CN112279892A - Improved gonadorelin solid-phase synthesis method - Google Patents
Improved gonadorelin solid-phase synthesis method Download PDFInfo
- Publication number
- CN112279892A CN112279892A CN202011087407.4A CN202011087407A CN112279892A CN 112279892 A CN112279892 A CN 112279892A CN 202011087407 A CN202011087407 A CN 202011087407A CN 112279892 A CN112279892 A CN 112279892A
- Authority
- CN
- China
- Prior art keywords
- gonadorelin
- gly
- resin
- synthesis
- fmoc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an improved gonadorelin solid-phase synthesis method, which comprises the following steps: 1) taking amide resin as a solid phase carrier, taking Fmoc protected amino acid as a monomer, taking Pyr with the end protected by Boc and taking piperazine as a decapping reagent, and sequentially and one by one connecting amino acid under the action of a condensing agent to synthesize gonadorelin; 2) cutting peptides by using a peptide cutting reagent, and concentrating to obtain a gonadorelin crude product; the method uses the piperazine solution as a uncapping reagent to synthesize the gonadorelin, the piperazine is a non-toxicity-making reagent and is in a solid form, the gonadorelin is convenient to purchase, transport and store, the quality guarantee period is long, the price is low, the purity of the synthesized gonadorelin crude product is high, the product can be more easily refined, the method is suitable for large-scale industrial production, no toxic and easily-making reagent is used, no violent chemical reaction exists, and the automatic production is convenient.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an improved gonadorelin solid-phase synthesis method.
Background
Gonadorelin, the amino acid sequence of which is: 5-Oxyprolinyl-L-a group of aminoacyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl-Lleucyl-L-arginyl-L-prolyl-glycinamide (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH)2) Molecular formula is C55H75N17O13And the molecular weight is 1182.33.
Gonadorelin is an artificially synthesized gonadotropin releasing hormone, belongs to a peptide compound, is decapeptide, can stimulate pituitary to synthesize and release follicle stimulating hormone and luteinizing hormone (FSH and LH), and is mainly clinically used for identifying and diagnosing male or female fertility disorders caused by hypothalamus or hypophysis dysfunction, gonadal atrophic gonadal insufficiency, galactorrhea amenorrhea, primary and secondary amenorrhea, menopause and premature menopause, pituitary tumor, organ injury of pituitary, actual hypothalamic dysfunction and the like. Indications include:
1) for diagnosing hypothalamic-pituitary-gonadal dysfunction;
2) treating amenorrhea, hypogonadotropic hormone hyposecretion and infertility caused by follicular ovary;
3) gonadorelin or its congeners buserelin, goserelin, leuprorelin, nafarelin and triptorelin are also useful for contraception, cryptorchidism, malignancies (especially prostate cancer), delayed and advanced puberty;
4) can also be used for endometriosis;
5) for use in ovulation induction in the treatment of infertility due to hypothalamic amenorrhea, primary ovarian dysfunction, particularly in patients who are clomiphene ineffective;
6) it is also used for treating infantile cryptorchidism, androgen excess, pituitary tumor, etc. As a veterinary drug, the compound can promote the adenohypophysis of animals to release follicle stimulating hormone and luteinizing hormone, is used for treating the ovarian function stop of cows and inducing the cows to have estrus synchronously.
The method for synthesizing gonadorelin in the prior art is less reported, Chinese patent application 201710439767.8 reports a method for synthesizing gonadorelin by a solid phase synthesis method, RinkAmideAM resin is adopted as a solid phase carrier, a condensing agent regulating member is HBTU/HOBt/DIPEA, Fmoc amino acid is a monomer, piperidine is a deprotection reagent, then coupling is carried out one by one from a C end to an N end until a peptide chain of the gonadorelin resin is synthesized and protected, and finally, the gonadorelin is obtained by cracking. The method adopts piperidine which is an easily toxic reagent, and the last amino acid 5-oxyproline is not protected, so that a large amount of byproducts are generated.
The Czech patent CZ2014976A3 adopts a similar amino acid monomer and Rink resin as a carrier synthesis method.
US patent No. 4024248 mentions that the gonadorelin analogs can be prepared by fragment condensation, which divides the fragments into Pyr-His-Trp and Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2Or Pyr-His-Trp-Ser and Tyr-Gly-Leu-Arg-Pro-Gly-NH2And then, carrying out condensation by adopting an azide method, wherein the condensation segment is not protected, side reactions are more, and the cost is higher.
