CN102816213A - Method for preparing pramlintide by using solid-phase and liquid-phase combined technology - Google Patents

Method for preparing pramlintide by using solid-phase and liquid-phase combined technology Download PDF

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CN102816213A
CN102816213A CN2012101713948A CN201210171394A CN102816213A CN 102816213 A CN102816213 A CN 102816213A CN 2012101713948 A CN2012101713948 A CN 2012101713948A CN 201210171394 A CN201210171394 A CN 201210171394A CN 102816213 A CN102816213 A CN 102816213A
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resin
pro
side chain
peptide
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苏贤斌
缪李鹏
毛怡春
王蔡典
董海军
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NANJING IMPACT BIOSCIENCE Ltd
Nanjing Tech University
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NANJING IMPACT BIOSCIENCE Ltd
Nanjing Tech University
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Abstract

The invention relates to a method for preparing pramlintide by utilizing a solid-phase and liquid-phase combination technology, which comprises the steps of respectively synthesizing 1-12, 13-20, 21-28 and 29-37 peptide intermediate fragments by utilizing solid-phase chemistry, and then obtaining pramlintide by utilizing liquid-phase chemical condensation. The pramlintide is prepared by the route of the invention, the operation steps are short, the post-treatment is simple, the byproducts are few, the yield is high, the pollution is small, and the method is suitable for large-scale industrial production.

