CN107417786B - Preparation method of thymosin alpha 1 - Google Patents
Preparation method of thymosin alpha 1 Download PDFInfo
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- CN107417786B CN107417786B CN201710309702.1A CN201710309702A CN107417786B CN 107417786 B CN107417786 B CN 107417786B CN 201710309702 A CN201710309702 A CN 201710309702A CN 107417786 B CN107417786 B CN 107417786B
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Abstract
The invention provides a preparation method of thymosin alpha 1, which synthesizes polypeptide by 3 segments at reasonable sites, avoids the main site formed by a beta folding structure in a segmentation manner, improves the bonding degree, reduces the chance of forming by-products and greatly improves the yield of thymosin alpha 1. Solves some problems in the synthesis process of the thymosin alpha 1, and provides the solid-phase synthesis of the thymosin alpha 1 with higher yield, lower cost and contribution to realizing industrialization.
Description
Technical Field
The invention relates to the technical field of thymosin alpha 1, in particular to a synthetic method of thymosin alpha 1.
Background
The thymulin is a protein and polypeptide hormone produced by thymus gland, is a biological response regulating factor, can promote lymphocyte maturation, regulate and enhance human body immunity mechanism, and has the functions of resisting aging, resisting virus replication and resisting tumor cell differentiation clinically. The thymosin preparation has been clinically applied for more than 20 years in China, and the main component is thymus tissue extract of animals such as healthy calves and the like. But the production process and the standard of various preparations are different, and the product quality and the clinical curative effect are also greatly different.
Thymopentin (TP-5) is composed of five amino acids of arginine, lysine, aspartic acid, valine and tyrosine, and is an effective part of thymopoietin II of thymus secretion. Thymin II is a single polypeptide compound separated from thymic hormone, and is composed of 49 amino acids, and a peptide chain fragment composed of 5 amino acids has the same all physiological functions as the thymopoietin II, so the pentapeptide fragment is called thymic pentapeptide. At present, the thymopentin preparation in China is a compound artificially synthesized by a high-tech means by taking amino acid as a raw material, and has a definite structure and high purity.
The thymosin alpha 1(T alpha 1) is a high-end product in the thymosin, and has the sequence of N-acetylation-L-seryl-L-aspartyl-L-alanyl-L-valyl-L-aspartyl-L-threonyl-L-seryl-L-glutamyl-L-isoleucyl-L-threonyl-L-lysyl-L-aspartyl-L-leucyl-L-lysyl-L-glutamyl-L-lysyl-L-glutamyl-L-valyl-L-glutamyl-L-valyl-L-glutamyl-L -alanyl-L-glutamyl-L-asparagine. The medicine is a small molecular bioactive polypeptide separated and purified from thymosin component 5(TF-5), is formed by arranging 28 amino acids, has molecular weight of 3108.37, accounts for about 0.6% of TF-5, and has high immune enhancement activity. The thymosin alpha 1 and the thymopoietin II are two thymus hormone components which are most clearly understood by human beings at present, so that the thymosin alpha 1 on the market has a clear structure as same as the thymopentin, and the purity is as high as more than 99%.
In general, thymosin, thymopentin and thymosin alpha 1 are thymic hormone immunomodulators, have the functions of regulating development, differentiation and maturation of T lymphocytes, can repair damaged T lymphocytes and play an important role in a human immune system. The thymopeptide is an animal thymus extract and contains various thymohormones, and the thymopentin and the thymopeptide alpha 1 are important active components in the human thymohormones, and are artificially synthesized, so that the active ingredients are exact, the action mechanism is clear, and a skin test is not needed. The biological activity of the thymosin alpha 1 is highest compared with that of single components of the thymosin or compared with that of the thymosin as a whole, the activity is 2000 times that of the thymosin and 10-1000 times that of the thymopentin, so that a high-yield and low-cost synthesis mode is particularly important.
The method for producing thymosin alpha 1 adopted in the market at present comprises the following steps: genetic engineering methods and chemical solid-phase synthesis methods. The genetic engineering method utilizes the biological engineering technology to recombine a plurality of thymosin genes and express the recombined genes in host cells so as to obtain a plurality of thymosin fusions with high expression; the fusion is cleaved and modified by chemical techniques to form the thymosin alpha 1 monomer. The genetic engineering method has the disadvantages of low expression level, high cost and difficult realization of industrialization.
