CN110317258A - A kind of novel polypeptide segment of Suo Malu peptide and preparation method thereof - Google Patents
A kind of novel polypeptide segment of Suo Malu peptide and preparation method thereof Download PDFInfo
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- CN110317258A CN110317258A CN201810269653.8A CN201810269653A CN110317258A CN 110317258 A CN110317258 A CN 110317258A CN 201810269653 A CN201810269653 A CN 201810269653A CN 110317258 A CN110317258 A CN 110317258A
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Abstract
The present invention relates to polypeptide drugs to synthesize field, and in particular to a kind of polypeptide fragment with protecting group for being used to prepare Suo Malu peptide.The novel polypeptide segment reduces the impurity of segment in synthesis Suo Malu peptide, improves the purity of final products.
Description
Technical field
The present invention relates to polypeptide drugs to synthesize field, and in particular to it is a kind of be used to prepare Suo Malu peptide with protecting group
Polypeptide fragment.The polypeptide fragment reduces missing peptide fragment impurity and racemization impurity in synthesis Suo Malu peptide, improves final products
Purity.
Technical background
Glucagon-like peptide 1 (GLP-1) is one kind " secretin " of human intestines and stomach's mucous membrane natural secretion, Ke Yiyu
Receptor on islet cells is combined and is stimulated insulin secretion, and then generates the effect for reducing blood glucose.GLP-1 receptor stimulating agent class
The advantages of hypoglycemic medicine, is that the incidence of hypoglycemic event is significantly lower than insulin, and can reduce food intake and delay
Gastric emptying is conducive to control weight, can protect islet beta cell function.
Suo Malu peptide (Sermaglutide) is a kind of new long-acting glucagon peptide -1 (GLP-1) analog, often
Week subcutaneous injection is primary, type 2 diabetes patient's blood glucose level can be made to greatly improve, and risk of hypoglycemia is lower.It is same in this
When, Suo Malu peptide can also be by reducing appetite and reducing food intake dose, induction weight-reducing.In addition, Suo Malu peptide can also be shown
Writing reduces type 2 diabetes patient's major cardiovascular event risk.
Suo Malu peptide is drawn after Liraglutide (Novo Nordisk, 2010/1/25), albiglutide (GSK, 2014/4/15), degree
On Shandong peptide (gift comes, 2014/9/18), Exenatide (AZ, 2015/4/28) and lixisenatide (Sino is luxuriant and rich with fragrance, 2016/7/28) 5
The another GLP-1 drug being widely noticed after city's drug, and on December 7th, 2017 FDA have approved NovoNorm Durso Ma Shandong peptide
Application for quotation.
The peptide sequence structure of Suo Malu peptide is as follows:
About the synthetic method of Suo Malu peptide, mainly with solid phase method, amino acid couplings obtain backbone sequence one by one at present, so
Amino acid access or the segment access one by one on the side chain of 20 Lys again afterwards.And about Suo Malu peptide fragment synthetic method
Document report is less.Patent WO2016046753, which is reported, utilizes segment [1-4], [5-31], [5-16], [17-31], [5-12]
The backbone sequence of [13-16] Lai Hecheng Suo Malu peptide, patent WO2016046753 do not report the synthesis side of segment [1-4]
Method, synthesis in solid state obtains other segments mainly on resin, is then coupled again with other segments.Synthesis in solid state is with guarantor
The polypeptide fragment of shield base can not reduce the naturally occurring high cost factor of solid phase polypeptide synthesis；The Fragment purity of acquisition is low,
It is directly reacted with other segments without purifying, the impurity for bringing product into also increases；It is condensed amino acid acquisition one by one with solid phase method
Segment, process is tedious for condensation and deprotection each time, and can all increase the possibility of amino acid racemization, generates racemization impurity.
Suo Malu peptide especially is synthesized come gradually coupling amino acid with solid phase polypeptide synthesis, after the amino acid Aib for introducing big steric hindrance,
Great challenge is brought to the coupling of subsequent His, steric hindrance causes coupling efficiency to reduce the piece for together and generating missing His
Section impurity, while coupling efficiency and generate be exactly histidine high racemization.These reasons can all cause product obtain purity and
The decline of yield, or even qualified product cannot be produced.
Summary of the invention
In view of existing Suo Malu peptide preparation method above shortcomings, the object of the present invention is to provide a kind of use
In the novel polypeptide segment with protecting group for preparing Suo Malu peptide.
Term explanation:
Side chain protecting group: refer to and the side chain of amino acid (i.e. amino acid general formula H2R yl in N-C (R) (H)-COOH) it is even
The chemical part of connection, help to prevent a part of side chain and Peptide systhesis, processing and etc. used in chemical substance it is anti-
It answers.
Condensing agent: can cause the reagent of condensation reaction, in Peptide systhesis, can espespecially amino be promoted to be coupled to be formed with carboxyl
The reagent of peptide bond.
Assistant activator: in polypeptide condensation reaction, can assist in the reagent that condensing agent preferably promotes condensation reaction, such as:
The generation of racemization impurity in condensation reaction, catalysis is inhibited to accelerate reaction speed etc..
First aspect present invention provides a kind of novel polypeptide segment for being used to prepare Suo Malu peptide, which has following knot
Structure:
R1-His(R2)-Aib-Glu(OtBu)-Gly-R
Wherein, R1Selected from Fmoc, Boc, Dde or Cbz；R2Selected from H, Fmoc, Boc, Mmt or Mtt；R be selected from OH, F, Cl,
Br、I、CN、-OSu。
Preferably, aforementioned polypeptides segment R1-His(R2)-Aib-Glu (OtBu)-Gly-R is selected from:
R1-His(R2)-Aib-Glu(OtBu)-Gly-OH
Or R1-His(R2)-Aib-Glu(OtBu)-Gly-Cl
Or R1-His(R2)-Aib-Glu(OtBu)-Gly-OSu
It is highly preferred that the polypeptide fragment is selected from Fmoc-His (Boc)-Aib-Glu (OtBu)-Gly-OH, Boc-His-
Aib-Glu(OtBu)-Gly-OSu、Fmoc-His(Fmoc)-Aib-Glu(OtBu)-Gly-OH、Fmoc-His(Fmoc)-Aib-
Glu(OtBu)-Gly-Cl。
Second aspect of the present invention provides a kind of polypeptide fragment R1-His(R2)-Aib-Glu (OtBu)-Gly-OH preparation side
Method, the method for using liquid phase condensations obtain, specifically includes the following steps:
(1) Cbz-Glu (OtBu)-OH and glycine methyl ester hydrochloride are dissolved in tetrahydrofuran, dicyclohexyl carbon is added
Reaction 18 hours is stirred at room temperature in diimine and assistant activator 1- hydroxy benzo triazole (HOBt).After reaction, filtering reaction
Liquid, filter cake are washed with tetrahydrofuran, are concentrated under reduced pressure after merging filtrate, then are redissolved in methylene chloride, and solution is at 0 DEG C
It after standing 30 minutes, is filtered, solid is washed with methylene chloride, and pressurization concentration is carried out after merging filtrate, obtains Cbz-Glu
(OtBu)-Gly-OMe。
(2) it takes Cbz-Glu (OtBu)-Gly-OMe to be dissolved in the methylene chloride containing a small amount of HCl, Pd/ is added after nitrogen purge
C catalyst, then be passed through hydrogen and carry out reduction reaction.Reaction terminates, Filtration of catalyst, and filtrate directly carries out anti-in next step
It answers.