Disclosure of Invention
In view of the above, there is a need to provide an improved method for the solid-phase synthesis of gonadorelin, which addresses the problems in the background art.
The technical scheme of the invention is to construct an improved gonadorelin solid-phase synthesis method, which comprises the following steps:
1) taking amide resin as a solid phase carrier, taking Fmoc protected amino acid as a monomer, taking Pyr with the end protected by Boc and taking piperazine as a decapping reagent, and sequentially and one by one connecting amino acid under the action of a condensing agent to synthesize gonadorelin;
2) and (4) cutting peptides by using a peptide cutting reagent, and concentrating to obtain a gonadorelin crude product.
In one embodiment, in step 1), the protected amino acid monomers are: Boc-Pyr-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (but) -OH, Fmoc-Tyr (but) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Arg (pbf) -OH, Fmoc-Pro-OH.
In one embodiment, in step 1), the solvent of the solution of the uncapping reagent piperazine is a dipolar aprotic solvent, and the mass concentration of piperazine is 2-5%.
In one embodiment, in step 1), the condensing agent is: one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC.
In one embodiment, in step 2), the peptide-cleaving reagent is: trifluoroacetic acid solution.
In one embodiment, in step 2), the purity of the gonadorelin crude product can reach 95%.
The invention has the following advantages and beneficial effects:
the method for synthesizing gonadorelin by taking the piperazine solution as the uncapping reagent has the advantages of convenience in purchase, transportation and storage, long shelf life, low price, high purity of the synthesized gonadorelin crude product, simplicity in the subsequent purification process, suitability for large-scale industrial production, no use of any toxic and easily-prepared reagents, no violent chemical reaction and convenience in automatic production.
Drawings
FIG. 1 is an RP-HPLC profile of a small test gonadorelin crude product of the invention.
Fig. 2 is a MS map of the experimental gonadorelin of the present invention.
FIG. 3 is an RP-HPLC profile of a pilot test gonadorelin crude product of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
Abbreviations appearing in the claims and in the specification have the following meanings:
the 5g resin charge table is as follows:
| name (R) | Molecular weight | Density of | Volume μ l | Mass mg of feed |
| Rink-Amidelesin substitution rate (0.8mmol/g) | 5000 | |||
| Fmoc-Gly-OH | 297 | \ | \ | 2376 |
| Fmoc-Pro-OH | 337 | \ | \ | 2696 |
| Fmoc-Arg(pbf)-OH | 648.8 | \ | \ | 5190.4 |
| Fmoc-Leu-OH | 353.4 | \ | \ | 2827.2 |
| Fmoc-Gly-OH | 297 | \ | \ | 2376 |
| Fmoc-Tyr(tBu)-OH | 459.5 | \ | \ | 3676 |
| Fmoc-Ser(tBu)-OH | 383.4 | \ | \ | 3067.2 |
| Fmoc-Trp(Boc)-OH | 526.6 | \ | \ | 4212.8 |
| Fmoc-His(Boc)-OH | 477.51 | \ | \ | 3820.08 |
| Boc-Pyr-OH | 229.2 | \ | \ | 1833.6 |
| HBTU | 379.24 | \ | \ | 2882.224 |
| DIEA | 129.24 | 0.782 | 1586.58 | 1240.704 |
An improved method for the solid-phase synthesis of gonadorelin, comprising the steps of:
1) taking amide resin as a solid phase carrier, taking Fmoc protected amino acid as a monomer, taking Pyr with the end protected by Boc and taking piperazine as a decapping reagent, and sequentially and one by one connecting amino acid under the action of a condensing agent to synthesize gonadorelin;
preferably, in step 1), the protected amino acid monomers are: Boc-Pyr-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (but) -OH, Fmoc-Tyr (but) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Arg (pbf) -OH, Fmoc-Pro-OH.
Preferably, in the step 1), the solvent of the solution of the uncapping reagent piperazine is a dipolar aprotic solvent, and the mass concentration of the solution of piperazine is 2-5%;
more preferably, the solvent is DMF and the mass concentration of piperazine is 3%.
Preferably, in step 1), the condensing agent is: one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC.