The chemical solid-phase synthesis method adopts the traditional polypeptide chemical solid-phase synthesis method and takes the Wang resin as a carrier, and synthesizes the thymosin alpha 1 by a symmetrical anhydride method and a DCC-HOBt condensation method. Although the thymosin alpha 1 product with relatively high purity and activity can be obtained, the yield of the finally prepared pure product is extremely low due to the continuous generation of byproducts by repeated condensation and a beta-sheet structure generated in synthesis, and industrial scale-up production is difficult.
In view of the above-mentioned drawbacks, the present designer has made active research and innovation to create a method for preparing thymosin alpha 1, which has industrial application value.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of the thymosin alpha 1 with the fragment solid-liquid phase, which optimizes the defects in the original chemical solid-phase synthesis and increases a fragment synthesis method and a liquid-phase synthesis method.
The preparation method of the thymosin alpha 1 is characterized by comprising the following steps:
(1) respectively preparing polypeptide tablets with the following amino acid sequences:
Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin and
Fmoc-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-OH。
(2) subjecting the polypeptide fragment obtained in step (1)
Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-OH was ligated to the amino terminus of Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin and the Fmoc protecting group at the amino terminus was removed, followed by ligation of site amino acids 15-19 using a first condensing agent to give a polypeptide fragment having the following amino acid sequence:
Fmoc-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
(3) removing the Fmoc protecting group at the amino terminal of the polypeptide fragment obtained in the step (2), and connecting the amino acids at the positions 20-24 by using a second condensing agent to obtain the polypeptide fragment with the following amino acid sequence:
Fmoc-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
(4) preparing a polypeptide fragment with the following amino acid sequence:
Fmoc-Ser(Trt)-Asp(Otbu)-Ala-Ala-OH。
(5) connecting the amino terminal of the polypeptide fragment obtained in the step (3) with the polypeptide fragment obtained in the step (4) to obtain the polypeptide fragment with the following amino acid sequence:
Fmoc-Ser(Trt)-Asp(Otbu)-Ala-Ala-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
(6) and (3) removing the Fmoc protecting group at the amino terminal of the polypeptide fragment obtained in the step (5), and acetylating the naked amino group to obtain the resin peptide with the following amino acid sequence:
Ac-Ser(Trt)-Asp(Otbu)-Ala-Ala-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
(7) and (3) splitting the resin peptide obtained in the step (6), and purifying to obtain the thymosin alpha 1 with the following amino acid sequence:
AC-SER-ASP-ALA-ALA-VAL-ASP-THR-SER-SER-GLU-ILE-THR-THR-LYS-ASP-LEU-LYS-GLU-LYS-LYS-GLU-VAL-VAL-GLU-GLU-ALA-GLU-ASN-OH。
further, in step (1), the polypeptide fragment
Fmoc-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)
-Linker2-Chlorotrityl Chloride resin preparation comprising the following steps:
(1-1) reacting the 2-Chlorotrityl Chloride resin with the activated amino acid Fmoc-Asn (Trt) -OH to obtain the resin connected with the amino acid.
And (1-2) removing the Fmoc protecting group of the resin connected with the amino acid obtained in the step (1-1).
(1-3) repeating the steps (1-1) and (1-2) by repeating the removal of Fmoc protecting groups and the sequential addition of activated amino acids for reaction, wherein the added amino acids are Fmoc-Glu (Otbu) -OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Val-OH in this order, to obtain a polypeptide fragment Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin.
Further, in the step (1-1), a catalyst is added in the reaction, and the catalyst is one or two of N, N-Diisopropylethylamine (DIEA) and N-methylmorpholine (NMM). DIEA is preferred.
Amount of DIEA to polypeptide fragments
Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin in a molar amount of about 10 times the molar amount.
In the step (1-1), a condensing agent 2- (7-azobenzotriazol) -tetramethyluronium Hexafluorophosphate (HBTU) is also added.
The amount of HBTU as the polypeptide fragment
Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin in a molar amount of about 3 times the molar amount.
The amino acid amount is polypeptide fragment
Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin in a molar amount of about 3 times the molar amount.
Further, in the step (1-1), the substitution degree of the 2-Chlorotrityl Chloride resin is 0.5-0.6 mmol/g. Preferably 0.55 mmol/g.