(3) Cbz-Aib-OH, (1- cyano -2- ethyoxyl -2- oxo ethyleneimino oxygen is added in filtrate one step up
Base) dimethylamino-morpholine-carbon hexafluorophosphate (COMU) and n,N-diisopropylethylamine be stirred at room temperature 24 hours, react
Terminate, be concentrated under reduced pressure remove methylene chloride, remaining grease is dissolved in ethyl acetate, then respectively with 10% potassium sulfate
Solution, 5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam, obtains Cbz-Aib- after column chromatography
Glu(OtBu)-Gly-OMe。
(4) above-mentioned product is dissolved in the methylene chloride containing a small amount of HCl, Pd/C catalyst is added after nitrogen purge, then be passed through
Hydrogen carries out reduction reaction.Reaction terminates, and Filtration of catalyst, filtrate is spin-dried for.Dissolve the residue in tetrahydrofuran, then plus
Enter the NaOH solution of 1mol/L, is reacted under ice bath 1 hour, adjust PH to neutrality with glacial acetic acid after reaction, vacuum rotary steam is used
Toluene vacuum distillation water removal.
(5) concentrate is dissolved in n,N-Dimethylformamide, R is added1- His (R2)-OH, (1- cyano -2- ethyoxyl -
2- oxo ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU) and N, N- diisopropylethylamine
It (is stirred at room temperature 24 hours, reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, remaining grease is dissolved in ethyl acetate, so
It is washed respectively with the sodium carbonate liquor of 10% potassium sulfate solution, 5% afterwards, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam,
R is obtained after column chromatography1- His (R2)-Aib-Glu (OtBu)-Gly-OH polypeptide fragment.
Third aspect present invention provides a kind of polypeptide fragment R1-His(R2)-Aib-Glu (OtBu)-Gly-OSu preparation side
Method, the method for using liquid phase condensations obtain, specifically includes the following steps:
By R1-His(R2)-Aib-Glu (OtBu)-Gly-OH and N- hydroxysuccinimide (HOSu) be dissolved in tetrahydrofuran
In, it is added dicyclohexylcarbodiimide (DCC), reaction 18 hours is stirred at room temperature.After reaction, filtering reacting liquid, filter cake are used
Tetrahydrofuran is washed, and is concentrated under reduced pressure after merging filtrate, then is redissolved in methylene chloride, and solution stands 30 points at 0 DEG C
Zhong Hou is filtered, and solid is washed with methylene chloride, and pressurization concentration is carried out after merging filtrate, obtains R after column chromatographs1-His
(R2)-Aib-Glu(OtBu)-Gly-OSu。
Fourth aspect present invention provides polypeptide fragment R described in one kind1-His(R2)-Aib-Glu (OtBu)-Gly-Cl
Preparation method, the method for using liquid phase condensations obtain, specifically includes the following steps:
By I compound represented R of formula1-His(R2)-Aib-Glu (OtBu)-Gly-OH is dissolved in methylene chloride, it is added suitable
N,N-Dimethylformamide is measured, thionyl chloride is added under ice bath, stirring removes ice bath after 15 minutes and is warming up to room temperature reaction.Reaction
Terminate, vacuum rotary steam is evaporated methylene chloride and thionyl chloride；Product R is obtained after silica gel column chromatography1-His(R2)-Aib-Glu
(OtBu)-Gly-Cl segment.
Novel polypeptide segment provided by the present invention can be purified and be controlled in the synthesis of every step, reduce the miscellaneous of segment
Matter improves purity, and segment is introduced after Suo Malu peptide to the purity that final products also can be improved.It takes full advantage of without chirality
The advantage of carboxyl in glycine (Gly)；His, Glu are followed by reducing in solid phase into main chain together with Aib liquid phase coupling
It is gradually coupled the racemization degree of His and Glu, ensure that the reproducibility between every batch of；Coupling His ratio is in solid phase in the liquid phase
Being coupled racemization degree reduces, and improves the coupling efficiency of His after big steric hindrance amino acid Aib, reduces the life of entire Suo Malu peptide
Produce cost.
Fragment condensation of the invention introduces four amino acid of Suo Malu peptide molecule nitrogen end, increases once for easy
The middle control chance of dl aminoadipic acid (histidine etc.), becoming solid phase to the passive detection of amino acid racemization after solid phase condensation
To the initiativequality control of the used polypeptide fragment purity (including racemization degree) with protecting group before condensation, keep away simultaneously
Exempt from or reduce [D-His1]-Suo Malu peptide, [Des His1]-Suo Malu peptide, [Des Gly4]-Suo Malu peptide, [+Gly4]-rope
The generation of Ma Shandong peptide impurity has great help for improving the product quality of Remedies for diabetes Suo Malu peptide in this way.
In the present invention, the Cbz refers to benzyloxycarbonyl group；Used reagent is conventional reagent, the raw material made
It is conventional chemical raw material, it can be by commercially available or be prepared according to published existing literature.Such as Fmoc-
Gly-Wang resin or Fmoc-Gly-2-chlorotrityl resin can be commercially available from gill biochemistry Co., Ltd.