Specifically, the synthesis process is as follows:
synthesis of Gly-resin:
(1) the resin is weighed into a 50ml polypeptide reactor, 40ml uncapping reagent (3% piperazine/DMF solution by mass concentration) is measured and poured into the resin, and the reaction is carried out for 20 min. The ninhydrin test was positive after four washes with DMF.
(2) Weighing Fmoc-Gly-OH/HBTU in a 50ml container, weighing DMF20ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Pro-Gly-resin:
(1) weighing Fmoc-Pro-OH/HBTU in a 50ml container, weighing DMF20ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring the mixture into a reactor for reaction for 2 hours, pumping out the reaction solution, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Arg (pbf) -OH/HBTU in a 50ml container, measuring DMF20ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 3 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Leu-OH/HBTU in a 50ml container, weighing DMF20ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring the mixture into a reactor for reaction for 2 hours, pumping out the reaction solution, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Gly-Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Gly-OH/HBTU in a 50ml container, weighing DMF20ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Tyr (tBu) -OH/HBTU in a 50ml container, measuring DMF20ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Ser (tBu) -OH/HBTU in a 50ml container, measuring DMF20ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-Trp (Boc) -OH/HBTU in a 50ml container, measuring DMF20ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
(1) weighing Fmoc-His (Boc) -OH/HBTU in a 50ml container, measuring DMF20ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
(2) 40ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Boc-Pyr-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Boc-Pyr-OH/HBTU in a 50ml container, weighing DMF20ml, dissolving, adding DIEA, mixing well, pouring into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
2) And (4) cutting peptides by using a peptide cutting reagent, and concentrating to obtain a gonadorelin crude product.
Preferably, in step 2), the peptide-cleaving reagent is: trifluoroacetic acid solution.
Preferably, in step 2), the purity of the gonadorelin crude product can reach 95%.
Specifically, the synthesis process is as follows:
washing the resinoid with methanol for 4 times, vacuum drying for 24 hr to obtain cleavage reagent trifluoroacetic acid lysate (TFA: H)2TIS 95:2.5:2.5), pouring 15ml of lysate into a reactor for reaction for 30min, and collecting reaction solutionThe reaction was repeated four times, 200ml of cold diethyl ether was added for precipitation, the precipitate was collected, dried in vacuo, and weighed to obtain 4.1g, yield 86.7%, purity 93.6%, and HPLC chromatogram shown in fig. 1 (analytical method: mobile phase: prepared solution a: 0.1M phosphoric acid solution, pH adjusted to 3 with triethylamine, solution a: acetonitrile: 85: 15, isocratic elution; analytical wavelength: 220 nm;), and MS chromatogram shown in fig. 2.
Example 2
The table for 100g resin charge is as follows:
an improved method for the solid-phase synthesis of gonadorelin, comprising the steps of:
1) taking amide resin as a solid phase carrier, taking Fmoc protected amino acid as a monomer, taking Pyr with the end protected by Boc and taking piperazine as a decapping reagent, and sequentially and one by one connecting amino acid under the action of a condensing agent to synthesize gonadorelin;
synthesis of Gly-resin:
(1) the resin is weighed in a 1000ml polypeptide reactor, 800ml uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into the resin, and the reaction is carried out for 20 min. The ninhydrin test was positive after four washes with DMF.
(2) Weighing Fmoc-Gly-OH/HBTU in a 1000ml container, weighing DMF400ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring into a reactor, reacting for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Pro-Gly-resin:
weighing Fmoc-Pro-OH/HBTU in a 1000ml container, weighing DMF400ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring the mixture into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Arg (pbf) -OH/HBTU in a 1000ml container, measuring DMF400ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 3 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Leu-OH/HBTU in a 1000ml container, weighing DMF400ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring the mixture into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Gly-OH/HBTU in a 1000ml container, weighing DMF400ml, adding the DMF, dissolving the DMF, adding DIEA, mixing uniformly, pouring into a reactor, reacting for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Tyr (tBu) -OH/HBTU in a 1000ml container, measuring DMF400ml, dissolving, adding DIEA, mixing, pouring into a reactor, reacting for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Ser (tBu) -OH/HBTU in a 1000ml container, measuring DMF400ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-Trp (Boc) -OH/HBTU in a 1000ml container, measuring DMF400ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Fmoc-His (Boc) -OH/HBTU in a 1000ml container, measuring DMF400ml, dissolving, adding DIEA, mixing, pouring into a reactor for reaction for 2 hours, removing reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative. 800ml of uncapping reagent (3% piperazine/DMF solution by mass concentration) is weighed and poured into resin for reaction for 20 min. The ninhydrin test was positive after four washes with DMF.