Further, in the step (1-2), the uncapping solution for removing the Fmoc protecting group is one or more of piperidine, DMF, diethylamine and triethylamine. Piperidine and DMF solutions are preferred. Specifically, 20% volume percent of piperidine and 80% volume percent of DMF solution.
Further, in the step (2), the first condensing agent is one or more of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), 1H-benzotriazole-1-oxatripyrrolidinium hexafluorophosphate and (7-azabenzotriazole-1-oxo) tripyrrolophosphonium hexafluorophosphate. HATU is preferred.
The amount of HATU is the polypeptide fragment obtained in step (1)
Fmoc-Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin in about 3-fold molar amount.
Further, in the step (2), a coupling agent of benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate (PyBop) is added.
PyBop amount of the polypeptide fragment obtained in step (1)
About 3-fold molar amount of Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin.
Further, in the step (3), the second condensing agent is one or two of 1-Hydroxybenzotriazole (HOBT) and 1-hydroxy-7-azabenzotriazole (HOAT). HOBT is preferred.
Further, in the step (3) and the step (2), a catalyst is added when the amino acids at the positions 15-19 are connected by using a second condensing agent, and the catalyst is N, N-diisopropylethylamine.
The amount of HOBT is about 3 times the molar amount of the polypeptide fragment obtained in step (2).
Further, in the step (3), a catalyst is added when the amino acids at the positions 20-24 are connected by a second condensing agent, and the catalyst is N, N' -Diisopropylcarbodiimide (DIC).
DIC is present in an amount of about 10-fold molar amount relative to the polypeptide fragment obtained in step (2).
Further, in the step (6), the acetylation solution of acetylated amino groups is one or more of acetic anhydride, pyridine, DMF, acetylation solvent and acetic acid. Acetic anhydride, pyridine and DMF are preferred.
The volume ratio of acetic anhydride, pyridine and DMF is 70-80: 60-70: 900-.
Further, in the step (7), the cleavage solution of the resinoid obtained in the cleavage step (6) is one or more of trifluoroacetic acid, 1, 2-Ethanedithiol (EDT), Triisopropylsilane (TIS), thioanisole, trifluoroethanol, anisole, phenol and m-cresol. Trifluoroacetic acid, EDT, TIS, thioanisole are preferred.
The volume ratio of trifluoroacetic acid to EDT to TIS to thioanisole is 92-98:1-3:1-3:0.7-1.3, preferably 95:2:2: 1.
Wherein the amount of the lysis solution is 10 ml/g.
By the scheme, the invention at least has the following advantages:
1. the invention provides a synthetic method of thymosin alpha 1, which provides a fragment solid-liquid phase synthetic method on the basis of chemical solid-phase synthesis, optimizes the defects in the original chemical solid-phase synthesis, increases the fragment synthetic method and the liquid phase synthetic method, solves some problems in the thymosin alpha 1 synthetic process, and provides a solid-phase synthetic process of thymosin alpha 1, which has higher yield and lower cost and is beneficial to realizing industrialization;
2. the fragment solid-liquid phase synthesis method adopted by the invention synthesizes the polypeptide by 3 fragments at reasonable sites, thereby shortening the synthesis time;
3. meanwhile, the main sites formed by beta-folded structures are reasonably segmented, the bonding degree is improved, the chance of forming byproducts is reduced, and the yield of thymosin alpha 1 is greatly improved.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a mass spectrum analysis chart of a thymosin alpha 1 pure product in example 1 of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
1. Preparation of Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin:
100g of 2-Chlorotrityl Chloride resin with a degree of substitution of 0.55mmol/g was weighed into a reactor, 500ml of DCM (dichloromethane) was added, the liquid and the resin were mixed by flushing from bottom to top with nitrogen and the blade stirrer was turned on to stir for 15 minutes to fully swell the resin, during which time a small amount of DCM was added to prevent the solution from evaporating to dryness. Filtering off solvent with sand core, adding 3 times molar excess of Fmoc-Asn (Trt) -OH amino acid, adding DCM for dissolving, adding 10 times molar excess of DIEA, and reacting for 60min. Blocking with methanol. Then, 500ml of 20% piperidine/DMF solution (decapping solution) was added to the reactor, and the mixture was stirred under nitrogen for 5min, the solution was removed, washed 2 times with 500ml DMF, and then 500ml decapping solution was added, and after stirring for 10 min, the solution was removed, washed 2 times with 500ml DMF, 2 times with 500ml methanol, and 2 times with 500ml DMF to remove the removed Fmoc protecting group. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
Adding 3 times molar excess of Fmoc-Glu (Otbu) -OH amino acid and HBTU with 3 times molar excess, dissolving with DMF as little as possible, adding into reaction column, adding 500ml DMF, immediately adding DIEA with tenfold excess, and reacting for 30min.
