CN105384799B - A kind of method that solid liquid phase combination prepares sinapultide - Google Patents
A kind of method that solid liquid phase combination prepares sinapultide Download PDFInfo
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Abstract
The present invention relates to Peptides Synthesis, in particular to a kind of solid liquid phase combines the method for preparing sinapultide.Method provided by the invention is as follows: preparing polypeptide fragment I: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH first; then it uses on Fmoc synthesis in solid state normal direction vector resin and is successively coupled four polypeptide fragments I; it is finally coupled protected amino acid Lys, sinapultide is made after cracking reaction.Preparing sinapultide only using this method needs by five step peptide reactions, and with short production cycle, thick peptide purity is high, production cost is low, easy to operate, is suitable for carrying out industrialized production.
Description
Technical field
The present invention relates to Peptides Synthesis, in particular to a kind of solid liquid phase combines the method for preparing sinapultide.
Background technique
There is progressive expiratory dyspnea after referring to newborn's birth soon and exhales in respiratory distress syndrome of newborn (RDS)
The symptoms such as failure are inhaled, mainly due to caused by lacking alveolar surfactant, cause alveolar progressive to wither, infant is in life
Occur progressive expiratory dyspnea, groan, cyanosis, air-breathing three depressions sign in 4-12 hours afterwards, respiratory failure occurs for serious person.Disease incidence
Related with gestational age, gestational age is smaller, and disease incidence is higher, and weight is lighter, and case fatality rate is higher.
On March 6th, 2012, the intratracheal suspension Surfaxin of Discovery Laboratories company
(Lucinactant, reed Xi Natan) obtains Food and Drug Adminstration of the US (FDA) approval, for treating premature's respiratory distress
Syndrome.Surfaxin is the production designed on the basis of sinapultide according to the characteristics of natural human Curosurf
Product, for simulating people lung surfactant protein B (SP-B).
Sinapultide, English name Sinapultide, molecular weight 2469.40 are that a kind of engineer contains 21
The peptide molecule of amino acid, structure are as follows:
The domestic synthetic method about sinapultide mainly has method of gene recombination and chemical method at present.Wherein United States Patent (USP)
US5260273 synthesizes sinapultide using gene recombination technology, but this method is cumbersome, and technical requirements and production cost pole
Height is unfavorable for carrying out industrialization large-scale production, it is most important that, once the nucleotide on recombination DNA molecular chain occurs
Mutation, it will cause amino acid mutations corresponding on peptide chain, reduce the purity and safety in utilization of drug.
102850440 A of Chinese patent CN is the domestic first patent in relation to sinapultide synthetic method, uses segment
Synthetic method.This method passes through method synthesis segment peptide resin Fmoc-Lys (Boc)-Leu-Leu- of synthesis in solid state first
Then Leu-Leu- resin is gradually coupled again with intermediate Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu- made from solid phase method
KLLLLKLLLLKLLLLKLLLL peptide resin is made in OH, and sinapultide analog is obtained after cracking, which is prepared using solid phase method
Polypeptide fragment needs to consume the vector resin of a large amount of valuableness, and production cost is higher, and in synthesis segment Fmoc-Lys (Boc)-
During Leu-Leu-Leu-Leu-OH, it is easy to produce impurity F moc-Lys-Leu-Leu-Leu-Leu-OH, and then leads to whole production
The impurity of upper segment H-Lys-Leu-Leu-Leu-Leu-OH is coupled in product containing Lys side-chain amino group.
104098656 A of Chinese patent CN uses Fmoc solid-phase synthesis, is successively coupled each protected amino acid, obtains
21 peptide resins must be protected, sinapultide, yield 47.9% is made through cracking reaction.The method production cycle is longer, undergoes more
Peptide reaction is walked, thick peptide purity is lower, causes total recovery lower, high production cost.
104817631 A of Chinese patent CN describes a kind of first by Fmoc solid-phase synthesis synthesis Fmoc-Lys
(Boc)-resin, then alternately coupling protected fragment Fmoc- (Leu)4- OH and protected amino acid Fmoc-Lys (Boc)-OH, system
Obtain sinapultide peptide resin, the method that sinapultide most is made through cracking reaction afterwards.The method still needs to experience multistep peptide reaction,
Crude product purity is lower, and for yield between 52.3%-57.1%, the production cycle is longer, high production cost.
In conclusion in the sinapultide synthetic method reported at present, it is longer to there is the production cycle, high production cost
Problem is unfavorable for amplification production, the lower problem of yield.
Summary of the invention
To solve the problems, such as that above-mentioned sinapultide synthesis process, the present invention provide a kind of solid liquid phase combination preparation west
The method of that general peptide: polypeptide fragment I is prepared using liquid phase method first:
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH, then using on Fmoc synthesis in solid state normal direction vector resin according to
Four polypeptide fragments I of secondary coupling are finally coupled protected amino acid Lys, and sinapultide is made after cracking reaction.Using this method
Preparing sinapultide only needs by five step peptide reactions, and with short production cycle, thick peptide purity is high, high income greatly reduces simultaneously
Production cost is suitable for carrying out industrialized production.
