CN104418948B - A method of preparing polypeptide drugs - Google Patents
A method of preparing polypeptide drugs Download PDFInfo
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- CN104418948B CN104418948B CN201310410636.9A CN201310410636A CN104418948B CN 104418948 B CN104418948 B CN 104418948B CN 201310410636 A CN201310410636 A CN 201310410636A CN 104418948 B CN104418948 B CN 104418948B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
The present invention relates to a kind of methods for preparing polypeptide drugs DiaPep277, include: Fmoc-Asp (OtBu)-OH and resin P being coupled, Fmoc-Asp (OtBu)-P is obtained；By Fmoc-Asp (OtBu)-P synthesis in solid state by the way of being coupled one by one, DiaPep277-P is obtained；DiaPep277-P is cracked, the thick peptide of DiaPep277 is obtained.
Description
Technical field
The present invention relates to a kind of methods for preparing polypeptide drugs, in particular to the preparation method of DiaPep277.
Background technique
DiaPep277 is derived from one section in human heat shock protein 60 (HSP60) sequence peptide fragment containing 24 amino acid.
The structure of DiaPep277 is as follows:
H-Val-Leu-Gly-Gly-Gly-Val-Ala-Leu-Leu-Arg-Val-Ile-Pro-Ala-Leu-Asp-
Ser-Leu-Thr-Pro-Ala-Asn-Glu-Asp-OH。
Research has shown that DiaPep277 can adjust immune system, makes islet cells damage and protects insulin secretion
Function.Therefore, the treatment zone that DiaPep277 is autoimmune diabetes patient carrys out substantive breakthrough, and can be used as
The preventive usage of this onset diabetes people at highest risk.This drug can significantly improve the existence matter of type-1 diabetes mellitus patient
Amount, delays or prevents the generation of various complication, to improve the life expectancy of patient.
Summary of the invention
For the small peptide containing 24 amino acid, since solid phase method is there are easy to operate, solvent usage amount is small, be easy to from
The advantages that dynamicization, therefore solid phase sequence coupling is often carried out using conventional protected amino acid and is synthesized.But through analyzing
After the peptide chain of DiaPep277, the inventors discovered that, it is some special which there is a situation where, a large amount of hydrophobic group is (especially
N-terminal) and-Gly-Gly-Gly- structure presence can make synthesis there are bigger difficulty.If using conventional Fmoc-Gly-
OH raw material is easy the impurity etc. of the generation ± Gly in synthesis-Gly-Gly-Gly- structure.
It is an object of the invention to solve above-mentioned difficulties using special amino acid starting material, the purity and receipts of thick peptide are improved
Rate.
The present invention provides a kind of method for preparing polypeptide drugs DiaPep277 comprising the steps of:
1) using a kind of resin P as carrier, Fmoc-Asp (OtBu)-OH and resin is coupled, Fmoc-Asp is obtained
(OtBu)-P；
2) using Fmoc-Asp (OtBu)-P as raw material, solid phase is carried out with other amino acid starting materials by the way of being coupled one by one
Synthesis, obtains DiaPep277-P；
3) DiaPep277-P is cracked, obtains the thick peptide of DiaPep277；
Wherein Fmoc is 9-fluorenylmethyloxycarbonyl, is connected with amino or is connected with the nitrogen-atoms on the connected carbon atom of carboxyl,
The synthesis material that Asp-Ser structure uses in the mode being wherein coupled one by one described in step 2) is Fmoc-Asp
(OtBu)-Ser(ψMe,MePro)-OH is a pseudo proline dipeptides, so that serine is protected into five-membered ring, structure is as follows:
The synthesis material that wherein-Gly-Gly-Gly- structure uses is Fmoc- (Hmb) Gly-OH, Fmoc- (Dmb) Gly-
One or both of OH, Fmoc-Gly- (Dmb) Gly-OH or Fmoc-Gly- (Hmb) Gly-OH.
Preferably, the method for the present invention further includes the steps that purifying DiaPep277.
Specific embodiment
Abbreviation used in the present invention and its meaning are described as follows:
Herein, " substitution degree " refers to that the quantity of the resin-carried substance of unit quantity, unit are " mmol/g ".
Herein, as without opposite explanation, then the reaction carries out at normal temperatures and pressures.
