CN102942625A - Solid-phase synthesis method of exenatide - Google Patents

Solid-phase synthesis method of exenatide Download PDF

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Publication number
CN102942625A
CN102942625A CN2012105049496A CN201210504949A CN102942625A CN 102942625 A CN102942625 A CN 102942625A CN 2012105049496 A CN2012105049496 A CN 2012105049496A CN 201210504949 A CN201210504949 A CN 201210504949A CN 102942625 A CN102942625 A CN 102942625A
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fmoc
ser
exenatide
pro
tbu
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苏贤斌
钱依冰
毛怡春
叶青
董海军
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NANJING IMPACT BIOSCIENCE TECHNOLOGY Co Ltd
Nanjing Tech University
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NANJING IMPACT BIOSCIENCE TECHNOLOGY Co Ltd
Nanjing Tech University
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides a solid-phase synthesis method of exenatide, which comprises the following steps: 1) preparing Fmoc-Ser(tBu)-amino resin from initial raw materials Fmoc-Ser(tBu)-OH and amino resin with the substitution degree of 0.3-1.2mmol/g; 2) after removing Fmoc protecting group from the Fmoc-Ser(tBu)-amino resin, sequentially connecting Fmoc amino resins and at least one amino-protected Fmoc amino resin or dipeptide with temporarily substituted amide N in a peptide sequence, and removing Fmoc to obtain the exenatide amino resin of which the side chain is fully protected; and 3) removing the side chain protecting group from the resin, and precipitating with ice aether, thereby obtaining the exenatide. The synthesis method has the advantages of high efficiency, simple reaction operation and easy after-treatment, and is suitable for industrial production.

Description

A kind of Exenatide solid phase synthesis process
Technical field
The present invention relates to the polypeptide drugs synthesis technical field, be specifically related to a kind of Exenatide solid phase synthesis process.
Background technology
Diabetes (diabetes mellitus) are because Regular Insulin is absolute or relative deficiency causes with hyperglycemia and many complication and save as endocrine metabolism common disease, the frequently-occurring disease of feature, show according to the up-to-date publish data of the World Health Organization (WHO), global diabetic subject's number had reached 1.8 hundred million in 2007, and sickness rate still increases year by year, predict 2025, whole world diabetic subject's number will reach 300,000,000, become the human the third-largest killer after cardiovascular and cerebrovascular and cancer.
Diabetes mainly are divided into I type (insulin-dependent, insulin dependent diabetes, IDDM) and II type (non insulin dependent diabetes, non-insulin dependent diabetes, NIDDM), wherein the type ii diabetes patient accounts for more than 90%.The type ii diabetes patient exists insulin resistant and hypoinsulinism two aspects unusual more, islet beta-cell apoptosis often occurs at the middle and advanced stage of morbidity.The mechanism of action of the oral antidiabetic drug of present clinical use mostly is and strengthens insulin sensitivity or promote insulin secretion with stabilizing blood sugar, all can't solve this difficult problem of β apoptosis.Yet, glucagon-like-peptide-1 (Glucagon-like peptide 1, GLP-1) and the application of analogue medicine so that the treatment of type ii diabetes obtains breakthrough, being expected to provides brand-new selection scheme for treating diabetes.But because GLP-1 is easily degraded rapidly by pepx (dipeptidyl pepidiase IV, DPP IV), the transformation period only had about 2 minutes in vivo, so external source GLP-1 is difficult to obtain clinical use.
Exendin-4 is that a kind of wishing from America drawn 39 the amino acid whose natural polypeptides hormones that contain that separation obtains huge lizard (Heloderma suspectum) saliva, with GLP-1 53% sequence homology is arranged, the function that not only has class GLP-1 insulin secretion accelerating, and its body internal stability is also far above GLP-1.According to these characteristics of Exendin-4, U.S. Amylin company and Eli Lilly company have developed jointly the medicine Exendin-4 (claiming again Exenatide, formula I compound, Chinese name Exenatide, trade(brand)name Byetta) of chemosynthesis.Byetta is first exendin-4 medicine in the world, goes on the market through drugs approved by FDA in April, 2005.Studies show that Exenatide can not only be controlled blood sugar well, also can promote the newborn and propagation of islet cells, reduce the β apoptosis, improve the β cell function, have very significantly clinical application advantage.In addition, find can also the slow down speed of gastrointestinal emptying of Exenatide in the clinical study, make the people not have hunger sensation and reduce food intake, thereby make people's weight loss.
Its structural formula is as follows:
1 5 10 15
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-
20 25 30 35
Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
The preparation method of Exenatide mainly contains biological synthesis process and chemical synthesis.Biological synthesis process utilizes the artificial gene recombination form to obtain Exenatide complete sequence (such as patent CN101501209A).Chemical synthesis can adopt conventional full solid phase synthesis (such as patent WO2008109079A2, CN101357938A, CN101538324A) technology usually.Full solid phase synthesis is simple to operate, and the peptide chain short for some fragments is the method for a quickness and high efficiency.Yet along with the prolongation of peptide chain, the possibility that the peptide section that wherein hydrophobicity is strong forms beta sheet is very large, and solid phase synthesis process often causes the sequence that encounters difficulties.In addition, also exist the molecule between peptide chain and the resin carrier to gather (aggregation) phenomenon during solid-phase peptide is synthetic, this all can cause coupling and deprotection efficient greatly to reduce.
In the solid phase synthesis of Exenatide, main challenge has two places.There are a plurality of hydrophobic amino acids in one place the from the 26th to the 19th the amino acid whose sequence in peptide chain middle part, form first " difficult sequences " district, the solid phase synthesis practice shows, the 26th Leu, the 23rd Ile, the 21st Leu and the 19th 's Val condensation is all very difficult, even if used the reaction conditions that improves, such as conversion condensation reaction system, prolong the reaction times, repeatedly repeat condensation etc., the result also produces little effect.Second " difficult sequences " district relates to three continuous glutaminic acid residues.In solid phase synthesis, behind the Side chain protective group, hydrophobicity increases the L-glutamic acid sequence of repetition greatly on tape, causes producing at resin the part of distortion, and this is so that be difficult to continue other amino acid of coupling to prolong peptide chain in the L-glutamic acid sequence of this repetition.