The solution is pumped out through a sand core, 500ml DMF is used for washing for 3 times, dozens of particles of resin are taken and washed for three times by ethanol, a detection reagent is added for detection, and the mixture is heated for 5min at the temperature of 105-110 ℃, and the colorless negative reaction is obtained.
Sequentially adding subsequent amino acids by repeatedly removing Fmoc protecting groups and putting the amino acids into reaction until the sequence is completed
Fmoc-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
2. Preparation of Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-OH:
the synthesis of the polypeptide fragment resin peptide is completed according to the method in the step 1, and the sequence is
Fmoc-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Linker2-Chlorotrityl Chloride resin。
The resinoid was washed 3 times with 500ml methanol and dried completely by suction filtration. The resinated peptide was placed in a 1000ml round-bottom flask and 750ml of polypeptide-protected lysate (trifluoroethanol 30% + DCM 70%) was added. Fully oscillate for 3 h.
And (3) leaching the lysate containing the polypeptide through a sand core funnel, and putting the residual resin into a chemical recycling bin for abandoning. The lysate containing the polypeptide was evaporated to a white dry powder using a rotary evaporator. And (5) storing at low temperature in a sealed manner for later use.
3. Preparation of Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin:
and (2) adding 750ml of 20% piperidine/DMF solution (decapping solution) into the polypeptide fragment obtained in the step 1, stirring and reacting for 5min under the protection of nitrogen, pumping out the liquid, washing for 2 times by 750ml DMF, adding 7500ml of decapping solution, stirring and reacting for 10 min, pumping out, washing for 2 times by 750ml DMF, washing for 2 times by 750ml methanol, and washing for 2 times by 750ml DMF to remove the removed Fmoc protecting group. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
And (2) taking out the polypeptide fragment obtained in the step (2), adding PyBop with 3 times molar excess, dissolving with DMF as little as possible, adding into a reaction column, adding 750ml of DMF, immediately adding DIEA with ten times excess, reacting for 60min, removing the solution through a sand core, washing with 750ml of DMF for 3 times, taking dozens of resin, washing with ethanol for three times, adding a detection reagent, detecting, heating at 105-110 ℃ for 5min, and obtaining a negative reaction if colorless. To obtain a sequence of
Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin.
4. Preparation of Fmoc-Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin:
and (3) adding 750ml of 20% piperidine/DMF solution (decapping solution) into the resin peptide obtained in the step (3), stirring and reacting for 5min under the protection of nitrogen, pumping out the liquid, washing for 2 times by 750ml DMF, adding 750ml decapping solution, stirring and reacting for 10 min, pumping out, washing for 2 times by 750ml DMF, washing for 2 times by 750ml methanol, and washing for 2 times by 750ml DMF to remove the removed Fmoc protecting group. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
Adding Fmoc-Lys (Boc) -OH amino acid with 3-fold molar excess, HATU with 3-fold molar excess, dissolving with DMF as little as possible, adding into a reaction column, adding 750ml of DMF, immediately adding DIEA with ten-fold excess, and reacting for 30min.
The solution is pumped out through a sand core, 750ml DMF is used for washing for 3 times, dozens of particles of resin are taken and washed for three times by ethanol, a detection reagent is added for detection, and the mixture is heated for 5min at the temperature of 105-110 ℃, and the colorless negative reaction is obtained.
Sequentially adding subsequent amino acids by repeatedly removing Fmoc protecting groups and putting the amino acids into reaction until the sequence is completed
Fmoc-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
The reaction of amino acids at positions 15-19 is particularly important, and a more efficient condensation reagent is selected for the reaction. Here the condensing reagent changed the previous HBTU to HATU.