Wide in range, preferred, preferred and most preferred definition and range in the present invention can be combined with each other.
" peptide " refers to the coupled product obtained similarly by the Fmoc solid phase synthesis process in Peptide systhesis in the present invention.
The vector resin is not particularly limited, as long as it has the active site that can be reacted with the carboxyl in substance
And it is not reacted with the other groups in the substance.The vector resin is preferably two class of 2-CTC resin or Wang Shuzhi, tree
Rouge substitution degree is 0.2-1.0mmol/g, preferably 0.3-0.8mmol/g, more preferably 0.4-0.6mmol/g.
The structure of Wang Shuzhi class used in the present invention is as follows:
The structure of 2-CTC resinae used is as follows:
WhereinIt represents the rest part of resin structure and the part is not involved in reaction.
Above two resin is available commercially (Tianjin Nankai Hecheng S&T Co., Ltd.) or by method known to document
Preparation.
In a specific embodiment, can according to before the dosage of resin, reaction substitution degree and required reaction after
Substitution degree select the dosage of Fmoc- amino acid-OH, usually can be with the molal quantity of the functional group of amino acid couplings on resin
1-5 equivalent, preferred 2-4 equivalent, more preferable 2.5-3.5 equivalent wherein can be with the functional group mole of amino acid couplings on resin
Number is the product of weight resin and substitution degree.
The solvent is, such as, but not limited to, DMF, DCM, DMSO, NMP, THF, ethyl acetate, methanol, ether etc. and
The mixed liquor (for example, the two volume ratio is 1:1) of its any mixture, preferably DMF, DCM or DMF and DCM.
Coupling, which prepares peptide resin, can be used the reality of Fmoc solid phase synthesis process known to technical staff in Peptides Synthesis
It is existing, such as may refer to document Fmoc Solid Phase Peptide Synthesis:A Practical Approach,
W.C.Chan, Peter D.White write, March 2,2000 (ISBN-10:0199637245), Britain Oxford
University Press。
The washing of resin and swelling can be used in coupling and realize that any reagent of the purpose carries out, preferably DMF.
Described be coupled in solid phase reaction column carries out.Solid phase reaction column is not particularly limited, can be that this purpose can be achieved
Any solid phase reaction column.
Preferably, by step 1) -2) in carboxyl in substance activate, activation preferably carries out in ice-water bath.Activation
Agent is preferably DIPEA or DIC.
It further include that one is added thereto wherein after amino acid Fmoc-Asp (OtBu)-OH is coupled on vector resin
Kind of confining liquid removes the step of excessive unreacted group in resin, to avoid its in next step coupling reaction with it is other to
It is coupled substance reaction and forms by-product, and the step of removing confining liquid be added.When the vector resin is 2-CTC resin
When, the confining liquid being added is preferably methanol；When the vector resin is Wang Shuzhi, the confining liquid being added is preferably mole
Than the acetic anhydride and pyridine for 1:1.
Coupling preferably carries out in the presence of coupling agent, and the coupling agent is preferably DIC, A or its combination, Huo Zhewei
DIPEA, A, B or its combination, wherein A is HOBt or HOAt, one of B PyBOP, PyAOP, HATU, HBTU and TBTU；Example
Such as, the coupling agent in step 1) is HOBt/DIC.
(the OtBu)-Ser of Fmoc-Asp described in step 2) (ψMe,MePro) coupling system of-OH is HATU/HOAt/
One of DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
The synthesis material that-Gly-Gly-Gly- structure uses in the mode being coupled one by one described in step 2) is Fmoc-
(Hmb) one in Gly-OH, Fmoc- (Dmb) Gly-OH, Fmoc-Gly- (Dmb) Gly-OH or Fmoc-Gly- (Hmb) Gly-OH
Kind or two kinds；Preferably, the raw material is one kind selected from Fmoc- (Hmb) Gly-OH and Fmoc- (Dmb) Gly-OH, Yi Jixuan
From one kind of Fmoc-Gly- (Dmb) Gly-OH and Fmoc-Gly- (Hmb) Gly-OH.
The coupling system of Gly synthesis material described in step 2) is DIC/HOAt, HATU/BOAt/DIPEA or PyBOP/
One of HOBt/DIPEA.