Specific embodiment
The present invention will be further described by the following examples.It is understood that these embodiments are only used for
It specifically describes and is used in more detail, and should not be construed as present invention is limited in any form.Herein, unless separately
External declaration, in which: (i) temperature indicates that operation carries out under room temperature environment, and the room temperature generally refers to 15- with degree Celsius (DEG C)
30 DEG C, preferential 15-25 DEG C, more preferable 15-20 DEG C.(ii) content and yield " % " are mass percent.(iii) product is pure
Spending % is high-efficient liquid phase color spectral purity HPLC.
The preparation of embodiment 1, Fmoc-His (Boc)-Aib-Glu (OtBu)-Gly-OH
Cbz-Glu (OtBu)-OH (67.5g, 200mmol) and glycine methyl ester hydrochloride (25.1g, 200mmol) is molten
In 550ml tetrahydrofuran, dicyclohexylcarbodiimide (45.4g, 220mmol) and HOBt (40.5g, 300mmol), room is added
Temperature is stirred to react 18 hours.After reaction, filtering reacting liquid, filter cake are washed with tetrahydrofuran, are carried out after merging filtrate
It is concentrated under reduced pressure, then is redissolved in methylene chloride, solution is filtered, solid is washed with methylene chloride after 0 DEG C stands 30 minutes
It washs, pressurization concentration is carried out after merging filtrate, obtain Cbz-Glu (OtBu)-Gly-OMe (76.6g, 93.8%), Ms=408.19
(M+H+)。
Cbz-Glu (OtBu)-Gly-OMe (75g, 183.6mmol) is taken to be dissolved in the methylene chloride of 600ml HCl containing 11.4ml
In, Pd/C catalyst is added after nitrogen purge, then be passed through hydrogen and carry out reduction reaction.Reaction terminates, Filtration of catalyst, filter
Liquid directly carries out next step reaction.
Cbz-Aib-OH (43.6g, 183.6mmol), (1- cyano -2- ethyoxyl -2- oxo is added in filtrate one step up
Ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU, 157.3g, 367.2mmol) and N, N- bis-
Wopropyl ethyl amine (71.2g, 550.8mmol) is stirred at room temperature 24 hours, and reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, will remain
Remaining grease is dissolved in ethyl acetate, is then washed respectively with the sodium carbonate liquor of 10% potassium sulfate solution, 5%, organic
Mutually dry with anhydrous sodium sulfate, vacuum rotary steam, column obtains Cbz-Aib-Glu (OtBu)-Gly-OMe (84.3g, 93%) after chromatographing
Ms=493.24 (M+H+)。
Cbz-Aib-Glu (OtBu)-Gly-OMe (80g, 162.1mmol) is taken to be dissolved in the dichloro of 800ml HCl containing 10.1ml
In methane, Pd/C catalyst is added after nitrogen purge, then is passed through hydrogen and carries out reduction reaction.Reaction terminates, and is filtered to remove catalysis
Agent, filtrate are spin-dried for.800ml tetrahydrofuran is dissolved the residue in, 1mol/L NaOH solution 180ml is then added, is reacted under ice bath
1 hour, PH is adjusted to neutrality with glacial acetic acid after reaction, vacuum rotary steam is evaporated under reduced pressure with toluene and is removed water, obtains concentrate
Aib-Glu(OtBu)-Gly-OH。
Above-mentioned concentrate is dissolved in n,N-Dimethylformamide, addition Fmoc-His (Boc)-OH (77.4g,
162.1mmol), (1- cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphoric acid
Salt (COMU, 146.6g, 342.2mmol) and n,N-diisopropylethylamine (62.9g, 486.3mmol) are stirred at room temperature 24 hours, instead
Should terminate, be concentrated under reduced pressure remove methylene chloride, remaining grease is dissolved in ethyl acetate, then respectively with 10% sulfuric acid
Potassium solution, 5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam, obtains Fmoc- after column chromatography
His (Boc)-Aib-Glu (OtBu)-Gly-OH (118.2g, yield 90.6%), Ms=804.37 (M+H+)。
The preparation of embodiment 2, Boc-His-Aib-Glu (OtBu)-Gly-OSu
Cbz-Glu (OtBu)-OH (50.6g, 150mmol) and glycine methyl ester (18.8g, 150mmol) are dissolved in 500ml
In tetrahydrofuran, dicyclohexylcarbodiimide (34g, 165mmol) and HOBt (30.4g, 225mmol) is added, is stirred at room temperature anti-
It answers 18 hours.After reaction, filtering reacting liquid, filter cake are washed with tetrahydrofuran, are concentrated under reduced pressure after merging filtrate,
It is redissolved in methylene chloride again, solution is filtered, solid is washed with methylene chloride, merging filtrate after 0 DEG C stands 30 minutes
After carry out pressurization concentration, obtain Cbz-Glu (OtBu)-Gly-OMe (57.2g, 93.3%).Ms=408.19 (M+H+)。
Cbz-Glu (OtBu)-Gly-OMe (56g, 137.1mmol) is taken to be dissolved in the methylene chloride of 500ml HCl containing 8.5ml
In, Pd/C catalyst is added after nitrogen purge, then be passed through hydrogen and carry out reduction reaction.Reaction terminates, Filtration of catalyst, filter
Liquid directly carries out next step reaction.
