CN105111303A - Solid-liquid combined preparation method for liraglutide - Google Patents

Abstract

The invention relates to the field of peptide synthesis, especially to a solid-liquid combined chemical preparation method for liraglutide. The method provided by the invention can simplify conventional preparation process for liraglutide and improve the quality of a final product. According to the invention, dipeptide monomer Fmoc-Lys(N-epsilon-(gamma-Glu(N-Boc)-OtBu)-OH is synthesized for the first time and is applied to preparation of liraglutide; as Fmoc-Ala-Ala-OH participates in preparation of liraglutide, generation of Ala impurity peptide in which position 24 or 25 is deleted is avoided, purification difficulty is reduced and yield is improved; and trifluoro-acetylated liraglutide (unmodified) has good water-solubility, which facilitates reversed phase chromatographic purification and preparation and protects an amino group at terminal N from side reaction in the process of palmitic acid modification, so the yield of the final product is greatly improved. Through implementation of the method, the problems that related impurity peptides in the final product exceed standard and overall yield is low in conventional chemical synthesis of liraglutide are overcome; the final product with purity of greater than 99.5% and simple impurity of less than 0.1% is obtained; and production cost is lowered.

Description

A kind of solid-liquid combination prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Technical field

The present invention relates to polypeptide drugs preparation method field, particularly a kind of solid phase and liquid phase combine the method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].

Background technology

Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide), trade(brand)name Victoza, is researched and developed by Novo Nordisk Co., Ltd of Denmark, on January 25th, 2010 in U.S.'s listing, obtains SFDA approval on March 4th, 2011, enters Chinese market; Control blood sugar for adults with type 2 diabetes, after being applicable to metformin alone or the treatment of sulfonylurea drugs maximum tolerable dose, blood sugar still controls not good patient, with N1,N1-Dimethylbiguanide or sulfonylurea drugs combined utilization.On December 23rd, 2014, Novo Nordisk Co., Ltd releases again and is used for the treatment of fat Saxenda (trade(brand)name), and dosage is 3mg/ days.

Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is a kind of GLP-1 analogue, has the sequence homology of 97% with people GLP-1, people GLP-1 can in conjunction with and activate GLP-1 acceptor.GLP-1 acceptor is the target spot of natural GLP-1, and GLP-1 is a kind of endogenous gut incretin hormones, can promote pancreatic beta cell glucose concn dependency ground excreting insulin.The dosage regimen being all applicable to once a day unlike pharmacokinetics in human body of, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and pharmacodynamic characteristics with natural GLP-1.After subcutaneous administrations, the mechanism of its extended durations of action comprises: make the self association that absorption is slowed down; With albumin bound; To DPP IV (DPP-IV) and neutral endopeptidase (NEP), there is higher enzyme stability, thus there is longer plasma half-life.

Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] chemistry is expressed as Arg³⁴lys²⁶-[N-ε-(γ-Glu (N-α-palmitoyl))]-GLP-1^7-37, molecular formula is C₁₇₂h₂₆₅n₄₃o₅₁, relative molecular mass is 3751.2, No. CAS is 204656-20-2, and sequence information is as follows:

At present, Novo Nordisk Co., Ltd (US6458924 and US6268343), mainly through gene recombination technology, utilizes yeast production Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; But main chain Arg can only be produced owing to adopting gene recombination technology³⁴-GLP-1^7-37, also need to react with N-α-palmitoyl-Glu (OSu)-OtBu, utilize chemical process at Lys²⁶connect side chain; Due to Arg³⁴-GLP-1^7-37side chain all do not protect, there is multiple avtive spot, so this process can produce more impurity, lose larger.