In addition, it is usually 1-6 hours, preferably 2-5 hours that every kind of substance, which carries out the time of coupling reaction,；Pressure is preferably normal
Pressure, can also properly increase or reduced pressure (such as 0.01-1.5 atmospheric pressure) under carry out；Temperature be preferably room temperature (refer to 20 ±
5 DEG C), it (such as 0-50 DEG C) can also be carried out at a temperature of properly increasing or is reduced.
In coupling process, detection agent used can be any reagent that can determine that the reaction end, preferably ninhydrin.
The reagent for removing Fmoc can be any reagent that the purpose can be achieved, preferably DBLK (i.e. 20% piperidines/DMF).
The deprotection reaction time is usually 5-30 minutes, preferably 10-20 minutes；Pressure is preferably normal pressure, can also be appropriate
It is carried out under the pressure (such as 0.01-1.5 atmospheric pressure) increased or decreased；Temperature is preferably room temperature (referring to 20 ± 5 DEG C), can also be suitable
(such as 0-50 DEG C) carries out at a temperature of increasing or decreasing.
The cracking preferably carries out at room temperature and atmospheric pressure, can also properly increase or reduced temperature and pressure under into
Row.
The mixed solvent of lytic reagent TFA, tri isopropyl silane (TIS) and water in step 3), wherein mixed solvent
Proportion are as follows: TFA volume ratio is 85-90%, and the volume ratio of tri isopropyl silane is 1-5%, and the volume ratio of water is 1-5%, three
The sum of be 100%.
In a specific embodiment, lytic reagent described in step 3) is TFA:TIS:H2O=90:5:5 (V:V).
Preferably, the method for preparing DiaPep277 compound further includes the steps that purifying DiaPep277.Purifying can be used
Fast protecting etc. is carried out conventionally used for the method for purifying DiaPep277.
In the present invention, it is preferable that be purified by recrystallization method and carry out, i.e., by dissolving DiaPep277 with solvent, so
The crystal of DiaPep277 is precipitated afterwards and realizes.
In a specific embodiment, purify DiaPep277 the step of by with good solvent dissolve DiaPep277,
Then poor solvent is added the crystal of DiaPep277 is precipitated and realizes." good solvent " refers to right at room temperature and atmospheric pressure herein
The solubility of DiaPep277 is greater than the solvent of 90 weight %, preferably greater than 95 weight %, more preferably greater than 99 weight %；It is " bad
Solvent " refer at room temperature and atmospheric pressure to the solubility of DiaPep277 less than 10 weight %, preferably smaller than 5%, more preferably less than
1% solvent.
Recrystallization preferably carry out at room temperature and atmospheric pressure, can also properly increase or reduced pressure under carry out.Recrystallization
It can be used and realized conventionally used for this purpose all devices.
Preferably, it is purified by RP-HPLC (for example, chromatographic column is reverse phase C18 column) purifying, freeze-drying obtains
DiaPep277, purity are greater than 98%.
Referring to embodiment the present invention is described in detail, it should be appreciated that following embodiments are intended to illustrate, not constitute to the present invention
Limitation.Compound or reagent used in the following embodiment can be bought by commercial sources, or pass through those skilled in the art
Known conventional method is prepared；Used laboratory apparatus can be bought by commercial sources.
Embodiment
Embodiment one: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.4mmol/g
The Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is washed with DMF
Wash 2 times, after DMF swellable resins 30 minutes, take 98.7g Fmoc-Asp (OtBu)-OH, 38.9g HOBt, 36.3g DIC,
2.93g DMAP is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added in solid phase reaction column, reacts at room temperature 2h.Reaction
After washed 4 times with DMF, DCM is washed 2 times.Then 189.8g pyridine is added and 245.04g acetic anhydride mixed liquor closes resin
6h.It is washed 4 times with DMF, after DCM is washed 2 times, methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-Wang resin, examines
Survey substitution degree is 0.402mmol/g.
Embodiment two: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.6mmol/g
The Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is washed with DMF
Wash 2 times, after DMF swellable resins 30 minutes, take 138.2g Fmoc-Asp (OtBu)-OH, 54.5g HOBt, 50.8g DIC,
4.1g DMAP is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added in solid phase reaction column, reacts at room temperature 2h.Reaction knot
Shu Houyong DMF is washed 4 times, and DCM is washed 2 times.Then 189.8g pyridine is added and 245.04g acetic anhydride mixed liquor closes resin 6h.