Cbz-Aib-OH (32.5g, 137.1mmol), (1- cyano -2- ethyoxyl -2- oxo is added in filtrate one step up
Ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU, 117.4g, 274.2mmol) and N, N- bis-
Wopropyl ethyl amine (53.2g, 411.3mmol) is stirred at room temperature 24 hours, and reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, will remain
Remaining grease is dissolved in ethyl acetate, is then washed respectively with the sodium carbonate liquor of 10% potassium sulfate solution, 5%, organic
It is mutually dry with anhydrous sodium sulfate, vacuum rotary steam, obtain after column chromatography Cbz-Aib-Glu (OtBu)-Gly-OMe (63.3g,
93.5%) Ms=493.24 (M+H+)。
Cbz-Aib-Glu (OtBu)-Gly-OMe (60g, 121.6mmol) is taken to be dissolved in the dichloro of 600ml HCl containing 7.6ml
In methane, Pd/C catalyst is added after nitrogen purge, then is passed through hydrogen and carries out reduction reaction.Reaction terminates, and is filtered to remove catalysis
Agent, filtrate are spin-dried for.600ml tetrahydrofuran is dissolved the residue in, 1mol/L NaOH solution 135ml is then added, is reacted under ice bath
1 hour, PH is adjusted to neutrality with glacial acetic acid after reaction, and vacuum rotary steam is evaporated, and obtains concentrate Aib-Glu (OtBu)-
Gly-OH。
Above-mentioned concentrate is dissolved in n,N-Dimethylformamide, Boc-His-OH (31g, 121.6mmol), (1- is added
Cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU,
104.2g, 243.2mmol) and n,N-diisopropylethylamine (47.2g, 486.3mmol) be stirred at room temperature 24 hours, reaction terminates,
Be concentrated under reduced pressure remove methylene chloride, remaining grease is dissolved in ethyl acetate, then respectively with 10% potassium sulfate solution,
5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, obtains Boc-His- through silica gel column chromatography after vacuum rotary steam
Aib-Glu (OtBu)-Gly-OH (64.6g, 91.1%) Ms=583.31 (M+H+)。
By above-mentioned Boc-His-Aib-Glu (OtBu)-Gly-OH (60g, 102.8mmol) and N- hydroxysuccinimide
(HOSu, 11.8g, 102.8mmol) is dissolved in 550ml tetrahydrofuran, addition dicyclohexylcarbodiimide (DCC, 21.2g,
102.8mmol), reaction 18 hours is stirred at room temperature.After reaction, filtering reacting liquid, filter cake are washed with tetrahydrofuran, are closed
And be concentrated under reduced pressure after filtrate, then be redissolved in methylene chloride, solution is filtered after 0 DEG C stands 30 minutes, and solid is used
Methylene chloride washing, pressurization concentration is carried out after merging filtrate, obtain Boc-His-Aib-Glu (OtBu)-Gly-OSu (62.2g,
Yield 89.4%).Ms=676.34 (M+H+)。
The preparation of embodiment 3, Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-OH
It weighs Cbz-Glu (OtBu)-OH (16.9g, 50mmol) and glycine methyl ester (6.3g, 50mmol) is dissolved in 150ml
In tetrahydrofuran, dicyclohexylcarbodiimide (11.4g, 55mmol) and HOBt (10.1g, 75mmol) is added, is stirred at room temperature anti-
It answers 18 hours.After reaction, filtering reacting liquid, filter cake are washed with tetrahydrofuran, are concentrated under reduced pressure after merging filtrate,
It is redissolved in methylene chloride again, solution is filtered, solid is washed with methylene chloride, merging filtrate after 0 DEG C stands 30 minutes
After carry out pressurization concentration, obtain Cbz-Glu (OtBu)-Gly-OMe (18.9g, 92.4%).Ms=408.19 (M+H+)。
Cbz-Glu (OtBu)-Gly-OMe (16g, 39.2mmol) is taken to be dissolved in the methylene chloride of 150ml HCl containing 2.4ml
In, Pd/C catalyst is added after nitrogen purge, then be passed through hydrogen and carry out reduction reaction.Reaction terminates, Filtration of catalyst, filter
Liquid directly carries out next step reaction.
Cbz-Aib-OH (9.3g, 39.2mmol), (1- cyano -2- ethyoxyl -2- oxo Asia is added in filtrate one step up
Ethylamino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU, 33.6g, 78.4mmol) and N, N- diisopropyl
Base ethamine (15.2g, 117.6mmol) is stirred at room temperature 24 hours, and reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, by remaining oil
Shape object is dissolved in ethyl acetate, is then washed respectively with the aqueous sodium carbonate of 10% potassium sulfate solution, 5%, organic
It is mutually dry with anhydrous sodium sulfate, vacuum rotary steam, obtain after column chromatography Cbz-Aib-Glu (OtBu)-Gly-OMe (18.1g,
93.4%) Ms=493.24 (M+H+)。
Cbz-Aib-Glu (OtBu)-Gly-OMe (17g, 34.5mmol) is taken to be dissolved in the dichloromethane of 200ml HCl containing 2.1ml
In alkane, Pd/C catalyst is added after nitrogen purge, then is passed through hydrogen and carries out reduction reaction.Reaction terminates, Filtration of catalyst,
Filtrate is spin-dried for.200ml tetrahydrofuran is dissolved the residue in, 1mol/L NaOH solution 38ml is then added, reaction 1 is small under ice bath
When, PH is adjusted to neutrality with glacial acetic acid after reaction, and vacuum rotary steam is evaporated under reduced pressure with toluene and is removed water, obtains concentrate Aib-
Glu(OtBu)-Gly-OH。
Above-mentioned concentrate is dissolved in n,N-Dimethylformamide, addition Fmoc-His (Fmoc)-OH (20.1g,
34.5mmol), (1- cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphoric acid
Salt (COMU, 31.1g, 34.5mmol) and n,N-diisopropylethylamine (13.4g, 103.5mmol) are stirred at room temperature 24 hours, reaction
Terminate, be concentrated under reduced pressure remove methylene chloride, remaining grease is dissolved in ethyl acetate, then respectively with 10% potassium sulfate
Solution, 5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam, obtains after silica gel column chromatography
Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-OH (28.8g, yield 90%).Ms=926.39 (M+H+)。
The preparation of embodiment 4, Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-Cl
It weighs Cbz-Glu (OtBu)-OH (33.8g, 100mmol) and glycine methyl ester (12.6g, 100mmol) is dissolved in
It in 300ml tetrahydrofuran, is added dicyclohexylcarbodiimide (22.8g, 110mmol), reaction 18 hours is stirred at room temperature.Reaction knot
Shu Hou, filtering reacting liquid, filter cake are washed with tetrahydrofuran, are concentrated under reduced pressure after merging filtrate, then are redissolved in dichloro
Methane, solution are filtered, solid is washed with methylene chloride, carries out pressurizeing after merging filtrate dense after 0 DEG C stands 30 minutes
Contracting obtains CbZ-Glu (OtBu)-Gly-OMe (36.9g, 90.4%).Ms=408.19 (M+H+)。
Cbz-Glu (OtBu)-Gly-OMe (35g, 85.8mmol) is taken to be dissolved in the methylene chloride of 350ml HCl containing 5.3ml
In, Pd/C catalyst is added after nitrogen purge, then be passed through hydrogen and carry out reduction reaction.Reaction terminates, Filtration of catalyst, filter
Liquid directly carries out next step reaction.