Description

Use solid phase and liquid phase combination technique to prepare the method for tripro-amylin
Technical field
The present invention relates to the polypeptide drugs synthesis technical field, be specifically related to the method that a kind of solid phase and liquid phase combination technique prepare tripro-amylin.
Background technology
Tripro-amylin (Pramlitide, structure is seen formula I, 2-7; Disulfide linkage) be a kind of pancreas opsonin (Amylin, amylin) analogue of synthetic, its constitutional features is that the amino acid of the 25th (L-Ala) of pancreas opsonin, the 28th (Serine) and the 29th (Serine) substitutes with proline(Pro) respectively.It has fully kept the physiological action of endogenous pancreas opsonin, has overcome people's pancreas opsonin instability, facile hydrolysis, agglutinophilic physical property in solution.Clinical study shows that tripro-amylin can suppress the release of glicentin, delays stomach emptying and inhibition is ingested; Use with insulin combination, effectively controlling blood sugar, lose weight, reduce insulin dosage.Tripro-amylin obtains drugs approved by FDA the earliest by U.S. Amylin Pharmaceuticals company development on March 16th, 2005, is applicable to use Regular Insulin but can't reach the I type of expected effect and type ii diabetes patient's assisting therapy.
Figure BDA0000169737421
The synthetic of known tripro-amylin can pass through solid-phase synthesis (WO-A-93/10146, US-A-5424394, CN102206266A; CN101747426A), the carboxyl of C being held tyrosine on insoluble resin, is a starting point with this amino group of amino acids with the form fix of covalent linkage again; Condensation amino acid successively; Prolong peptide chain, after accomplishing peptide chain synthetic on the resin, the sulfydryl formation intramolecular disulfide bond of the 2nd and the 7th halfcystine of oxidation; Again this peptide chain is cut down and remove Side chain protective group from resin, finally prepare tripro-amylin.Obviously, for containing 37 amino acid whose tripro-amylins, this route of synthesis needs repetition tens step coupling and deprotection steps, and synthetic route is tediously long, and operating procedure is loaded down with trivial details; And along with the prolongation of peptide chain and the influence of the secondary structure that possibly exist, condensation efficiency causes impurity to become many and be difficult to and separates worse and worse, and target product yield is extremely low, and cost is very high, is not suitable for suitability for industrialized production.
Use solid phase and liquid phase bonded fragment condensation technology, be suitable for the synthetic polypeptide that contains longer aminoacid sequence, its advantage is that by product is less and is easy to purifying that product purity is high.Yet the fragment condensation strategy is hampered by the danger that (i) has increases racemization sometimes; (ii) big protection fragment poor solubility; (iii) intersegmental low condensation rate of sheet and the danger that takes place with side reaction.In order to eliminate or to reduce racemization, generally select Gly or Pro as segmental C end residue, if can not realize this point, then select Ala or Arg to hold residue, because they are difficult for racemization as C.When application fragment condensation strategy, target sequence is carried out rational segmentation can improve the result greatly.Except choose reasonable C end residue, reduce the risk of racemization as far as possible, also the segmental purity of considered synthetic on solid phase wants high, and solvability will be got well; The segmental C end and the N that participate in condensation hold the sterically hindered little of residue, to improve the intersegmental condensation rate of sheet.
Tripro-amylin is made up of 37 amino-acid residues; Thereby there are possible fragment of many kinds and a condensation order; For example CN101824086A adopts 1-8 (fragment 1), 9-19 (fragment 2), 20-28 (fragment 3); Four fragments of 29-37 (fragment 4) are with the method for the order condensation of [(4+ (3+2))+1]; CN102164608A (patent families US2010/0081788A1) reported method is similar with it; Adopt 1-8 (fragment 1), 9-19 (fragment 2), 20-29 (fragment 3); Four fragments of 30-37 (fragment 4) are held the condensation successively of N end [((4+3)+2)+1] order from C; CN101790535A (patent families WO2009/00366A1) has reported employing 1-12 (fragment 1), 13-24 (fragment 2), and three fragments of 25-37 (fragment 3) are held the method for N end order condensation from C; CN102180943A then adopts 1-16 (fragment 1), 17-28 (fragment 2), and three fragments of 29-37 (fragment 3) hold N to hold the method for condensation successively from C.In the above fragment condensation synthesis strategy; In preceding two kinds of methods in the 19th Ser (C end) and the 20th Ser (N end) and the 4th kind of method the 16th Leu (C end) and the 17th Val (N end) be not the ideal breakpoint all because the intersegmental condensation efficiency of these sheets is not high in actually operating; Though the third method Cut Selection is comparatively suitable, fragment 3 purity on solid phase synthesis is not good, will influence fragment condensation reaction efficient.
The pharmaceutical application important in view of tripro-amylin is worth, and is necessary to provide more effective tripro-amylin synthetic schemes, can overcome the defective of above method and be fit to plant-scale production.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of new tripro-amylin, and this method can overcome existing preparing method's deficiency and defective, and suitable suitability for industrialized production.