In chemical synthesis, WO2009053315A1 discloses a kind of solid phase and liquid phase of adopting in conjunction with the method for synthesizing Exenatide.The method of solid-liquid combination has been because shortened the length of fragment, so synthetic comparatively easy on solid phase of individual chip, and purity is higher, is conducive to the purifying of final product.Yet, the problems such as condensation difficulty that fragment dissolving difficulty, C-end racemization and secondary structure cause appear when carrying out fragment condensation in liquid phase easily.In addition, the synthetic of each fragment must be used relatively expensive full guard peptide resin, greatly increased cost.
The pharmaceutical application important in view of Exenatide is worth, and is necessary to provide more effective Exenatide synthetic schemes, can overcome the defective of above method and be fit to plant-scale production.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide that a kind of cost is low, the synthetic method of the Exenatide that is suitable for suitability for industrialized production.
Technical scheme: the invention provides a kind of Exenatide solid phase synthesis process, it is characterized in that: may further comprise the steps:
(1) be that the aminoresin of 0.3-1.2mmol/g is starting raw material with solid phase synthesis process by Fmoc-Ser (tBu)-OH and substitution value, reaction makes Fmoc-Ser (tBu)-aminoresin;
(2) Fmoc-Ser (tBu)-aminoresin is after sloughing Fmoc under the effect of Fmoc deprotecting regent, the interim dipeptides that replaces on the Fmoc amino acid that in coupling solvent, connects successively Fmoc amino acid and at least one amido protecting under the coupling system effect according to the peptide order or acid amides N, and under the effect of Fmoc deprotecting regent, slough successively Fmoc, make the Exenatide aminoresin of side chain full guard;
(3) Exenatide aminoresin cracking under the effect of cracking system of side chain full guard removed Side chain protective group, the ice ether sedimentation namely gets Exenatide.
Wherein, in the step (1), described aminoresin is Rink Amide resin, Rink Amide-AM resin, RinkAmide-MBHA resin or Sieber Amide resin; The quality mol ratio of aminoresin and Fmoc-Ser (tBu)-OH is 1g:(0.5-5) mmol; Reaction times 0.5-2h, temperature of reaction is 10-40 ℃.
Wherein, in the step (2), the Fmoc amino acid of described amido protecting is 2-hydroxyl-4-methoxy-benzyl (Hmb), 2, the amino acid of 4-dimethoxy-benzyl (Dmb) or 2,4,6-trimethoxy benzyl (Tmb) protection, the upper interim dipeptides that replaces of described acid amides N is that the upper interim substituting group of pseudo-proline(Pro) or acid amides N is 2-hydroxyl-4-methoxy-benzyl, 2,4-dimethoxy-benzyl, 2,4, the dipeptides of 6-trimethoxy benzyl protection; Wherein, the structural formula of Hmb is
Figure BDA00002498172500031
The structural formula of Dmb is The structural formula of Tmb is
Figure BDA00002498172500033
Among the present invention, " pseudo-proline(Pro) (Pseudoprolines) " refers in the prolyl ring γCH 2By the proline analogs that Sauerstoffatom or sulphur atom replaced.The dipeptides that contains Ser, Thr and Cys, its side chain beta-hydroxy or β-sulfydryl and aldehyde or ketone cyclocondensation form the pseudo-proline(Pro) structure that is similar to proline(Pro) De oxazolidine five-membered ring structure, and this oxazole ring is unstable to TFA.By adopting the protection strategy of this provisional protection framework amide key, formation and the molecule between peptide chain and the resin carrier that can effectively overcome beta sheet in the solid phase synthesis gather (aggregation) phenomenon.
Can replace corresponding two amino-acid residues in the pseudo-proline(Pro) solid phase synthesis, be positioned at pseudo-proline(Pro) part common amino acid A(such as Ser, Thr or the Cys corresponding to the oxazole ring protection of C-end), and adjacent part can corresponding arbitrarily other amino acid B.Be expressed as: R 1-B (R 2)-A (ψ R 3 , R 4Pro)-and OH, its structural formula is:
Figure BDA00002498172500041
R 1Be amino protecting group, be preferably Fmoc; R2 ForThe side chain of amino acid B determines whether to contain corresponding protecting group according to different amino acid; R 3, R 4Separately independently methyl or H.Preferably, R 3, R 4It is methyl.For the Ser of oxazole ring protection, R 5=H; For the Thr of oxazole ring protection, R 5=Me.
Behind the deprotection, pseudo-proline(Pro) reduction becomes corresponding dipeptides, is expressed as: R 1-B (R 2)-A-OH.At this moment, R 2Be the side chain of amino acid B, the unprotect base, its structural formula is:
Figure BDA00002498172500042
Further preferred as Exenatide solid phase synthesis process of the present invention, described pseudo-proline(Pro) comprises Fmoc-Gly-Thr (ψ Me, MePro)-OH, Fmoc-Phe-Thr (ψ Me, MePro)-OH, Fmoc-Thr (tBu)-Ser (ψ Me, MePro)-OH, Fmoc-Leu-Ser (ψ Me, MePro)-OH, Fmoc-Pro-Ser (ψ Me, MePro)-OH, Fmoc-Ser (tBu)-Ser (ψ Me, MePro)-OH.
Its structural formula is as follows:
Figure BDA00002498172500051
Among the present invention, " Hmb ", " Dmb " and " Tmb " refer to the provisional protecting group of connected on the polypeptide backbone amido linkage N atom, i.e. benzyl derivative, the different substituents of corresponding its ortho para.The ability of the provisional protection framework amide key of Hmb, Dmb or Tmb form is extensive, can not be limited in and only contain Ser, uses in the fragment of Thr and Cys, reaches the effect that is similar to pseudo-proline(Pro).The provisional protecting group of Hmb, Dmb or Tmb form is unstable to TFA, adds a certain amount of triethyl silicane or tri isopropyl silane in the TFA of cracking Linker, can remove.
Among the present invention, the amino acid structure formula of the Hmb that adopts, Dmb or Tmb protection is:
Figure BDA00002498172500052
R 6Be amino acid whose side chain, determine whether to contain corresponding protecting group according to different amino acid; R 7Be amino protecting group, be preferably Fmoc.
