CN105111303B - A kind of method that solid-liquid combination prepares Liraglutide - Google Patents

A kind of method that solid-liquid combination prepares Liraglutide Download PDF

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CN105111303B
CN105111303B CN201510347921.XA CN201510347921A CN105111303B CN 105111303 B CN105111303 B CN 105111303B CN 201510347921 A CN201510347921 A CN 201510347921A CN 105111303 B CN105111303 B CN 105111303B
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fmoc
liraglutide
glu
ala
otbu
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CN105111303A (en
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张颖
李同金
王仁友
石鑫磊
南勤坤
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JINAN KANGHE MEDICAL TECHNOLOGY Co Ltd
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JINAN KANGHE MEDICAL TECHNOLOGY Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

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Abstract

The present invention relates to Peptides Synthesis, in particular to the method that a kind of chemical method combined using solid liquid phase prepares Liraglutide, the present invention can simplify the preparation process of Liraglutide, improve the quality standard of finished product；The present invention synthesizes two peptide monomer Fmoc-Lys (N- ε-(γ-Glu (N-Boc)-OtBu)-OH for the first time, and it is used for the preparation of Liraglutide, Liraglutide is prepared using Fmoc-Ala-Ala-OH participation simultaneously, avoid the generation of 24 or 25 missing Ala impurity peptides, purifying difficulty is reduced, yield is improved；The Liraglutide of trifluoroacetylation is (unmodified) water-soluble preferable, is conducive to Reverse phase chromatography and prepares, while also protecting the amino of N-terminal that side reaction does not occur in palmitinic acid modification, greatly improves the yield of finished product；Implementation of the invention solves the problems, such as that finished product related impurities peptide is exceeded during chemical synthesis Liraglutide, total recovery is low, obtains purity greater than 99.5%, single miscellaneous finished product less than 0.1% reduces production cost.

Description

A kind of method that solid-liquid combination prepares Liraglutide
Technical field
The present invention relates to polypeptide drugs preparation method field, in particular to a kind of solid phase and liquid phase combine and prepare Liraglutide Method.
Background technique
Liraglutide (Liraglutide), trade name Victoza were researched and developed by danish novo nordisk, in 2010 1 The moon 25 listed in the U.S., SFDA approval was obtained on March 4th, 2011, into Chinese market；For adults with type 2 diabetes control Blood glucose processed still controls bad trouble suitable for blood glucose after metformin alone or the treatment of sulfonylurea drugs maximum tolerable dose Person, with melbine or sulfonylurea drugs use in conjunction.On December 23rd, 2014, Novo Nordisk Co., Ltd releases for treating fertilizer again Fat Saxenda (trade name), dosage are 3mg/ days.
Liraglutide is a kind of GLP-1 analog, has 97% sequence homology with people GLP-1, and people GLP-1 can be tied Merge activation GLP-1 receptor.GLP-1 receptor is the target spot of natural GLP-1, and GLP-1 is a kind of endogenous gut incretin hormones, With can promoting pancreatic beta cell concentration of glucose dependence excreting insulin.Unlike natural GLP-1, Liraglutide exists Pharmacokinetics and pharmacodynamic characteristics in human body are suitable for dosage regimen once a day.After subcutaneous administrations, The mechanism of extended durations of action includes: to make to absorb the self association slowed down；In conjunction with albumin；To DPP IV (DPP- IV) and neutral endopeptidase (NEP) has higher enzyme stability, to have longer plasma half-life.
Liraglutide chemistry is expressed as Arg34Lys26[N- ε-(γ-Glu (N- α-hexadecanoyl group))]-GLP-17-37, molecule Formula is C172H265N43O51, relative molecular mass 3751.2, No. CAS is 204656-20-2, and sequence information is as follows:
Currently, Novo Nordisk Co., Ltd (US6458924 and US6268343) utilizes yeast mainly by gene recombination technology Produce Liraglutide；But since main chain Arg can only be produced using gene recombination technology34-GLP-17-37, it is also necessary to and N- α-ten six Acyl group-Glu (OSu)-OtBu reaction, using chemical method in Lys26Connect side chain；Due to Arg34-GLP-17-37Side chain not Protection, there are multiple active sites, so this process can generate more impurity, lose larger.
