CN103288951A - Preparation method of liraglutide - Google Patents
Preparation method of liraglutide Download PDFInfo
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- CN103288951A CN103288951A CN2013102432043A CN201310243204A CN103288951A CN 103288951 A CN103288951 A CN 103288951A CN 2013102432043 A CN2013102432043 A CN 2013102432043A CN 201310243204 A CN201310243204 A CN 201310243204A CN 103288951 A CN103288951 A CN 103288951A
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Abstract
The invention relates to the field of polypeptide synthesis, and particularly relates to a preparation method of liraglutide. The preparation method comprises the following steps: coupling Gly and resin to obtain Gly-resin; carrying out primary successive coupling on the Gly-resin to obtain polypeptide segment-resin; and carrying out side chain modification, secondary successive coupling, cracking, purification and freeze-drying on the polypeptide segment-resin to obtain the liraglutide, wherein the sequence of the polypeptide segment in the polypeptide segment-resin is disclosed as SEQ ID NO:1, and the sequence of the liraglutide is disclosed as SEQ ID NO:2. The side chain modification process comprises the following steps: removing the protective group on the Lys side chain in the polypeptide segment-resin, and sequentially coupling Fmoc-Glu-OtBu and palmityl chloride. The liraglutide prepared by the preparation method has fewer impurities, is easy to purify and can enhance the yield of the final product.
Description
Technical field
The present invention relates to polypeptide synthetic field, particularly a kind of preparation method of Li Lalu peptide.
Background technology
Along with the change of growth in the living standard and mode of life, in recent years, the sickness rate of China's diabetes has the trend that increases year by year.Diabetes are by inherited genetic factors, immunologic function disorder, infected by microbes and toxin thereof, the free radical toxin, various virulence factors such as mental element act on body, cause hypoinsulinism, insulin resistant etc., then sugared in the primosome, protein, fat, a series of metabolism disorder syndromes such as power and water Xie Zhi, be principal feature clinically with the hyperglycemia, diuresis can appear in model case, many drinks, many foods, performance such as become thin, i.e. " three-many-one-little " symptom, cause severe complications in case control bad meeting, cause kidney, eye, the depleted pathology at positions such as foot, and can't cure.Diabetes are divided into gestational diabetes, specificity diabetes, type i diabetes and type ii diabetes.Wherein, type ii diabetes has another name called non insulin dependent diabetes, and characteristics are that human body self can produce Regular Insulin, but cell can't react to it, and the effect of Regular Insulin is had a greatly reduced quality.
Type ii diabetes is mainly by oral or subcutaneous injection antidiabetic drug treatment.Antidiabetic drug kind at type ii diabetes is a lot, comprises receptor agonism element of N1,N1-Dimethylbiguanide, sulfonylurea drugs and pancreas hyperglycemia sample peptide-1(GLP-1) etc., and the receptor agonism element of GLP-1 is the focus of Recent study.Wherein, Li Lalu peptide (liraglutide) is one of receptor agonism element of GLP-1, on January 25th, 2010, drugs approved by FDA is gone on the market in the U.S. by Li Lalu peptide (commodity the are called Victoza) injection of Denmark Novo Nordisk Co.,Ltd research and development, on March 4th, 2011, the Li Lalu peptide obtains SFDA approval listing.The chemistry of Li Lalu peptide is expressed as Arg
34Lys
26-[N-ε-(γ-Glu-(N-hexadecanoyl))]-GLP-1
7-37, molecular formula is C
172H
265N
43O
51, relative molecular mass 3751.2, the CAS registration number is 204656-20-2, and molecular formula is compared with natural GLP-1 suc as formula shown in the I, and drug effect is suitable and action time is longer.
Formula I
At present, Novo Nordisk Co.,Ltd mainly is by gene recombination technology, utilizes yeast to produce the Li Lalu peptide; Have report to utilize the solid-liquid synthesis method to synthesize the Li Lalu peptide, its intermediate GLP-1 (7-37)-OH all needs through the reversed-phase HPLC purifying, reacts with N α-alkanoyl-Glu (ONSu)-OtBu under liquid-phase condition again, and purifying obtains the Li Lalu peptide; There is report to utilize the method for synthetic its analogue of a kind of solid-liquid method, ultimate principle is to adopt the synthetic GLP-1 (7-37) of solid phase method-OH, under liquid-phase condition, use NMP and water dissolution again, add alkali (DIPEA), add N α-alkanoyl-Glu (ONSu)-OtBu again, reaction finishes the back and adds the glycine termination reaction, obtains target product through the RP-HPLC purifying again; Also have report to utilize a kind of full solid phase synthesis process, adopt 2-CTC resin or king's resin, according to Li Lalu peptide peptide order coupling amino acid one by one, obtain the Li Lalu peptide finally by crossing reverse purifying, or adopt the synthetic Li Lalu peptide of method of 5 fragment couplings.