It is washed 4 times with DMF, after DCM is washed 2 times, methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-Wang resin, detection is replaced
Dai Du is 0.603mmol/g.
Embodiment three: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.5mmol/g
The Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is washed with DMF
Wash 2 times, after DMF swellable resins 30 minutes, take 118.4g Fmoc-Asp (OtBu)-OH, 46.7g HOBt, 43.6g DIC,
3.5g DMAP is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added in solid phase reaction column, reacts at room temperature 2h.Reaction knot
Shu Houyong DMF is washed 4 times, and DCM is washed 2 times.Then 189.8g pyridine is added and 245.04g acetic anhydride mixed liquor closes resin 6h.
It is washed 4 times with DMF, after DCM is washed 2 times, methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-Wang resin, detection is replaced
Dai Du is 0.505mmol/g.
Example IV: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.4mmol/g
The 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is used
DMF is washed 2 times, after DMF swellable resins 30 minutes, takes 29.6g (72mmol) Fmoc-Asp (OtBu)-OH to be dissolved with DMF, ice
After 13.9g (108mmol) DIPEA activation is added under water-bath, it is added in the above-mentioned reaction column equipped with resin, after reacting 5min, then
Secondary addition 9.3g (72mmol) DIPEA reacts at room temperature 60min.182mL anhydrous methanol is added in reaction solution and closes 30min.With
DMF is washed 3 times, and DCM is washed 3 times, and methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-CTC resin, and detection substitution degree is
0.407mmol/g。
Embodiment five: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.6mmol/g
The 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is used
DMF is washed 2 times, after DMF swellable resins 30 minutes, 44.4g (108mmol) Fmoc-Asp (OtBu)-OH is taken to be dissolved with DMF,
After 20.9g (162mmol) DIPEA activation is added under ice-water bath, it is added in the above-mentioned reaction column equipped with resin, after reacting 5min,
13.9g (108mmol) DIPEA is added again, reacts at room temperature 60min.The closing of 182mL anhydrous methanol is added in reaction solution
30min.It is washed 3 times with DMF, DCM is washed 3 times, and methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-CTC resin, is detected
Substitution degree is 0.601mmol/g.
Embodiment six: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.5mmol/g
The 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g is weighed, is added in solid phase reaction column, is used
DMF is washed 2 times, after DMF swellable resins 30 minutes, takes 37.0g (90mmol) Fmoc-Asp (OtBu)-OH to be dissolved with DMF, ice
After 17.4g (135mmol) DIPEA activation is added under water-bath, it is added in the above-mentioned reaction column equipped with resin, after reacting 5min, then
Secondary addition 11.6g (90mmol) DIPEA reacts at room temperature 60min.182mL anhydrous methanol is added in reaction solution and closes 30min.
It is washed 3 times with DMF, DCM is washed 3 times, and methanol is collected concentration and drained, and obtains Fmoc-Asp (OtBu)-CTC resin, detects substitution degree
For 0.508mmol/g.
The preparation of seven: DiaPep277-Wang resin of embodiment
Weigh Fmoc-Asp (the OtBu)-Wang resin 198.0g that substitution degree in embodiment three is 0.505mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7g Fmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol；Purchased from gill biochemistry),
114.1g HATU (300mmol), 49.0g HOAt (360mmol) are dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, ice
It is added in solid phase reaction column after 104.3ml DIPEA (600mmol) activation 3min is added under water-bath, is detected and judged with ninhydrin method
Reaction end, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, need to be coupled anti-again
It answers 1 hour.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide sequence,
It is sequentially completed Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg
(pbf) coupling of-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 130.1g
Fmoc- (Hmb) Gly-OH (300mmol), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), is dissolved in volume ratio
Solid phase is added after 104.3ml DIPEA (600mmol) activation 3min is added for DCM the and DMF mixed solution of 1:1, under ice-water bath
In reaction column, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
Then indicate that reaction not exclusively, needs coupling reaction 1 hour again.It repeats above-mentioned removing Fmoc protection and corresponding amino acid couplings is added
Step is sequentially completed the coupling of Fmoc- (Hmb) Gly-OH, Fmoc- (Hmb) Gly-OH according to DiaPep277 main chain peptide sequence.Claim
It takes 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), it is mixed to be dissolved in DCM and DMF that volume ratio is 1:1
Solution is closed, is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, room temperature reaction 2 is small
When.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that
Reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria detects judgement reaction suitable for subsequent content with ninhydrin method
Terminal.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, it is complete according to DiaPep277 main chain peptide sequence
At the coupling of Fmoc-Val-OH.It is collected and is concentrated with methanol after reaction, resin vacuum is dried overnight, and weighing obtains
DiaPep277-Wang resin 451.6g.