It also has been reported that and Liraglutide is prepared using mechanochemical method, be concentrated mainly in the connection strategy of side chain modification: such as Patent CN102286092A participates in peptide reaction, Pd (PPh) using Fmoc-Lys (Alloc)-OH3Alloc is taken off, then connecting side Chain；CN103087181A participates in peptide reaction using Fmoc-Lys (Mtt)-OH or Fmoc-Lys (Mmt)-OH, uses 1%TFA/5% TIS/DCM takes off Side chain protective group, connects side chain；CN103145828A participates in peptide reaction using Fmoc-Lys (ivDde)-OH, Hydrazine hydrate takes off Side chain protective group, then connects side chain reaction；CN103980358A uses elder generation liquid phase synthesis monomer Fmoc-Lys (N- ε- (γ-Glu (N- α-hexadecanoyl group)-OtBu)-OH, then participates in peptide reaction；CN103275209A is using first synthesis N-terminal protection Liraglutide main chain use N then under liquid-phase conditionαCoupling reaction occurs therewith for-Pal- γ-Glu (OtBu)-OSu, so Tert-butyl is taken off by sour TFA, Fmoc or the post-processing of two step of Dde (or ivDde) are taken off under alkaline condition, then purified obtains Li Lalu Peptide.
The existing synthetic method of the present inventor prepares Liraglutide, it is found that purity and yield be not high, be unsuitable for industry Large-scale production.In view of problem above, the present inventor studies the synthetic method of Liraglutide, to obtain of the invention Technical solution.
Summary of the invention
In order to solve the deficiencies in the prior art, the present invention provides a kind of solid phase of Liraglutide and liquid phase combines preparation Method.
In the present invention using synthesis two peptide monomer Fmoc-Lys (N- ε-(γ-Glu (N-Boc)-OtBu)-OH and Fmoc-Ala-Ala-OH participation prepares Liraglutide, and using Liraglutide (unmodified) mode of trifluoroacetylation, greatly Improve the yield of finished product；The present invention reduces synthesis difficulty and production cost, improves the purity and yield of fine peptide, is conducive to Scale industrial production.A kind of skill book scheme that solid-liquid combination prepares Liraglutide method of the present invention includes the following steps:
(a) Boc-Glu (OSu)-OtBu and Fmoc-Lys-OH is coupled under alkaline solution generates two peptide monomer Fmoc- Lys (N-ε-(γ-Glu(N-α-Boc)-OtBu)-OH；
(b) using Wang resin or CTC resin as solid phase carrier, using Fmoc chemoproection strategy, according to Liraglutide peptide Sequence successively synthesizes the full guard peptide resin of N-terminal trifluoroacetylation, wherein Fmoc protected amino acid 26 with Fmoc protected amino acid Using monomer Fmoc-Lys, (N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH, 24 and 25 use two peptide monomers to Lys Fmoc-Ala-Ala-OH；The full guard peptide resin structure of N-terminal trifluoroacetylation is as follows: Tfa-His (Trt)-Ala-Glu (OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)- Ser(tBu)- Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε-(γ-Glu(N-α-Boc) -OtBu)- Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resin；
(c) Liraglutide (unmodified) of trifluoroacetylation is obtained with lytic reagent peptide resin, and purified, obtained Fine peptide: Tfa-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr- Leu-Glu-Gly-Gln -Ala- Ala -Lys(γ-Glu –OH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-OH；
(d) under alkaline condition, Liraglutide (unmodified) fine peptide of Pal- OSu and step (c) gained trifluoroacetylation Coupling reaction occurs, obtains the product of palmitinic acid modification；
(e) Liraglutide is obtained through alkaline hydrolysis, purifying, freeze-drying.
Wherein Fmoc-Lys (the specific synthetic method of N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH in above step (a) Are as follows:
Fmoc-Lys-OH or its salt and alkali A are dissolved in water according to the ratio of molar ratio 1:1~2,5~20% bodies are added Long-pending organic solvent B hydrotropy is added dropwise to 1~1.2 times of mole (with the amount of Fmoc-Lys-OH until completely dissolved, under stirring Meter) Boc-Glu (OSu)-OtBu organic solvent B solution；TLC monitors reaction end, is evaporated under reduced pressure and removes to the end of reacting After organic solvent, 10% aqueous citric acid solution tune solution ph is added to 2~3, ethyl acetate extraction, crystallization obtains Fmoc-Lys (N-ε-(γ-Glu(N-α-Boc)-OtBu)-OH。
Alkali A is sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethylamine, N- ethyl two in above step Isopropylamine, N, one of N- diisopropylethylamine etc.;
Organic solvent B be tetrahydrofuran, dioxane, N,N-dimethylformamide, acetone, n-methyl-2-pyrrolidone, One or more of acetonitrile.