Because the peptide order of Li Lalu peptide is longer, wherein contain 15 hydrophobic amino acids, and in the Fmoc solid phase synthesis, the existence of hydrophobic amino acid can cause the βZhe Die of peptide chain, cause the amino acid coupling efficiency low, thereby produce default peptide (non-purpose peptide), in purge process, be difficult to purifying, cause the yield of Li Lalu peptide on the low side.In addition, when existing synthetic method is synthesized the Li Lalu peptide, in removing the step of lysine side-chain, used expensive palladium catalyst, made that synthetic cost is higher.Therefore, provide a kind of product yield height, Li Lalu peptide preparation method with low cost to have important practical significance.
Summary of the invention
In view of this, the invention provides a kind of preparation method of Li Lalu peptide.Impurity is few in the Li Lalu peptide that this method makes, and easy purifying can improve the yield of the finished product.
In order to realize the foregoing invention purpose, the invention provides following technical scheme:
The invention provides a kind of preparation method of Li Lalu peptide, comprise the steps:
Steps A: get Gly and resin coupling, obtain the Gly-resin;
Step B: get the Gly-resin through the first progressively coupling, obtain polypeptide fragment-resin;
Step C: get polypeptide fragment-resin through side chain modification, the second progressively coupling, cracking, purifying, freeze-drying, namely;
The sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin;
The sequence of Li Lalu peptide is shown in SEQ ID NO:2;
Side chain is modified and is specially: remove the protecting group of Lys side chain in polypeptide fragment-resin, successively coupling Fmoc-Glu-OtBu, palmityl chloride.
As preferably, resin is CLEAR-Acid Resin in the steps A.
As preferably, the substitution degree of resin is 0.2～1.0mmol/g;
As preferably, the substitution degree of Gly-resin is 0.15～0.32mmol/g.
As preferably, to hold from N, the amino acid starting material that the Li Lalu peptide is the 11st, the 12nd is Fmoc-Ser (tBu)-Ser (Psi (Me, Me) pro)-OH.
As preferably, to hold from N, the amino acid starting material that the Li Lalu peptide is the 7th, the 8th is Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH.
As preferably, the reagent that removes employing is the mixture of DCM, TES and TFA.
As preferably, remove that the volume ratio of DCM, TES and TFA is (85～90) in the reagent of employing: (3～8): (1～5).
As preferably, the coupling reagent of palmityl chloride is DIPEA.
As preferably, the reagent that cracking is adopted among the step C is selected from TFA, TES, EDT and H
2The mixture of O, TFA, PhSMe, TES, EDT and H
2The mixture of O.
As preferably, TFA, PhSMe, TES, EDT and H in the reagent that cracking is adopted
2The volume ratio of O is (85～90): (0～4): (1～3): (1～3): (2～5).
As preferably, purifying is the RPLC purifying among the step C.
The invention provides a kind of preparation method of Li Lalu peptide.This preparation method comprises the steps: to get Gly and resin coupling, obtains the Gly-resin; Get the Gly-resin through the first progressively coupling, obtain polypeptide fragment-resin; Get polypeptide fragment-resin through side chain modification, the second progressively coupling, cracking, purifying, freeze-drying, namely; The sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin; The sequence of Li Lalu peptide is shown in SEQ ID NO:2; Side chain is modified and is specially: remove the protecting group of Lys side chain in polypeptide fragment-resin, successively coupling Fmoc-Glu-OtBu, palmityl chloride.Preparation method provided by the invention is by earlier synthetic polypeptide fragment-resin, modify through side chain, the remaining amino acid of coupling progressively again, obtain the Li Lalu peptide, the thick peptide yield that utilizes preparation method provided by the invention to make can reach more than 84%, and smart peptide yield reaches more than 30%, and impurity is few, easy purifying has improved the yield of the finished product.This shows that impurity is few in the Li Lalu peptide that preparation method provided by the invention makes, easy purifying can improve the yield of the finished product.