For achieving the above object, the present invention the following technical schemes are provided:
A kind of method that solid liquid phase combination prepares sinapultide, includes the following steps:
(a) polypeptide fragment I: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH is prepared;
(b) it is successively coupled using the polypeptide fragment I on Fmoc synthesis in solid state normal direction vector resin successively in coupling step (a)
After 4 times, it is then coupled protected amino acid Lys, prepares sinapultide peptide resin;
(c) sinapultide peptide resin prepares the thick peptide of sinapultide through cracking reaction;
(d) the purified process of the thick peptide of sinapultide prepares sinapultide fine peptide.
Wherein, in step (a), the preparation process of polypeptide fragment I are as follows:
(1) in organic solvent, using DCC as activator, reaction is made by synthesis Boc-Leu-OSu:Boc-Leu-OH and HOSu
Standby Boc-Leu-OSu;Wherein the molar ratio of Boc-Leu-OH and HOSu is 1:1.0~1.2;Mole of Boc-Leu-OH and DCC
Than for 1:1.0~1.2;
(2) synthesis H-Leu-Leu-OH:H-Leu-OH and Boc-Leu-OSu reacts in sodium carbonate liquor, prepares Boc-
Then Leu-Leu-OH is handled through TFA, prepare H-Leu-Leu-OH;Wherein the molar ratio of H-Leu-OH and sodium carbonate is 1:1.0
~2.0;The molar ratio of H-Leu-OH and Boc-Leu-OSu is 1:1.1~1.5;
(3) synthesis Fmoc-Leu-OSu:Fmoc-Leu-OH and HOSu in organic solvent, using DCC as activator, react
Prepare Fmoc-Leu-OSu;Wherein the molar ratio of Fmoc-Leu-OH and HOSu is 1:1.0~1.2;Fmoc-Leu-OH's and DCC
Molar ratio is 1:1.0~1.2;
(4) preparation of Fmoc-Leu-Leu-OH: H-Leu-OH and Fmoc-Leu-OSu react in sodium carbonate liquor, system
Standby Fmoc-Leu-Leu-OH;Wherein the molar ratio of H-Leu-OH and sodium carbonate is 1:1.0~2.0;H-Leu-OH and Fmoc-
The molar ratio of Leu-OSu is 1:1.1~1.5;
(5) preparation of Fmoc-Leu-Leu-OSu: Fmoc-Leu-Leu-OH and HOSu are to live with DCC in organic solvent
Agent, reaction preparation Fmoc-Leu-Leu-OSu;Wherein the molar ratio of Fmoc-Leu-Leu-OH and HOSu is 1:1.0~1.2;
The molar ratio of Fmoc-Leu-Leu-OH and DCC is 1:1.0~1.2;
(6) preparation of Fmoc-Leu-Leu-Leu-Leu-OH: H-Leu-Leu-OH and Fmoc-Leu-Leu-OSu are in carbonic acid
It is reacted in sodium solution, prepares Fmoc-Leu-Leu-Leu-Leu-OH;Wherein the molar ratio of H-Leu-Leu-OH and sodium carbonate is 1:
1.0~2.0;The molar ratio of H-Leu-Leu-OH and Fmoc-Leu-Leu-OSu is 1:1.1~1.5;
(7) preparation of Fmoc-Leu-Leu-Leu-Leu-OSu: Fmoc-Leu-Leu-Leu-Leu-OH and HOSu are organic
In solvent, preparation Fmoc-Leu-Leu-Leu-Leu-OSu is reacted by activator of DCC;Wherein Fmoc-Leu-Leu-Leu-
The molar ratio of Leu-OH and HOSu is 1:1.0~1.2;The molar ratio of Fmoc-Leu-Leu-Leu-Leu-OH and DCC is 1:1.0
~1.2;
(8) preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH: H-Lys (Boc)-OH and Fmoc-Leu-
Leu-Leu-Leu-OSu reacts in sodium carbonate liquor, prepares Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH;Wherein H-
The molar ratio of Lys (Boc)-OH and sodium carbonate is 1:1.0~2.0;H-Lys (Boc)-OH and Fmoc-Leu-Leu-Leu-Leu-
The molar ratio of OSu is 1:1.1~1.5.
Preferably, in step (a), the preparation process of polypeptide fragment I are as follows:
The preparation of Boc-Leu-OSu: Boc-Leu-OH and HOSu are dissolved in organic solvent, and DCC is then added dropwise thereto
Organic solvent solution, be stirred to react, TLC monitor reaction end.It filters after reaction, concentration of reaction solution, into concentrate
The petroleum ether of 5~6 times of volumes is added, white solid is precipitated, filters, volume after filter cake is re-dissolved with ethyl acetate to concentration,
5~6 times of volume petroleum ethers are added, crystallization filters, and Vacuum dry filter cake obtains Boc-Leu-OSu.Boc-Leu-OH's and HOSu
Molar ratio is 1:1.0~1.2;The molar ratio of Boc-Leu-OH and DCC is 1:1.0~1.2.
The preparation of H-Leu-Leu-OH: H-Leu-OH and sodium carbonate is soluble in water, the organic molten of Boc-Leu-OSu is added
Agent solution is stirred to react, and TLC monitors reaction end, and after reaction, 10% aqueous citric acid solution is added into filtrate for filtering
Solution ph is adjusted to 2~3, ethyl acetate extraction is added 5~6 times of volume petroleum ether crystallizations and obtains Boc-Leu-Leu- after concentration
OH.Boc-Leu-Leu-OH is settled out after TFA is handled in cold ether to get H-Leu-Leu-OH.H-Leu-OH and carbonic acid
The molar ratio of sodium is 1:1~2;The molar ratio of H-Leu-OH and Boc-Leu-OSu is 1:1.1~1.5.