The full guard peptide resin of N-terminal trifluoroacetylation in above technical scheme step (b) specific the preparation method comprises the following steps: with Wang resin or CTC resin are solid phase carrier, with 2-5 times of feed ratio (synthesize the substance meter of scale), are added corresponding Fmoc protected amino acid carries out coupling reaction, and it is anti-that each coupling reaction is that the solid phase carried out in the presence of condensing agent connects peptide It answers, removes Fmoc with deprotecting regent after completion of the reaction, then carry out coupling reaction with next Fmoc protected amino acid；It repeats to grasp After making until being blended into 1 His, is blocked with trifluoroacetic anhydride, obtain the full guard peptide resin Tfa-His of N-terminal trifluoroacetylation (Trt)- Ala-Glu(OtBu)-Gly -Thr(tBu)-Phe- Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser (tBu)-Ser(tBu)-Tyr(tBu)-Leu -Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Lys(N-ε-(γ-Glu (N-α-Boc)-OtBu) -Glu(OtBu)- Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly -Arg (Pbf)-Gly- resin.The Fmoc protected amino acid that wherein 26 Lys are used is (the N- ε-of Fmoc-Lys made from step (a) (γ-Glu (N- α-Boc)-OtBu)-OH, 24 and 25 Ala use Fmoc protected amino acid for two peptide monomer Fmoc-Ala- Ala-OH；Other sites are all made of conventional Fmoc protected amino acid coupling.Preferably, Wang resin described in step (b) Or the substitution degree of CTC resin is 0.3-0.5mmol/g；The condensing agent is DIC/HOBT, DIC/HOAT, TBTU/ HOBT/ One kind of DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA；Deprotecting regent is 15~25%(volume content) piperazine Pyridine/DMF solution.
Lytic reagent described in step (c) is the TFA solution that volume ratio 1-5% scavenger is added, institute in above-mentioned technical proposal Stating scavenger is one of methyl phenyl ethers anisole, thioanisole, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane or several Kind.
The concrete operation step of step (d) in above-mentioned technical proposal are as follows:
Fine peptide obtained by step (c) and alkali A is taken to be dissolved in water according to the ratio of molar ratio 1:4~6, addition volume ratio 5~ 20% organic solvent B hydrotropy is added dropwise to 1~2.0 times of mole (with the meter of fine peptide) Pal- until completely dissolved, under stirring The organic solvent B solution of OSu；Continue to be stirred to react, TLC monitors reaction end；After reaction reaches terminal, it is added 1~2.0 times and rubs The glycine (with the meter of fine peptide) of your amount terminates reaction.
Wherein alkali A described in step (d) is sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethyl Amine, N- ethyl diisopropyl amine, N, one of N- diisopropylethylamine etc.；The organic solvent B is tetrahydrofuran, dioxy six One or more of ring, N,N-dimethylformamide, acetone, n-methyl-2-pyrrolidone, acetonitrile.
In technical solution in step (e) alkaline hydrolysis concrete operation method are as follows: measure volume ratio 10-12% piperidines (with step (d) resulting liquor capacity meter), it is added in step (d) resulting solution, is configured to the piperidine solution that concentration is 1M, stirring is anti- It should be complete to trifluoroacetyl group alkaline hydrolysis；Then through ultrafiltration apparatus desalination, it is continuously added pure water dilution peptide solution, keeps liquor capacity It is constant；When solution ph is down to 12~10, peptide solution is diluted using 0.2-1.0% aqueous acetic acid, until pH value is down to 5~6 When, stop filtering.