The preparation of eight: DiaPep277-Wang resin of embodiment
Weigh Fmoc-Asp (the OtBu)-Wang resin 198.0g that substitution degree in embodiment three is 0.505mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7g Fmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol)、156.1g PyBOP
(300mmol), 48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, is sequentially completed
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、
The coupling of Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 134.3gFmoc- (Dmb)
Gly-OH (300mmol), 156.1g PyBOP (300mmol), 48.6g HOBt (360mmol), being dissolved in volume ratio is 1:1's
Solid phase reaction column is added after 104.3ml DIPEA (600mmol) activation 3min is added under ice-water bath in DCM and DMF mixed solution
In, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that
Reaction not exclusively, needs coupling reaction 1 hour again.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added,
According to DiaPep277 main chain peptide sequence, it is sequentially completed the coupling of Fmoc- (Dmb) Gly-OH, Fmoc- (Dmb) Gly-OH.It weighs
106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol) are dissolved in DCM and DMF that volume ratio is 1:1 and mix
Solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, reacts at room temperature 2 hours.
Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that anti-
Coupling reaction 1 hour again not exclusively should be needed, this judgment criteria detects judgement reaction eventually with ninhydrin method suitable for subsequent content
Point.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, completes
The coupling of Fmoc-Val-OH.It is collected and is concentrated with methanol after reaction, resin vacuum is dried overnight, and weighing obtains
DiaPep277-Wang resin 455.6g.
The preparation of nine: DiaPep277-Wang resin of embodiment
Weigh Fmoc-Asp (the OtBu)-Wang resin 198.0g that substitution degree in embodiment three is 0.505mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7g Fmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol)、96.3g TBTU
(300mmol), 48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, is sequentially completed
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、
The coupling of Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 134.3gFmoc- (Dmb)
Gly-OH (300mmol), 49.0g HOAt (360mmol) are dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, ice-water bath
It is added in solid phase reaction column after lower addition 56.3ml DIC (360mmol) activation 3min, judgement reaction is detected eventually with ninhydrin method
Point, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs again coupling reaction 1 small
When.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide sequence, successively
Complete the coupling of Fmoc- (Dmb) Gly-OH, Fmoc- (Dmb) Gly-OH.106.1g Fmoc-Leu-OH (300mmol) is weighed,
48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1,56.3ml DIC is added under ice-water bath
(360mmol) is added in solid phase reaction column after activating 3min, reacts at room temperature 2 hours.Reaction end is judged with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again,
This judgment criteria judges reaction end suitable for subsequent content with ninhydrin method detection.It repeats above-mentioned removing Fmoc protection and adds
The step of entering corresponding amino acid couplings completes the coupling of Fmoc-Val-OH according to DiaPep277 main chain peptide sequence.After reaction
It is collected and is concentrated with methanol, resin vacuum is dried overnight, and weighing obtains DiaPep277-Wang resin 458.1g.