Also have report to adopt mechanochemical method to prepare Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], mainly concentrate in the connection strategy of side chain modification: as patent CN102286092A adopts Fmoc-Lys (Alloc)-OH to participate in connecing reactive polypeptide, Pd (PPh)₃de-Alloc, then connects side chain; CN103087181A adopts Fmoc-Lys (Mtt)-OH or Fmoc-Lys (Mmt)-OH to participate in connecing reactive polypeptide, takes off Side chain protective group, connect side chain with 1%TFA/5%TIS/DCM; CN103145828A adopts Fmoc-Lys (ivDde)-OH to participate in connecing reactive polypeptide, and hydrazine hydrate takes off Side chain protective group, then connects side chain reaction; CN103980358A adopts first liquid phase synthesis monomer Fmoc-Lys, and (N-ε-(γ-Glu (N-α-palmitoyl)-OtBu)-OH, then participates in connecing reactive polypeptide; CN103275209A adopts the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain first synthesizing the protection of N end, then under liquid-phase condition, uses N^αthere is linked reaction with it in-Pal-γ-Glu (OtBu)-OSu, then takes off the tertiary butyl through sour TFA, de-Fmoc or Dde (or ivDde) two step aftertreatment under alkaline condition, more purifiedly obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].

The present inventor uses existing synthetic method, prepares Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], find purity and yield all not high, be unsuitable for industrial-scale production.In view of above problem, the synthetic method of the present inventor to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is studied, thus obtains technical scheme of the present invention.

Summary of the invention

In order to solve the deficiencies in the prior art, the invention provides the preparation method that a kind of solid phase of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and liquid phase combine.

(N-ε-(γ-Glu (N-Boc)-OtBu)-OH and Fmoc-Ala-Ala-OH participates in preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] to utilize two peptide monomer Fmoc-Lys of synthesis in the present invention, and adopt Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) pattern of trifluoroacetylation, greatly improve the yield of finished product; The present invention reduces synthesis difficulty and production cost, improves purity and the yield of smart peptide, is conducive to large-scale industrial and produces.The skill book scheme that a kind of solid-liquid combination of the present invention prepares Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] method comprises the steps:

A () Boc-Glu (OSu)-OtBu and Fmoc-Lys-OH coupling under basic solution generates two peptide monomer Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH;

(b) with Wang resin or CTC resin for solid phase carrier, adopt Fmoc chemoproection strategy, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence, the full guard peptide resin that N holds trifluoroacetylation is synthesized successively with Fmoc protected amino acid, that wherein Fmoc protected amino acid 26 Lys adopt is monomer Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH, 24 and 25 employing two peptide monomer Fmoc-Ala-Ala-OH, N holds the full guard peptide resin structure of trifluoroacetylation 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 () obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) of trifluoroacetylation by lytic reagent peptide resin, and purified, obtain smart 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 () in the basic conditions, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) the smart peptide generation linked reaction of Pal-OSu and the trifluoroacetylation of step (c) gained, obtains the product that palmitinic acid is modified;

E () obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] through alkaline hydrolysis, purifying, freeze-drying.

Wherein Fmoc-Lys in above step (a) (the concrete synthetic method of N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH is:

Fmoc-Lys-OH or its salt and alkali A are dissolved in water according to the ratio of mol ratio 1:1 ~ 2, add the organic solvent B hydrotropy of 5 ~ 20% volumes, until completely dissolved, the organic solvent B solution of 1 ~ 1.2 times of molar weight (gauge with Fmoc-Lys-OH) Boc-Glu (OSu)-OtBu is added dropwise under stirring; TLC monitors reaction end, after question response terminates decompression removed by evaporation organic solvent, add 10% aqueous citric acid solution again and adjust solution ph to 2 ~ 3, extraction into ethyl acetate, crystallization obtains Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH.

In above step, alkali A is the one in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA etc.;

Organic solvent B is one or more in tetrahydrofuran (THF), dioxane, DMF, acetone, METHYLPYRROLIDONE, acetonitrile.

N holds the concrete preparation method of the full guard peptide resin of trifluoroacetylation to be in above technical scheme steps (b): with Wang resin or CTC resin for solid phase carrier, with 2-5 feed ratio (to synthesize the material gauge of scale) doubly, add corresponding Fmoc protected amino acid and carry out linked reaction, each linked reaction is all that the solid phase of carrying out under the existence of condensing agent connects reactive polypeptide, remove Fmoc with deprotecting regent after completion of the reaction, then carry out linked reaction with next Fmoc protected amino acid; Repetitive operation is until after being blended into 1 His; use trifluoroacetic anhydride end-blocking, obtain full guard peptide resin 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 that N holds trifluoroacetylation.The Fmoc protected amino acid that wherein 26 Lys adopt be the obtained Fmoc-Lys of step (a) (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH, 24 and 25 Ala employing Fmoc protected amino acids are two peptide monomer Fmoc-Ala-Ala-OH; Other sites all adopt conventional Fmoc protected amino acid coupling.Preferably, the Wang resin described in step (b) or the substitution degree of CTC resin are 0.3-0.5mmol/g; Described condensing agent is the one of DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA; Deprotecting regent is 15 ~ 25%(volume content) piperidines/DMF solution.

