Tymlos, at 28, 4, 2017.
Postmenopausal osteoporosis (postmenopausal osteoporosis, POP) is a common frequently occurring occurrence associated with aging. The estrogen levels in postmenopausal women decrease and the coupling of bone formation to bone resorption is imbalanced, resulting in a systemic metabolic bone disease with reduced bone mass and damaged bone tissue microstructure. With the aging of the population of China, POPs gradually become serious public health and social problems with high morbidity and high disability rate.
The existing synthesis process of the abamectin has the problems of more impurities, low purity and yield, high cost, complicated operation steps, excessive waste liquid and adverse industrial production, and has important significance in researching a novel synthesis method of the abamectin.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides an optimized solid-phase synthesis method of abamectin. The method improves the purity of the crude peptide, greatly reduces the material cost and the purification cost, and is beneficial to industrialized amplified production.
The technical scheme adopted by the invention for achieving the purpose is as follows: the invention discloses a solid-phase synthesis method of abamectin, which comprises the following steps:
A. sequentially coupling to form Fmoc-Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala amino resin according to an Fmoc protection strategy, and removing Fmoc protecting groups to obtain a peptide resin fragment 1;
sequentially coupling to form Fmoc-Gly-Lys (Boc) -Ser (tBu) -Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Arg (Pbf) -Arg (Pbf) -Glu (OtBu) -2CTC resin according to Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 2;
sequentially coupling to form Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -2-CTC resin according to Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 3;
B. coupling reaction is carried out on the C end of the peptide fragment 2 and the N end of the peptide resin fragment 1 to obtain peptide resin I;
C. removing N-terminal protecting group of the peptide resin I, and performing coupling reaction with C-terminal of the peptide fragment 3 to obtain peptide resin II;
D. adding the lysate to remove the resin of the peptide resin II and all protecting groups to obtain the abamectin.
The solid-phase synthesis method of the abamectin provided by the invention is further preferred in the technical scheme that: condensing agents used for synthesizing the respective amino acids are one or more of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
The solid-phase synthesis method of the abamectin provided by the invention is further preferred in the technical scheme that: the reaction solvent used is one or more of DCM, DMF, NMP, DMSO.
The solid-phase synthesis method of the abamectin provided by the invention is further preferred in the technical scheme that: the cleavage reagent was TFA: EDT: tis=95:2.5:2.5.
Compared with the prior art, the invention has the following beneficial effects.
The method adopts a fragment method for coupling to prepare, synthesizes 1-12 fragments, 13-23 fragments and 24-34 fragments according to the amino acid sequence from the C end to the N end of a peptide chain of the abamectin, and couples 3 fragments to obtain the abamectin. The method improves the purity of the crude peptide, greatly reduces the material cost and the purification cost, and is beneficial to industrialized amplified production.
Drawings
FIG. 1 is a chromatogram of a crude peptide obtained in example 1 of the present invention;
FIG. 2 is a chromatogram of a crude peptide obtained in example 2 of the present invention;
FIG. 3 is a chromatogram of a crude peptide obtained in example 3 of the present invention.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1: preparation of Abaparib peptide resin (sequential Synthesis)
30.00g (sub=0.33 mmol/g) of Rink Amide-MBHA resin was weighed into a solid phase reactor and 200mL of DCM swollen resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 7+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Ala-OH 31.15g,HOBt 4.82g,DIC 5.5mL,200mL DMF solution was weighed and activated for 10 minutes in an ice bath at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
Repeating the above steps according to the amino acid sequences Fmoc-Ala-OH, fmoc-Thr (tBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Aib-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Arg (Pbf) -OH,
Fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Asp (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ile-OH, fmoc-Ser (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Gly-OH, fmoc-Lys (Boc) -OH, fmoc-Asp (OtBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Gln (Trt) -OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Ala-OH, and the coupling reaction was performed. After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by 200mL of v/v 20% piperidine/DMF solution and reaction for 7+15min. The solution is drained, DCM and methanol are alternately washed for 3 times, 400mL each time, and the indene detection result is positive. Vacuum drying to obtain the Abapatitin resin.
Cleavage of
300mL of cleavage reagent is prepared as TFA: EDT: TIS=95:2.5:2.5, full-protection peptide is added under ice bath condition, reaction is continued for 2h at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The product obtained after drying is the crude peptide of the abamectin, 20.21g of crude peptide is obtained, the ration of the crude peptide by a reference substance is 5.26g, the total yield is 5.82%, and the purity is 59.26%. The chromatogram of the crude peptide is shown in FIG. 1.
Example 2: preparation of Abaparib peptide resin (Fmoc-Aib-Lys (Boc) -OH Fmoc-Leu-Leu-OH the dipeptide fragments described above were all commercially available and met the pharmaceutical manufacturing criteria)
30.00g (sub=0.33 mmol/g) of Rink Amide-MBHA resin was weighed into a solid phase reactor and 200mL of DCM swollen resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 7+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Ala-OH 31.15g,HOBt 4.82g,DIC 5.5mL,200mL DMF solution was weighed and activated for 10 minutes in an ice bath at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
Repeating the above steps according to the amino acid sequences Fmoc-Ala-OH, fmoc-Thr (tBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Aib-Lys (Boc) -OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Arg (Pbf) -OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Asp (OtBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ile-OH, fmoc-Ser (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Gly-OH, fmoc-Lys (Boc) -OH, fmoc-Asp (OtBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Gln (Trt) -OH, fmoc-His (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Ala-OH. After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by 200mL of v/v 20% piperidine/DMF solution and reaction for 7+15min. The solution is drained, DCM and methanol are alternately washed for 3 times, 400mL each time, and the indene detection result is positive. Vacuum drying to obtain the Abapatitin resin.