Description of drawings
Fig. 1 shows the HPLC collection of illustrative plates of the thick peptide of Li Lalu peptide that embodiment 1 provides;
Fig. 2 shows the HPLC collection of illustrative plates of the thick peptide of Li Lalu peptide that Comparative Examples 1 provides.
Embodiment
The invention discloses a kind of preparation method of Li Lalu peptide, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The concrete implication of employed abbreviation is as follows in specification sheets and claims:
Raw materials used and reagent all can be buied by market among the preparation method of Li Lalu peptide provided by the invention.
Below in conjunction with embodiment, further set forth the present invention:
The preparation of embodiment 1 Li Lalu peptide
Taking by weighing substitution degree is the CLEAR-Acid Resin 25g(5mmol of 0.2mmol/g), join in the solid state reaction post, with DMF washing 2 times, use DMF swelling resin 30 minutes.
Take by weighing 2.97g Fmoc-Gly-OH(10mmol) and 1.48g HOBt(11mmol) dissolve with 20mL DMF, adding 1.68mL DIPCDI(11mmol under the ice-water bath) activation is after 3 minutes, join in the above-mentioned solid state reaction post that resin is housed, react after 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency then reacts completely; If developing the color, resin continues reaction 1 hour).After reaction finishes, use DMF washing resin 3 times, obtain Fmoc-Gly-CLEAR-Acid Resin, the mensuration substitution degree is 0.15mmol/g.Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Take by weighing 6.49g Fmoc-Arg (Pbf)-OH and 1.5g HOBt, dissolve with 25mL DMF, add 1.7mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, uses DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, the time of deprotection was respectively 5 minutes and 10 minutes; after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color, obtains H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
Adopt the method for above-mentioned coupling Fmoc-Arg (Pbf)-OH, 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 (mmt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH successively on H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
After the Gly coupling end of the 16th at N end, with DMF washing resin 3 times, DCM washing resin 6 times, the mixing solutions 100mL that at every turn adds DCM, TES and TFA again, DCM:TES:TFA=85:8:5(V:V wherein), reacted 3 minutes, react 10 times, remove fully up to mmt, detecting resin with triketohydrindene hydrate has color.After reaction finishes, use DCM washing resin 6 times, DMF washing resin 3 times obtains polypeptide fragment-resin, and the sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin.
4.2g Fmoc-Glu-OtBu, 1.4g HOBt and 5.2g PyBOP are dissolved among the 25mL DMF, add 3.5mL DIPEA activation 3 minutes under the condition of ice bath, the solution that activation is good joins the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, drain DMF washing 4 times, DCM washing 2 times.
In above-mentioned solid state reaction post, add 40mL DCM and 5mL DIPEA, slowly drip the 3.2mL palmityl chloride, dropwise the back and continue reaction 2.5h, triketohydrindene hydrate detects and is negative, drain, DCM washing 6 times obtains Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Resin.
Taking by weighing 12.75g Fmoc-Glu (OtBu)-OH and 4.46g HOAt is dissolved among the 60mL DMF, add 5.2mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, use DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Adopt the method for above-mentioned coupling Fmoc-Glu (OtBu)-OH, coupling Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-Ser (Psi (Me successively, Me) pro)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH.
18.6g Boc-His (Boc)-OH and 4.45g HOBt are dissolved among the 150mL DMF, add 5.2mL DIC activation 5 minutes under the condition of ice bath, the solution that activation is good joins in the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, DMF washing 4 times, DCM washing 2 times, normal hexane washing 3 times is drained, and obtains Li Lalu peptide peptide resin 78.2g.
Li Lalu peptide peptide resin 78.2g with obtaining joins in the 1000mL flask, preparation 780mL lysate TFA:PhSMe:TES:EDT:H
2O=90:1:2:2:5(V:V), lysate is joined in the flask, room temperature reaction 3 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate joined in the 7800mL anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 36.7g of Li Lalu peptide, and HPLC purity is 78.3%, thick peptide yield 97.9%.MALDI-TOF：(M+H)
+=3752.5。The thick peptide HPLC of Li Lalu peptide collection of illustrative plates as shown in Figure 1.