The preparation of ten: DiaPep277-Wang resin of embodiment
Weigh Fmoc-Asp (the OtBu)-Wang resin 198.0g that substitution degree in embodiment three is 0.505mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7gFmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol)、96.3g TBTU
(300mmol), 48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, is sequentially completed
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、
The coupling of Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 134.3gFmoc- (Dmb)
Gly-OH (300mmol), 49.0g HOAt (360mmol) are dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, ice-water bath
It is added in solid phase reaction column after lower addition 56.3ml DIC (360mmol) activation 3min, judgement reaction is detected eventually with ninhydrin method
Point, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs again coupling reaction 1 small
When.With DBLK removing Fmoc protection, is then washed 6 times with DMF, detect color of resin with ninhydrin method, resin has color, indicates
Fmoc has been removed.151.4g Fmoc-Gly- (Dmb) Gly-OH (purchased from Merck), 49.0g HOAt (360mmol) are weighed, it is molten
It is added after 56.3ml DIC (360mmol) activation 3min is added under DCM the and DMF mixed solution that volume ratio is 1:1, ice-water bath
In solid phase reaction column, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin
Colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.With DBLK removing Fmoc protection, then washed 6 times with DMF,
Color of resin is detected with ninhydrin method, resin has color, indicates that Fmoc has been removed.Weigh 106.1g Fmoc-Leu-OH
(300mmol), 48.6g HOBt (360mmol) are dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, are added under ice-water bath
56.3ml DIC (360mmol) is added in solid phase reaction column after activating 3min, reacts at room temperature 2 hours.Sentenced with ninhydrin method detection
Disconnected reaction end, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, need to be coupled again
Reaction 1 hour, this judgment criteria judge reaction end suitable for subsequent content with ninhydrin method detection.Repeat above-mentioned removing
The step of Fmoc is protected and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, completes the idol of Fmoc-Val-OH
Connection.It is collected and is concentrated with methanol after reaction, resin vacuum is dried overnight, and weighing obtains DiaPep277-Wang resin
448.3g。
The preparation of 11: DiaPep277-CTC resin of embodiment
Weigh Fmoc-Asp (the OtBu)-CTC resin 196.9g that substitution degree in embodiment six is 0.508mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7g Fmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol)、114.1g HATU
(300mmol), 49.0g HOAt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, is sequentially completed
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、
The coupling of Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 130.1g Fmoc- (Hmb)
Gly-OH (300mmol), 49.0g HOAt (360mmol) are dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, ice-water bath
It is added in solid phase reaction column after lower addition 56.3ml DIC (360mmol) activation 3min, judgement reaction is detected eventually with ninhydrin method
Point, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs again coupling reaction 1 small
When.With DBLK removing Fmoc protection, is then washed 6 times with DMF, detect color of resin with ninhydrin method, resin has color, indicates
Fmoc has been removed.Weigh 147.2g Fmoc-Gly- (Hmb) Gly-OH (purchased from Merck), 156.1g PyBOP (300mmol),
48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1,104.3ml is added under ice-water bath
DIPEA (600mmol) is added in solid phase reaction column after activating 3min, reaction end is judged with ninhydrin method detection, if resin
It is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.It is de- with DBLK
It except Fmoc is protected, is then washed 6 times with DMF, detects color of resin with ninhydrin method, resin has color, indicates that Fmoc has been removed.
It weighs 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), is dissolved in the DCM and DMF that volume ratio is 1:1
Mixed solution is added in solid phase reaction column, room temperature reaction 2 after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath
Hour.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin develops the color, then table
Show reaction not exclusively, need coupling reaction 1 hour again, this judgment criteria is anti-with ninhydrin method detection judgement suitable for subsequent content
Answer terminal.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide sequence,
Complete the coupling of Fmoc-Val-OH.It is collected and is concentrated with methanol after reaction, resin vacuum is dried overnight, and weighing obtains
DiaPep277-Wang resin 449.3g.