Synthesis of Boc-Pyr-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin:
weighing Boc-Pyr-OH/HBTU in a 1000ml container, weighing DMF400ml, dissolving, adding DIEA, mixing well, pouring into a reactor for reaction for 2 hours, pumping out reaction liquid, washing with DMF for four times, and detecting ninhydrin as negative.
2) And (4) cutting peptides by using a peptide cutting reagent, and concentrating to obtain a gonadorelin crude product.
Specifically, the crude product (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH)2) Synthesis of (2)The process is as follows:
the resinoid was washed with methanol 4 times, dried in vacuo for 24 hours, and prepared as trifluoroacetic acid lysate (TFA: H)2And (3) TIS (95: 2.5:2.5), pouring 300ml of lysate into a reactor for reaction for 30min, collecting the reaction solution, repeating the reaction for four times, collecting all lysate, concentrating, adding 500ml of cold diethyl ether for precipitation when the amount of lysate is small, collecting the precipitate, washing the precipitate for 3 times by using ethyl acetate, filtering, collecting the precipitate, drying in vacuum, weighing 84.25g, obtaining 89.1% of yield and 96.5% of purity, and obtaining an HPLC analysis chart shown in the attached figure 3 (analysis method: mobile phase: preparing a solution a: 0.1M phosphoric acid solution, triethylamine to pH 3, solution a: acetonitrile 85: 15, isocratic elution; analyzing the wavelength: 220 nm).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. An improved gonadorelin solid-phase synthesis method is characterized by comprising the following steps:
1) taking amide resin as a solid phase carrier, taking Fmoc protected amino acid as a monomer, taking Pyr with the end protected by Boc and taking piperazine as a decapping reagent, and sequentially and one by one connecting amino acid under the action of a condensing agent to synthesize gonadorelin;
2) and (4) cutting peptides by using a peptide cutting reagent, and concentrating to obtain a gonadorelin crude product.
2. The improved solid phase synthesis of gonadorelin according to claim 1, wherein: in the step 1), the protected amino acid monomer is: Boc-Pyr-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (but) -OH, Fmoc-Tyr (but) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Arg (pbf) -OH, Fmoc-Pro-OH.
3. The improved solid phase synthesis of gonadorelin according to claim 1, wherein: in the step 1), the solvent of the uncapping reagent piperazine solution is a dipolar aprotic solvent, and the mass concentration of piperazine is 2-5%.
4. The improved solid phase synthesis of gonadorelin according to claim 1, wherein: in the step 1), the condensing agent is: one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC.
5. The improved solid phase synthesis of gonadorelin according to claim 1, wherein: in the step 2), the peptide cutting reagent is as follows: trifluoroacetic acid solution.
6. The improved solid phase synthesis of gonadorelin according to claim 1, wherein: in the step 2), the purity of the gonadorelin crude product can reach 95%.
7. The improved gonadorelin solid phase synthesis method according to any one of claims 1 to 6, characterized in that: the synthesis steps in the step 1) are as follows:
1) synthesis of Gly-resin;
2) synthesis of Pro-Gly-resin;
3) synthesis of Arg (pbf) -Pro-Gly-resin;
4) synthesis of Leu-Arg (pbf) -Pro-Gly-resin;
5) synthesis of Gly-Leu-Arg (pbf) -Pro-Gly-resin;
6) synthesis of Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin;
7) synthesis of Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin;
8) synthesis of Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin;
9) his (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin synthesis.
10) Synthesis of Boc-Pyr-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Gly-Leu-Arg (pbf) -Pro-Gly-resin.