5. Preparation of the resin peptide Fmoc-Val-Asp (Otbu) -Thr (Tbu) -Ser (Trt) -Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Lin 2-Chlorotrityl Chloride resin:
and (3) adding 1000ml of 20% piperidine/DMF solution (decapping solution) into the polypeptide fragment obtained in the step 3, stirring and reacting for 5min under the protection of nitrogen, removing the liquid, washing for 2 times by 1000ml of DMF, adding 1000ml of decapping solution, stirring and reacting for 10 min, removing the solution, washing for 2 times by 1000ml of DMF, washing for 2 times by 1000ml of methanol, and washing for 2 times by 1000ml of DMF to remove the removed Fmoc protecting group. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
Adding Fmoc-Ser (Trt) -OH amino acid in 3-fold molar excess, HOBT in 3-fold molar excess, dissolving with DMF as little as possible, adding into a reaction column, adding 1000ml DMF, and immediately adding DIC in ten-fold excess. The reaction was carried out for 60min.
The solution is pumped out through a sand core, 1000ml DMF is used for washing for 3 times, dozens of particles of resin are taken and washed for three times by ethanol, a detection reagent is added for detection, and the mixture is heated for 5min at the temperature of 105-110 ℃, and the colorless negative reaction is obtained.
Sequentially adding subsequent amino acids by repeatedly removing Fmoc protecting groups and putting the amino acids into reaction until the sequence is completed
Fmoc-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin。
The sites 20-24 are prone to residual peptides and side reactions, and we use more stable condensation reagents for the reaction. Here, the condensation reagent was HOBT and the catalyst was DIC.
6. Preparation of Fmoc-Ser (Trt) -Asp (Otbu) -Ala-Ala-OH:
resin peptide synthesis of the polypeptide fragment was performed according to the procedure of step 1, with the sequence Fmoc-Ser (Trt) -Asp (Otbu) -Ala-Ala-Linker2-Chlorotrityl Chloride resin.
The resinoid was washed 3 times with 500ml methanol and dried completely by suction filtration. The resinated peptide was placed in a 1000ml round-bottom flask and 750ml of polypeptide-protected lysate (trifluoroethanol 30% + DCM 70%) was added. Fully oscillate for 3 h.
And (3) leaching the lysate containing the polypeptide through a sand core funnel, and putting the residual resin into a chemical recycling bin for abandoning. The lysate containing the polypeptide was evaporated to a white dry powder using a rotary evaporator. And (5) storing at low temperature in a sealed manner for later use.
7. Ac-Ser (Trt) -Asp (Otbu) -Ala-Val-Asp (Otbu) -Thr (Tbu) -Ser (Trt) -Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin preparation:
and (3) adding 1000ml of 20% piperidine/DMF solution (decapping solution) into the resin peptide obtained in the step (5), stirring and reacting for 5min under the protection of nitrogen, pumping out the liquid, washing for 2 times by 1000ml of DMF, adding 1000ml of decapping solution, stirring and reacting for 10 min, pumping out, washing for 2 times by 1000ml of DMF, washing for 2 times by 1000ml of methanol, and washing for 2 times by 1000ml of DMF to remove the removed Fmoc protecting group. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
And (3) taking out the polypeptide fragment obtained in the step (6), adding PyBop with 3 times molar excess, dissolving with DMF as little as possible, adding into a reaction column, adding 1000ml of DMF, immediately adding DIEA with ten times excess, reacting for 60min, removing the solution through a sand core, washing with 500ml of DMF for 3 times, taking over more than ten grains of resin, washing with ethanol for three times, adding a detection reagent, detecting, heating at 105-110 ℃ for 5min, and taking out colorless negative reaction. To obtain a sequence of
Fmoc-Ser (Trt) -Asp (Otbu) -Ala-Val-Asp (Otbu) -Thr (Tbu) -Ser (Trt) -Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (OtbOtbu) -Asn-Lys (Asn Trt) -Linker2-Chlorotrityl Chloride resin, which is added to a 20% piperidine/solution (decapping solution) 1000ml of DMF and stirred for reaction under nitrogen protection for 5min, the liquid is aspirated, the DMF is washed 2 times with 1000ml of DMF, 1000ml of decapping solution is added, the reaction is aspirated after stirring for 10 min, the DMF is aspirated for 2 ml of DMF, the Fmoc protecting group removed was washed off by washing 2 times with 1000ml methanol and 2 times with 1000ml DMF. Taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
Preparing acetylation solution of 76ml of acetic anhydride, 65ml of pyridine and 1000ml of DMF1000ml, uniformly mixing, adding into a reaction column, reacting for 120min under the protection of nitrogen, performing suction filtration, washing for 2 times by 1000ml of DMF, washing for 2 times by 1000ml of DCM, washing for 6 times by 1000ml of methanol, and performing suction filtration to completely dry the resin peptide. Obtaining the resin peptide
Ac-Ser(Trt)-Asp(Otbu)-Ala-Ala-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin 342.62g
8. Preparation of thymosin alpha 1 crude product Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH:
preparing a polypeptide lysate: trifluoroacetic acid, EDT, TIS and thioanisole are prepared into 3500ml of lysate with the ratio of 95:2:2: 1.