Through the RP-HPLC purifying, freeze-drying obtains the smart peptide 14.3g of Li Lalu peptide, and total recovery is 38.2%.The smart peptide HPLC of Li Lalu peptide collection of illustrative plates as shown in Figure 2.
The preparation of embodiment 2 Li Lalu peptides
Taking by weighing substitution degree is the CLEAR-Acid Resin20g(10mmol of 0.5mmol/g), join in the solid state reaction post, with DMF washing 2 times, use DMF swelling resin 30 minutes.
Take by weighing 5.98g Fmoc-Gly-OH(20mmol) and 2.96g HOBt(22mmol) dissolve with 50mL DMF, adding 3.42mL DIPCDI(22mmol under the ice-water bath) activation is after 3 minutes, join in the above-mentioned solid state reaction post that resin is housed, react after 1 hour, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency then reacts completely; Continue to react 1 hour if resin develops the color), after reaction finished, DMF washing resin 3 times were used in adding 35mL acetic anhydride and the sealing of 30mL pyridine 30 minutes, obtain Fmoc-Gly-CLEAR-Acid Resin, and the mensuration substitution degree is 0.25mmol/g.Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Take by weighing 13.1g Fmoc-Arg (Pbf)-OH and 2.97g HOBt, dissolve with 50mL DMF, add 3.4mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, uses DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, the time of deprotection was respectively 5 minutes and 10 minutes; after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color, obtains H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
Adopt the method for above-mentioned coupling Fmoc-Arg (Pbf)-OH, 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 (mmt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH successively on H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
After the Gly coupling end of the 16th at N end, with DMF washing resin 3 times, DCM washing resin 6 times, the mixing solutions 80mL that at every turn adds DCM, TES and TFA again, DCM:TES:TFA=90:3:1(V:V wherein), reacted 3 minutes, react 10 times, remove fully up to mmt, detecting resin with triketohydrindene hydrate has color.After reaction finishes, use DCM washing resin 6 times, DMF washing resin 3 times obtains polypeptide fragment-resin, and the sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin.
8.4g Fmoc-Glu-OtBu, 2.98g HOBt and 10.4g PyBOP are dissolved among the 80mL DMF, add 7.2mL DIPEA activation 3 minutes under the condition of ice bath, the solution that activation is good joins the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, drain DMF washing 4 times, DCM washing 2 times.
In above-mentioned solid state reaction post, add 80mL DCM and 10mL DIPEA, slowly drip the 6.4mL palmityl chloride, dropwise the back and continue reaction 2.5h, triketohydrindene hydrate detects and is negative, drain, DCM washing 6 times obtains Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Resin.
Taking by weighing 12.75g Fmoc-Glu (OtBu)-OH and 4.46g HOAt is dissolved among the 60mLDMF, add 5.2mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, use DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Adopt the method for above-mentioned coupling Fmoc-Glu (OtBu)-OH, coupling Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-Ser (Psi (Me successively, Me) pro)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH.
18.6g Boc-His (Boc)-OH and 4.45g HOBt are dissolved among the 150mL DMF, add 5.2mL DIC activation 5 minutes under the condition of ice bath, the solution that activation is good joins in the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, DMF washing 4 times, DCM washing 2 times, normal hexane washing 3 times is drained, and obtains Li Lalu peptide peptide resin 75.3g.
Li Lalu peptide peptide resin 75.3g with obtaining joins in the 1000mL flask, preparation 750mL lysate TFA:PhSMe:TES:EDT:H
2O=87:4:2:3:4(V:V), lysate is joined in the flask, room temperature reaction 3 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate joined in the 7500mL anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 35.6g of Li Lalu peptide, and HPLC purity is 71.5%.Thick peptide yield 94.6%.MALDI-TOF：(M+H)
+=3752.2。
Through the RP-HPLC purifying, freeze-drying obtains the smart peptide 12.2g of Li Lalu peptide, and total recovery is 32.6%.
The preparation of embodiment 3 Li Lalu peptides
Taking by weighing substitution degree is the CLEAR-Acid Resin10g(10mmol of 1.0mmol/g), join in the solid state reaction post, with DMF washing 2 times, use DMF swelling resin 30 minutes.