For the upper temporary protection base Hmb of polypeptide backbone acid amides N, R 8(or R 9)=OH, R 9(or R 8)=H; For the upper temporary protection base Dmb of polypeptide backbone acid amides N, R 8(or R 9)=OCH 3, R 9(or R 8)=H; For the upper temporary protection base Tmb of framework amide N, R 8=R 9=OCH 3
R wherein 7Behind required next aminoacid replacement, Hmb (or Dmb, Tmb) amino acid can also be the form of dipeptides, and its structural formula is:
Figure BDA00002498172500061
Wherein, R 10Be amino acid whose side chain, determine whether to contain corresponding protecting group according to different amino acid; R 11Be amino protecting group, be preferably Fmoc; R 6, R 8And R 9Definition the same.
Further preferred as Exenatide solid phase synthesis process of the present invention, the amino acid of described Hmb, Dmb or Tmb protection comprises Fmoc-(Hmb) Gly-OH, Fmoc-(Hmb) Leu-OH, Fmoc-(Hmb) Ala-OH; Fmoc-(Dmb) Gly-OH, Fmoc-(Dmb) Leu-OH, Fmoc-(Dmb) Ala-OH, Fmoc-(Tmb) Gly-OH, Fmoc-(Tmb) Leu-OH and Fmoc-(Tmb) Ala-OH.
Still more preferably, at the monamino acid that adopts the Dmb protection with upper/lower positions: Fmoc-(Dmb) Ala 18-OH, Fmoc-(Dmb) Leu 21-OH, Fmoc-(Dmb) Leu 26-OH, Fmoc-(Dmb) Gly 29-OH; Its structure is as follows:
Figure BDA00002498172500062
Particularly, in the Exenatide fragment, optionally adopt at least one Hmb, Dmb, or Tmb protection monamino acid or the upper interim dipeptides that replaces of acid amides N, be Gly-Thr (4-5 position), Phe-Thr (6-7 position), Thr-Ser (7-8 position), Leu-Ser (10-11 position), the Pro-Ser(31-32 position) and the Ser-Ser(32-33 position) at least one two peptide fragment replaced by pseudo-proline(Pro), perhaps at least one amino acid is Hmb by the upper temporary protection base of corresponding framework amide N in the sequence, the amino acid of Dmb or Tmb or dipeptides displacement, described fragment randomly contains Side chain protective group; Preferably, be used in combination for the amino acid that adopts pseudo-proline(Pro) fragment and Dmb protection; More preferably, at Gly-Thr 5, Leu 10-Ser 11The pseudo-proline(Pro) fragment of middle employing is simultaneously at Ala 18, Leu 21, Leu 26, Gly 29The amino acid of the corresponding Dmb protection of middle employing.
Wherein, in the step (2), coupling solvent is DCM, any one among DMF or the NMP or more than one combination; Coupling system is any one or the combination of a plurality of and DIC among HOBt, HOAt, HOOBt or the Cl-HOBt, or is any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt; The linked reaction temperature is 10-50 ℃, and the linked reaction time is 0.5-3h; The Fmoc deprotecting regent is piperidines, and the deprotection reaction temperature is 10-50 ℃, and the reaction times is 5-60min, and the volume ratio of Fmoc deprotecting regent and coupling solvent is 1:(1-5).
Wherein, in the step (3), described cracking system comprises the mixing solutions of trifluoroacetic acid (TFA), 1，2-ethandithiol (EDT), water and tri isopropyl silane (TIS), and its volume ratio is (90-95): (2-5): (2-5): (1-5); The cracking system is (5-50) mL:1g with the volume mass ratio of the Exenatide aminoresin of side chain full guard; The scission reaction temperature is 10-50 ℃, and the reaction times is 1-3h.
Wherein, in the step (3), behind the ice ether sedimentation, also comprise purification step, be specially: Exenatide is dissolved in the acetic acid aqueous solution, filters, filtrate is through C18 or C8 post gradient elution, and moving phase is water and acetonitrile, desalination, and freeze-drying namely gets Exenatide.Preferably, elution requirement is: gradient is water consumption from 30% to 50%, and elution time is 20-40min, and flow velocity is 10-500mL/min; Add respectively properties-correcting agent in water and the acetonitrile, described properties-correcting agent is acetate, phosphoric acid salt or trifluoroacetate, and the amount of described properties-correcting agent is 0.1 ‰-1.0 ‰.
Particularly, preparation method of the present invention is take aminoresin as starting raw material, according to the method for solid phase synthesis successively Fmoc amino acid or the upper interim dipeptides that replaces of acid amides N of coupling amido protecting, obtains 39 peptide fragment of full guard, comprises following steps:
1) take aminoresin such as Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin or SieberAmide resin as starting raw material, first amino acid of coupling Fmoc-Ser (tBu)-OH obtains Fmoc-Ser (tBu)-aminoresin;
2) take corresponding amino acid as starting raw material, synthetic pseudo-proline(Pro).Such as Fmoc-Gly 4-Thr (ψ Me, MePro) 5-OH, Fmoc-Phe 6-Thr (ψ Me, MePro) 7-OH, Fmoc-Thr (tBu) 7-S er (ψ Me, MePro) 8-OH, Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH, Fmoc-Pro 31-Ser (ψ Me, MePro) 32-OH, Fmoc-Ser (tBu) 32-Ser (ψ Me, MePro) 33-OH.
3) take corresponding amino acid as starting raw material, the upper interim dipeptides that replaces of synthetic Hmb (or Dmb, Tmb) protection monamino acid or acid amides N is such as Fmoc-(Hmb) Gly-OH, Fmoc-(Hmb) Leu-OH, Fmoc-(Hmb) Ala-OH; Fmoc-(Dmb) Gly-OH, Fmoc-(Dmb) Leu-OH, Fmoc-(Dmb) Ala-OH or Fmoc-(Tmb) Gly-OH, Fmoc-(Tmb) Leu-OH, Fmoc-(Tmb) Ala-OH.
4) continue successively Fmoc amino acid or the upper interim dipeptides that replaces of acid amides N of coupling amido protecting in the step 1) on the resin of gained, and remove successively the Fmoc protecting group, obtain Exenatide--the aminoresin of full guard.Coupling system is any one or the more than one combination among DIC and HOBt, HOAt, HOOBt or the Cl-HOBt, also can be any one or more than one combination and HBTU, the HATU among DPIEA or NMM and HOBt, HOAt, HOOBt or the Cl-HOBt, the condensation system that any one among PyBOP or the PyAOP or more than one combination form.
4) deprotection base, cracking resin get the Exenatide crude product; Pass through the HPLC purifying, lyophilize obtains sterling again.