The preparation of 12: DiaPep277-CTC resin of embodiment
Weigh Fmoc-Asp (the OtBu)-CTC resin 196.9g that substitution degree in embodiment six is 0.508mmol/g
(100mmol) is added in solid phase reaction column, is washed 2 times with DMF, after DMF swellable resins 30 minutes, is removed with DBLK
Fmoc protection, is then washed 6 times with DMF, detects color of resin with ninhydrin method, and resin has color, indicates that Fmoc has been removed.It takes
127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in volume ratio be 1:1 DCM and
DMF mixed solution is added in solid phase reaction column after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath, and room temperature is anti-
It answers 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing,
It then indicates reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is sentenced suitable for subsequent content with ninhydrin method detection
Disconnected reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to DiaPep277 main chain peptide
Sequence is sequentially completed Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-
The coupling of Leu-OH.Take 166.7g Fmoc-Asp (OtBu)-Ser (ψMe,MePro)-OH(300mmol)、156.1g PyBOP
(300mmol), 48.6g HOBt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings are added, according to DiaPep277 main chain peptide sequence, is sequentially completed
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、
The coupling of Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH.Weigh 147.2g Fmoc-Gly-
(Hmb) Gly-OH (300mmol), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), being dissolved in volume ratio is 1:1
DCM and DMF mixed solution, solid phase reaction column is added after 104.3ml DIPEA (600mmol) activation 3min is added under ice-water bath
In, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that
Reaction not exclusively, needs coupling reaction 1 hour again.With DBLK removing Fmoc protection, is then washed 6 times with DMF, examined with ninhydrin method
Color of resin is surveyed, resin has color, indicates that Fmoc has been removed.Weigh 134.3g Fmoc- (Dmb) Gly-OH, 114.1g HATU
(300mmol), 49.0g HOAt (360mmol) is dissolved in DCM the and DMF mixed solution that volume ratio is 1:1, is added under ice-water bath
104.3ml DIPEA (600mmol) is added in solid phase reaction column after activating 3min, judges reaction end with ninhydrin method detection,
If resin is colorless and transparent, then it represents that fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again.
With DBLK removing Fmoc protection, is then washed 6 times with DMF, detect color of resin with ninhydrin method, resin has color, indicates
Fmoc has been removed.It weighs 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), being dissolved in volume ratio is 1:
Solid phase reaction column is added after 56.3ml DIC (360mmol) activation 3min is added under ice-water bath in 1 DCM and DMF mixed solution
In, it reacts at room temperature 2 hours.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that fully reacting；
Resin colour developing, then it represents that reaction not exclusively, needs coupling reaction 1 hour again, this judgment criteria is suitable for subsequent content with indenes three
The detection of ketone method judges reaction end.The step of repeating above-mentioned removing Fmoc protection and corresponding amino acid couplings be added, according to
DiaPep277 main chain peptide sequence, completes the coupling of Fmoc-Val-OH.It is collected and is concentrated with methanol after reaction, resin vacuum is dry
Overnight, weighing obtains DiaPep277-Wang resin 450.7g.
The preparation of the thick peptide of embodiment 13: DiaPep277
The peptide resin prepared in Example seven to 12 is placed in cracking reaction wherein, is added with the ratio of 10ml/g resin
Lytic reagent (TFA:TIS: water=90:5:5 (V/V)), is stirred at room temperature 2h.Reactant is filtered with sand core funnel, collects filtrate, tree
Rouge is washed 3 times with a small amount of TFA again, is concentrated under reduced pressure after merging filtrate.The anhydrous ether precipitating of frost is added, is washed with anhydrous ether
3 times, vacuum drying obtains white powder solid, the i.e. thick peptide of DiaPep277, and weight yield is 98.6%~103.9%.HPLC is pure
Degree is greater than 55%.Single impurity is less than 5%.
The preparation of 14: DiaPep277 fine peptide of embodiment
It weighs in embodiment 13 after any thick peptide 4000ml water dissolution of 80.0g DiaPep277, using Waters
2545RP-HPLC system, wavelength 230nm, chromatographic column are 50 × 250mm reverse phase C18 column, conventional 0.2%TFA/ acetonitrile mobile phase
Purpose peak fraction is collected in purifying, obtains purity greater than 98.5% fine peptide.Rotary evaporation concentration, freeze-drying obtain DiaPep277 fine peptide
23.5g, RP-HPLC purity are greater than 98.5%.