Take by weighing 5.98g Fmoc-Gly-OH(20mmol) and 2.96g HOBt(22mmol) dissolve with 50mL DMF, adding 3.42mL DIPCDI(22mmol under the ice-water bath) activation is after 3 minutes, join in the above-mentioned solid state reaction post that resin is housed, react after 1 hour, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency then reacts completely; If developing the color, resin continues reaction 1 hour).After reaction finishes, add 35mL acetic anhydride and 30mL pyridine sealing 30 minutes, use DMF washing resin 3 times, obtain Fmoc-Gly-CLEAR-Acid Resin, the mensuration substitution degree is 0.32mmol/g.Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Take by weighing 13.1g Fmoc-Arg (Pbf)-OH and 2.97g HOBt, dissolve with 50mL DMF, add 3.4mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, uses DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, the time of deprotection was respectively 5 minutes and 10 minutes; after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color, obtains H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
Adopt the method for above-mentioned coupling Fmoc-Arg (Pbf)-OH, 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 (mmt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH successively on H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
After the Gly coupling end of the 16th at N end, with DMF washing resin 3 times, DCM washing resin 6 times, the mixing solutions 80mL that at every turn adds DCM, TES and TFA again, DCM:TES:TFA=88:5:3(V:V wherein), reacted 3 minutes, react 10 times, remove fully up to mmt, detecting resin with triketohydrindene hydrate has color.After reaction finishes, use DCM washing resin 6 times, DMF washing resin 3 times obtains polypeptide fragment-resin, and the sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin.
8.4g Fmoc-Glu-OtBu, 2.98g HOBt and 10.4g PyBOP are dissolved among the 80mL DMF, add 7.2mL DIPEA activation 3 minutes under the condition of ice bath, the solution that activation is good joins the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, drain DMF washing 4 times, DCM washing 2 times.
In above-mentioned solid state reaction post, add 80mL DCM and 10mL DIPEA, slowly drip the 6.4mL palmityl chloride, dropwise the back and continue reaction 2.5h, triketohydrindene hydrate detects and is negative, drain, DCM washing 6 times obtains Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Resin.
Taking by weighing 12.75g Fmoc-Glu (OtBu)-OH and 4.46g HOAt is dissolved among the 60mL DMF, add 5.2mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, use DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Adopt the method for above-mentioned coupling Fmoc-Glu (OtBu)-OH, coupling Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-Ser (Psi (Me successively, Me) pro)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH.
18.6g Boc-His (Boc)-OH and 4.45g HOBt are dissolved among the 150mL DMF, add 5.2mL DIC activation 5 minutes under the condition of ice bath, the solution that activation is good joins in the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, DMF washing 4 times, DCM washing 2 times, normal hexane washing 3 times is drained, and obtains Li Lalu peptide peptide resin 70.6g.
Li Lalu peptide peptide resin 70.6g with obtaining joins in the 100mL flask, preparation 780mL lysate TFA:PhSMe:TES:EDT:H
2O=87:4:2:3:4(V:V), lysate is joined in the flask, room temperature reaction 3 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate joined in the 7000mL anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 31.8g of Li Lalu peptide, and HPLC purity is 67.6%, thick peptide yield 84.7%.MALDI-TOF：(M+H)
+=3752.7。
Through the RP-HPLC purifying, freeze-drying obtains the smart peptide 11.3g of Li Lalu peptide, and total recovery is 30.1%.
The preparation of embodiment 4 Li Lalu peptides
Taking by weighing substitution degree is the CLEAR-Acid Resin25g(5mmol of 0.2mmol/g), join in the solid state reaction post, with DMF washing 2 times, use DMF swelling resin 30 minutes.
Take by weighing 2.97g Fmoc-Gly-OH(10mmol) and 1.48g HOBt(11mmol) dissolve with 20mL DMF, adding 1.68mL DIPCDI(11mmol under the ice-water bath) activation is after 3 minutes, join in the above-mentioned solid state reaction post that resin is housed, react after 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency then reacts completely; If developing the color, resin continues reaction 1 hour).After reaction finishes, use DMF washing resin 3 times, obtain Fmoc-Gly-CLEAR-Acid Resin, the mensuration substitution degree is 0.15mmol/g.Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Take by weighing 6.49g Fmoc-Arg (Pbf)-OH and 1.5g HOBt, dissolve with 25mL DMF, add 1.7mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, uses DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, the time of deprotection was respectively 5 minutes and 10 minutes; after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color, obtains H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
Adopt the method for above-mentioned coupling Fmoc-Arg (Pbf)-OH, 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 (mmt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH successively on H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
After the Gly coupling end of the 16th at N end, with DMF washing resin 3 times, DCM washing resin 6 times, the mixing solutions 100mL that at every turn adds DCM, TES and TFA again, DCM:TES:TFA=85:8:5(V:V wherein), reacted 3 minutes, react 10 times, remove fully up to mmt, detecting resin with triketohydrindene hydrate has color.After reaction finishes, use DCM washing resin 6 times, DMF washing resin 3 times obtains polypeptide fragment-resin, and the sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin.