8. The improved gonadorelin solid phase synthesis method according to any one of claims 1 to 7, characterized in that: the synthesis steps in the step 2) are as follows:
1. washing the resin peptide with methanol for 4 times, and drying in vacuum for 24 hours;
2. preparation of trifluoroacetic acid lysate (TFA: H)2TIS (95: 2.5:2.5), pouring 15ml of lysate into a reactor for reacting for 30min, collecting reaction liquid, and repeating the reaction for four times;
3. adding 200ml cold ether for precipitation, collecting the precipitate, and vacuum drying;
4. to obtain the gonadorelin crude product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011087407.4A CN112279892A (en) | 2020-10-12 | 2020-10-12 | Improved gonadorelin solid-phase synthesis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011087407.4A CN112279892A (en) | 2020-10-12 | 2020-10-12 | Improved gonadorelin solid-phase synthesis method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112279892A true CN112279892A (en) | 2021-01-29 |
Family
ID=74496672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011087407.4A Withdrawn CN112279892A (en) | 2020-10-12 | 2020-10-12 | Improved gonadorelin solid-phase synthesis method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112279892A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254701A (en) * | 2015-11-03 | 2016-01-20 | 江苏诺泰生物制药股份有限公司 | Synthesis method of deslorelin acetate |
| CN105646671A (en) * | 2016-02-25 | 2016-06-08 | 吉尔生化（上海）有限公司 | Gonadorelin purification method |
| CN105884865A (en) * | 2016-05-18 | 2016-08-24 | 江苏开元药业有限公司 | Synthesis method of goserelin |
| CN106243194A (en) * | 2016-09-12 | 2016-12-21 | 兰州凯博药业股份有限公司 | A kind of method of the Fmoc Solid-phase synthesis peptides oxytocin of improvement |
| CN107176975A (en) * | 2017-06-12 | 2017-09-19 | 丹东中科润华生物科技有限公司 | A kind of method of synthesis in solid state Gonadorelin |
-
2020
- 2020-10-12 CN CN202011087407.4A patent/CN112279892A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254701A (en) * | 2015-11-03 | 2016-01-20 | 江苏诺泰生物制药股份有限公司 | Synthesis method of deslorelin acetate |
| CN105646671A (en) * | 2016-02-25 | 2016-06-08 | 吉尔生化（上海）有限公司 | Gonadorelin purification method |
| CN105884865A (en) * | 2016-05-18 | 2016-08-24 | 江苏开元药业有限公司 | Synthesis method of goserelin |
| CN106243194A (en) * | 2016-09-12 | 2016-12-21 | 兰州凯博药业股份有限公司 | A kind of method of the Fmoc Solid-phase synthesis peptides oxytocin of improvement |
| CN107176975A (en) * | 2017-06-12 | 2017-09-19 | 丹东中科润华生物科技有限公司 | A kind of method of synthesis in solid state Gonadorelin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20060276626A1 (en) | Methods for the production of peptide derivatives | |
| AU4199397A (en) | Improved solid-phase peptide synthesis and agent for use in such synthesis | |
| US20130060004A1 (en) | Novel Process For The Preparation Of Leuprolide And Its Pharmaceutically Acceptable Salts Thereof | |
| US6627609B1 (en) | LHRH antagonists having improved solubility properties | |
| CN112279894A (en) | Method for synthesizing alarelin by polypeptide solid-liquid combination | |
| CN104177490B (en) | Method for preparing salmon calcitonin acetate by fragment condensation | |
| CN107056894B (en) | Method for solid-phase synthesis of ganirelix acetate by fragment method | |
| CN105408344B (en) | Peptide-resin conjugates and uses thereof | |
| CN107022002B (en) | Method for preparing degarelix by solid-liquid combination | |
| CN112500455A (en) | Method for synthesizing gonadorelin by polypeptide solid-liquid combination | |
| CN110922453B (en) | Method for synthesizing goserelin | |
| CN112279892A (en) | Improved gonadorelin solid-phase synthesis method | |
| CN108383896B (en) | Method for synthesizing goserelin by fragment method | |
| CN112279891A (en) | Improved solid-phase synthesis method of triptorelin | |
| CN112409458A (en) | Preparation method of carbetocin | |
| US7176282B1 (en) | Solid-phase peptide synthesis and agent for use in such synthesis | |
| CN107778355B (en) | Method for synthesizing cetrorelix | |
| CN104402977B (en) | A kind of solid-liquid synthetic method of Buserelin | |
| CN113698458A (en) | Method for synthesizing triptorelin in full liquid phase | |
| CN113603752A (en) | Method for synthesizing gonadorelin in full liquid phase | |
| CN112175046A (en) | Method for synthesizing triptorelin by polypeptide solid-liquid combination | |
| CN107337717A (en) | A kind of method of fragment method synthesis Cetrorelix | |
| CN113603750A (en) | Method for synthesizing nafarelin in full liquid phase | |
| CN109293736B (en) | Dipeptide for synthesizing relin medicines | |
| CN110386964B (en) | Solid-liquid synthesis method of leuprorelin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20210129 |