CN103497245B - Method for synthesizing thymalfasin - Google Patents

Method for synthesizing thymalfasin Download PDF

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CN103497245B
CN103497245B CN201310256576.XA CN201310256576A CN103497245B CN 103497245 B CN103497245 B CN 103497245B CN 201310256576 A CN201310256576 A CN 201310256576A CN 103497245 B CN103497245 B CN 103497245B
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fmoc
otbu
tbu
resin
asp
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CN103497245A (en
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肖庆
刘建
马亚平
袁建成
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention relates to the field of medicine synthesis, and discloses a method for synthesizing thymalfasin. According to the method of the invention, based on the amino acid sequence from the C terminal to the N terminal of the thymalfasin peptide chain, fragments of 1-8, 9-19 and 20-28 are synthesized, and the three polypeptide fragments are coupled to obtain thymalfasin. According to the invention, a plurality of fragments are synthesized simultaneously; the synthetic period is reduced by 2/3; intermediates are easy to purify; the cost is low; the purity of the finished products is high; by-products are few; the product yield is high; and the method facilitates large-scale production of thymalfasin.

Description

A kind of method of synthesizing Thymosin-Alpha1
Technical field
The present invention relates to medical synthesis field, be specifically related to a kind of method of synthesizing Thymosin-Alpha1.
Background technology
Thymosin-Alpha1 (Thymosin α 1, also claims Thymosin alpha 1) is the main bioactive ingredients of thymosin, is immune regulator important in body, and be the polypeptide of acetylizad 28 amino acid composition of N end, its peptide sequence is as follows:
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-COOH
Though the mechanism of action of this product Treatment chronic Hepatitis B and enhancing immune system response is illustrated not yet completely, but multinomial in vitro tests display, this product promotes the lymphocytic maturation of T by stimulating peripheral blood lymphocyte mitogen, interferon alpha, interferon-gamma and the lymphokine such as interleukin-22, interleukin-13 level that after increasing antigen or mitogen activation, T cell is secreted, increase T cell surface lymphokine receptor level simultaneously.This product, also by the activation to cd4 cell, strengthens allosome and autologous mankind's mixed lymphocyte reacion.This product may increase the gathering of front NK cell, and Interferon, rabbit can make its cytotoxicity strengthen.In vivo test shows, and this product can improve the expression level of mouse lymphocyte IL-2R after concanavalin A activates, and improves the secretion level of interleukin-22 simultaneously.
Current Thymosin-Alpha1 preparation technology has gene engineering research (as CN1431311) and solid-phase synthesis (as CN102199205), wherein gene engineering research because of technique is loaded down with trivial details, the three wastes are many unfavorable factor general not as solid-phase synthesis application.Patent CN102199205 discloses the synthetic method of a peptide species Thymosin-Alpha1, and it adopts the synthesis mode of coupling one by one.But, with coupling method is in the process preparing Thymosin-Alpha1 one by one, synthesis cycle is long, impurity more (easily producing the impurity such as default peptide in the synthesis of long peptide), impurity is similar with desired polypeptides causes follow-up purifying to be difficult to carry out for major part, and yield and purity cannot reach higher level.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of synthesizing Thymosin-Alpha1, make the method for the invention can improve its purity and synthesis weight yield.
For achieving the above object, the invention provides following technical scheme:
Synthesize a method for Thymosin-Alpha1, comprise the following steps:
The aminoacid sequence coupling on Lys side chain, on Glu side chain shown in SEQ ID NO:1 of step 1, solid phase synthesis has protecting group and holds coupling to have the polypeptide resin 1 of resin at C;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:2, Ser side chain, on Glu side chain, on Thr side chain, on Lys side chain, on Asp side chain has the polypeptide fragment 1 of protecting group;
Solid phase synthesis aminoacid sequence N shown in SEQ ID NO:3 holds the polypeptide fragment 2 that coupling has ethanoyl, coupling has protecting group on Ser side chain, on Thr side chain, on Asp side chain;
Step 2, the C section of polypeptide fragment 1 and the N of polypeptide resin 1 held and carries out linked reaction and obtain polypeptide resin 2;
Step 3, the N sloughing polypeptide resin 2 hold protecting group and carry out linked reaction with the C section of polypeptide fragment 2 and obtains polypeptide resin 3, and the resin and all protecting groups that add lysate removal polypeptide resin 3 obtain Thymosin-Alpha1 crude product;