Compared with the existing technology, the beneficial effects of the present invention are:
The present invention synthesizes two peptide monomer Fmoc-Lys for the first time, and (N- ε-(γ-Glu (N-Boc)-OtBu)-OH, is used in combination In the preparation of Liraglutide, synthesis process and purification process are simple and easy, detect convenient for middle control, are easy to amplify production；Simultaneously Liraglutide is prepared using Fmoc-Ala-Ala-OH participation, the generation of 24 or 25 missing Ala impurity peptides is avoided, reduces Purifying difficulty, improves yield；The Liraglutide of trifluoroacetylation is (unmodified) water-soluble preferable, and it is pure to be conducive to reverse-phase chromatography Change preparation, while also protecting the amino of N-terminal that side reaction does not occur in palmitinic acid modification, greatly improves finished product Yield；Implementation of the invention, solves during chemical synthesis Liraglutide that finished product related impurities peptide is exceeded, total recovery is low Problem, the purity of finished product is increased to 99.5% or more, and single miscellaneous control is below 0.1%.
Specific embodiment
With specific embodiment, the present invention is described in detail below, but does not limit this patent；Change according to the present invention former The feed ratio or reaction dissolvent of material or and condensing agent etc., be within the scope of the invention.
Abbreviation meaning used in specification and claims is as follows:
Fmoc 9-fluorenylmethyloxycarbonyl
CTC resin 2- chlorine trityl chloride resin
Wang Resins Wang Shuzhi
tBu Tert-butyl
Pbf 2,2,4,6,7- pentamethyl benzofuran -5- sulfonyl
Trt Trityl
Tfa Trifluoroacetyl group
Boc Tertbutyloxycarbonyl
EDPA N- ethyl diisopropyl amine
NMP N-methyl-2-pyrrolidone
DCM Methylene chloride
DMF N,N-dimethylformamide
DMAP 4-dimethylaminopyridine
DIPEA N, N- diisopropylethylamine
DIC N, N- diisopropylcarbodiimide
HBTU Benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate
HATU 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
TBTU O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid
HOBT I-hydroxybenzotriazole
HOAT 1- hydroxyl -7- azo benzotriazole
TFA Trifluoroacetic acid
TIS Tri isopropyl silane
Pal- OSu Palmitinic acid active ester
Embodiment 1:Fmoc-Lys (the synthesis of N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH
It accurately weighs Fmoc-Lys-OH 183.8g (0.5mol) and sodium carbonate 63.6 (0.6mol) is dissolved in 1200mL water, (2-8 DEG C) is slowly added to tetrahydrofuran solution (200.3g, 0.5mol)/1000ml of Boc-Glu (OSu)-OtBu under low temperature, stirs Reaction is mixed, TLC monitors reaction end, after fully reacting, rotates THF, 10% aqueous citric acid solution tune solution is added under ice-water bath PH value to 2~3,1000ml ethyl acetate extracts 3 times, merges organic phase, and 200ml saturated common salt is washed 3 times, and anhydrous sodium sulfate is dry It is dry, concentrated by rotary evaporation to 1000ml, stand crystallization, obtain Fmoc-Lys (N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH 253.6g, Yield 73.5%.
The synthesis of embodiment 2:Fmoc-Gly-Wang Resins
Carrier Wang resin 400.0g (sub=0.47mmol/g) is placed in synthesis column, washs two with 2400mL DMF It is secondary, 4000mL DCM is added and is swollen 30min；After leaching out DCM, the mixing DCM solution of Fmoc-Gly-OH/DIC/HOBT is added It [weighs 118.8g (400mmol) Fmoc-Gly-OH and 64.8g (480mmol) HOBT and is placed in glycine activated bottle, be added 76.4ml (480mmol) is added under low temperature (0 DEG C) in DMF the and DCM mixed solution stirring and dissolving that 2000mL volume ratio is 1: 1 DIC is activated 5 minutes], 4.8g (4mmol) DMAP is added after reacting 10min；3h is reacted, reaction solution is taken out, with 4000mL DMF It washes twice, capping reagent 2400mL (480ml acetic anhydride and 408ml pyridinium dissolution are in 1512mL DMF) is added and reacts 2h, Reaction solution is leached out, is washed 2 times with DMF, DCM, methanol respectively, Fmoc-Gly-Wang Resins 436.6 is obtained after vacuum drying G；It is 0.30mmol/g that substitution degree is surveyed in sampling.