342.62g of resin peptide is placed in a round-bottom flask, 3500ml of prepared lysate is added under the ice bath state, the ice bath is removed, and the mixture is stirred and cracked for 3 hours under the normal temperature state. And then filtering resin through a sand core, adding 10 times of volume of glacial ethyl ether, fully and uniformly stirring, settling for 2 hours, placing in a centrifuge with 3600 revolutions per minute, and washing for 6 times by using ethyl ether.
The precipitate was removed, dried under reduced pressure and rolled continuously until 152.47g of crude peptide of thymosin alpha 1 was obtained as a white dry powder.
9. Dissolving and purifying preparation of thymosin alpha 1:
1500ml of a mixed solution of pure water and acetonitrile (ratio 8: 2) is added into the crude product of the thymosin alpha 1, and the mixture is stirred for 30min by ultrasonic oscillation until the thymosin alpha is completely dissolved. The solution of thymosin alpha 1 was filtered through a 0.45um pore size microfiltration membrane.
Using 0.1% trifluoroacetic acid (TFA) solution as a mobile phase A, and acetonitrile as a mobile phase B; preparation of liquid chromatography system using 10 μm
Reversed phase C18 (100X 650mm) packing, ultraviolet detector set at 220nm, flow rate adjusted at 80ml/min, 5% acetonitrile, balance for 15 minutes, injection. Gradient elution was used: 0-2min, 5% -5%; 2-42min, 17% -23%; 42-48min, 50% -50%. The peak emergence time is about 26 min. Three sections of the liquid before the peak, the peak top and the liquid after the peak are respectively collected, and the purity of the collected liquid at the peak top is more than 98 percent. Concentrating the collected liquid before and after the peak to proper amount, and purifying and collecting by the same method to obtain purified liquid with purity of more than 98%. All the collected liquid with the content of more than 98 percent is combined, and the acetonitrile in the collected liquid is removed by rotary concentration.
Pure water for injection is taken as a mobile phase A, and acetonitrile is taken as a mobile phase B; preparing a preparative liquid chromatography system using a 10 μm reverse reaction
A column of phase C18 (100X 650mm) was packed, the UV detector set at 220nm, and the flow rate was adjusted to 60ml/min, first with 80% acetonitrile and then with 2% acetonitrile and then with 15 minutes. And taking the purified target peptide collecting liquid and injecting a sample.
Gradient elution was used: 0-15min, 5% -5%; 15-65min, 5% -40%. Beginning to collect when the main peak appears and ending when no peak appears. The collected solutions were combined, and acetonitrile and most of water were removed by rotary concentration to obtain 1205ml of a purified solution of thymosin alpha 1.
10. Lyophilized thymosin alpha 1:
1205ml of the purified and purified thymosin alpha 1 solution was dispensed into a plurality of clean eggplant-shaped bottles, and the purified and purified thymosin alpha 1 solution was frozen on the walls of the eggplant-shaped bottles by liquid nitrogen. Placing into a freeze dryer. 78.92g of pure thymosin alpha 1 peptide with the purity of 98.97% is obtained.
11. Detection of thymosin alpha 1:
dissolving a small sample of thymosin alpha 1 pure polypeptide by using pure water, analyzing purity and confirming mass spectrum, wherein ESI negative ion spectrum shows that the product mass spectrum is correct (a negative ion trivalent target peak value 1035.1 and a negative ion divalent target peak value 1553.3) as shown in figure 1.