Cbz-Aib-OH (20.4g, 85.8mmol), (1- cyano -2- ethyoxyl -2- oxo is added in filtrate one step up
Ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate (COMU, 73.6g, 171.6mmol) and N, N- bis- it is different
Propylethylamine (33.3g, 257.4mmol) is stirred at room temperature 24 hours, and reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, will be remaining
Grease is dissolved in ethyl acetate, is then washed respectively with the sodium carbonate liquor of 10% potassium sulfate solution, 5%, organic phase
Dry, the vacuum rotary steam with anhydrous sodium sulfate, column obtain Cbz-Aib-Glu (OtBu)-Gly-OMe (39.9g, 94.1%) after chromatographing
Ms=493.24 (M+H+)。
Cbz-Aib-Glu (OtBu)-Gly-OMe (36g, 73.1mmol) is taken to be dissolved in the dichloromethane of 400ml HCl containing 4.5ml
In alkane, Pd/C catalyst is added after nitrogen purge, then is passed through hydrogen and carries out reduction reaction.Reaction terminates, Filtration of catalyst,
Filtrate is spin-dried for.400ml tetrahydrofuran is dissolved the residue in, 1mol/L NaOH solution 80.6ml is then added, reacts 1 under ice bath
Hour, PH is adjusted to neutrality with glacial acetic acid after reaction, and vacuum rotary steam is evaporated under reduced pressure with toluene and is removed water, obtains concentrate
Aib-Glu(OtBu)-Gly-OH。
Above-mentioned concentrate is dissolved in n,N-Dimethylformamide, addition Fmoc-His (Fmoc)-OH (42.6g,
73.1mmol), (1- cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphoric acid
Salt (COMU, 62.6g, 146.2mmol) and n,N-diisopropylethylamine (28.3g, 219.3mmol) are stirred at room temperature 24 hours, instead
Should terminate, be concentrated under reduced pressure remove methylene chloride, remaining grease is dissolved in ethyl acetate, then respectively with 10% sulfuric acid
Potassium solution, 5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, obtains after vacuum rotary steam through silica gel column chromatography
Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-OH (60.5g, yield 89.3%).Ms=926.39 (M+H+)。
Above-mentioned Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-OH (50g, 53.9mmol) is dissolved in 500ml dichloro
In methane, appropriate n,N-Dimethylformamide is added, thionyl chloride (4.3ml, 59.3mmol) is added under ice bath, stirs 15 points
Ice bath is removed after clock, is warming up to room temperature reaction.Reaction terminates, and vacuum rotary steam is evaporated methylene chloride and thionyl chloride.After purification
To product Fmoc-His (Fmoc)-Aib-Glu (OtBu)-Gly-Cl (45.1g, yield 88.4%).Ms=945.36.
The preparation of embodiment 5, side chain all risk insurance guard wire Ma Shandong peptide [5-31] peptide-resin
It weighs Fmoc-Gly-2-chlorotrityl resin 33.3g (capacity value 0.3mmol/g, 10mmol) and solid phase is added
In reactor, 20% piperidines n,N-Dimethylformamide solution 150ml is added, 25~30 DEG C are stirred to react 10min, repeat de-
Protection 3 times, filters after completion of the reaction, resin is washed with 200ml n,N-Dimethylformamide, filters, and repeated washing totally 6 times,
By Fmoc-Arg (Pbf)-OH (molecular weight: 648.8,30mmol) 19.5g, 1- hydroxy benzo triazole (HOBt) (molecular weight:
135.1,30mmol) 3.9g is dissolved in 100ml n,N-Dimethylformamide, is added in solid phase reactor, adds N, N- diisopropyl
(molecular weight: 126.2,30mmol) 3.6ml, 25~35 DEG C are reacted about 2 hours base carbodiimide (DIC), and reaction end is with indenes three
Ketone detection washs resin with 200ml n,N-Dimethylformamide subject to being negative, and filters, and washes repeatedly 3 times.More than repeating
Step is coupled with corresponding Fmoc protected amino acid one by one according to Suo Malu peptide sequence, protected amino acid and condensing agent equivalent with it is aforementioned
Fmoc-Arg (Pbf)-OH is identical；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(AEEA-AEEA-
Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、
Side chain all risk insurance guard wire Ma Shandong peptide [5-31] peptide-resin is made in Fmoc-Thr (tBu)-OH, and structure is as follows:
Fmoc-Thr(tBu)-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr
(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(EAAE-AEEA-Alloc)-Glu(OtBu)-Phe-
Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-chlorotrityl resin.
The preparation of embodiment 6, side chain all risk insurance guard wire Ma Shandong peptide [5-31] peptide-resin
It weighs Fmoc-Gly-Wang resin 25.0g (capacity value 0.4mmol/g, 10mmol) to be added in solid phase reactor, add
Enter 20% piperidines n,N-Dimethylformamide solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, instead
It is filtered after answering, resin is washed with 200ml n,N-Dimethylformamide, filtered, repeated washing totally 6 times, by Fmoc-Arg
(Pbf)-OH (molecular weight: 648.8,30mmol) 19.5g, 1- hydroxy benzo triazole (HOBt) (molecular weight: 135.1,
30mmol) 3.9g is dissolved in 100ml n,N-Dimethylformamide, is added in solid phase reactor, adds N, N- diisopropyl carbon two
(molecular weight: 126.2,30mmol) 3.6ml, 25~35 DEG C are reacted about 2 hours imines (DIC), and reaction end is detected with ninhydrin
Subject to being negative, resin is washed with 200ml n,N-Dimethylformamide, filtered, washed repeatedly 3 times.Above step is repeated,
It is coupled one by one with corresponding Fmoc protected amino acid according to Suo Malu peptide sequence, protected amino acid and condensing agent equivalent and aforementioned Fmoc-
Arg (Pbf)-OH is identical；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(AEEA-AEEA-Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-
OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-
OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr
(tBu) side chain all risk insurance guard wire Ma Shandong peptide [5-31] peptide-resin is made in-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH,
Structure is as follows:
Fmoc-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(AEEA-EAAE-Alloc)-Glu(OtBu)-
Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin.
The preparation of embodiment 7,2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor
Suo Malu peptide [5-31] peptide-resin prepared by Example 5 is added in solid phase reactor, 20% piperidines is added
N,N-Dimethylformamide solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, Fmoc-His prepared by embodiment 1
(Boc)-Aib-Glu (OtBu)-Gly-OH (molecular weight: 805.89,30mmol) 24.2g, 1- hydroxy benzo triazole (HOBt)
(molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml n,N-Dimethylformamide, is added in solid phase reactor, adds N,
(molecular weight: 126.2,30mmol) 3.6ml, 25~35 DEG C are reacted about 2 hours N- diisopropylcarbodiimide (DIC), and reaction is eventually
Point be subject to ninhydrin detection be negative, resin wash with 200ml n,N-Dimethylformamide, filter, repeated washing 3 times.