The synthesis of embodiment 3:Fmoc-Gly-CTC Resins
It weighs CTC resin 50.0g (sub=0.40mmol/g) to be placed in synthesis column, is washed twice, added with 240mL DMF Enter 240mL DCM swelling 30min；After leaching out DCM, the DCM/DMF dissolved with 5.94g (20mmol) Fmoc-Gly-OH is added DIPEA 6.6ml (40mmol) is added after stirring in (3/1, volume ratio) solution 150ml, drum N260min is reacted, reaction solution is taken out, DCM/CH is added3OH/DIPEA (volume ratio 17:2:1) mixed solution 300ml is blocked 3 times, each 10min；Then with DMF, DCM, methanol wash 2 times respectively, are dried in vacuo to obtain Fmoc-Gly-CTC Resins 53.80g.Survey substitution degree is 0.29mmol/ g.
Embodiment 4: the preparation of peptide resin
Accurately weigh Fmoc-Gly-Wang Resins 100g (the synthesis rule that 2 substitution degree of embodiment is 0.30mmol/g Mould 30mmol) it is placed in synthesis column, 1000ml DCM is added and is swollen 30min；After leaching out DCM, 800ml DMF is washed 2 times, is added Enter 20% piperidines/DMF solution 1000ml to be deprotected 2 times, reacts 10min and 10min respectively；Then 800ml DMF, DCM, DMF are used It washs 2 times respectively；Fmoc-Arg (Pbf)-OH 53.7g (90mmol), HOBT 13.4g (99mmol) and DIC 15.4ml is added The DMF solution 500ml of (99mmol), drum N2It is stirred to react 2h, reaction end is subject to Kaiser reagent testing result, and reaction reaches After terminal, reaction solution is taken out, is washed respectively 2 times with 800ml DMF, DCM, DMF；Then it is deprotected again.Circulation behaviour repeatedly Make, according to Liraglutide peptide sequence, is coupled one by one with protected amino acid；Sequentially connected protected amino acid are as follows: 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(N-ε-(γ-Glu(N-α-Boc)-OtBu)-OH、 Fmoc-Ala-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、 Fmoc-Tyr(tBu)-OH 、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-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、Fmoc-Ala-OH、 Fmoc-His(Trt)--OH、(Tfa)2O obtains N-terminal Full guard Liraglutide (unmodified) peptide resin of trifluoroacetylation: Tfa-His (Trt)-Ala-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu (OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε-(γ-Glu(N-α-Boc)-OtBu)-Glu(OtBu)-Phe-Ile- Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)- Gly-Wang resins；It is vacuum dried, weighing Are as follows: 245.3g, resin weight gain 145.3g, rate of body weight gain 99.2%.
Embodiment 5: peptide resin cracking
Full guard Liraglutide (unmodified) peptide resin for the 240.0g N-terminal trifluoroacetylation that embodiment 4 is obtained, adds Enter 2400ml lysate (the volume proportion TFA/TIS/H to freezing20=95/2.5/2.5) in, it is stirred to react 3h at room temperature； Cracking reaction terminates, and filters resin, and 200mlTFA is washed resin 2 times, merging filtrate and washing lotion, concentrated by rotary evaporation to 1600ml, Enter in the 16L freezing tertiary ether of first, white precipitate is precipitated；After standing 30min, filtering, the tertiary ether of first is washed 6 times, is dried in vacuo to obtain N-terminal three The thick peptide 104.1g of fluoroacetylation Liraglutide (unmodified), thick peptide yield 94.9%, purity 76.8%.