Beneficial effect: Exenatide solid phase synthesis process efficient provided by the invention is high, operation is simple, aftertreatment is easy, is suitable for suitability for industrialized production.
The method adopts the Fmoc amino acid of amido protecting or the protection strategy of the upper interim dipeptides that replaces of acid amides N; at Exenatide sequence Gly-Thr (4-5); Phe-Thr (6-7); Thr-Ser (7-8); Leu-Ser (10-11); Pro-Ser(31-32) and at least one two peptide fragment Ser-Ser(32-33) replaced by pseudo-proline(Pro); perhaps/with sequence at least one amino acid by corresponding amino Hmb; Dmb; or Fmoc amino acid or the upper interim substituting group of acid amides N that Tmb protects are Hmb; Dmb; or the displacement of Tmb protection dipeptides, can effectively overcome the formation of beta sheet in the solid phase synthesis and the molecule aggregation phenomenon between peptide chain and the resin carrier.
Particularly; in the Exenatide fragment; optionally adopt at least one Hmb; Dmb; or Tmb protection monamino acid or the upper interim dipeptides that replaces of acid amides N; be Gly-Thr (4-5 position); Phe-Thr (6-7 position); Thr-Ser (7-8 position); Leu-Ser (10-11 position); the Pro-Ser(31-32 position) and the Ser-Ser(32-33 position) at least one two peptide fragment replaced by pseudo-proline(Pro), perhaps at least one amino acid is Hmb by the upper temporary protection base of corresponding framework amide N in the sequence; Dmb; or the amino acid of Tmb or dipeptides displacement.
Embodiment
The present invention is including but not limited to following examples.
The reagent name abbreviation:
DMF:N, dinethylformamide
DCM: methylene dichloride
The NMP:N-methyl-2-pyrrolidone
DIC:N, N '-DIC
TFA: trifluoroacetic acid
DIPEA:N, the N-diisopropylethylamine
The NMM:N-methylmorpholine
The HOBt:1-hydroxybenzotriazole
HOAt:N-hydroxyl-7-azepine benzotriazole
HOOBt:3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone
Cl-HOBt:6-chloro-1-hydroxy benzo triazole
HBTU: benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU:2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus
PyAOP:(3H-1,2,3-triazolo [4,5-b] pyridine-3-oxygen base) three-1-pyrrolidyl phosphorus hexafluorophosphate
PPTS: para-methylbenzenepyridinsulfonate sulfonate
DMP:2, the 2-Propanal dimethyl acetal
EDT:1, the 2-dithioglycol
PIP: piperidines
TIS: tri isopropyl silane
Reagent source:
Fmoc-Ser (tBu)-OH, N-(9-fluorenes methoxy carbonyl the acyl group)-O-tertiary butyl-Serine: the biochemical (Shanghai) Co., Ltd. of gill
Fmoc-Leu-OH: the biochemical (Shanghai) Co., Ltd. of gill
H-Ser-OBzlHCl: the biochemical (Shanghai) Co., Ltd. of gill
Rink Amide resin: Tianjin Nankai Hecheng S﹠T Co., Ltd.
Rink Amide-AM resin: Tianjin Nankai Hecheng S﹠T Co., Ltd.
Rink Amide-MBHA resin: Tianjin Nankai Hecheng S﹠T Co., Ltd.
Sieber Amide resin: Tianjin Nankai Hecheng S﹠T Co., Ltd.
The pseudo-proline(Pro) Fmoc-Leu-Ser (ψ of embodiment 1 preparation Me, MePro)-OH
(1) preparation Fmoc-Leu-Ser (ψ Me, MePro)-OH
1) preparation Fmoc-Leu-Ser-OBzl
In flask, add Fmoc-Leu-OH(3.54g, 10mmol), H-Ser-OBzlHCl(2.32g, 10mmol), HOBt(1.42g, 10.5mmol), add DMF(100mL under the ice bath), behind dissolution of solid, add DIPEA(3.8mL, 22mmol) activation 5min adds HBTU(3.98g, 10.5mmol at last), reaction 10min recession deicing is bathed, and room temperature reaction is complete to the TLC detection reaction.Use subsequently ethyl acetate extraction, organic phase 1N HCl, saturated NaHCO 3, saturated NaCl washing, anhydrous Na 2SO 4Drying is spin-dried for solvent and gets the 5.5g white solid, is Fmoc-Leu-Ser-OBzl.
2) preparation Fmoc-Leu-Ser (ψ Me, MePro)-OBzl
In flask, add Fmoc-Leu-Ser-OBzl(3.2g, 5mmol), DMP(3.07mL, 25mmol), PPTS(0.377g, 1.5mmol), toluene (100mL), back flow reaction is complete to the TLC detection reaction.Use subsequently ethyl acetate extraction, the saturated NaHCO of organic phase 3, saturated NaCl washing, anhydrous Na 2SO 4Drying is spin-dried for solvent and gets brown oil.Cross column purification and get yellow oil 2.9g, be Fmoc-Leu-Ser (ψ Me, MePro)-OBzl.
3) preparation Fmoc-Leu-Ser (ψ Me, MePro)-OH
In flask, add Fmoc-Leu-Ser (ψ Me, MePro)-OBzl(2.9g), the MeOH dissolving, (wherein, palladium massfraction 10% 0.29g), passes into H to add Pd/C 2Compressive reaction is complete to the TLC detection reaction.Filtering Pd/C, liquid are spin-dried for the rear post of crossing, and get white solid 2.1g, are Fmoc-Leu-Ser (ψ Me, MePro)-OH.
(2) other pseudo-proline(Pro) are such as Fmoc-Gly-Thr (ψ Me, MePro)-OH, Fmoc-Phe-Thr (ψ Me, MePro)-OH, Fmoc-Thr (tBu)-Ser (ψ Me, MePro)-OH, Fmoc-Pro-Ser (ψ Me, MePro)-OH, Fmoc-Ser (tBu)-Ser (ψ Me, MePro the preparation method of) – OH is with (1).