The preparation of Fmoc-Leu-OSu: Fmoc-Leu-OH and HOSu are dissolved in organic solvent, are then added dropwise thereto
The organic solvent solution of DCC, is stirred to react, and TLC monitors reaction end.It filters after reaction, concentration of reaction solution, to concentrate
The middle petroleum ether that 5~6 times of volumes are added, is precipitated white solid, filters, body after filter cake is re-dissolved with ethyl acetate to concentration
5~6 times of volume petroleum ethers are added in product, and crystallization filters, and Vacuum dry filter cake obtains Fmoc-Leu-OSu.Fmoc-Leu-OH and
The molar ratio of HOSu is 1:1.0~1.2;The molar ratio of Fmoc-Leu-OH and DCC is 1:1.0~1.2.
The preparation of Fmoc-Leu-Leu-OH: H-Leu-OH and sodium carbonate is soluble in water, and Fmoc-Leu-OSu is added has
Solvent solution, is stirred to react, and TLC monitors reaction end, and after reaction, 10% citric acid water is added into filtrate for filtering
Solution adjusts solution ph to 2~3, and ethyl acetate extraction is added 5~6 times of volume petroleum ether crystallizations and obtains Fmoc-Leu- after concentration
Leu-OH.The molar ratio of H-Leu-OH and sodium carbonate is 1:1~2;The molar ratio of H-Leu-OH and Fmoc-Leu-OSu is 1:1.1
~1.5.
.Fmoc-Leu-Leu-OSu preparation: Fmoc-Leu-Leu-OH and HOSu are dissolved in organic solvent, then to
The organic solvent solution of DCC is wherein added dropwise, is stirred to react, TLC monitors reaction end.It filters, adds into filtrate after reaction
Enter the petroleum ether of its 5~6 times of volumes, white solid be precipitated, filters, volume after filter cake is re-dissolved with ethyl acetate to concentration,
5~6 times of volume petroleum ethers are added, crystallization filters, and Vacuum dry filter cake obtains Fmoc-Leu-Leu-OSu.Fmoc-Leu-Leu-
The molar ratio of OH and HOSu is 1:1.0~1.2;The molar ratio of Fmoc-Leu-Leu-OH and DCC is 1:1.0~1.2.
The preparation of Fmoc-Leu-Leu-Leu-Leu-OH: H-Leu-Leu-OH and sodium carbonate is soluble in water, then it is added
The organic solvent solution of Fmoc-Leu-Leu-OSu, is stirred to react, and TLC monitors reaction end, after reaction, filtering, to filter
10% aqueous citric acid solution is added in liquid and adjusts solution ph to 2~3, solid is precipitated, filters to obtain white solid, washes 3 times, does
Recrystallization is after dry to get Fmoc-Leu-Leu-Leu-Leu-OH.The molar ratio of H-Leu-Leu-OH and sodium carbonate is 1:1~2;
The molar ratio of H-Leu-Leu-OH and Fmoc-Leu-Leu-OSu is 1:1.1~1.5.
The preparation of Fmoc-Leu-Leu-Leu-Leu-OSu: Fmoc-Leu-Leu-Leu-Leu-OH and HOSu, which are dissolved in, to be had
In solvent, the organic solvent solution of DCC is then added dropwise thereto, is stirred to react, TLC monitors reaction end.After reaction
It filters, concentration of reaction solution, the petroleum ether of 5~6 times of volumes is added into concentrate, white solid is precipitated, filter, filter cake is with acetic acid
5~6 times of volume petroleum ethers are added in volume after ethyl ester is re-dissolved to concentration, and crystallization filters, and Vacuum dry filter cake obtains Fmoc-
Leu-Leu-Leu-Leu-OSu.The molar ratio of Fmoc-Leu-Leu-Leu-Leu-OH and HOSu is 1:1.0~1.2;Fmoc-
The molar ratio of Leu-Leu-Leu-Leu-OH and DCC is 1:1.0~1.2.
The preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH: H-Lys (Boc)-OH and sodium carbonate are dissolved in water
In, the organic solvent solution of Fmoc-Leu-Leu-Leu-Leu-OSu is then added, is stirred to react, TLC monitors reaction end, instead
After answering, 10% aqueous citric acid solution is added into filtrate and adjusts solution ph to 2~3, solid is precipitated, filters for filtering
White solid is washed 3 times, is recrystallized after dry to get Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH.H-Lys
(Boc) molar ratio of-OH and sodium carbonate is 1:1~2;H-Lys (Boc)-OH's and Fmoc-Leu-Leu-Leu-Leu-OSu rubs
You are than being 1:1.1~1.5.
More preferably, organic solvent is tetrahydrofuran, dioxane, mixed liquor one or more kinds of in acetone.