Embodiment 6: the preparation of peptide resin
Accurately weighing 3 substitution degree of embodiment is 0.29mmol/g Fmoc-Gly-CTC Resins 51.7g (synthesis scale It 15mmol) is placed in synthesis column, 500ml DCM is added and is swollen 30min；After leaching out DCM, 400ml DMF is washed 2 times, is added 20% piperidines/DMF solution 500ml is deprotected 2 times, reacts 10min and 10min respectively；Then with 400ml DMF, DCM, DMF points It Xi Di not be 2 times；26.9g (45mmol) Fmoc-Arg (Pbf)-OH, 6.7g (49.5mmol) HOBT and 7.7ml is added The DMF solution 300ml of (49.5mmol) DIC, drum N2It is stirred to react 2h, reaction end is subject to Kaiser reagent testing result, After reaction reaches terminal, reaction solution is taken out, is washed respectively 2 times with 400ml DMF, DCM, DMF；Then it is deprotected again.Repeatedly Circulate operation is coupled with protected amino acid one by one according to Liraglutide peptide sequence；Sequentially connected protected amino acid are as follows: 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(N-ε-(γ-Glu(N-α-Boc)- OtBu)-OH、Fmoc-Ala-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc- Leu-OH、Fmoc-Tyr(tBu)-OH 、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc- 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, Fmoc-Ala-OH, Fmoc-His (Trt)-OH, (Tfa)2O obtains N Hold full guard Liraglutide (unmodified) peptide resin of trifluoroacetylation: Tfa-His (Trt)-Ala-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val- Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu (OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε-(γ-Glu(N-α-Boc)-OtBu)-Glu(OtBu)-Phe-Ile- Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)- Gly-CTC resins；It is vacuum dried, weighing are as follows: 116.9g, resin weight gain 65.2g, rate of body weight gain 89.1%.
Embodiment 7: peptide resin cracking
Full guard Liraglutide (unmodified) peptide resin for the 115.0g N-terminal trifluoroacetylation that embodiment 6 is obtained, adds Enter 1150ml lysate (the volume proportion TFA/TIS/H to freezing20=95/2.5/2.5) in, it is stirred to react 3h at room temperature； Cracking reaction terminates, and filters resin, and 100mlTFA is washed resin 2 times, and merging filtrate and washing lotion, concentrated by rotary evaporation to 800ml are poured into 8L is freezed in the tertiary ether of first, and white precipitate is precipitated；After standing 30min, filtering, the tertiary ether of first is washed 6 times, is dried in vacuo to obtain N-terminal trifluoro The thick peptide 47.6g of acetylation Liraglutide (unmodified), thick peptide yield 85.0%, purity 77.6%.
The purification of embodiment 8:N end trifluoroacetylation Liraglutide (unmodified)
The thick peptide 100.0g of 5 gained of embodiment is weighed to be dissolved in 10% acetonitrile solution 2500ml, after concussion dissolution, 0.45um It is spare after membrane filtration.
Internal diameter is that 100mm C18 prepares column, and mobile phase is 0.1%TFA/ water -0.1%TFA/ acetonitrile system, and applied sample amount is 6.0g/ times, flow velocity 300ml/min, gradient elution；Recycle sample introduction behind before peak and peak, interception middle control analysis purity be 99.5% with On, fine peptide 56.2g is lyophilized to obtain in single miscellaneous fine peptide solution less than 0.1% after concentration, purifying yield is 56.2%, and purity 99.6% is single It is miscellaneous to be respectively less than 0.1%.
The purification of embodiment 9:N end trifluoroacetylation Liraglutide (unmodified)
The thick peptide 45.0g of 7 gained of embodiment is weighed to be dissolved in 10% acetonitrile solution 1200ml, after concussion dissolution, 0.45um filter It is spare after film filtering.
Internal diameter is that 100mm C18 prepares column, and mobile phase is 0.1%TFA/ water -0.1%TFA/ acetonitrile system, and applied sample amount is 6.0g/ times, flow velocity 300ml/min, gradient elution；Recycle sample introduction behind before peak and peak, interception middle control analysis purity be 99.5% with On, fine peptide 25.7g is lyophilized to obtain in single miscellaneous fine peptide solution less than 0.1% after concentration, purifying yield is 57.1%, and purity 99.6% is single It is miscellaneous to be respectively less than 0.1%.
Embodiment 10: the preparation of Liraglutide
8 gained fine peptide 56.0g (15mmol) of embodiment is added in tri- mouthfuls of reaction flasks of 2L, 10% acetonitrile water of 1000ml is added Under ice-water bath, 10% Na is slowly added dropwise in solution stirring and dissolving2CO3Aqueous solution adjusts pH value of solution to 11, stops being added dropwise；In ice water Lower THF solution 7.1g (the 30mmol)/150ml that Pal-OSu is added dropwise of bath, is added dropwise, removes ice bath, react 3h at room temperature;It is added 2.25g (30mmol) glycine, the reaction was continued 30min, TLC monitor reaction end；
150ml piperidines is added under high degree of agitation into reaction solution, continues to stir at room temperature, HPLC monitors alkaline hydrolysis terminal；Instead It should stop stirring, G3 sand core funnel filters out insoluble matter, and three times with pure water washing insoluble matter to after terminal；Merge washing lotion and Filtrate is continuously added pure water dilution peptide solution, is kept liquor capacity constant with ultrafiltration apparatus desalination；When solution ph is down to 11 When, peptide solution is diluted using 0.5% aqueous acetic acid, until stopping filtering when pH value is down to 5.Filtrate through 0.45um membrane filtration, For use.