The amino acid of embodiment 2 preparation Hmb, Dmb or Tmb protection
(1) preparation Fmoc-(Dmb) Gly-OH
1) preparation H-(Dmb) Gly-OH
First with amino acid Gly(7.5g, 100mmol) be dissolved in KOH(5.6g, 100mmol) the aqueous solution in, the ethanolic soln of 2,4-dimethoxy benzaldehyde (16.6g, 100mmol) is added in the aqueous solution, slowly add NaBH after at room temperature stirring 30min 4In the aqueous solution of (4.1g, 101mmol) (NaOH that contains a small amount of 1M).Be stirred to and react completely.Regulate PH=4 with concentrated hydrochloric acid, have solid to separate out.With the washing of the methanol/water solution of frozen water and 50%, get the Gly-OH 14g of compound H-(Dmb), productive rate 62.2%.
2) preparation Fmoc-(Dmb) Gly-OH
To H-(Dmb) Gly-OH(11.25g, add dioxane in frozen water solution 50mmol) to fully dissolving, add subsequently yellow soda ash (17g, 160mmol), the dioxane solution that slowly adds again Fmoc-Cl (27.2g, 105mmol), rear ethyl acetate extraction 3 times of using react completely, HCl(0.5N) washing is 3 times, merges organic phase, anhydrous Na 2SO 4Drying is spin-dried for behind the solvent to get yellow oil.Cross column purification and get Fmoc-(Dmb) Gly-OH 7.2g, productive rate 31.8%.
(2) amino acid of other Hmb, Dmb or Tmb protection is such as the preparation method same (1) of Fmoc-(Hmb) Gly-OH, Fmoc-(Hmb) Leu-OH, Fmoc-(Hmb) Ala-OH, Fmoc-(Dmb) Leu-OH, Fmoc-(Dmb) Ala-OH, Fmoc-(Tmb) Gly-OH, Fmoc-(Tmb) Leu-OH and Fmoc-(Tmb) Ala-OH.
Embodiment 3 preparation Fmoc-Gly-(Hmb) Gly-OH
1) preparation H-(Hmb) Gly-OH
First with amino acid Gly(7.5g, 100mmol) be dissolved in KOH(5.6g, 100mmol) the aqueous solution in, the ethanolic soln of 2,4-dimethoxy benzaldehyde (15.2g, 100mmol) is added in the aqueous solution, slowly add NaBH after at room temperature stirring 30min 4In the aqueous solution of (4.1g, 101mmol) (NaOH that contains a small amount of 1M).Be stirred to and react completely.Regulate PH=4 with concentrated hydrochloric acid, have solid to separate out.With the washing of the methanol/water solution of frozen water and 50%, get the Gly-OH 13.8g of compound H-(Hmb), productive rate 65.4%.
2) preparation Fmoc-Gly-OSu
With Fmoc-Gly-OH(2.97g, 10mmol), N-hydroxy-succinamide (1.15g, 10mmol) is dissolved among the DMF, adds EDCHCl(2.30g, 10mmol after the ice-water bath cooling).Room temperature reaction 24h, the TLC monitoring reaction is complete.Use subsequently ethyl acetate extraction, the saturated NaHCO of organic phase 3, saturated NaCl washing, anhydrous Na 2SO 4Drying is spin-dried for solvent and gets white solid 3.6g, productive rate 91.3%.
3) preparation Fmoc-Gly-(Hmb) Gly-OH
With H-(Hmb) Gly-OH(1.06g, 5mmol) be dissolved in DMF(200mL) and H 2O(20mL) in the mixed solution, with adding DIPEA in the backward suspension, stirring and dissolving.Add at last Fmoc-Gly-OSu(1.97g, 5mmol).Reaction solution is used, the EA extraction.The HCl of 1M regulates pH to 1-2, organic phase H 2O washes 3 times, Na 2SO 4Drying is revolved and is steamed to get oily matter, SiO 2Cross post and get product 1.3g, productive rate 53.1%.
(2) the dipeptides preparation method of other Hmb, Dmb or Tmb protection is with (1), such as Fmoc-Asn (Trt)-(Hmb) Gly-OH, Fmoc-Glu (OtBu)-(Hmb) Ala-OH.
Embodiment 4
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide resin of 0.81mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain.In solid phase reactor, add Fmoc-Ser (tBu)-OH(1mmol), HOBt(0.162g, 1.2mmol), DIC(0.19mL, 1.2mmol), DMF(10mL), 20 ℃ the reaction 1.5h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (tBu) 5-Phe-Thr (tBu) 7-Ser (tBu) 8-Asp (OtBu)-Leu 10-Ser (ψ Me, MePro) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu) 15-Glu (OtBu) 16-Glu (OtBu)-Ala-Val-Arg (pbf) 20-Leu-Phe-Ile-Glu (OtBu)-Trp (Boc) 25-Leu-Lys (Boc)-Asn (Trt)-(Dmb) Gly-Gly 30-Pro-Ser (tBu) 32-Ser (tBu) 33-Gly-Ala 35-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin
Get H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(2mmol), HOBt(0.162g, 2.4mmol), DIC(0.19mL, 2.4mmol), DMF(10mL), 30 ℃ of reaction 1h.The coupling completeness can use the Kaiser test to detect.By known in this field, alternatively, coupling reagent can be selected from any one or the combination of a plurality of and DIC among HOBt, HOAt, HOOBt or the Cl-HOBt.
After detection was passed through, 60min removed the Fmoc protecting group with 10 ℃ of reactions of 20%PIP/DMF solution, wherein contains PIP10mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With the remaining amino acid of this method successively coupling.Wherein, Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH replaces Fmoc-Leu-OH and Fmoc-Ser (tBu)-OH on the corresponding position.Gly 29Adopt Fmoc-(Dmb) Gly 29-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and namely gets the Exenatide aminoresin 6.4g of side chain full guard.
(3) preparation Exenatide
Configuration lysate 100mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=92.5:2.5:2.5:2.5.The ice bath cooling, to the Exenatide aminoresin 6.4g that wherein adds the side chain full guard, 25 ℃ are stirred 2h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 800mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.5g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (30min), eluent A:0.5 ‰ TFA/ water, B:0.5 ‰ TFA/ acetonitrile; Flow velocity is 10mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.4g.
Embodiment 5
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide-AM resin of 0.30mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group, washs repeatedly with DCM, MeOH and DMF respectively, drains.In solid phase reactor, add Fmoc-Ser (tBu)-OH(4mmol), HOAt(5mmol), DIC(5mmol) and DCM(5mL), 40 ℃ of reaction 1.5h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (tBu) 5-Phe-Thr (ψ Me, MePro) 7-Ser (tBu) 8-Asp (OtBu)-Leu 10-Ser (ψ Me, MePro) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu) 15-Glu (OtBu) 16-Glu (OtBu)-Ala-Val-Arg (pbf) 20-(Dmb) Leu-Phe-Ile-Glu (OtBu)-Trp (Boc) 25-(Dmb) Leu-Lys (Boc)-Asn (Trt)-(Dmb) Gly-Gly 30-Pro-Ser (tBu) 32-Ser (tBu) 33-Gly-Ala 35-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin.