The preparation method of tripro-amylin among the present invention, employing be the combination technique of solid phase synthesis fragment and the condensation of fragment liquid phase.At first utilize the intermediate fragments peptide of solid state chemistry 4 side chain protected of synthesizing pramlintide on resin carrier: fragment a (1-12); Fragment b (13-20); Fragment c (21-28); Fragment d (29-37), then in liquid phase with the intermediate fragments peptide condensation of side chain protected, remove aminoterminal at last and Side chain protective group obtains tripro-amylin.Selected 4 suitable fragment peptides in present method, through conventional solid-phase synthesis all can high-level efficiency, high yield, highly purified acquisition each fragment peptide; Each fragment peptide solubleness in liquid phase condensation system is high, and the segmental C end and the N end spaces steric hindrance of participation condensation are little, and the condensation productive rate is high, and product is easy to purifying.
Particularly, tripro-amylin preparation method provided by the invention may further comprise the steps:
(1) utilizing the solid state chemistry method, is carrier with aminoresin, the peptide intermediate fragments a:AA of synthetic side chain protected 29-AA 37-aminoresin, the instance of fragment a:
Figure BDA0000169737422
The instance of aminoresin is: the amino xanthene of 9-Fmoc--3-oxygen base polystyrene resin [9-Fmoc-Aminoxanthen-3 – yloxy-polystyrene (Sieber Amide resin)] or the amino xanthene of 9-Fmoc--3-oxygen base acetylmethyl resin (9-Fmoc-Aminoxanthen-3-yloxy-acetamidomethyl Resin).
(2) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments b:AA of synthetic side chain protected 21-AA 28-trityl resin, the instance of fragment b:
(3) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments c:AA of synthetic side chain protected 13-AA 20-trityl resin, the instance of fragment c:
Figure BDA0000169737424
(4) utilizing the solid state chemistry method, is carrier with the trityl resin, and the peptide midbody of synthetic side chain protected forms 2,7 disulfide bridge bonds then, obtains peptide intermediate fragments d:AA 1-AA 12-trityl resin, the instance of fragment d:
Figure BDA0000169737425
P representes pseudo-proline(Pro) protection base (ψ among fragment a, c, the d Me, MePro) or tBu protection base, preferred pseudo-proline(Pro) protection base (ψ Me, MePro); K among the fragment d representes Fmoc protection base or Boc protection base, preferred Boc protection base.
Synthetic 2, the instance of the cyclization reagent of 7 disulfide bridge bonds is to use I 2, solvent is DMF, and the cyclisation time is 1-4h, and the cyclisation temperature is 15-25 ℃.
Trityl resin instance in step (2), (3), (4) is a 2-chloro-trityl chloride resin (CTC resin), chlorinated triphenyl methyl resin, 4-methyl chlorination trityl resin or 4-methoxyl group chlorination trityl resin.The synthetic method (SPPS) that adopts conventional solid phase synthesis of peptide of fragment.For example; Can be employed in step (1)-(4) on the resin according to peptide preface coupling protection amino acid progressively; Coupling system is DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the coupling system that any one or more combination among PyBOP or the PyAOP is formed.
(5) the peptide intermediate fragments a of side chain protected, b, c, d cracking from the resin is got off, in solution, carry out condensation reaction, obtain side chain protected peptide (d-c-b-a);
The method of liquid phase condensation can with reference to the document Chemical Synthesis of Proteins in Solution of Shumpei Sakakibara (Biopolymers (Peptide Science). 1999; 51 (4): 279-296); For example used solvent can be NMP or DMF, and coupling system is DCC and HOBt, any one or more combination among HOAt or the Cl-HOBt; Perhaps DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the condensation system that any one or more combination among PyBOP or the PyAOP is formed.
(6) aminoterminal that removes side chain protected peptide (d-c-b-a) is protected base and Side chain protective group, obtains the thick peptide of tripro-amylin;
(7) obtain tripro-amylin after purified.
Employed term among the present invention " pseudo-proline(Pro) (Pseudoprolines) " is meant C in the prolyl ring γH 2Base is by oxygen or sulphur atom substituted proline analogs (CN101472939A).Pseudo-proline(Pro) is that Serine, Threonine and halfcystine and aldehyde or ketone cyclocondensation form.The pseudo-proline dipeptides structure of Serine of using in the embodiment of the invention and Threonine is following:
Figure BDA0000169737427
Through adopting pseudo-proline(Pro) protection strategy, not only improved the combined coefficient of difficult sequences in solid phase synthesis, and reduced the danger of C end racemization.
In above-mentioned steps (5), fragment a, b, c, d can be through different order condensations.For example, can distinguish condensation fragment b and fragment a, obtain fragment (b-a), condensation fragment d and fragment c obtain fragment (d-c), and fragment (b-a) obtains side chain protected peptide (d-c-b-a) with fragment (d-c) condensation more then; Also can first condensation fragment c and fragment b, obtain fragment (c-b), and then obtain side chain protected peptide (d-c-b-a) with fragment a and fragment d condensation respectively.Preferred scheme is according to the order condensation successively of holding the N end from C, and is specific as follows:
(5.1) fragment a cracking from the resin is got off, remove aminoterminal protection base; Fragment b cracking from the resin is got off; The two is condensation in solution; Obtain fragment (b-a), the instance of fragment (b-a):
Figure BDA0000169737428