2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor is made, structure is as follows:
Fmoc-His(Boc)-Aib-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
(AEEA-EAAE-Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg
(Pbf)-Gly-2-chlorotrityl resin.
The preparation of embodiment 8,2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor
Suo Malu peptide [5-31] peptide-resin prepared by Example 6 is added in solid phase reactor, 20% piperidines is added
N,N-Dimethylformamide solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, Boc-His prepared by embodiment 2
(Boc)-Aib-Glu (OtBu)-Gly-OSu (molecular weight: 780.84,30mmol) 23.4g is dissolved in 100ml N, N- dimethyl methyl
Amide, add n,N-diisopropylethylamine (n,N-diisopropylethylamine) (molecular weight: 129.25,30mmol) 5ml, 25~35
DEG C reaction about 2 hours, reaction end be subject to ninhydrin detection be negative, resin is washed with 200ml n,N-Dimethylformamide
It washs, filters, wash repeatedly 3 times.2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor is made, structure is as follows:
Boc-His(Boc)-Aib-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
(AEEA-EAAE-Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg
(Pbf)-Gly-Wang resin.
The preparation of embodiment 9,2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor
Suo Malu peptide [5-31] peptide-resin prepared by Example 5 is added in solid phase reactor, 20% piperidines is added
N,N-Dimethylformamide solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, Fmoc-His prepared by embodiment 3
(Fmoc)-Aib-Glu (OtBu)-Gly-OH (molecular weight: 928.02,30mmol) 27.8g, 1- hydroxy benzo triazole (HOBt)
(molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml n,N-Dimethylformamide, is added in solid phase reactor, adds N,
(molecular weight: 126.2,30mmol) 3.6ml, 25~35 DEG C are reacted about 2 hours N- diisopropylcarbodiimide (DIC), and reaction is eventually
Point be subject to ninhydrin detection be negative, resin wash with 200ml n,N-Dimethylformamide, filter, repeated washing 3 times.
2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor is made, structure is as follows:
Fmoc-His(Fmoc)-Aib-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
(AEEA-EAAE-Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg
(Pbf)-Gly-2-chlorotrityl resin.
The preparation of embodiment 10,2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor
Suo Malu peptide [5-31] peptide-resin prepared by Example 6 is added in solid phase reactor, 20% piperidines is added
N,N-Dimethylformamide solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, Fmoc-His prepared by embodiment 4
(Fmoc)-Aib-Glu (OtBu)-Gly-Cl (molecular weight: 946.46,30mmol) 28.4g, be dissolved in 100ml N, N- dimethyl methyl
Amide, add n,N-diisopropylethylamine (n,N-diisopropylethylamine) (molecular weight: 129.25,30mmol) 5ml, 25~35
DEG C reaction about 2 hours, reaction end be subject to ninhydrin detection be negative, resin is washed with 200ml n,N-Dimethylformamide
It washs, filters, wash repeatedly 3 times.2 peptide resin of side chain all risk insurance guard wire Ma Shandong peptide precursor is made, structure is as follows:
Fmoc-His(Fmoc)-Aib-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
(AEEA-EAAE-Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg
(Pbf)-Gly-Wang resin.
Embodiment 11, the preparation of side chain all risk insurance guard wire Ma Shandong peptide-resin
It prepares deprotecting regent: weighing 3.47g Pd (PPh3)4(molecular weight: 1155.6,3mmol) is dissolved in 150ml dichloromethane
In alkane, PhSiH is added312.3ml (molecular weight: 108.2,100mmol), is uniformly mixed.
In 2 peptide resin of precursor prepared by prepared deprotecting regent addition embodiment 7,30min is reacted at room temperature, then
It filters, repeats plus deprotecting regent reacts 30min.It filters after completion of the reaction, by resin 200ml n,N-Dimethylformamide
Washing filters, and repeated washing totally 6 times.By the octadecane diacid list tert-butyl ester-Glu-OtBu (molecular weight: 555.8,30mmol)
16.7g, 1- hydroxy benzo triazole (HOBt) (molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml N, N- dimethyl formyl
Amine is added in solid phase reactor, adds N, N- diisopropylcarbodiimide (DIC) (molecular weight: 126.2,30mmol)
3.6ml, 25~35 DEG C react about 2 hours, reaction end be subject to ninhydrin detection be negative.Reaction solution is filtered, resin is used
The washing of 200ml n,N-Dimethylformamide, filters, and repeated washing totally 6 times.20% piperidines N,N-dimethylformamide is added
Solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, remove the protecting group of His, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, obtains side chain all risk insurance guard wire Ma Shandong peptide-
Resin.
Embodiment 12, the preparation of side chain all risk insurance guard wire Ma Shandong peptide-resin
It prepares deprotecting regent: weighing 3.47g Pd (PPh3)4(molecular weight: 1155.6,3mmol) is dissolved in 150ml dichloromethane
In alkane, PhSiH is added312.3ml (molecular weight: 108.2,100mmol), is uniformly mixed.
In 2 peptide resin of precursor prepared by prepared deprotecting regent addition embodiment 8,30min is reacted at room temperature, then
It filters, repeats plus deprotecting regent reacts 30min.It filters after completion of the reaction, by resin 200ml n,N-Dimethylformamide
Washing filters, and repeated washing totally 6 times.By the octadecane diacid list tert-butyl ester-Glu-OtBu (molecular weight: 555.8,30mmol)
16.7g, 1- hydroxy benzo triazole (HOBt) (molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml N, N- dimethyl formyl
Amine is added in solid phase reactor, adds N, N- diisopropylcarbodiimide (DIC) (molecular weight: 126.2,30mmol)
3.6ml, 25~35 DEG C react about 2 hours, reaction end be subject to ninhydrin detection be negative.Reaction solution is filtered, resin is used
The washing of 200ml n,N-Dimethylformamide, filters, and repeated washing totally 6 times.Obtain side chain all risk insurance guard wire Ma Shandong peptide-resin.