Preferably, in step (b), vector resin is the Wang resin or CTC that substitution degree is 0.6-1.2mmol/g
resin。
Preferably, in step (b), the preparation process of sinapultide peptide resin are as follows: effect of the polypeptide fragment I in condensation reagent
Lower and vector resin carries out coupling reaction, prepares Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Wang resin or Fmoc-
Then Leu-Leu-Leu-Leu-Lys (Boc)-CTC resin is successively coupled three polypeptide fragments I again, finally coupling protection ammonia
Sinapultide peptide resin is made in base acid Lys.
Preferably, polypeptide fragment I is added with 2-5 times of feed ratio or protected amino acid Lys carries out coupling reaction, each
Coupling reaction is the solid phase peptide reaction carried out in the presence of condensation reagent, and each step coupling reaction is with Kaiser reagent
Reaction end is detected, removes Fmoc with deprotecting regent after completion of the reaction, then carry out occasionally with next segment or protected amino acid
Connection reaction.
More preferably, deprotecting regent is the mixed solution of piperidines and DMF volume ratio 1:4 composition;Protected amino acid Lys
For Fmoc-Lys (Boc)-OH or Boc-Lys (Boc)-OH;Condensation reagent is DIC/HOBt, DIC/HOAt, TBTU/HOBt/
One of DIEA, HBTU/HOBt/DIEA, HATU/HOAt/DIEA.
Preferably, in step (c), the preparation of the thick peptide of sinapultide are as follows: sinapultide peptide resin is added to lysate
In, at 15~30 DEG C, 2~5h of cracking reaction, reaction terminates, and lysate is filled into methyl tertiary butyl ether(MTBE) or ether and is settled
And wash, precipitating is collected by centrifugation, the thick peptide of sinapultide is obtained after vacuum drying.
More preferably, lysate is the TFA solution that volume ratio 1-5% scavenger is added, and scavenger is methyl phenyl ethers anisole, benzene first
One or more of thioether, dithioglycol, mercaptoethanol, phenol, water.
Preferably, a kind of solid liquid phase combines the method for preparing sinapultide, which comprises the steps of: step
A: liquid phase method prepares polypeptide fragment I: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH;
Step B: excessive polypeptide fragment I is taken to prepare I peptide resin of polypeptide fragment with resin coupling under coupling reagent effect;
Step C: in a manner of Excess quantities, continue successively to be coupled three polypeptide fragments I on I peptide resin of polypeptide fragment;
Step D: coupling protected amino acid Lys prepares sinapultide peptide resin on peptide resin obtained by step C
Step E: sinapultide peptide resin prepares the thick peptide of sinapultide through cracking reaction;
Step F: the thick peptide of sinapultide is purified to prepare sinapultide fine peptide.
Preferably, protected amino acid Lys is Fmoc-Lys (Boc)-OH or Boc-Lys (Boc)-OH;Condensation reagent is
DIC/HOBt or DIC/HOA;Lysate be added volume ratio 1-5% scavenger TFA solution, the scavenger be methyl phenyl ethers anisole,
One or more of thioanisole, water.
Compared with the existing technology, the beneficial effects of the present invention are:
1, liquid phase method prepares polypeptide fragment I: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH, compared to synthesis in solid state
Method, the more cheap raw materials and reagents of use, reduces production cost;Simultaneously synthesizing process reaction is high-efficient, high income, every step
Reaction yield reaches 80% or more, and product purity is high, and impurity content is few;
1, peptide reaction step is greatly reduced using segment synthetic method, improves thick peptide purity, shortens production
Period, while further reduced production cost;
2, simple operation of process is suitable for amplification production.
Specific embodiment
With specific embodiment, the present invention is described in detail below, but does not limit this patent;Change according to the present invention former
The feed ratio or reaction dissolvent of material or and condensing agent etc., be within the scope of the invention.
Abbreviation meaning used in specification and claims is as follows:
The preparation of embodiment 1:Boc-Leu-OSu
It accurately weighs Boc-Leu-OH 1156g (5.0mol) and HOSu 633g (5.5mol) is dissolved in 12L tetrahydrofuran,
Ice-water bath stirring.DCC 1238g (6.0mol) is accurately weighed, is dissolved in 7L tetrahydrofuran, is slowly dropped in above-mentioned solution, stirs
Reaction is mixed, TLC monitors reaction end.After the reaction was completed, it filters, concentration of reaction solution to 6~7L, 35L stone is added into concentrate
A large amount of white solids, -20 DEG C of standing 1h of solution are precipitated in oily ether.It is filtered after standing, 35L is added with the dissolution of 6L ethyl acetate in filter cake
Petroleum ether, crystallization filter, and after filter cake vacuum drying, obtain Boc-Leu-OSu 1538g, purity is greater than 99%, yield 93.7%.
The preparation of embodiment 2:H-Leu-Leu-OH
It accurately weighs H-Leu-OH 511g (3.9mol) and sodium carbonate 615g (5.8mol) is dissolved in 7L water, under ice-water bath
It is slowly added to the tetrahydrofuran solution 8L of gained Boc-Leu-OSu 1538g (4.7mol) in embodiment 1, is stirred to react, TLC prison
Survey reaction end.It after fully reacting, filters, 10% aqueous citric acid solution is added under ice-water bath into filtrate, adjusts pH value of solution
Value to 2~3,10L ethyl acetate extracts, and merges organic phase, and 30L petroleum ether is added into concentrate for concentrated by rotary evaporation to 5~6L,
It is placed in crystallization in -20 DEG C, Boc-Leu-Leu-OH is obtained, is drained after filtering, with 60% TFA/ aqueous dissolution, is stirred to react
2h, cold ether sedimentation washing, is dried in vacuo to obtain H-Leu-Leu-OH 883g, and purity is greater than 99%, yield 92.7%.