4.2g Fmoc-Glu-OtBu, 1.4g HOBt and 5.2g PyBOP are dissolved among the 25mL DMF, add 3.5mL DIPEA activation 3 minutes under the condition of ice bath, the solution that activation is good joins the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, drain DMF washing 4 times, DCM washing 2 times.
In above-mentioned solid state reaction post, add 40mL DCM and 5mL DIPEA, slowly drip the 3.2mL palmityl chloride, dropwise the back and continue reaction 2.5h, triketohydrindene hydrate detects and is negative, drain, DCM washing 6 times obtains Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Resin.
Taking by weighing 12.75g Fmoc-Glu (OtBu)-OH and 4.46g HOAt is dissolved among the 60mL DMF, add 5.2mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, use DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Adopt the method for above-mentioned coupling Fmoc-Glu (OtBu)-OH, coupling Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-Ser (Psi (Me successively, Me) pro)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH.
18.6g Boc-His (Boc)-OH and 4.45g HOBt are dissolved among the 150mL DMF, add 5.2mL DIC activation 5 minutes under the condition of ice bath, the solution that activation is good joins in the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, DMF washing 4 times, DCM washing 2 times, normal hexane washing 3 times is drained, and obtains Li Lalu peptide peptide resin 78.2g.
Li Lalu peptide peptide resin 78.2g with obtaining joins in the 1000mL flask, preparation 780mL lysate TFA:TES:EDT:H
2O=85:3:1:2(V:V), lysate is joined in the flask, room temperature reaction 3 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate joined in the 7800mL anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 17.45g of Li Lalu peptide, and HPLC purity is 68.3%, thick peptide yield 93.1%.MALDI-TOF：(M+H)
+=3752.5。
Through the RP-HPLC purifying, freeze-drying obtains the smart peptide 5.1g of Li Lalu peptide, and total recovery is 31.2%.
The preparation of embodiment 5 Li Lalu peptides
Taking by weighing substitution degree is the CLEAR-Acid Resin20g(10mmol of 0.5mmol/g), join in the solid state reaction post, with DMF washing 2 times, use DMF swelling resin 30 minutes.
Take by weighing 5.98g Fmoc-Gly-OH(20mmol) and 2.96g HOBt(22mmol) dissolve with 50mL DMF, adding 3.42mL DIPCDI(22mmol under the ice-water bath) activation is after 3 minutes, join in the above-mentioned solid state reaction post that resin is housed, react after 1 hour, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency then reacts completely; Continue to react 1 hour if resin develops the color), after reaction finished, DMF washing resin 3 times were used in adding 35mL acetic anhydride and the sealing of 30mL pyridine 30 minutes, obtain Fmoc-Gly-CLEAR-Acid Resin, and the mensuration substitution degree is 0.25mmol/g.Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Take by weighing 13.1g Fmoc-Arg (Pbf)-OH and 2.97g HOBt, dissolve with 50mL DMF, add 3.4mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, uses DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, the time of deprotection was respectively 5 minutes and 10 minutes; after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color, obtains H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
Adopt the method for above-mentioned coupling Fmoc-Arg (Pbf)-OH, 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 (mmt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH successively on H-Arg (Pbf)-Gly-CLEAR-Acid Resin.
After the Gly coupling end of the 16th at N end, with DMF washing resin 3 times, DCM washing resin 6 times, the mixing solutions 80mL that at every turn adds DCM, TES and TFA again, DCM:TES:TFA=90:3:1(V:V wherein), reacted 3 minutes, react 10 times, remove fully up to mmt, detecting resin with triketohydrindene hydrate has color.After reaction finishes, use DCM washing resin 6 times, DMF washing resin 3 times obtains polypeptide fragment-resin, and the sequence of polypeptide fragment is shown in SEQ ID NO:1 in polypeptide fragment-resin.