Embodiment 13, the preparation of side chain all risk insurance guard wire Ma Shandong peptide-resin
It prepares deprotecting regent: weighing 3.47g Pd (PPh3)4(molecular weight: 1155.6,3mmol) is dissolved in 150ml dichloromethane
In alkane, PhSiH is added312.3ml (molecular weight: 108.2,100mmol), is uniformly mixed.
In 2 peptide resin of precursor prepared by prepared deprotecting regent addition embodiment 9,30min is reacted at room temperature, then
It filters, repeats plus deprotecting regent reacts 30min.It filters after completion of the reaction, by resin 200ml n,N-Dimethylformamide
Washing filters, and repeated washing totally 6 times.By the octadecane diacid list tert-butyl ester-Glu-OtBu (molecular weight: 555.8,30mmol)
16.7g, 1- hydroxy benzo triazole (HOBt) (molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml N, N- dimethyl formyl
Amine is added in solid phase reactor, adds N, N- diisopropylcarbodiimide (DIC) (molecular weight: 126.2,30mmol)
3.6ml, 25~35 DEG C react about 2 hours, reaction end be subject to ninhydrin detection be negative.Reaction solution is filtered, resin is used
The washing of 200ml n,N-Dimethylformamide, filters, and repeated washing totally 6 times.20% piperidines N,N-dimethylformamide is added
Solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, remove the protecting group of His, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, obtains side chain all risk insurance guard wire Ma Shandong peptide-
Resin.
Embodiment 14, the preparation of side chain all risk insurance guard wire Ma Shandong peptide-resin
It prepares deprotecting regent: weighing 3.47g Pd (PPh3)4(molecular weight: 1155.6,3mmol) is dissolved in 150ml dichloromethane
In alkane, PhSiH is added312.3ml (molecular weight: 108.2,100mmol), is uniformly mixed.
In 2 peptide resin of precursor prepared by prepared deprotecting regent addition embodiment 10,30min is reacted at room temperature, so
After filter, repeat plus deprotecting regent react 30min.It filters after completion of the reaction, by resin 200ml N, N- dimethyl formyl
Amine washing, filters, and repeated washing totally 6 times.By the octadecane diacid list tert-butyl ester-Glu-OtBu (molecular weight: 555.8,30mmol)
16.7g, 1- hydroxy benzo triazole (HOBt) (molecular weight: 135.1,30mmol) 3.9g is dissolved in 100ml N, N- dimethyl formyl
Amine is added in solid phase reactor, adds N, N- diisopropylcarbodiimide (DIC) (molecular weight: 126.2,30mmol)
3.6ml, 25~35 DEG C react about 2 hours, reaction end be subject to ninhydrin detection be negative.Reaction solution is filtered, resin is used
The washing of 200ml n,N-Dimethylformamide, filters, and repeated washing totally 6 times.20% piperidines N,N-dimethylformamide is added
Solution 150ml, 25~30 DEG C are stirred to react 10min, repeat deprotection 3 times, remove the protecting group of His, filter after completion of the reaction,
Resin is washed with 200ml n,N-Dimethylformamide, is filtered, repeated washing totally 6 times, obtains side chain all risk insurance guard wire Ma Shandong peptide-
Resin.
The preparation of embodiment 15, Suo Malu peptide crude product
Side chain all risk insurance guard wire Ma Shandong peptide-resin 20g prepared by embodiment 11 is added in 500ml round-bottomed flask；It prepares
Lytic reagent 200ml, wherein trifluoroacetic acid 192ml, tri isopropyl silane 2ml, thioanisole 2ml, 1,2- dithioglycol 2ml,
Water 2ml is cooled to 0 DEG C in advance；Lytic reagent is added in institute Suo Malu peptide-resin, acidolysis reaction temperature rose to 25 in 20 minutes
DEG C, and react 2 hours at this temperature, resin is filtered, washs resin, merging filtrate with a small amount of trifluoroacetic acid.It is being vigorously stirred
In the lower ether that filtrate is slowly added to 2L precooling, there is white precipitate, after standing 1 hour, filters, and washed with ice ether
Filter cake 5 times, vacuum drying obtains thick peptide 5.74g.Thick peptide yield 92.6%.
The preparation of embodiment 16, Suo Malu peptide crude product
Side chain all risk insurance guard wire Ma Shandong peptide-resin 40g prepared by embodiment 13 is added in 500ml round-bottomed flask；It prepares
Lytic reagent 400ml, wherein trifluoroacetic acid 384ml, tri isopropyl silane 4ml, thioanisole 4ml, 1,2- dithioglycol 4ml,
Water 4ml is cooled to 0 DEG C in advance；Lytic reagent is added in institute Suo Malu peptide-resin, acidolysis reaction temperature rose to 25 in 20 minutes
DEG C, and react 2 hours at this temperature, resin is filtered, washs resin, merging filtrate with a small amount of trifluoroacetic acid.It is being vigorously stirred
In the lower ether that filtrate is slowly added to 4L precooling, there is white precipitate, after standing 1 hour, filters, and washed with ice ether
Filter cake 5 times, vacuum drying obtains thick peptide 11.5g.Thick peptide yield 93%.
The purifying of embodiment 17, Suo Malu peptide crude product
The Suo Malu peptide crude powder 10.0g for weighing the preparation of embodiment 16, is dissolved, solution is micro- with 0.45 μm using suitable quantity of water
Hole membrane filtration, it is spare.
Condition when high performance liquid chromatography is purified, chromatographic column: being solid with the eight alkyl silane bonded silica gels of 10um
Determine phase, pillar diameter and length are as follows: 50mm × 250mm；Mobile phase: 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solution；It washes
De- flow velocity 60ml/min；Using gradient elution, input mode loading is recycled.The sample solution of above-mentioned processing is splined on chromatography
In column, starting mobile phase elution collects main peak and detects purity with analysis liquid phase, merges main peak solution, in less than 40 DEG C water-baths
Under the conditions of be concentrated under reduced pressure, boil off most of acetonitrile, get Suo Malu peptide trifluoroacetic acid salting liquid with Rotary Evaporators.It is freeze-dried
Suo Malu peptide sterling 3.25g purifies yield 32.5%, product purity 99.7%, [D-His1]-Suo Malu peptide impurity is less than
0.1%.