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1.2mmol), HOAt(2mmol), DIC(1.8mmol) and DCM(5mL) 50 ℃ of reaction 0.5h.The coupling completeness can use the Kaiser test to detect.
After detection was passed through, 30min removed the Fmoc protecting group with 20 ℃ of reactions of 25%PIP/DMF solution, wherein contains PIP 2mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH replaces Fmoc-Leu-OH and Fmoc-Ser (tBu)-OH on the corresponding position; FmocPhe 6-Thr (ψ Me, MePro) 7-OH replaces Fmoc-Phe-OH and Fmoc-Thr (tBu) on the corresponding position 7-OH.Leu 21Adopt Fmoc-(Dmb) Leu 21-OH; Leu 26Adopt Fmoc-(Dmb) Leu 26-OH; Gly 29Adopt Fmoc-(Dmb) Gly 29-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and namely gets the Exenatide aminoresin 6.0g of side chain full guard.
(3) preparation Exenatide
Configuration lysate 30mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=90:2:2:1.The ice bath cooling, to the Exenatide aminoresin 6.0g that wherein adds the side chain full guard, 50 ℃ are stirred 1h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 250mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.3g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (20min), eluent A:0.8 ‰ TFA/ water, B:0.8 ‰ TFA/ acetonitrile; Flow velocity is 500mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.6g.
Embodiment 6
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide-MBHAAmide-AM resin of 1.2mmol/g in solid phase reactor; add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group; wash repeatedly with DCM, MeOH and DMF respectively, drain.In solid phase reactor, add Fmoc-Ser (tBu)-OH(6mmol) HOOBt(7mmol), DIC(7mmol) and NMP(10mL), 10 ℃ of reaction 2h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:5), reaction 0.5h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (ψ Me, MePro) 5-Phe-Thr (tBu) 7-Ser (tBu) 8-Asp (OtBu)-Leu 10-Ser (ψ Me, MePro) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu) 15-Glu (OtBu) 16-Glu (OtBu)-(Dmb) Ala-Val-Arg (pbf) 20-(Dmb) Leu-Phe-Ile-Glu (OtBu)-Trp (Boc) 25-(Dmb) Leu-Lys (Boc)-Asn (Trt)-(Dmb) Gly-Gly 30-Pro-Ser (tBu) 32-Ser (tBu) 33-Gly-Ala 35-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin.
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1mmol), HOOBt(1.6mmol), DIC(1.6mmol) and NMP(10mL) 10 ℃ of reaction 3h.The coupling completeness can use the Kaiser test to detect.
After detection was passed through, 5min removed the Fmoc protecting group with 50 ℃ of reactions of 30%PIP/DMF solution, wherein contains PIP 6mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH replaces Fmoc-Leu-OH and Fmoc-Ser (tBu)-OH on the corresponding position; FmocGly 4-Thr (ψ Me, MePro) 5-OH replaces Fmoc-Gly-OH and Fmoc-Thr (tBu)-OH on the corresponding position.Ala 18Adopt Fmoc-(Dmb) Ala 18-OH; Leu 21Adopt Fmoc-(Dmb) Leu 21-OH; Leu 26Adopt Fmoc-(Dmb) Leu 26-OH; Gly 29Adopt Fmoc-(Dmb) Gly 29-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and gets thick peptide resin 6.8g.
(3) preparation Exenatide
Configuration lysate 300mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=95:5:5:5.The ice bath cooling, to the Exenatide aminoresin 1g that wherein adds the side chain full guard, 10 ℃ are stirred 3h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 2000mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.6g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (40min), eluent A:0.1 ‰ acetate/water, B:0.1 ‰ TFA/ acetonitrile; Flow velocity is 100mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.8g.
Embodiment 7
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Sieber Amide resin of 0.6mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group, washs repeatedly with DCM, MeOH and DMF respectively, drains.In solid phase reactor, add Fmoc-Ser (tBu)-OH(8mmol), Cl-HOBt(9mmol), DIC(9mmol) in DMF(10mL), 30 ℃ the reaction 0.5h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:10), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Leu-Ser (ψ Me, MePro) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Ala-Val-Arg (pbf)-Leu-Phe-Ile-Glu (OtBu)-Trp (Boc)-Leu-Lys (Boc)-Asn (Trt)-Gly-Gly-Pro-Ser (tBu)-Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser (tBu)-Rink Amide resin.
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1mmol), Cl-HOBt(2mmol), DIC(2mmol) in DMF(10mL) 35 ℃ of reaction 1h.The coupling completeness can use the Kaiser test to detect.
After detection was passed through, 20min removed the Fmoc protecting group with 30 ℃ of reactions of 20%PIP/DMF solution, wherein contains PIP 8mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH replaces Fmoc-Leu-OH and Fmoc-Ser (tBu)-OH on the corresponding position.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and gets thick peptide resin 5.7g.
(3) preparation Exenatide
Configuration lysate 60mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=92:3:3:3.The ice bath cooling, to the Exenatide aminoresin 4g that wherein adds the side chain full guard, 35 ℃ are stirred 1.5h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 500mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.1g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (30min), eluent A:0.5 ‰ TFA/ water, B:0.5 ‰ TFA/ acetonitrile; Flow velocity is 50mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.0g.
Embodiment 8
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide-AM resin of 0.92mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group, washs repeatedly with DCM, MeOH and DMF respectively, drains.In solid phase reactor, add Fmoc-Ser (tBu)-OH(10mmol), HOBt(12mmol), DPIEA(3mmol), HBTU(12mmol) and DMF(10mL), 20 ℃ of reaction 1.5h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol)=1:1), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (tBu) 5-Phe-Thr (tBu) 7-Ser (tBu) 8-Asp (OtBu)-Leu 10-Ser (tBu) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu) 15-Glu (OtBu) 16-Glu (OtBu)-Ala-Val-Arg (pbf) 20-Leu-Phe-Ile-Glu (OtBu)-Trp (Boc) 25-Leu-Lys (Boc)-Asn (Trt)-(Dmb) Gly-Gly 30-Pro-Ser (tBu) 32-Ser (tBu) 33-Gly-Ala 35-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1mmol), HOBt(1.2mmol), DPIEA(1.2mmol), HBTU(1.2mmol) and DMF(10mL) 20 ℃ of reaction 2h.The coupling completeness can use the Kaiser test to detect.By known in this field, alternatively, coupling reagent can be selected from any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.