8.4g Fmoc-Glu-OtBu, 2.98g HOBt and 10.4g PyBOP are dissolved among the 80mL DMF, add 7.2mL DIPEA activation 3 minutes under the condition of ice bath, the solution that activation is good joins the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, drain DMF washing 4 times, DCM washing 2 times.
In above-mentioned solid state reaction post, add 80mL DCM and 10mL DIPEA, slowly drip the 6.4mL palmityl chloride, dropwise the back and continue reaction 2.5h, triketohydrindene hydrate detects and is negative, drain, DCM washing 6 times obtains Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Resin.
Taking by weighing 12.75g Fmoc-Glu (OtBu)-OH and 4.46g HOAt is dissolved among the 60mLDMF, add 5.2mL DIPCDI activation 5 minutes under the ice-water bath, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge, after reaction finishes, use DMF washing resin 3 times.
Add the solution 100mL of 20% piperidines/DMF(V:V), carry out deprotection 2 times, 5 minutes and the 10 minutes respectively time of deprotection, after reaction finishes, with DMF washing resin 6 times, adopt triketohydrindene hydrate detection resin that color is arranged.
Adopt the method for above-mentioned coupling Fmoc-Glu (OtBu)-OH, coupling Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-Ser (Psi (Me successively, Me) pro)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH.
18.6g Boc-His (Boc)-OH and 4.45g HOBt are dissolved among the 150mL DMF, add 5.2mL DIC activation 5 minutes under the condition of ice bath, the solution that activation is good joins in the above-mentioned solid phase reaction column, room temperature reaction 2h, triketohydrindene hydrate detects and is negative, DMF washing 4 times, DCM washing 2 times, normal hexane washing 3 times is drained, and obtains Li Lalu peptide peptide resin 75.3g.
Li Lalu peptide peptide resin 75.3g with obtaining joins in the 1000mL flask, preparation 750mL lysate TFA:PhSMe:TES:EDT:H
2O=88:2:1:1:3(V:V), lysate is joined in the flask, room temperature reaction 3 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate joined in the 7500mL anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 36.0g of Li Lalu peptide, and HPLC purity is 65.2%.Thick peptide yield 95.8%.MALDI-TOF：(M+H)
+=3752.3。
Through the RP-HPLC purifying, freeze-drying obtains the smart peptide 12.97g of Li Lalu peptide, and total recovery is 34.6%.
The preparation of Comparative Examples 1 Li Lalu peptide
Taking by weighing substitution degree is Fmoc-Gly-king's resin 12.5g of 0.12mmol/g; add in the solid state reaction post; with DMF washing 2 times; after 30 minutes, remove Fmoc protection with 20%DBLK with DMF swelling Fmoc-Gly-king resin, then with DMF washing 4 times; DCM washes 2 times; detect color of resin with ninhydrin method, resin has color, and expression Fmoc removes.
With 3.89g Fmoc-Arg (Pbf)-OH(6.0mmol), 0.97g HOBt(7.2mmol), 0.91g DIC(7.2mmol) be dissolved in DCM and DMF mixing solutions that volume ratio is 1:1, join in the above-mentioned solid phase reaction column, room temperature reaction 2 hours detects the judgement reaction end with ninhydrin method.
According to the method for coupling Fmoc-Arg (Pbf)-OH, finish Fmoc-Gly-OH successively, 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-Ala-OH, Fmoc-Gln (Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, the coupling of Fmoc-Ala-OH and Boc-His (Trt)-OH.