Internal diameter is that 100mm C8 prepares column, and mobile phase is 20mM ammonium acetate aqueous solution-acetonitrile system, and applied sample amount is 3.0g/ times, flow velocity 300ml/min, gradient elution；Recycle sample introduction behind before peak and peak, interception middle control analysis purity be 99.5% with On, single miscellaneous fine peptide solution less than 0.1% is concentrated freeze-dried that fine peptide 39.3g, yield 70.2%, purity 99.6% are single after desalination It is miscellaneous to be respectively less than 0.1%；Preparing total recovery is 37.5%.
Embodiment 11: the preparation of Liraglutide
9 gained fine peptide 24.3g (6.5mmol) of embodiment is added in tri- mouthfuls of reaction flasks of 1L, 10% acetonitrile water of 500ml is added Under ice-water bath, 10% EDPA/NMP solution is slowly added dropwise in solution stirring and dissolving, is adjusted pH value of solution to 11, is stopped being added dropwise；In ice water Lower THF solution 4.6g (the 13mmol)/80ml that Pal-OSu is added dropwise of bath, is added dropwise, removes ice bath, react 3h at room temperature;It is added 0.98g (13mmol) glycine, the reaction was continued 30min, TLC monitor reaction end；
61ml piperidines is added under high degree of agitation into reaction solution, continues to stir at room temperature, HPLC monitors alkaline hydrolysis terminal；Reaction To after terminal, stop stirring, G3 sand core funnel filters out insoluble matter, and three times with pure water washing insoluble matter；Merge washing lotion and filter Liquid is continuously added pure water dilution peptide solution, is kept liquor capacity constant with ultrafiltration apparatus desalination；When solution ph is down to 11, Peptide solution is diluted using 0.5% aqueous acetic acid, until stopping filtering when pH value is down to 5.Filtrate through 0.45um membrane filtration, to With.
Internal diameter is that 100mm C8 prepares column, and mobile phase is 20mM ammonium acetate aqueous solution-acetonitrile system, and applied sample amount is 3.0g/ times, flow velocity 300ml/min, gradient elution；Recycle sample introduction behind before peak and peak, interception middle control analysis purity be 99.5% with On, single miscellaneous fine peptide solution less than 0.1% is concentrated freeze-dried that fine peptide 17.3g, yield 71.1%, purity 99.6% are single after desalination It is miscellaneous to be respectively less than 0.1%；Preparing total recovery is 34.5%.

Claims (7)

1. a kind of method that solid-liquid combination prepares Liraglutide, which comprises the steps of:
(a) Boc-Glu (OSu)-OtBu and Fmoc-Lys-OH is coupled under alkaline solution generates two peptide monomer Fmoc-Lys (N- ε-(γ-Glu(N-α-Boc)-OtBu)-OH；Concrete operations are as follows: by Fmoc-Lys-OH or its salt and alkali A according to molar ratio 1:1 ~2 ratio is dissolved in water, and the organic solvent B hydrotropy of 5~20% volumes is added, until completely dissolved, with Fmoc-Lys-OH Meter, the organic solvent B solution of 1~1.2 times of mole Boc-Glu (OSu)-OtBu is added dropwise under stirring；It is anti-to continue stirring It answers, TLC monitors reaction end, and after reaction, decompression evaporates organic solvent, and 10% aqueous citric acid solution tune solution ph is added To 2~3, ethyl acetate extraction, crystallization obtains Fmoc-Lys (N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH；
(b) using Wang resin or CTC resin as solid phase carrier, using Fmoc chemoproection strategy, according to Liraglutide peptide sequence, according to The secondary full guard peptide resin that N-terminal trifluoroacetylation is synthesized with Fmoc protected amino acid, wherein 26 Lys of Fmoc protected amino acid are adopted It is that (N- ε-(γ-Glu (N- α-Boc)-OtBu)-OH, 24 and 25 use Fmoc-Ala-Ala- to monomer Fmoc-Lys OH, other sites are all made of conventional Fmoc protected amino acid coupling；The full guard peptide resin structure of N-terminal trifluoroacetylation is such as Under:
Tfa-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε- (γ-Glu(N-α-Boc)-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly- Arg (Pbf)-Gly- resin；
(c) the unmodified Liraglutide of trifluoroacetylation is obtained with lytic reagent peptide resin, and purified, obtains essence Peptide:
Tfa-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-Lys(γ-Glu-OH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH；
(d) under alkaline condition, Pal-OSu and the unmodified Liraglutide fine peptide of step (c) gained trifluoroacetylation occur Coupling reaction obtains the product of palmitinic acid modification；
(e) Liraglutide is obtained through alkaline hydrolysis, purifying, freeze-drying；
The alkali A be sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethylamine, N- ethyl diisopropyl amine, One of N, N- diisopropylethylamine etc.；The organic solvent B be tetrahydrofuran, dioxane, N,N-dimethylformamide, One of acetone, n-methyl-2-pyrrolidone, acetonitrile or more than one mixed liquor.