In technique scheme, lytic reagent described in step (c) is the TFA solution adding volume ratio 1-5% scavenging agent, and described scavenging agent is one or more in methyl-phenoxide, thioanisole, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane.

In technique scheme, the concrete operation step of step (d) is:

Get step (c) gained essence peptide and alkali A be dissolved in water according to the ratio of mol ratio 1:4 ~ 6, add the organic solvent B hydrotropy of volume ratio 5 ~ 20%, until completely dissolved, the organic solvent B solution of 1 ~ 2.0 times of molar weight (gauge with smart peptide) Pal-OSu is added dropwise under stirring; Continue stirring reaction, TLC monitors reaction end; After reaction reaches terminal, add the glycine (gauge with smart peptide) of 1 ~ 2.0 times of molar weight, termination reaction.

Alkali A wherein described in step (d) is the one in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA etc.; Described organic solvent B is one or more in tetrahydrofuran (THF), dioxane, DMF, acetone, METHYLPYRROLIDONE, acetonitrile.

In technical scheme, in step (e), the concrete operation method of alkaline hydrolysis is: the piperidines (liquor capacity in step (d) gained) measuring volume ratio 10-12%, add in the solution of step (d) gained, be mixed with the piperidine solution that concentration is 1M, stirring reaction is complete to trifluoroacetyl group alkaline hydrolysis; Then through ultra-filtration equipment desalination, constantly add pure water dilution peptide solution, keep liquor capacity constant; When solution ph is down to 12 ~ 10, adopts 0.2-1.0% aqueous acetic acid dilution peptide solution, when being down to 5 ~ 6 to pH value, stop suction filtration.

Relative to prior art, the invention has the beneficial effects as follows:

The present invention synthesizes two peptide monomer Fmoc-Lys (N-ε-(γ-Glu (N-Boc)-OtBu)-OH first, and use it for the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], its building-up process and purge process simple, be convenient to middle control detect, be easy to amplify produce; Adopt Fmoc-Ala-Ala-OH to participate in preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] simultaneously, avoid the generation of 24 or 25 disappearance Ala impurity peptides, reduce purifying difficulty, improve yield; The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) of trifluoroacetylation is better water-soluble, is beneficial to Reverse phase chromatography preparation, and the amino simultaneously also protecting N end side reaction does not occur in palmitinic acid modification, greatly improves the yield of finished product; Enforcement of the present invention, solve the difficult problem that in chemosynthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] process, finished product related impurities peptide exceeds standard, total recovery is low, the purity of finished product brings up to more than 99.5%, and single mixing controls below 0.1%.

Embodiment

The present invention is described in detail to use specific embodiment below, but do not limit this patent; According to the feed ratio of feed change of the present invention or reaction solvent or and condensing agent etc., all in protection scope of the present invention.

The abbreviation implication used in specification sheets and claims is as follows:

Fmoc	9-fluorenylmethyloxycarbonyl
		CTC resin	2-chlorine trityl chloride resin
Wang Resins	King's resin
		tBu	The tertiary butyl
Pbf	2,2,4,6,7-pentamethyl-cumarone-5-alkylsulfonyl
		Trt	Trityl
Tfa	Trifluoroacetyl group
		Boc	Tertbutyloxycarbonyl
EDPA	N-ethyl diisopropyl amine
		NMP	METHYLPYRROLIDONE
DCM	Methylene dichloride
		DMF	DMF
DMAP	DMAP
		DIPEA	DIPEA
DIC	N, N-DIC
		HBTU	Benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU	2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
		TBTU	O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric 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