Claims (6)
1. a kind of polypeptide fragment, which is had the following structure:
R1-His(R2)-Aib-Glu(OtBu)-Gly-R
Wherein, R1Selected from Fmoc, Boc, Dde or Cbz；R2Selected from H, Fmoc, Boc, Mmt or Mtt；R be selected from OH, F, Cl, Br, I,
CN、-Osu。
2. polypeptide fragment according to claim 1, which is characterized in that the polypeptide fragment is selected from:
R1-His(R2)-Aib-Glu(OtBu)-Gly-OH
Or R1-His(R2)-Aib-Glu(OtBu)-Gly-Cl
Or R1-His(R2)-Aib-Glu(OtBu)-Gly-OSu
3. polypeptide fragment according to claim 1, which is characterized in that the polypeptide fragment is selected from Fmoc-His (Boc)-
Aib-Glu(OtBu)-Gly-OH、Boc-His-Aib-Glu(OtBu)-Gly-OSu、Fmoc-His(Fmoc)-Aib-Glu
(OtBu)-Gly-OH、Fmoc-His(Fmoc)-Aib-Glu(OtBu)-Gly-Cl。
4. a kind of polypeptide fragment R1-His(R2)-Aib-Glu (OtBu)-Gly-OH preparation method comprising following steps:
(1) Cbz-Glu (OtBu)-OH and glycine methyl ester hydrochloride are dissolved in tetrahydrofuran, it is sub- that dicyclohexyl carbon two is added
Reaction 18 hours is stirred at room temperature in amine and assistant activator 1- hydroxy benzo triazole；After reaction, filtering reacting liquid, filter cake are used
Tetrahydrofuran is washed, and is concentrated under reduced pressure after merging filtrate, then is redissolved in methylene chloride, and solution stands 30 points at 0 DEG C
Zhong Hou is filtered, and solid is washed with methylene chloride, and pressurization concentration is carried out after merging filtrate, is obtained Cbz-Glu (OtBu)-
Gly-OMe；
(2) it takes Cbz-Glu (OtBu)-Gly-OMe to be dissolved in the methylene chloride containing a small amount of HCl, Pd/C is added after nitrogen purge and urges
Agent, then be passed through hydrogen and carry out reduction reaction；Reaction terminates, Filtration of catalyst, and filtrate directly carries out next step reaction；
(3) Cbz-Aib-OH, (1- cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) two is added in filtrate one step up
Methylamino-morpholine-carbon hexafluorophosphate and n,N-diisopropylethylamine are stirred at room temperature 24 hours, and reaction terminates, and depressurize dense
Contracting removes methylene chloride, and remaining grease is dissolved in ethyl acetate, then respectively with 10% potassium sulfate solution, 5%
Sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam, obtains Cbz-Aib-Glu (OtBu)-after column chromatography
Gly-OMe；
(4) above-mentioned product is dissolved in the methylene chloride containing a small amount of HCl, Pd/C catalyst is added after nitrogen purge, then be passed through hydrogen
Carry out reduction reaction；Reaction terminates, and Filtration of catalyst, filtrate is spin-dried for；Tetrahydrofuran is dissolved the residue in, is then added
The NaOH solution of 1mol/L, reacts 1 hour under ice bath, adjusts PH to neutral with glacial acetic acid after reaction, vacuum rotary steam uses first
Benzene vacuum distillation water removal；
(5) concentrate is dissolved in n,N-Dimethylformamide, R is added1- His (R2)-OH, (1- cyano -2- ethyoxyl -2- oxygen
For ethyleneimino oxygroup) dimethylamino-morpholine-carbon hexafluorophosphate and N, N- diisopropylethylamine (be stirred at room temperature 24
Hour, reaction terminates, and is concentrated under reduced pressure and removes methylene chloride, remaining grease is dissolved in ethyl acetate, is then used respectively
10% potassium sulfate solution, 5% sodium carbonate liquor washing, organic phase is dry with anhydrous sodium sulfate, vacuum rotary steam, after column chromatography
Obtain R1- His (R2)-Aib-Glu (OtBu)-Gly-OH polypeptide fragment.
5. a kind of polypeptide fragment R1-His(R2)-Aib-Glu (OtBu)-Gly-OSu preparation method, use liquid phase condensations
Method obtains, specifically includes the following steps:
By R1-His(R2)-Aib-Glu (OtBu)-Gly-OH and N- hydroxysuccinimide be dissolved in tetrahydrofuran, two rings are added
Reaction 18 hours is stirred at room temperature in hexyl carbodiimide；After reaction, filtering reacting liquid, filter cake are washed with tetrahydrofuran,
It is concentrated under reduced pressure after merging filtrate, then is redissolved in methylene chloride, solution is filtered, solid after 0 DEG C stands 30 minutes
It is washed with methylene chloride, pressurization concentration is carried out after merging filtrate, obtain R after column chromatographs1-His(R2)-Aib-Glu(OtBu)-
Gly-OSu。
6. polypeptide fragment R described in one kind1-His(R2)-Aib-Glu (OtBu)-Gly-Cl preparation method, use liquid phase to contract
The method of conjunction obtains, specifically includes the following steps:
By I compound represented R of formula1-His(R2)-Aib-Glu (OtBu)-Gly-OH is dissolved in methylene chloride, appropriate N is added,
Dinethylformamide, thionyl chloride is added under ice bath, and stirring removes ice bath after 15 minutes and is warming up to room temperature reaction；Reaction knot
Beam, vacuum rotary steam are evaporated methylene chloride and thionyl chloride；Product R is obtained after silica gel column chromatography1-His(R2)-Aib-Glu
(OtBu)-Gly-Cl segment.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759972A (en) * | 2019-10-31 | 2020-02-07 | 成都圣诺生物制药有限公司 | Preparation method of atosiban |
| CN110759990A (en) * | 2019-10-31 | 2020-02-07 | 成都圣诺生物制药有限公司 | Preparation method of linagliptin |
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| CN113759048A (en) * | 2021-10-14 | 2021-12-07 | 成都普赛唯新生物科技有限公司 | Inspection method of mono-tert-butyl octadecanedioate |
| CN113759048B (en) * | 2021-10-14 | 2022-06-21 | 成都普康唯新生物科技有限公司 | Inspection method of mono-tert-butyl octadecanedioate |
| CN115160430A (en) * | 2022-07-27 | 2022-10-11 | 成都普康唯新生物科技有限公司 | Synthesis method of somaglutide backbone fragment |
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