Figure BDA0000169737429
(5.2) aminoterminal that removes fragment (b-a) is protected base, and the c fragment condensation solution with cracking from resin is got off obtains fragment (c-b-a), instance (c-b-a):
Figure BDA00001697374210
(5.3) remove the aminoterminal protection base of fragment (c-b-a); D fragment condensation solution of getting off with cracking from resin; Obtain side chain protected peptide (d-c-b-a), the instance of side chain protected peptide (d-c-b-a):
Figure BDA00001697374211
In the above structural formula, Boc (tertbutyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Trt (trityl), tBu (tertiary butyl), Pbf (2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl) are the protection bases.But the present invention is not limited to these several kinds protection bases, and those skilled in the art also can adopt other suitable protection bases.
Description of drawings
Fig. 1: the ESI-MS of tripro-amylin analyzes collection of illustrative plates.
Embodiment
The present invention comprises but is not limited to following examples.
The reagent name abbreviation
DMF:N, dinethylformamide
The NMP:N-SL 1332
DCC: NSC 57182
TFA: trifluoroacetic acid
DCM: methylene dichloride
TIS: tri isopropyl silane
DIEA:N, the N-diisopropylethylamine
The HOBt:1-hydroxybenzotriazole
HOAt:N-hydroxyl-7-azepine benzotriazole
Cl-HOBt:6-chloro-1-hydroxy benzo triazole
HBTU: benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU:2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus
PyAOP: (3H-1,2,3-triazolo [4,5-b] pyridine-3-oxygen base) three-1-Bi coughs up Wan Ji Phosphonium hexafluorophosphate
Embodiment 1: the preparation of fragment a
Fmoc-Tyr (tBu)-OH is coupled on the Seiber Amide insoluble resin that the 50g substitution value is 0.69mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.Divide with the TFA/DCM solution of 2-5% after the fragment coupling is accomplished and elute the synthetic polypeptide from resin for three times.Concentrate the back with ether wash white solid 12g.
The alternate fragment a ' with pseudo-proline(Pro) that can prepare 30 Thr (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Pro-Thr (ψ Me, MePro)-importing of OH dipeptides.
Embodiment 2: the preparation of fragment b
Fmoc-Pro-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF of the piperidines with 20% removed Fmoc protection base in room temperature.TFA/DCM solution with 1% after the fragment coupling is accomplished elutes the synthetic polypeptide from resin.Concentrate the back with ether wash white solid 25g.
Embodiment 3: the preparation of fragment c
Fmoc-Ser (tBu)-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.TFA/DCM solution with 1% after the fragment coupling is accomplished elutes the synthetic polypeptide from resin.Concentrate the back with ether wash white solid 11g.
The alternate fragment c ' with pseudo-proline(Pro) that can prepare 20 Ser (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Ser (tBu)-Ser (ψ Me, MePro)-importing of OH dipeptides.
Embodiment 4: the preparation of fragment d
Fmoc-Leu-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.The fragment coupling is accomplished the back with 10 normal iodine Synthetic 2s, the disulfide bridge bond of 7 two halfcystines, and the TFA/DCM solution of following with 1% elutes the synthetic polypeptide from resin.Concentrate the back with ether wash faint yellow solid 12g.
The alternate fragment d ' with pseudo-proline(Pro) that can prepare 4 Thr (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Asn (Trt)-Thr (ψ Me, MePro)-importing of OH dipeptides; The alternate fragment d ' ' with pseudo-proline(Pro) that perhaps prepares 6 Thr (tBu), pseudo-proline(Pro) unit adopts Fmoc-Ala-Thr (ψ Me, MePro)-importing of OH dipeptides.
Embodiment 5: the preparation of fragment (b-a)
Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds 3.31g fragment a then, carries out coupling with 3.12g fragment b.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 5.83g, productive rate 90.6%.
Embodiment 6: the preparation of fragment (c-b-a)
3.22g fragment (b-a) is dissolved in the DMF solution of 20% piperidines, question response is poured mixture into after fully has deposition to separate out in the cold water, and filtration washing gets white solid.Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds white solid then, carries out coupling with 1.70g fragment c.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 4.42g, productive rate 94.6%.
Embodiment 7: the preparation of side chain protected peptide (d-c-b-a)
2.34g fragment (c-b-a) is dissolved in the DMF solution of 20% piperidines, question response is poured mixture into after fully has deposition to separate out in the cold water, and filtration washing gets white solid.Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds white solid then, carries out coupling with 1.35g fragment d.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 3.09g, productive rate 90.0%.
Embodiment 8: the preparation of tripro-amylin
The volume ratio that 2.10g side chain protected peptide (d-c-b-a) is dissolved in phenol (Phenol): TIS:TFA=2.5:2.5:95 disposes in the 20ml solution that forms.Add a large amount of cold diethyl ethers behind the reaction 2.5h, have solid to separate out, filtration washing gets the tripro-amylin bullion.The tripro-amylin bullion is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP (10um); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:28%-48% (20 minutes), eluent A:0.8 ‰ TFA/ water, B:0.8 ‰ TFA/ acetonitrile.Removing freeze-drying behind the acetonitrile, to get purity be 95% the pure article 1.06g of tripro-amylin, productive rate 87.9%.
Confirm its structure through the ESI-MS collection of illustrative plates, see Fig. 1.