Accurately take Fmoc-Lys-OH183.8g (0.5mol) and sodium carbonate 63.6 (0.6mol) is dissolved in 1200mL water, under low temperature, (2-8 DEG C) slowly adds the tetrahydrofuran solution (200.3g of Boc-Glu (OSu)-OtBu, 0.5mol)/1000ml, stirring reaction, TLC monitors reaction end, after reacting completely, revolve and evaporate THF, add 10% aqueous citric acid solution under ice-water bath and adjust solution ph to 2 ~ 3, 1000ml extraction into ethyl acetate 3 times, merge organic phase, 200ml saturated common salt washes 3 times, anhydrous sodium sulfate drying, concentrated by rotary evaporation is to 1000ml, leave standstill 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-WangResins

Carrier Wang resin 400.0g (sub=0.47mmol/g) is placed in synthesis post, washes twice with 2400mLDMF, add the swelling 30min of 4000mLDCM; After suction filtration falls DCM, the mixing DCM solution adding Fmoc-Gly-OH/DIC/HOBT [takes 118.8g (400mmol) Fmoc-Gly-OH and 64.8g (480mmol) HOBT and is placed in glycine activated bottle, add DMF and the DCM mixing solutions stirring and dissolving that 2000mL volume ratio is 1: 1,76.4ml (480mmol) DIC is added under low temperature (0 DEG C), activate 5 minutes], add 4.8g (4mmol) DMAP after reaction 10min; Reaction 3h, take out reaction solution, wash twice with 4000mLDMF, add capping reagent 2400mL (480ml diacetyl oxide and 408ml pyridinium dissolution are in 1512mLDMF) and react 2h, suction filtration falls reaction solution, use DMF, DCM, methanol wash 2 times respectively, after vacuum-drying, obtain Fmoc-Gly-WangResins 436.6g;it is 0.30mmol/g that substitution degree is surveyed in sampling.

the synthesis of embodiment 3:Fmoc-Gly-CTCResins

Take CTC resin 50.0g (sub=0.40mmol/g) and be placed in synthesis post, wash twice with 240mLDMF, add the swelling 30min of 240mLDCM; After suction filtration falls DCM, add DCM/DMF (3/1, volume ratio) the solution 150ml being dissolved with 5.94g (20mmol) Fmoc-Gly-OH, after stirring, add DIPEA6.6ml (40mmol), drum N₂reaction 60min, takes out reaction solution, adds DCM/CH₃oH/DIPEA (volume ratio 17:2:1) mixing solutions 300ml end-blocking 3 times, each 10min; Then wash 2 times respectively with DMF, DCM, methyl alcohol, vacuum-drying obtains Fmoc-Gly-CTCResins53.80g.Survey substitution degree is 0.29mmol/g.

embodiment 4:the preparation of peptide resin

Accurately take the Fmoc-Gly-WangResins100g (synthesis scale 30mmol) that embodiment 2 substitution degree is 0.30mmol/g and be placed in synthesis post, add the swelling 30min of 1000mlDCM; After suction filtration falls DCM, 800mlDMF washs 2 times, adds 20% piperidines/DMF solution 1000ml deprotection 2 times, reacts 10min and 10min respectively; Then 2 times are washed respectively with 800mlDMF, DCM, DMF; Add the DMF solution 500ml of Fmoc-Arg (Pbf)-OH53.7g (90mmol), HOBT13.4g (99mmol) and DIC15.4ml (99mmol), drum N₂stirring reaction 2h, reaction end is as the criterion with Kaiser reagent detected result, after reaction reaches terminal, takes out reaction solution, washs 2 times respectively with 800mlDMF, DCM, DMF; Deprotection more subsequently.Iterative cycles operation like this, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence, one by one with protected amino acid coupling, the protected amino acid connected successively is: 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 full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) peptide resin: 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-Wangresins that N holds trifluoroacetylation, through vacuum-drying, be weighed as: 245.3g, resin weightening finish 145.3g, rate of body weight gain is 99.2%.