The preparation of embodiment 3:Fmoc-Leu-OSu
It accurately weighs Fmoc-Leu-OH 1767g (5.0mol) and HOSu 633g (5.5mol) is dissolved in 14L tetrahydrofuran
In, ice-water bath stirring.DCC 1238g (6.0mol) is accurately weighed, is dissolved in 7L tetrahydrofuran, above-mentioned solution is slowly dropped to
In, it is stirred to react, TLC monitors reaction end.After the reaction was completed, it filters, concentration of reaction solution to 6~7L is added into concentrate
A large amount of white solids, -20 DEG C of standing 1h of solution are precipitated in 35L petroleum ether.It is filtered after standing, filter cake is added with the dissolution of 6L ethyl acetate
Enter 35L petroleum ether, crystallization filters, and after filter cake vacuum drying, obtains Fmoc-Leu-OSu 2020g, purity is greater than 99%, yield
89.7%.
The preparation of embodiment 4:Fmoc-Leu-Leu-OH
It accurately weighs H-Leu-OH486g (3.7mol) and sodium carbonate 431g (4.1mol) is dissolved in 6L water, delay under ice-water bath
The slow tetrahydrofuran solution 9L that gained Fmoc-Leu-OSu 2020g (4.5mol) in embodiment 3 is added, is stirred to react, TLC prison
Survey reaction end.It after fully reacting, filters, 10% aqueous citric acid solution is added under ice-water bath into filtrate, adjusts pH value of solution
Value to 2~3,10L ethyl acetate extracts, and merges organic phase, and 30L petroleum ether is added into concentrate for concentrated by rotary evaporation to 5~6L,
It is placed in crystallization in -20 DEG C, is drained after filtering, Fmoc-Leu-Leu-OH 1512g is dried in vacuo to obtain, purity is greater than 99%, yield
87.6%.
The preparation of embodiment 5:Fmoc-Leu-Leu-OSu
Accurately weigh gained Fmoc-Leu-Leu-OH 1512g (3.2mol) and HOSu 546g (3.5mol) in embodiment 4
It is dissolved in 12L tetrahydrofuran, ice-water bath stirring.DCC792g (3.8mol) is accurately weighed, is dissolved in 4.5L tetrahydrofuran, slowly
It is added drop-wise in above-mentioned solution, is stirred to react, TLC monitors reaction end.After the reaction was completed, it filters, concentration of reaction solution to 4~5L,
25L petroleum ether is added into concentrate, a large amount of white solids, -20 DEG C of standing 1h of solution are precipitated.It is filtered after standing, filter cake is with 4L
20L petroleum ether is added in ethyl acetate dissolution, and crystallization filters, and after filter cake vacuum drying, obtains Fmoc-Leu-Leu-OSu 1690g,
Purity is greater than 99%, yield 93.7%.
The preparation of embodiment 6:Fmoc-Leu-Leu-Leu-Leu-OH
It is molten to accurately weigh gained H-Leu-Leu-OH 611g (2.5mol) and sodium carbonate 300g (2.8mol) in embodiment 2
The tetrahydro furan of gained Fmoc-Leu-Leu-OSu 1690g (3.0mol) in embodiment 5 is slowly added in 6L water, under ice-water bath
Mutter solution 7.5L, is stirred to react, and TLC monitors reaction end.It after fully reacting, filters, 10% is added into filtrate under ice-water bath
Aqueous citric acid solution, adjust solution ph to 2~3, white solid be precipitated, filter, filter cake is washed with water 3 times, uses after dry
7L ethyl acetate re-dissolves, and 30L petroleum ether crystallization is added, drains after filtering, is dried in vacuo to obtain Fmoc-Leu-Leu-Leu-
Leu-OH 1545g, purity are greater than 99%, yield 89.2%.
The preparation of embodiment 7:Fmoc-Leu-Leu-Leu-Leu-OSu
Accurately weigh gained Fmoc-Leu-Leu-Leu-Leu-OH 1525g (2.2mol) and HOSu 300g in embodiment 6
(2.6mol) is dissolved in 10L tetrahydrofuran, ice-water bath stirring.DCC 536g (2.6mol) is accurately weighed, 4.5L tetrahydro furan is dissolved in
It in muttering, is slowly dropped in above-mentioned solution, is stirred to react, TLC monitors reaction end.After the reaction was completed, it filters, concentration of reaction solution
To 4~5L, 25L petroleum ether is added into concentrate, a large amount of white solids, -20 DEG C of standing 1h of solution are precipitated.It is filtered after standing,
20L petroleum ether is added with the dissolution of 4L ethyl acetate in filter cake, and crystallization filters, and after filter cake vacuum drying, obtains Fmoc-Leu-Leu-
Leu-Leu-OSu 1577g, purity are greater than 99%, yield 90.7%.