Claims (17)
1. a kind of method for preparing polypeptide drugs DiaPep277 comprising the steps of:
1) using a kind of resin P as carrier, Fmoc-Asp (OtBu)-OH and resin is coupled, Fmoc-Asp (OtBu)-P is obtained；
2) using Fmoc-Asp (OtBu)-P as raw material, solid phase conjunction is carried out with other amino acid starting materials by the way of being coupled one by one
At obtaining DiaPep277-P；
3) DiaPep277-P is cracked, obtains the thick peptide of DiaPep277；
Wherein Fmoc is 9-fluorenylmethyloxycarbonyl, is connected with amino or is connected with the nitrogen-atoms on the connected carbon atom of carboxyl,
The synthesis material that Asp-Ser structure uses in the mode being wherein coupled one by one described in step 2) is Fmoc-Asp
(OtBu)-Ser(ψMe,MePro)-OH is a pseudo proline dipeptides, so that serine is protected into five-membered ring, structure is as follows:
The synthesis material that wherein-Gly-Gly-Gly- structure uses for Fmoc- (Hmb) Gly-OH, Fmoc- (Dmb) Gly-OH,
One or both of Fmoc-Gly- (Dmb) Gly-OH or Fmoc-Gly- (Hmb) Gly-OH；
Wherein Glu, Asn, Ala, Pro, Thr, Leu, Ile, Arg use synthesis material be respectively Fmoc-Glu (OtBu)-OH,
Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Pro-OH、Fmoc-Thr(tBu)-OH、Fmoc-Leu-OH、Fmoc-
Ile-OH,Fmoc-Arg(pbf)-OH；
(the OtBu)-Ser of Fmoc-Asp described in step 2) (ψMe,MePro) coupling system of-OH be HATU/HOAt/DIPEA or
PyBOP/HOBt/DIPEA；
The coupling system of Gly synthesis material described in step 2) is DIC/HOAt or HATU/HOAt/DIPEA.
2. method of claim 1, further include the steps that purifying DiaPep277, be purified by recrystallization method or HPLC and into
Row.
3. method for claim 2, HPLC is reversed-phase HPLC.
4. the method for claim 1 wherein steps 1) -3) carry out at room temperature and atmospheric pressure；Step 1) -2) coupling reaction exist
It is carried out in solid phase reaction column.
5. the method for claim 1 wherein the resin P is 2-CTC resin or Wang Shuzhi；The wherein substitution degree of the resin P
For 0.2-1.0mmol/g.
6. method for claim 5, wherein the substitution degree of the resin P is 0.3-0.8mmol/g.
7. method for claim 5, wherein the substitution degree of the resin P is 0.4-0.6mmol/g.
8. the method for claim 1 wherein-Gly-Gly-Gly- structure uses in the mode being coupled one by one described in step 2)
Synthesis material is one kind selected from Fmoc- (Hmb) Gly-OH and Fmoc- (Dmb) Gly-OH, and is selected from Fmoc-Gly- (Dmb)
One kind of Gly-OH and Fmoc-Gly- (Hmb) Gly-OH.
9. the method for claim 1 wherein the coupling agent in step 1) is HOBt/DIC.
10. the method for claim 1 wherein carried out to the washing of resin and swelling using DMF in coupling；By step 1) -2) in
Carboxyl in substance is activated.
11. method for claim 10, activation carries out in ice-water bath；Activator is DIPEA or DIC.
12. the method for claim 1 wherein steps 1) -2) in the reagent of removing Fmoc be DBLK；The deprotection reaction time is 5-
30 minutes.
13. the method for claim 12, wherein the deprotection reaction time is 10-20 minutes.
14. the method for claim 1 wherein the mixed solvent of lytic reagent TFA, tri isopropyl silane and water in step 3),
The wherein proportion of mixed solvent are as follows: TFA volume ratio is 85-90%, and the volume ratio of tri isopropyl silane is 1-5%, the volume of water
Than for 1-5%, the sum of three is 100%.
15. the method for claim 1 wherein the lytic reagent in step 3) is TFA:TIS:H2O volume ratio=90:5:5.
16. also being wrapped the method for claim 1 wherein after amino acid Fmoc-Asp (OtBu)-OH is coupled on vector resin
It includes and the step of a kind of confining liquid removes excessive unreacted group in resin is added thereto.
17. the method for claim 16, when the vector resin is 2-CTC resin, the confining liquid being added is methanol；Work as institute
State vector resin be Wang Shuzhi when, the confining liquid being added is the acetic anhydride and pyridine that molar ratio is 1:1.
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| CN1175214A (en) * | 1994-12-21 | 1998-03-04 | 耶达研究与发展有限公司 | Petide P227 analogs, and pharmaceutical compositions comprising them for treatment or diagnosis of diabetes |
| CN101484467A (en) * | 2006-05-10 | 2009-07-15 | Bcn肽类股份有限公司 | Method for synthesizing thymosins |
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| CN101484467A (en) * | 2006-05-10 | 2009-07-15 | Bcn肽类股份有限公司 | Method for synthesizing thymosins |
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