embodiment 5: peptide resin cracking

240.0gN embodiment 4 obtained holds full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) peptide resin of trifluoroacetylation, and (volume proportion is TFA/TIS/H to join freezing 2400ml lysate₂0=95/2.5/2.5), stirred at ambient temperature reaction 3h; Scission reaction terminates, and filter resin, 200mlTFA washing resin 2 times, merging filtrate and washing lotion, concentrated by rotary evaporation, to 1600ml, is poured in the tertiary ether of the freezing first of 16L, separates out white precipitate; After leaving standstill 30min, filter, the tertiary ether of first washs 6 times, and vacuum-drying obtains N and holds the thick peptide 104.1g of trifluoroacetylation Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified), thick peptide yield 94.9%, purity 76.8%.

embodiment 6:the preparation of peptide resin

Accurately taking embodiment 3 substitution degree is that 0.29mmol/gFmoc-Gly-CTCResins51.7g (synthesis scale 15mmol) is placed in synthesis post, adds the swelling 30min of 500mlDCM; After suction filtration falls DCM, 400mlDMF washs 2 times, adds 20% piperidines/DMF solution 500ml deprotection 2 times, reacts 10min and 10min respectively; Then 2 times are washed respectively with 400mlDMF, DCM, DMF; Add the DMF solution 300ml of 26.9g (45mmol) Fmoc-Arg (Pbf)-OH, 6.7g (49.5mmol) HOBT and 7.7ml (49.5mmol) DIC, drum N₂stirring reaction 2h, reaction end is as the criterion with Kaiser reagent detected result, after reaction reaches terminal, takes out reaction solution, washs 2 times respectively with 400mlDMF, DCM, DMF; Deprotection more subsequently.Iterative cycles operation like this, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence, one by one with protected amino acid coupling, the protected amino acid connected successively is: 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 full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) peptide resin: 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-CTCresins that N holds trifluoroacetylation, through vacuum-drying, be weighed as: 116.9g, resin weightening finish 65.2g, rate of body weight gain is 89.1%.

embodiment 7: peptide resin cracking

115.0gN embodiment 6 obtained holds full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) peptide resin of trifluoroacetylation, and (volume proportion is TFA/TIS/H to join freezing 1150ml lysate₂0=95/2.5/2.5), stirred at ambient temperature reaction 3h; Scission reaction terminates, and filter resin, 100mlTFA washing resin 2 times, merging filtrate and washing lotion, concentrated by rotary evaporation, to 800ml, is poured in the tertiary ether of the freezing first of 8L, separates out white precipitate; After leaving standstill 30min, filter, the tertiary ether of first washs 6 times, and vacuum-drying obtains N and holds the thick peptide 47.6g of trifluoroacetylation Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified), thick peptide yield 85.0%, purity 77.6%.

embodiment 8:n holds the refining of trifluoroacetylation Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified)

Taking the thick peptide 100.0g of embodiment 5 gained is dissolved in 10% acetonitrile solution 2500ml, after concussion is dissolved, for subsequent use after 0.45um membrane filtration.

Internal diameter is 100mmC18 preparative column, and moving phase is 0.1%TFA/ water-0.1%TFA/ acetonitrile system, and applied sample amount is 6.0g/ time, flow velocity 300ml/min, gradient elution; With peak Posterior circle sample introduction before peak, intercepting middle control analysis purity is more than 99.5%, the assorted smart peptide solution being less than 0.1% of list, and concentrated rear freeze-drying obtains smart peptide 56.2g, and purification yield is 56.2%, purity 99.6%, and single mixing all is less than 0.1%.

embodiment 9:n holds the refining of trifluoroacetylation Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified)

Taking the thick peptide 45.0g of embodiment 7 gained is dissolved in 10% acetonitrile solution 1200ml, after concussion is dissolved, for subsequent use after 0.45um membrane filtration.

Internal diameter is 100mmC18 preparative column, and moving phase is 0.1%TFA/ water-0.1%TFA/ acetonitrile system, and applied sample amount is 6.0g/ time, flow velocity 300ml/min, gradient elution; With peak Posterior circle sample introduction before peak, intercepting middle control analysis purity is more than 99.5%, the assorted smart peptide solution being less than 0.1% of list, and concentrated rear freeze-drying obtains smart peptide 25.7g, and purification yield is 57.1%, purity 99.6%, and single mixing all is less than 0.1%.