The preparation of embodiment 8:Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH
It accurately weighs H-Lys (Boc)-OH 249g (1.7mol) and sodium carbonate 216g (2.0mol) is dissolved in 3L water, ice water
The tetrahydrofuran solution of gained Fmoc-Leu-Leu-Leu-Leu-OSu 1577g (2.0mol) in embodiment 7 is slowly added under bath
7L is stirred to react, and TLC monitors reaction end.It after fully reacting, filters, 10% citric acid is added under ice-water bath into filtrate
Aqueous solution adjusts solution ph to 2~3, white solid is precipitated, filters, filter cake is washed with water 3 times, uses 6L acetic acid second after dry
Ester re-dissolves, and 30L petroleum ether crystallization is added, drains after filtering, is dried in vacuo to obtain Fmoc-Leu-Leu-Leu-Leu-Lys
(Boc)-OH 1350g, purity are greater than 99%, yield 86.2%.
The preparation of embodiment 9:Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Wang resin
Wang resin 165g (initial substitution degree is 0.605mmol/g, and synthesis scale is 100mmol) is accurately weighed to set
It in synthesis column, is washed twice with 1L DMF, 1.2L DCM is added and is swollen 30min;After leaching out DCM, Fmoc-Leu- is added
The mixed solution of Leu-Leu-Leu-Lys (Boc)-OH/DIC/HOBt [weighs gained Fmoc-Leu-Leu-Leu- in embodiment 8
Leu-Lys (Boc)-OH 276g (300mmol) and 45g (330mmol) HOBt is placed in dissolution bottle, and 700ml volume ratio is added
For 1: 1 DMF and DCM mixed solution stirring and dissolving, 51ml (330mmol) DIC is added under ice-water bath, activates 5min], reaction
3.6g (30mmol) DMAP is added after 10min;5h is reacted, reaction solution is taken out, is washed 3 times with 1L DMF, capping reagent 1L is added
(250ml acetic anhydride and 210ml pyridinium dissolution are in 540ml DMF) reacts 1h, leaches out reaction solution, uses DMF, DCM, first respectively
Alcohol washs 3 times, and Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Wang resin 254g is obtained after vacuum drying.
Embodiment 10: the preparation of sinapultide peptide resin
Accurately weigh Fmoc-Leu-Leu-Leu-Leu-Lys (the Boc)-Wang resin 254g (synthesis in embodiment 9
Scale 100mmol) it is placed in synthesis column, 2L DCM is added and is swollen 30min;After leaching out DCM, 2LDMF is washed 2 times, is added
20% piperidines/DMF solution 2L is deprotected 2 times, reacts 10min and 10min respectively;Then it is washed 6 times with DMF, each 2L;It weighs
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH 185g (200mmol), HOBT 30g (220mmol) are dissolved with 1LDMF,
15-20min is pre-chilled in ice-water bath, DIC 34ml (220mmol) is then added and activates 5min, solution is added in resin, instead
Answer 2h, reaction end is subject to Kaiser reagent testing result, and reaction takes out reaction solution up to after terminal, washs 6 times with DMF, often
Secondary 2L;Then it is deprotected again.Circulate operation repeatedly, successively be coupled Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH,
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH, Boc-Lys (Boc)-OH, after final step coupling reaction, resin
It is washed respectively 3 times with 2L DMF, DCM and methanol, is dried in vacuo to obtain sinapultide peptide resin 501g
Embodiment 11: the preparation of the thick peptide of sinapultide
The 501g sinapultide peptide resin (100mmol) that embodiment 10 is obtained is added to the 4.5L lysate (body of freezing
Product proportion is TFA/TIS/H2O=90/5/5 in), it is stirred to react 3h at room temperature;Cracking reaction terminates, and filters resin,
100mlTFA is washed resin 2 times, merging filtrate and washing lotion, concentrated by rotary evaporation to 1L, and concentrate pours into the 10L freezing tertiary ether of first, analysis
White precipitate out;After standing 1h, filtering, the tertiary ether of first is washed 6 times, is dried in vacuo to obtain the thick peptide 262g of sinapultide, yield
106.2%, purity 90.2%.
Embodiment 12: the thick peptide purification purification of sinapultide
The thick peptide 262g of sinapultide in Example 11, uses internal diameter for the C of 100mm18Column is prepared, mobile phase A is
0.1%TFA/ water, Mobile phase B are 0.1%TFA/ acetonitrile, and applied sample amount is 10g/ needle, flow velocity 300ml/min, Detection wavelength
210nm, gradient elution recycle sample introduction behind before peak and peak, obtain the fine peptide solution that middle control analysis purity is 98.5% or more;With vinegar
Sinapultide 197g is lyophilized to obtain after turning salt in acid/acetonitrile/water system, and purity is greater than 99.5%, total recovery 79.8%.