With DMF washing 3 times, DCM washes 5 times.Add the 30mL methylene dichloride, add the 2.21mL phenyl silane again, reacted 3 minutes, add 467mg Pd (PPh3) 4 again, room temperature reaction 45 minutes is taken out reaction solution, and triketohydrindene hydrate detects color of resin, and resin has color, and expression Fmoc removes.Take by weighing Fmoc-Glu-OtBu3.18g, PyBOP3.9g, HOBt1.22g with the dissolving of 25mL methylene dichloride, adds 2.7mL DIEA activation 3 minutes under the ice-water bath, add above-mentioned solid phase reaction column reaction 2 hours, detects with ninhydrin method and judges reaction end.Reaction finishes, and removes Fmoc, and DMF washing 6 times adds palmityl chloride 1.22g and DIEA2.1mL, and room temperature reaction 2 hours is judged reaction end with the ninhydrin method detection.Reaction finishes the back shrinks with methyl alcohol, and resin vacuum-drying is spent the night.Weigh and obtain Li Lalu peptide resin 19.8g(resin rate of body weight gain 91.1%).Use TFA: thioanisole: methyl-phenoxide: the cracking of EDT=90:5:3:2 lytic reagent obtains thick peptide 6.5g, thick peptide yield 80.5%, HPLC purity 56.2%.MALDI-TOF：(M+H)
+=3752.1。
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the preparation method of Yi Zhong Li Lalu peptide is characterized in that, comprises the steps:
Steps A: get Gly and resin coupling, obtain the Gly-resin;
Step B: get described Gly-resin through the first progressively coupling, obtain polypeptide fragment-resin;
Step C: get described polypeptide fragment-resin through side chain modification, the second progressively coupling, cracking, purifying, freeze-drying, namely;
The sequence of polypeptide fragment is shown in SEQ ID NO:1 in described polypeptide fragment-resin;
The sequence of described Li Lalu peptide is shown in SEQ ID NO:2;
Described side chain is modified and is specially: remove the protecting group of Lys side chain in described polypeptide fragment-resin, successively coupling Fmoc-Glu-OtBu, palmityl chloride.
2. preparation method according to claim 1 is characterized in that, resin described in the steps A is CLEAR-Acid Resin.
3. preparation method according to claim 1 is characterized in that, from the N end, the amino acid starting material that described Li Lalu peptide is the 11st, the 12nd is Fmoc-Ser (tBu)-Ser (Psi (Me, Me) pro)-OH.
4. preparation method according to claim 1 is characterized in that, from the N end, the amino acid starting material that described Li Lalu peptide is the 7th, the 8th is Fmoc-Thr (tBu)-Ser (Psi (Me, Me) pro)-OH.
5. preparation method according to claim 1 is characterized in that, the described reagent that removes employing is the mixture of DCM, TES and TFA.
6. preparation method according to claim 5 is characterized in that, the volume ratio of DCM, described TES and described TFA is (85～90) described in the described reagent that removes employing: (3～8): (1～5).
7. preparation method according to claim 1 is characterized in that, the coupling reagent of described palmityl chloride is DIPEA.
8. preparation method according to claim 1 is characterized in that, the reagent that cracking described in the step C is adopted is selected from TFA, TES, EDT and H
2The mixture of O, TFA, PhSMe, TES, EDT and H
2The mixture of O.
9. preparation method according to claim 8 is characterized in that, TFA, described PhSMe, described TES, described EDT and described H described in the reagent that described cracking is adopted
2The volume ratio of O is (85～90): (0～4): (1～3): (1～3): (2～5).
10. preparation method according to claim 1 is characterized in that, purifying described in the step C is the RPLC purifying.
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| CN103980358A (en) * | 2014-01-03 | 2014-08-13 | 杭州诺泰制药技术有限公司 | Preparation method of liraglutide |
| CN104004083A (en) * | 2014-06-13 | 2014-08-27 | 成都圣诺生物科技股份有限公司 | Method for synthesizing liraglutide |
| CN107022021A (en) * | 2017-03-24 | 2017-08-08 | 吉尔生化（上海）有限公司 | A kind of solid-phase synthesis of Liraglutide |
| CN107525880A (en) * | 2017-08-15 | 2017-12-29 | 浙江美华鼎昌医药科技有限公司 | A kind of ultra performance liquid chromatography analysis method of Liraglutide |
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| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
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| CN112851747A (en) * | 2019-11-12 | 2021-05-28 | 深圳翰宇药业股份有限公司 | Method for synthesizing quaternary ammonium base modified polypeptide |
| CN112851747B (en) * | 2019-11-12 | 2024-03-05 | 深圳翰宇药业股份有限公司 | Method for synthesizing polypeptide modified by quaternary ammonium base derivative |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN111018963A (en) * | 2019-12-27 | 2020-04-17 | 中肽生化有限公司 | Preparation method of glucagon |
| CN111018963B (en) * | 2019-12-27 | 2023-11-24 | 中肽生化有限公司 | Preparation method of glucagon |
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