Cracking:
300mL of cleavage reagent is prepared as TFA: EDT: TIS=95:2.5:2.5, full-protection peptide is added under ice bath condition, reaction is continued for 2h at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The product obtained after drying is the crude peptide of the abamectin, 25.83g of crude peptide is obtained, the ration of the crude peptide by a reference substance is 10.46g, the total yield is 23.14%, and the purity is 67.44%. The chromatogram of the crude peptide is shown in FIG. 2.
Example 3: preparation of Abaparib peptide resins (peptide resin fragment 1, peptide fragment 2, peptide fragment 3)
30.00g (sub=0.33 mmol/g) of Rink Amide-MBHA resin was weighed into a solid phase reactor and 200mL of DCM swollen resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 7+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Ala-OH 31.15g,HOBt 4.82g,DIC 5.5mL,200mLDMF solution was weighed and activated for 10 minutes in an ice bath at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The above steps were repeated, and the coupling reaction was performed according to the amino acid sequences Fmoc-Ala-OH, fmoc-Thr (tBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Aib-OH, fmoc-Leu-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Leu-OH. After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by 200mL of v/v 20% piperidine/DMF solution and reaction for 7+15min. And (3) pumping, washing the DCM and the methanol alternately for 3 times, wherein 400mL each time, and the indene detection result is positive, so as to obtain a peptide resin fragment 1, sequentially coupling the peptide resin fragment 1 and the Fmoc-Gly-Lys (Boc) -Ser (tBu) -Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Arg (Pbf) -Arg (Pbf) -Glu (OtBu) -2CTC resin according to an Fmoc protection strategy, and adding a lysate for full-protection cleavage to remove the 2-CTC resin, thereby obtaining the peptide fragment 2.
Sequentially coupling according to Fmoc protection strategy to form Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -2CTC resin, adding a lysate to perform full-protection cleavage to remove the 2-CTC resin, obtaining a peptide fragment 3, and performing a coupling reaction between the C end of the peptide fragment 2 and the N end of the peptide resin fragment 1 to obtain peptide resin I; removing N-terminal protecting group of peptide resin I, and coupling with C-terminal of peptide fragment 3 to obtain peptide resin II so as to obtain the invented Abaparib peptide resin.
Cracking:
300mL of cleavage reagent is prepared as TFA: EDT: TIS=95:2.5:2.5, full-protection peptide is added under ice bath condition, reaction is continued for 2h at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The crude peptide was 34.72g, the crude peptide was quantified by 25.33g as a control, the total yield was 61.32%, the purity was 98.13%, and the chromatogram of the crude peptide was shown in FIG. 3.
Claims (4)
1. A method for solid phase synthesis of abamectin, characterized in that it comprises the following steps:
A. sequentially coupling to form Fmoc-Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala amino resin according to an Fmoc protection strategy, and removing Fmoc protecting groups to obtain a peptide resin fragment 1;
sequentially coupling to form Fmoc-Gly-Lys (Boc) -Ser (tBu) -Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Arg (Pbf) -Arg (Pbf) -Glu (OtBu) -2CTC resin according to Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 2;
sequentially coupling to form Fmoc-Ala-Val-Ser (tBu) -Glu (OtBu) -His (Trt) -Gln (Trt) -Leu-Leu-His (Trt) -Asp (OtBu) -Lys (Boc) -2-CTC resin according to Fmoc protection strategy, adding a lysate to remove the 2-CTC resin, and obtaining a peptide fragment 3;
B. coupling reaction is carried out on the C end of the peptide fragment 2 and the N end of the peptide resin fragment 1 to obtain peptide resin I;
C. removing N-terminal protecting group of the peptide resin I, and performing coupling reaction with C-terminal of the peptide fragment 3 to obtain peptide resin II;
D. adding the lysate to remove the resin of the peptide resin II and all protecting groups to obtain the abamectin.
2. The solid phase synthesis method of abamectin according to claim 1, characterized in that the condensing agent used for synthesizing each amino acid is one or more of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
3. The solid phase synthesis method of abamectin as claimed in claim 1, wherein the reaction solvent used is one or a combination of DCM, DMF, NMP, DMSO.
4. The solid phase synthesis method of abamectin according to claim 1, characterized in that the cleavage reagent is TFA: EDT: tis=95:2.5:2.5.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108047329A (en) * | 2018-02-01 | 2018-05-18 | 润辉生物技术（威海）有限公司 | A kind of preparation method of A Bapa peptides |
| WO2019175173A1 (en) * | 2018-03-12 | 2019-09-19 | Fresenius Kabi Ipsum S.R.L. | Process for the manufacture of pthrp analogue |
| WO2020047994A1 (en) * | 2018-09-06 | 2020-03-12 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method of abaloparatide |
| CN113880936A (en) * | 2021-12-07 | 2022-01-04 | 浙江湃肽生物有限公司南京分公司 | Solid-phase synthesis method of abamectin |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108047329A (en) * | 2018-02-01 | 2018-05-18 | 润辉生物技术（威海）有限公司 | A kind of preparation method of A Bapa peptides |
| WO2019175173A1 (en) * | 2018-03-12 | 2019-09-19 | Fresenius Kabi Ipsum S.R.L. | Process for the manufacture of pthrp analogue |
| WO2020047994A1 (en) * | 2018-09-06 | 2020-03-12 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method of abaloparatide |
| CN110878119A (en) * | 2018-09-06 | 2020-03-13 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method of abamectin |
| CN113880936A (en) * | 2021-12-07 | 2022-01-04 | 浙江湃肽生物有限公司南京分公司 | Solid-phase synthesis method of abamectin |
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