After detection was passed through, 10min removed the Fmoc protecting group with 40 ℃ of reactions of 20%PIP/DMF solution, wherein contains PIP4mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Gly 29Adopt Fmoc-(Dmb) Gly 29-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and namely gets the Exenatide aminoresin 6.1g of side chain full guard.
(3) preparation Exenatide
Configuration lysate 60mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=92.5:2.5:2.5:2.5.The ice bath cooling, to the Exenatide aminoresin 6.1g that wherein adds the side chain full guard, 15 ℃ are stirred 2.5h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 500mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.5g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (30min), eluent A:0.5 ‰ TFA/ water, B:0.5 ‰ TFA/ acetonitrile; Flow velocity is 50mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.0g.
Embodiment 9
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide-AM resin of 0.58mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group, washs repeatedly with DCM, MeOH and DMF respectively, drains.In solid phase reactor, add Fmoc-Ser (tBu)-OH(1mmol), HOAt(1.6mmol), NMM(1.6mmol), PyBOP(1.6mmol) and DMF(10mL), 35 ℃ of reaction 0.5h.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His(Trt) 1-Gly-Glu(OtBu)-Gly-Thr(tBu) 5-Phe-Thr(tBu) 7-Ser(ψ Me,MePro) 8-
Asp(OtBu)-Leu 10-Ser(tBu) 11-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu) 15-Glu(OtBu) 16-Glu(OtBu)-
Ala-Val-Arg(pbf) 20-Leu-(Dmb)Phe-Ile-Glu(OtBu)-Trp(Boc) 25
-Leu-Lys(Boc)-Asn(Trt)-(Dmb)Gly-Gly-Pro-Ser(ψ Me,MePro) 32-Ser(tBu) 33-Gly-Ala 35
-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin.
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1mmol), HOAt(1.6mmol), NMM(1.6mmol), PyBOP(1.6mmol) and DMF(10mL), 30 ℃ of reaction 1.5h.The coupling completeness can use the Kaiser test to detect.
After detection was passed through, 10min removed the Fmoc protecting group with 30 ℃ of reactions of 20%PIP/DMF solution, wherein contains PIP 8mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Fmoc-Thr (tBu) 7-Ser (ψ Me, MePro) 8-OH replaces Fmoc-Thr (tBu) on the corresponding position 7-OH and Fmoc-Ser (tBu)-OH; Fmoc – Pro 31-Ser (ψ Me, MePro) 32-OH replaces Fmoc-Pro-OH and Fmoc-Ser (tBu)-OH on the corresponding position.Phe 22Adopt Fmoc-(Dmb) Phe 22-OH; Gly 29Adopt Fmoc-(Dmb) Gly 29-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and gets thick peptide resin 5.5g.
(3) preparation Exenatide
Configuration lysate 30mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=95:5:5:5.The ice bath cooling, to the Exenatide aminoresin 5.5g that wherein adds the side chain full guard, 40 ℃ are stirred 1.5h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 250mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.4g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (30min), eluent A:0.1 ‰ acetate/water, B:0.1 ‰ TFA/ acetonitrile; Flow velocity is 100mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.7g.
Embodiment 10
(1) preparation H-Ser (tBu)-resin
Take by weighing the 2g substitution value and be the Rink Amide resin of 0.6mmol/g in solid phase reactor, add DCM swelling 20min, add subsequently 20%PIP/DMF solution, reaction 20min removes the Fmoc protecting group, washs repeatedly with DCM, MeOH and DMF respectively, drains.In solid phase reactor, add Fmoc-Ser (tBu)-OH(1mmol), HOAt(1.2mmol), DPIEA(3mmol), HATU(1mmol) and DMF(30mL), 25 ℃ of reaction 30min.The resin washing is drained, namely get Fmoc-Ser (tBu)-resin, recording the resin carrying capacity is 0.25mmol/g.Add closed reagent 10mL(acetic anhydride (mmol) in resin: DIPEA(mmol): DMF=1:1:8), reaction 1h seals remaining amino, washs repeatedly with DCM, MeOH and DMF respectively, drains.Add 20%PIP/DMF solution reaction 20min and remove the Fmoc protecting group, wash repeatedly with DCM, MeOH and DMF respectively, drain, get H-Ser (tBu)-resin.
(2) preparation
Fmoc-His (Trt) 1-Gly-Glu (OtBu)-Gly-Thr (tBu) 5-Phe-Thr (tBu) 7-Ser (tBu) 8-Asp (OtBu)-Leu 10-Ser (ψ Me, MePro) 11-Lys (Boc)-Gln (Trt)-Met-Glu (OtBu) 15-Glu (OtBu) 16-Glu (OtBu)-(Hmb) Ala-Val-Arg (pbf) 20-Leu-Phe-Ile-Glu (OtBu)-Trp (Boc) 25-Leu-Lys (Boc)-Asn (Trt)-Gly-(Dmb) Gly 30-Pro-Ser (tBu) 32-Ser (ψ Me, MePro) 33-Gly-Ala 35-Pro-Pro-Pro-Ser (tBu) 39-Rink Amide resin
Take by weighing H-Ser (tBu)-resin (2.0g) in solid phase reactor, add amino acid Fmoc-Pro-OH(1mmol), HOBt(1.2mmol), DPIEA(3.6mmol), HBTU(1.2mmol) and DMF(10mL) 20 ℃ of reaction 2h.The coupling completeness can use the Kaiser test to detect.By known in this field, alternatively, coupling reagent can be selected from any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.
After detection was passed through, 20min removed the Fmoc protecting group with 25 ℃ of reactions of 20%PIP/DMF solution, wherein contains PIP5mL, washed repeatedly with DCM, MeOH and DMF respectively, drained.