The preparation of embodiment 13:Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-CTC resin
It accurately weighs CTC resin 70g (initial substitution degree is 0.710mmol/g, and synthesis scale is 50mmol) and is placed in conjunction
Cheng Zhuzhong is washed twice with 500ml DMF, and 600ml DCM is added and is swollen 30min;After leaching out DCM, Fmoc-Leu- is added
The mixed solution of Leu-Leu-Leu-Lys (Boc)-OH/DIC/HOBt [weighs gained Fmoc-Leu-Leu-Leu- in embodiment 8
Leu-Lys (Boc)-OH 138g (150mmol) and 23g (165mmol) HOBt is placed in dissolution bottle, and 300ml volume ratio is added
For 1: 1 DMF and DCM mixed solution stirring and dissolving, 26ml (165mmol) DIC is added under ice-water bath, activates 5min], reaction
1.8g (15mmol) DMAP is added after 10min;4h is reacted, reaction solution is taken out, is washed 3 times with 1L DMF, capping reagent is added
500ml (DCM/ methanol/DIEA=17/2/1, volume ratio) end capping reaction 1h, leaches out reaction solution, uses DMF, DCM, methanol respectively
Washing 3 times obtains Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-CTC resin 117g after vacuum drying.
Embodiment 14: the preparation of sinapultide peptide resin
Accurately weigh Fmoc-Leu-Leu-Leu-Leu-Lys (the Boc)-CTC resin 117g (synthesis in embodiment 13
Scale 50mmol) it is placed in synthesis column, 1L DCM is added and is swollen 30min;After leaching out DCM, 1LDMF is washed 2 times, is added 20%
Piperidines/DMF solution 2L is deprotected 2 times, reacts 10min and 10min respectively;Then it is washed 6 times with DMF, each 1L;It weighs
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH 921g (100mmol), HOBT 15g (110mmol) are molten with 500ml DMF
It solves, 15-20min is pre-chilled in ice-water bath, DIC 17ml (110mmol) is then added and activates 5min, solution is added in resin,
2h is reacted, reaction end is subject to Kaiser reagent testing result, and reaction is taken out reaction solution, washed 6 times with DMF up to after terminal,
Each 1L;Then it is deprotected again.Circulate operation repeatedly is successively coupled Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-
OH, Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH take off after final step coupling reaction
Protection, resin are washed 4 times with DMF, are then washed 3 times, each 1L with DCM and methanol respectively again, be dried in vacuo to obtain sinapultide peptide
Resin 225g
Embodiment 15: the preparation of the thick peptide of sinapultide
The 225g sinapultide peptide resin (50mmol) that embodiment 14 is obtained is added to the 2L lysate (volume of freezing
Proportion is TFA/TIS/H2O=90/5/5 in), it is stirred to react 3h at room temperature;Cracking reaction terminates, and filters resin, 100mlTFA
Washing resin 2 times, merging filtrate and washing lotion, concentrated by rotary evaporation to 500ml, concentrate pour into the 5L freezing tertiary ether of first, and white is precipitated
Precipitating;After standing 1h, filtering, the tertiary ether of first is washed 6 times, is dried in vacuo to obtain the thick peptide 127g of sinapultide, yield 102.8%, purity
89.5%.
Embodiment 16: the thick peptide purification purification of sinapultide
The thick peptide 262g of sinapultide in Example 15, uses internal diameter for the C of 100mm18Column is prepared, mobile phase A is
0.1%TFA/ water, Mobile phase B are 0.1%TFA/ acetonitrile, and applied sample amount is 10g/ needle, flow velocity 300ml/min, Detection wavelength
210nm, gradient elution recycle sample introduction behind before peak and peak, obtain the fine peptide solution that middle control analysis purity is 98.5% or more;With vinegar
Sinapultide 95g is lyophilized to obtain after turning salt in acid/acetonitrile/water system, and purity is greater than 99.5%, total recovery 76.9%.
Claims (8)
1. a kind of solid liquid phase combines the method for preparing sinapultide, which comprises the steps of:
(a) liquid phase method prepares polypeptide fragment I: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH;
(b) using the polypeptide fragment I in coupling step (a) on Fmoc synthesis in solid state normal direction vector resin, after being successively coupled four times
It is coupled again with protected amino acid Lys, prepares sinapultide peptide resin;
(c) sinapultide peptide resin prepares the thick peptide of sinapultide through cracking reaction;
(d) the purified process of the thick peptide of sinapultide prepares sinapultide fine peptide.