embodiment 10:the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Embodiment 8 gained essence peptide 56.0g (15mmol) is added in 2L tri-mouthfuls of reaction flasks, adds 1000ml10% acetonitrile solution stirring and dissolving, under ice-water bath, slowly drip 10%Na₂cO₃the aqueous solution, regulator solution pH to 11, stops dripping; Under ice-water bath, drip THF solution 7.1g (the 30mmol)/150ml of Pal-OSu, dropwise, remove ice bath, under room temperature, react 3h; Add 2.25g (30mmol) glycine, continue reaction 30min, TLC and monitor reaction end;

In reaction solution, add 150ml piperidines under high degree of agitation, continue under room temperature to stir, HPLC monitors alkaline hydrolysis terminal; After being reacted to terminal, stopping stirring, G3 sand core funnel filtering insolubles, and wash insolubles three times with pure water; Merge washing lotion and filtrate, use ultra-filtration equipment desalination, constantly add pure water dilution peptide solution, keep liquor capacity constant; When solution ph is down to 11, adopts 0.5% aqueous acetic acid dilution peptide solution, when being down to 5 to pH value, stop suction filtration.Filtrate is through 0.45um membrane filtration, stand-by.

Internal diameter is 100mmC8 preparative column, and moving phase is 20mM ammonium acetate aqueous solution-acetonitrile system, and applied sample amount is 3.0g/ time, flow velocity 300ml/min, gradient elution; Before peak and peak Posterior circle sample introduction, intercepting middle control analysis purity is more than 99.5%, single assorted smart peptide solution being less than 0.1%, concentrated freeze-dried smart peptide 39.3g after desalination, and yield is 70.2%, purity 99.6%, is singly assortedly all less than 0.1%; Preparation total recovery is 37.5%.

embodiment 11:the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Embodiment 9 gained essence peptide 24.3g (6.5mmol) is added in 1L tri-mouthfuls of reaction flasks, adds 500ml10% acetonitrile solution stirring and dissolving, under ice-water bath, slowly drip 10%EDPA/NMP solution, regulator solution pH to 11, stop dripping; Under ice-water bath, drip THF solution 4.6g (the 13mmol)/80ml of Pal-OSu, dropwise, remove ice bath, under room temperature, react 3h; Add 0.98g (13mmol) glycine, continue reaction 30min, TLC and monitor reaction end;

In reaction solution, add 61ml piperidines under high degree of agitation, continue under room temperature to stir, HPLC monitors alkaline hydrolysis terminal; After being reacted to terminal, stopping stirring, G3 sand core funnel filtering insolubles, and wash insolubles three times with pure water; Merge washing lotion and filtrate, use ultra-filtration equipment desalination, constantly add pure water dilution peptide solution, keep liquor capacity constant; When solution ph is down to 11, adopts 0.5% aqueous acetic acid dilution peptide solution, when being down to 5 to pH value, stop suction filtration.Filtrate is through 0.45um membrane filtration, stand-by.

Internal diameter is 100mmC8 preparative column, and moving phase is 20mM ammonium acetate aqueous solution-acetonitrile system, and applied sample amount is 3.0g/ time, flow velocity 300ml/min, gradient elution; Before peak and peak Posterior circle sample introduction, intercepting middle control analysis purity is more than 99.5%, single assorted smart peptide solution being less than 0.1%, concentrated freeze-dried smart peptide 17.3g after desalination, and yield is 71.1%, purity 99.6%, is singly assortedly all less than 0.1%; Preparation total recovery is 34.5%.

Claims (10)

1. solid-liquid combination prepares a method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, comprises the steps:

A () Boc-Glu (OSu)-OtBu and Fmoc-Lys-OH coupling under basic solution generates two peptide monomer Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH;

(b) with Wang resin or CTC resin for solid phase carrier, adopt Fmoc chemoproection strategy, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence, the full guard peptide resin that N holds trifluoroacetylation is synthesized successively with Fmoc protected amino acid, that wherein Fmoc protected amino acid 26 Lys adopt is monomer Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH, 24 and 25 employing two peptide monomer Fmoc-Ala-Ala-OH; N holds the full guard peptide resin structure of trifluoroacetylation 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 () obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) of trifluoroacetylation by lytic reagent peptide resin, and purified, obtain smart 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 () in the basic conditions, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (unmodified) the smart peptide generation linked reaction of Pal-OSu and the trifluoroacetylation of step (c) gained, obtains the product that palmitinic acid is modified;

E () obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] through alkaline hydrolysis, purifying, freeze-drying.