2. the method that solid-liquid combination according to claim 1 prepares Liraglutide, which is characterized in that N-terminal three in step (b) The full guard peptide resin of fluoroacetylation specific is the preparation method comprises the following steps: using Wang resin or CTC resin as solid phase carrier, to synthesize rule The substance meter of mould is added corresponding Fmoc protected amino acid with 2-5 times of feed ratio and carries out coupling reaction, each coupling is anti- Should be the solid phase peptide reaction carried out in the presence of condensing agent, after completion of the reaction with deprotecting regent remove Fmoc, then with Next Fmoc protected amino acid carries out coupling reaction；After repetitive operation is until be blended into 1 His, blocked with trifluoroacetic anhydride, Obtain full guard peptide resin Tfa-His (Trt)-Ala-Glu (OtBu)-Gly-Thr (tBu)-Phe- of N-terminal trifluoroacetylation Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly- Gln(Trt)-Ala-Ala-Lys(N-ε-(γ-Glu(N-α-Boc)-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resin.
3. the method that solid-liquid combination according to claim 2 prepares Liraglutide, which is characterized in that Wang resin or CTC The substitution degree of resin is 0.3-0.5mmol/g.
4. the method that solid-liquid combination according to claim 2 prepares Liraglutide, which is characterized in that condensing agent DIC/ One kind of HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA；Deprotection examination Agent is piperidines/DMF solution of volume content 15~25%.
5. the method that solid-liquid combination prepares Liraglutide according to claim 1, which is characterized in that split described in step (c) Solution reagent be added volume ratio 1-5% scavenger TFA solution, the scavenger be methyl phenyl ethers anisole, thioanisole, dithioglycol, One or more of mercaptoethanol, phenol, water, tri isopropyl silane.
6. the method that solid-liquid combination according to claim 1 prepares Liraglutide, which is characterized in that step (d) it is specific Operating procedure are as follows: take fine peptide obtained by step (c) and alkali A to be dissolved in water according to the ratio of molar ratio 1:4~6, volume ratio 5 is added ~20% organic solvent B hydrotropy with the meter of fine peptide, is added dropwise to 1~2.0 times of mole until completely dissolved under stirring The organic solvent B solution of Pal-OSu；Continue to be stirred to react, TLC monitors reaction end；After reaction reaches terminal, with the amount of fine peptide The glycine of 1~2.0 times of mole is added in meter, terminates reaction.
7. the method that solid-liquid combination according to claim 1 prepares Liraglutide, which is characterized in that alkaline hydrolysis in step (e) Concrete operation step are as follows: in terms of the resulting liquor capacity of step (d), measure volume ratio 10-12% piperidines, be added step (d) it is configured to the piperidine solution that concentration is 1M in resulting solution, is stirred to react complete to trifluoroacetyl group alkaline hydrolysis；Then through super Device desalination is filtered, pure water dilution peptide solution is continuously added, keeps liquor capacity constant；When solution ph is down to 12~10, adopt Peptide solution is diluted with 0.2-1.0% aqueous acetic acid, until stopping filtering when pH value is down to 5~6.
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