2. a kind of solid liquid phase according to claim 1 combines the method for preparing sinapultide, which is characterized in that step (a)
In, the preparation process of polypeptide fragment I are as follows:
(1) in organic solvent, using DCC as activator, prepared by reaction by synthesis Boc-Leu-OSu:Boc-Leu-OH and HOSu
Boc-Leu-OSu;The molar ratio of the Boc-Leu-OH and HOSu is 1:1.0~1.2;The Boc-Leu-OH's and DCC rubs
You are than being 1:1.0~1.2;
(2) synthesis H-Leu-Leu-OH:H-Leu-OH and Boc-Leu-OSu reacts in sodium carbonate liquor, prepares Boc-Leu-
Then Leu-OH is handled through TFA, prepare H-Leu-Leu-OH;The molar ratio of the H-Leu-OH and sodium carbonate be 1:1.0~
2.0;The molar ratio of the H-Leu-OH and Boc-Leu-OSu is 1:1.1~1.5;
(3) in organic solvent, using DCC as activator, prepared by reaction by synthesis Fmoc-Leu-OSu:Fmoc-Leu-OH and HOSu
Fmoc-Leu-OSu;The molar ratio of the Fmoc-Leu-OH and HOSu is 1:1.0~1.2;The Fmoc-Leu-OH's and DCC
Molar ratio is 1:1.0~1.2;
(4) preparation of Fmoc-Leu-Leu-OH: H-Leu-OH and Fmoc-Leu-OSu react in sodium carbonate liquor, preparation
Fmoc-Leu-Leu-OH;The molar ratio of the H-Leu-OH and sodium carbonate is 1:1.0~2.0;The H-Leu-OH and Fmoc-
The molar ratio of Leu-OSu is 1:1.1~1.5;
(5) preparation of Fmoc-Leu-Leu-OSu: Fmoc-Leu-Leu-OH and HOSu are activation with DCC in organic solvent
Agent, reaction preparation Fmoc-Leu-Leu-OSu;The molar ratio of the Fmoc-Leu-Leu-OH and HOSu is 1:1.0~1.2;Institute
The molar ratio for stating Fmoc-Leu-Leu-OH and DCC is 1:1.0~1.2;
(6) preparation of Fmoc-Leu-Leu-Leu-Leu-OH: H-Leu-Leu-OH and Fmoc-Leu-Leu-OSu is molten in sodium carbonate
It is reacted in liquid, prepares Fmoc-Leu-Leu-Leu-Leu-OH;The molar ratio of the H-Leu-Leu-OH and sodium carbonate is 1:1.0
~2.0;The molar ratio of the H-Leu-Leu-OH and Fmoc-Leu-Leu-OSu is 1:1.1~1.5;
(7) preparation of Fmoc-Leu-Leu-Leu-Leu-OSu: Fmoc-Leu-Leu-Leu-Leu-OH and HOSu are in organic solvent
In, preparation Fmoc-Leu-Leu-Leu-Leu-OSu is reacted by activator of DCC;The Fmoc-Leu-Leu-Leu-Leu-OH
Molar ratio with HOSu is 1:1.0~1.2;The molar ratio of the Fmoc-Leu-Leu-Leu-Leu-OH and DCC be 1:1.0~
1.2;
(8) preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH: H-Lys (Boc)-OH and Fmoc-Leu-Leu-
Leu-Leu-OSu reacts in sodium carbonate liquor, prepares Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-OH;The H-Lys
(Boc) molar ratio of-OH and sodium carbonate is 1:1.0~2.0;H-Lys (the Boc)-OH and Fmoc-Leu-Leu-Leu-Leu-
The molar ratio of OSu is 1:1.1~1.5.
3. a kind of solid liquid phase according to claim 2 combines the method for preparing sinapultide, which is characterized in that described organic
Solvent is tetrahydrofuran, dioxane, mixed liquor one or more kinds of in acetone.
4. a kind of solid liquid phase according to claim 1 combines the method for preparing sinapultide, which is characterized in that step (b)
In, the vector resin is Wang resin or the CTC resin that substitution degree is 0.6-1.2mmol/g.
5. a kind of solid liquid phase according to claim 1 combines the method for preparing sinapultide, which is characterized in that step (b)
In, the preparation process of the sinapultide peptide resin are as follows:
(1) polypeptide fragment I is coupled under condensation reagent with vector resin, prepares segment peptide resin Fmoc-Leu-Leu-Leu-
Leu-Lys (Boc)-Wang resin or Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-CTC resin;
(2) segment peptide resin is successively coupled three polypeptide fragments I again, is finally coupled protected amino acid Lys, and sinapultide peptide is made
Resin.
6. a kind of solid liquid phase according to claim 5 combines the method for preparing sinapultide, which is characterized in that the protection
Amino acid Lys is Fmoc-Lys (Boc)-OH or Boc-Lys (Boc)-OH;The condensation reagent be DIC/HOBt, DIC/HOAt,
One of TBTU/HOBt/DIEA, HBTU/HOBt/DIEA.
7. a kind of solid liquid phase according to claim 1 combines the method for preparing sinapultide, which is characterized in that step (c)
In, the preparation of the thick peptide of sinapultide are as follows: sinapultide peptide resin is added in lysate, at 15~30 DEG C, cracking is anti-
2~5h is answered, reaction terminates, and lysate is filled into methyl tertiary butyl ether(MTBE) or ether and settles and washs, and precipitating is collected by centrifugation, very
The thick peptide of sinapultide is obtained after sky is dry.
8. a kind of solid liquid phase according to claim 7 combines the method for preparing sinapultide, which is characterized in that the cracking
Liquid is the TFA solution that volume ratio 1-5% scavenger is added;The scavenger is methyl phenyl ethers anisole, thioanisole, dithioglycol, sulfydryl
One or more of ethyl alcohol, phenol, water.
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CN104817631A (en) * | 2015-05-26 | 2015-08-05 | 成都圣诺生物科技股份有限公司 | Method for synthesizing sinapultide |
CN104987382A (en) * | 2015-06-30 | 2015-10-21 | 济南康和医药科技有限公司 | Method for preparing thymalfasin through dipeptide fragment liquid-solid bonding |
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CN104817631A (en) * | 2015-05-26 | 2015-08-05 | 成都圣诺生物科技股份有限公司 | Method for synthesizing sinapultide |
CN104987382A (en) * | 2015-06-30 | 2015-10-21 | 济南康和医药科技有限公司 | Method for preparing thymalfasin through dipeptide fragment liquid-solid bonding |
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