To summarize: the total yield of the thymosin alpha 1 product synthesized by the method is 51.76 percent and is far beyond the technical level of the prior art.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A preparation method of thymosin alpha 1 is characterized by comprising the following steps:
(1) polypeptide fragments with the following amino acid sequences were prepared:
Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker 2-chlorotritylChlororin and Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-OH;
(2) subjecting the polypeptide fragment obtained in step (1)
Fmoc-Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-OH was ligated to the amino terminus of Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin and the Fmoc protecting group at the amino terminus was removed, followed by ligation of site amino acids 15-19 using a first condensing agent to give a polypeptide fragment having the following amino acid sequence:
Fmoc-Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker 2-ChlorotritylChlorhride resin; the first condensing agent is HATU;
(3) removing the Fmoc protecting group at the amino terminal of the polypeptide fragment obtained in the step (2), and connecting the amino acids at the positions 20-24 by using a second condensing agent to obtain the polypeptide fragment with the following amino acid sequence:
Fmoc-Val-Asp (Otbu) -Thr (Tbu) -Ser (Trt) -Glu (Otbu) -Ile-Thr (Tbu) -Lys (Boc) -Asp (Otbu) -Leu-Lys (Boc) -Glu-Lys (Boc) -Glu (Otbu) -Val-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin; the second condensing agent is HOBT;
(4) preparing a polypeptide fragment with the following amino acid sequence:
Fmoc-Ser(Trt)-Asp(Otbu)-Ala-Ala-OH；
(5) connecting the amino terminal of the polypeptide fragment obtained in the step (3) with the polypeptide fragment obtained in the step (4) to obtain ammonia
A polypeptide fragment having the amino acid sequence:
Fmoc-Ser(Trt)-Asp(Otbu)-Ala-Ala-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin；
(6) removing the Fmoc protecting group at the amino terminal of the polypeptide fragment obtained in the step (5), acetylating the naked amino,
obtaining the resin peptide with the amino acid sequence as follows:
Ac-Ser(Trt)-Asp(Otbu)-Ala-Ala-Val-Asp(Otbu)-Thr(Tbu)-Ser(Trt)-Ser(Trt)-Glu(Otbu)-Ile-Thr(Tbu)-Thr(Tbu)-Lys(Boc)-Asp(Otbu)-Leu-Lys(Boc)-Glu-Lys(Boc)-Lys(Boc)-Glu(Otbu)-Val-Val-Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride resin；
(7) and (3) splitting the resin peptide obtained in the step (6), and purifying to obtain the thymosin alpha 1 with the following amino acid sequence:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-
Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH；
in the step (1), the polypeptide fragment Fmoc-Val-
Glu(Otbu)-Glu(Otbu)-Ala-Glu(Otbu)-Asn(Trt)-Linker2-Chlorotrityl Chloride
The preparation of the resin comprises the following steps:
(1-1) reacting the 2-Chlorotrityl Chloride resin with activated amino acid Fmoc-Asn (Trt) -OH to obtain resin connected with the amino acid;
(1-2) removing the Fmoc protecting group of the resin connected with the amino acid obtained in the step (1-1);
(1-3) repeating the steps (1-1) and (1-2), and repeatedly removing the Fmoc protecting group and sequentially adding activated amino acids for reaction, wherein the added amino acids sequentially are as follows:
Fmoc-Glu (Otbu) -OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Val-OH to obtain polypeptide fragments Fmoc-Val-Glu (Otbu) -Ala-Glu (Otbu) -Asn (Trt) -Linker2-Chlorotrityl Chloride resin.
2. The method of claim 1, wherein: in the step (1-1), the activating agent for activating the amino acid is one or more of N, N-diisopropylethylamine, N-diisopropylcarbodiimide and pyridine.
3. The method of claim 1, wherein: in the step (1-1), the 2-Chlorotrityl
The substitution degree of the Chloride resin is 0.5-0.6 mmol/g.
4. The method of claim 1, wherein: in the step (1-2), the uncapping solution for removing the Fmoc protecting group is one or more of piperidine + DMF, diethylamine and triethylamine.
5. The method of claim 1, wherein: in the step (3), a catalyst is also added when the second condensing agent is used for connecting the amino acid at the position of 20-24, and the catalyst is N, N' -diisopropylcarbodiimide.
6. The method of claim 1, wherein: in the step (6), the acetylation solution of acetylated amino is one or more of acetic anhydride, pyridine, DMF and acetic acid.
7. The method of claim 1, wherein: in the step (7), the cleavage solution of the resinoid obtained in the cleavage step (6) is one or more of trifluoroacetic acid, 1, 2-ethanedithiol, triisopropylsilane, thioanisole, trifluoroethanol, anisole, phenol and m-cresol.
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