Claims (10)

1. method of utilizing solid phase and liquid phase combination technique to prepare tripro-amylin, tripro-amylin structural formula be suc as formula shown in the I,
Figure FDA0000169737411
Said method comprising the steps of:
(1) utilizing the solid state chemistry method, is carrier with aminoresin, the peptide intermediate fragments a:AA of synthetic side chain protected 29-AA 37-aminoresin;
(2) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments b:AA of synthetic side chain protected 21-AA 28-trityl resin;
(3) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments c:AA of synthetic side chain protected 13-AA 20-trityl resin;
(4) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments d:AA of synthetic side chain protected 1-AA 12-trityl resin;
(5) the peptide intermediate fragments a of side chain protected, b, c, d cracking from the resin is got off, in solution, carry out condensation reaction, obtain side chain protected peptide (d-c-b-a);
(6) aminoterminal that removes side chain protected peptide (d-c-b-a) is protected base and Side chain protective group, obtains the thick peptide of tripro-amylin;
(7) obtain tripro-amylin behind the purifying.
2. according to the described method of claim 1; Used aminoresin is the amino xanthene of 9-Fmoc--3-oxygen base polystyrene resin [9-Fmoc-Aminoxanthen-3-yloxy-polystyrene (Sieber Amide resin)] or the amino xanthene of 9-Fmoc--3-oxygen base acetylmethyl resin (9-Fmoc-Aminoxanthen-3-yloxy-acetamidomethyl Resin) in the step (1), preferred Sieber Amide resin.
3. according to the described method of claim 1; Used trityl resin is a 2-chloro-trityl chloride resin (CTC resin) in step (2)-(4); Chlorinated triphenyl methyl resin, 4-methyl chlorination trityl resin or 4-methoxyl group chlorination trityl resin, preferred CTC resin.
4. according to the described method of claim 1; Step (1)-(4) are employed on the resin according to peptide preface coupling protection amino acid progressively; Coupling system is DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the coupling system that any one or more combination among PyBOP or the PyAOP is formed.
5. according to the described method of claim 1, step (4) Synthetic 2, the cyclization reagent of the disulfide bridge bond of 7 two halfcystines is I 2, solvent is DMF, and the cyclisation time is 1-4h, and the cyclisation temperature is 15-25 ℃.
6. according to the described method of claim 1; Used solvent is NMP or DMF in the step (5), and the condensation system is DCC and HOBt, any one or more combination among HOAt or the Cl-HOBt; Perhaps DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the condensation system that any one or more combination among PyBOP or the PyAOP is formed.
7. according to the described method of claim 1, the method that removes aminoterminal Fmoc protection base in step (5)-(6) is carried out in the solution of DMF that contains piperidines or NMP.
8. according to the described method of claim 1, step (5) comprising:
(5.1) fragment a cracking from the resin is got off, remove aminoterminal protection base; Fragment b cracking from the resin is got off, and the two is condensation in solution, obtains fragment (b-a);
(5.2) aminoterminal that removes fragment (b-a) is protected base, and the c fragment condensation solution with cracking from resin is got off obtains fragment (c-b-a);
(5.3) aminoterminal that removes fragment (c-b-a) is protected base, and the d fragment condensation solution with cracking from resin is got off obtains side chain protected peptide (d-c-b-a).
9. described according to Claim 8 method, wherein:
Fragment (b-a) is
Figure FDA0000169737412
Figure FDA0000169737413
Fragment (c-b-a) is
Figure FDA0000169737414
Fragment (d-c-b-a) is
P among fragment a, c and the d representes pseudo-proline(Pro) protection base (ψ Me, MePro) or tBu protection base, preferred pseudo-proline(Pro) protection base (ψ Me, MePro).
10. method according to claim 1, wherein:
Fragment a is
Figure FDA0000169737416
Fragment b is
Figure FDA0000169737417
Fragment c is
Figure FDA0000169737418
Fragment d is
Figure FDA0000169737419
P among fragment a, c and the d representes pseudo-proline(Pro) protection base (ψ Me, MePro) or tBu protection base, preferred pseudo-proline(Pro) protection base (ψ Me, MePro); K among the fragment d representes Fmoc protection base or Boc protection base, preferred Boc protection base.
CN2012101713948A 2012-05-29 2012-05-29 Method for preparing pramlintide by using solid-phase and liquid-phase combined technology Pending CN102816213A (en)

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CN111499719A (en) * 2020-03-19 2020-08-07 杭州固拓生物科技有限公司 Method for synthesizing pramlintide
CN114249808A (en) * 2021-12-27 2022-03-29 杭州诺泰澳赛诺医药技术开发有限公司 Synthesis method of Cagrilintide
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东圆珍 等: "Fmoc法固相合成普兰林肽", 《中国医药工业杂志》 *

Cited By (6)

* Cited by examiner, † Cited by third party
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CN104530214A (en) * 2014-12-23 2015-04-22 扬子江药业集团四川海蓉药业有限公司 Preparation method of pramlintide acetate
CN104530214B (en) * 2014-12-23 2018-07-20 扬子江药业集团四川海蓉药业有限公司 A kind of preparation method of pramlintide acetate
CN111499719A (en) * 2020-03-19 2020-08-07 杭州固拓生物科技有限公司 Method for synthesizing pramlintide
CN114249808A (en) * 2021-12-27 2022-03-29 杭州诺泰澳赛诺医药技术开发有限公司 Synthesis method of Cagrilintide
CN115785192A (en) * 2022-12-06 2023-03-14 生工生物工程(上海)股份有限公司 Method for synthesizing polypeptide
CN118530332A (en) * 2024-07-26 2024-08-23 南京羚诺生物医药技术研究院有限公司 Preparation method of pramlintide

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