2. solid-liquid combination according to claim 1 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (a), (concrete operations of N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH are preparation two peptide monomer Fmoc-Lys: be dissolved in water by Fmoc-Lys-OH and alkali A according to the ratio of mol ratio 1:1 ~ 2, add the organic solvent B hydrotropy of 5 ~ 20% volumes, until completely dissolved, the organic solvent B solution of 1 ~ 1.2 times of molar weight (gauge with Fmoc-Lys-OH) Boc-Glu (OSu)-OtBu is added dropwise under stirring; Continue stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates organic solvent, add 10% aqueous citric acid solution and adjust solution ph to 2 ~ 3, extraction into ethyl acetate, crystallization obtains Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH.

3. solid-liquid combination according to claim 1 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, N holds the concrete preparation method of the full guard peptide resin of trifluoroacetylation to be in step (b): with Wang resin or CTC resin for solid phase carrier, with 2-5 feed ratio (to synthesize the material gauge of scale) doubly, add corresponding Fmoc protected amino acid and carry out linked reaction, each linked reaction is all that the solid phase of carrying out under the existence of condensing agent connects reactive polypeptide, Fmoc is removed after completion of the reaction with deprotecting regent, linked reaction is carried out again with next Fmoc protected amino acid, repetitive operation is until after being blended into 1 His, use trifluoroacetic anhydride end-blocking, obtain full guard peptide resin 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 that N holds trifluoroacetylation.

4. solid-liquid combination according to claim 3 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, the substitution degree of Wang resin or CTC resin is 0.3-0.5mmol/g.

5. solid-liquid combination according to claim 3 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, the Fmoc protected amino acid that 26 Lys adopt be Fmoc-Lys (N-ε-(γ-Glu (N-α-Boc)-OtBu)-OH, 24 and 25 Ala employing Fmoc protected amino acids are two peptide monomer Fmoc-Ala-Ala-OH; Other sites all adopt conventional Fmoc protected amino acid coupling.

6. solid-liquid combination prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 3, it is characterized in that, described condensing agent is the one of DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA; Deprotecting regent is 15 ~ 25%(volume content) piperidines/DMF solution.

7. solid-liquid combination prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 1, it is characterized in that, lytic reagent described in step (c) is the TFA solution adding volume ratio 1-5% scavenging agent, and described scavenging agent is one or more in methyl-phenoxide, thioanisole, dithioglycol, mercaptoethanol, phenol, water, tri isopropyl silane.

8. solid-liquid combination according to claim 1 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, the concrete operation step of step (d) is: get step (c) gained essence peptide and alkali A be dissolved in water according to the ratio of mol ratio 1:4 ~ 6, add the organic solvent B hydrotropy of volume ratio 5 ~ 20%, until completely dissolved, the organic solvent B solution of 1 ~ 2.0 times of molar weight (gauge with smart peptide) Pal-OSu is added dropwise under stirring; Continue stirring reaction, TLC monitors reaction end; After reaction reaches terminal, add the glycine (gauge with smart peptide) of 1 ~ 2.0 times of molar weight, termination reaction.

9. the solid-liquid combination according to any one of claim 2 or 8 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that: described alkali A is one or more in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA etc.; Described organic solvent B is one or more the mixed solution in tetrahydrofuran (THF), dioxane, DMF, acetone, METHYLPYRROLIDONE, acetonitrile.

10. solid-liquid combination according to claim 1 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (e), the concrete operation step of alkaline hydrolysis is: the piperidines (liquor capacity in step (d) gained) measuring volume ratio 10-12%, add in the solution of step (d) gained, be mixed with the piperidine solution that concentration is 1M, stirring reaction is complete to trifluoroacetyl group alkaline hydrolysis; Then through ultra-filtration equipment desalination, constantly add pure water dilution peptide solution, keep liquor capacity constant; When solution ph is down to 12 ~ 10, adopts 0.2-1.0% aqueous acetic acid dilution peptide solution, when being down to 5 ~ 6 to pH value, stop suction filtration.