With this remaining amino acid of coupling successively.Wherein, Gly 30Adopt Fmoc-(Dmb) Gly 30-OH; Fmoc-Leu 10-Ser (ψ Me, MePro) 11-OH replaces Fmoc-Leu-OH and Fmoc-Ser (tBu)-OH on the corresponding position; Fmoc-Ser (tBu) 32-Ser (ψ Me, MePro) 33-OH replaces Fmoc-Ser (tBu)-OH and Fmoc-Ser (tBu)-OH on the corresponding position; Fmoc-Glu (OtBu) 17-(Hmb) Ala 18-OH replaces Fmoc-Glu (OtBu) on the corresponding position 17-OH and Fmoc-Ala 18-OH.After coupling is finished, wash repeatedly with DCM, MeOH and DMF, MeOH shrinks and drains, and namely gets the Exenatide aminoresin 6.1g of side chain full guard.
(3) preparation Exenatide
Configuration lysate 50mL in round-bottomed flask, its each component volume ratio is TFA:EDT: methyl-phenoxide: thioanisole=92.5:2.5:2.5:2.5.The ice bath cooling, to the Exenatide aminoresin 6.1g that wherein adds the side chain full guard, 15 ℃ are stirred 2.5h; Filter, with a small amount of TFA drip washing resin, collect filtrate; Gained filtrate is slowly dropped in the 500mL ice ether, the adularescent Precipitation, centrifugal, namely get Exenatide crude product 2.5g.
(4) purifying
The Exenatide crude product is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP(3mm); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:30%-50% (30min), eluent A:0.5 ‰ TFA/ water, B:0.5 ‰ TFA/ acetonitrile; Flow velocity is 50mL/min.Removing freeze-drying behind the acetonitrile, to get purity be 95% Exenatide sterling 1.8g.

Claims (9)

1. Exenatide solid phase synthesis process is characterized in that may further comprise the steps:
(1) be that the aminoresin of 0.3-1.2mmol/g is starting raw material with solid phase synthesis process by Fmoc-Ser (tBu)-OH and substitution value, reaction makes Fmoc-Ser (tBu)-aminoresin;
(2) Fmoc-Ser (tBu)-aminoresin is after sloughing Fmoc under the effect of Fmoc deprotecting regent, the interim dipeptides that replaces on the Fmoc amino acid that in coupling solvent, connects successively Fmoc amino acid and at least one amido protecting under the coupling system effect according to the peptide order or acid amides N, and under the effect of Fmoc deprotecting regent, slough successively Fmoc, make the Exenatide aminoresin of side chain full guard;
(3) Exenatide aminoresin cracking under the effect of cracking system of side chain full guard removed Side chain protective group, the ice ether sedimentation, and get final product.
2. a kind of Exenatide solid phase synthesis process according to claim 1, it is characterized in that: in the step (1), described aminoresin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin or Sieber Amide resin.
3. a kind of Exenatide solid phase synthesis process according to claim 1, it is characterized in that: in the step (2), the Fmoc amino acid of described amido protecting is 2-hydroxyl-4-methoxy-benzyl, 2, the amino acid of 4-dimethoxy-benzyl or 2,4,6-trimethoxy benzyl protection, the upper interim dipeptides that replaces of described acid amides N is that the upper interim substituting group of pseudo-proline(Pro) or acid amides N is 2-hydroxyl-4-methoxy-benzyl, 2,4-dimethoxy-benzyl, 2,4, the dipeptides of 6-trimethoxy benzyl protection; Wherein, the structural formula of 2-hydroxyl-4-methoxy-benzyl is
Figure FDA00002498172400011
The structural formula of 2,4-dimethoxy-benzyl is
Figure FDA00002498172400012
The structural formula of 2,4,6-trimethoxy benzyl is
Figure FDA00002498172400013
4. a kind of Exenatide solid phase synthesis process according to claim 3, it is characterized in that: described pseudo-proline(Pro) comprises Fmoc-Gly-Thr (ψ Me, MePro)-OH, Fmoc-Phe-Thr (ψ Me, MePro)-OH, Fmoc-Thr (tBu)-Ser (ψ Me, MePro)-OH, Fmoc-Leu-Ser (ψ Me, MePro)-OH, Fmoc-Pro-Ser (ψ Me, MePro)-OH and Fmoc-Ser (tBu)-Ser (ψ Me, MePro)-OH.
5. a kind of Exenatide solid phase synthesis process according to claim 3, it is characterized in that: described 2-hydroxyl-4-methoxy-benzyl, 2,4-dimethoxy-benzyl or 2, the amino acid of 4,6-trimethoxy benzyl protection comprises Fmoc-(Hmb) Gly-OH, Fmoc-(Hmb) Leu-OH, Fmoc-(Hmb) Ala-OH; Fmoc-(Dmb) Gly-OH, Fmoc-(Dmb) Leu-OH, Fmoc-(Dmb) Ala-OH, Fmoc-(Tmb) Gly-OH, Fmoc-(Tmb) Leu-OH and Fmoc-(Tmb) Ala-OH.
6. a kind of Exenatide solid phase synthesis process according to claim 1, it is characterized in that: in the step (2), coupling solvent is DCM, any one among DMF or the NMP or more than one combination; Coupling system is any one or the combination of a plurality of and DIC among HOBt, HOAt, HOOBt or the Cl-HOBt, or is any one or a plurality of combinations among any one or two and HBTU, HATU, PyBOP or the PyAOP among any one or a plurality of and DPIEA or the NMM among HOBt, HOAt, HOOBt or the Cl-HOBt.
7. a kind of Exenatide solid phase synthesis process according to claim 1; it is characterized in that: in the step (2), the Fmoc deprotecting regent is piperidines, and the deprotection reaction temperature is 10-50 ℃; reaction times is 5-60min, and the volume ratio of Fmoc deprotecting regent and coupling solvent is 1:(3-5).
8. a kind of Exenatide solid phase synthesis process according to claim 1, it is characterized in that: in the step (3), described cracking system comprises trifluoroacetic acid, 1, the mixing solutions of 2-dithioglycol, water and tri isopropyl silane, its volume ratio are (90-95): (2-5): (2-5): (1-5).
9. a kind of Exenatide solid phase synthesis process according to claim 1, it is characterized in that: in the step (3), behind the ice ether sedimentation, also comprise purification step, be specially: Exenatide is dissolved in the acetic acid aqueous solution, filters, filtrate is through C18 or C8 post gradient elution, moving phase is water and acetonitrile, and freeze-drying namely gets Exenatide.
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