Thymosin-Alpha1 sterling is obtained after step 4, Thymosin-Alpha1 crude product micro-filtration, RP-HPLC purifying.
Wherein, as preferably, described in step 2 and step 3, linked reaction carries out coupling with HOBt/DIC double reagent coupling system, PyBOP/HOBt/DIPEA tri-reagent coupling system or HBTU/HOBt/DIPEA tri-reagent coupling system.Further preferably, linked reaction described in step 2 carries out coupling with HBTU/HOBt/DIPEA tri-reagent coupling system, and linked reaction described in step 3 carries out coupling with PyBOP/HOBt/DIPEA tri-reagent coupling system;
Mol ratio between each coupling reagent of the coupling system that step 2 and step 3 adopt is preferably:
PyBOP:HOBt:DIPEA is 1:1:2, HBTU:HOBt:DIPEA be 1:1:2, HOBt:DIC is 1:1.
As preferably, lysate described in step 3 is volume ratio TFA: phenol: thioanisole: dithioglycol: water is the mixed pyrolysis liquid of 80-85:1-5:1-5:1-5:1-5.
As preferably, described in step 2 and step 3, linked reaction is with one or more in DCM, NMP, DMF, DMSO for solvent, and wherein, as further preferred version, step 2 and step 3 are the mixed solvent that volume ratio DMF:DMSO is 1:1.
As preferably, described in step 1, solid-phase synthetic peptide resin 1 is:
Steps A 1, Fmoc-β-Asp(OtBu)-OH carries out linked reaction and obtains Fmoc-β-Asp(OtBu under coupling system effect with aminoresin)-aminoresin;
Steps A 2, remove Fmoc protecting group and obtain H-β-Asp(OtBu)-aminoresin, Fmoc-Glu(OtBu)-OH under coupling system effect with H-β-Asp(OtBu)-aminoresin carries out linked reaction and obtains Fmoc-Glu(OtBu)-β-Asp(OtBu)-aminoresin;
Steps A 3, according to the order that aminoacid sequence C shown in SEQ ID NO:1 holds N to hold, successively one by one by Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH carries out amino acid according to steps A 2 coupling mode and extends coupling, finally removing N holds Fmoc protecting group to obtain H-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-β-Asp(OtBu)-aminoresin, i.e. polypeptide resin 1,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, Boc are amino acid side chain protecting group.
In the preferred version of solid-phase synthetic peptide resin 1, further preferably, described aminoresin is the Rink amide resin of 0.2-1.0mmol/g, Rink amide AM resin or Rink amide mbha resin;
In the preferred version of solid-phase synthetic peptide resin 1, further preferably, described coupling system is HOBt/DIC double reagent coupling system, PyBOP/HOBt double reagent coupling system, HATU/HOAT double reagent coupling system or TBTU/HOBt double reagent coupling system, and the mol ratio between each coupling reagent of the coupling system adopted is preferably:
PyBOP:HOBt is 1:1, TBTU:HOBt be 1:1, HOBt:DIC be 1:1, HATU:HOAT is 1:1.
In the preferred version of solid-phase synthetic peptide resin 1, further preferably, described linked reaction is with one or more in DCM, NMP, DMF, DMSO for solvent, and being preferably volume ratio DCM:DMF is the mixed solvent of 1:1.
As preferably, described in step 1, solid-phase synthetic peptide fragment 1 is:
Step B1, Fmoc-Lys(Boc)-OH carries out linked reaction with 2-CTC resin and obtains Fmoc-Lys(Boc under DIPEA effect)-CTC resin;
Step B2, remove Fmoc protecting group and obtain H-Lys(Boc)-CTC resin, Fmoc-Glu(OtBu)-OH under coupling system effect with H-Lys(Boc)-CTC resin carries out linked reaction and obtains Fmoc-Glu(OtBu)-Lys(Boc)-CTC resin;
Step B3, according to the order that aminoacid sequence C shown in SEQ ID NO:2 holds N to hold, successively one by one by Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid according to step B2 coupling mode and extend coupling, obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-CTC resin, add lysate and remove 2-CTC resin, obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-OH, be polypeptide fragment 1,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, tBu, Boc are amino acid side chain protecting group.
In the preferred version of solid-phase synthetic peptide fragment 1, further preferably, described coupling system is HOBt/DIC double reagent coupling system, PyBOP/HOBt double reagent coupling system, HATU/HOAT double reagent coupling system or TBTU/HOBt double reagent coupling system, and the mol ratio between each coupling reagent of the coupling system adopted is preferably:
PyBOP:HOBt is 1:1, TBTU:HOBt be 1:1, HOBt:DIC be 1:1, HATU:HOAT is 1:1.
In the preferred version of solid-phase synthetic peptide fragment 1, further preferably, described linked reaction is with one or more in DCM, NMP, DMF, DMSO for solvent, and being preferably volume ratio DCM:DMF is the mixed solvent of 1:1.
In the preferred version of solid-phase synthetic peptide fragment 1, further preferably, the mixed pyrolysis liquid of described lysate to be volume ratio TFE:DCM be 1:4.
As preferably, described in step 1, solid-phase synthetic peptide fragment 2 is:
Step C1, Fmoc-Ser(tBu)-OH carries out linked reaction with 2-CTC resin and obtains Fmoc-Ser(tBu under DIPEA effect)-CTC resin;
Step C2, remove Fmoc protecting group and obtain H-Ser(tBu)-CTC resin, Fmoc-Thr(tBu)-OH under coupling system effect with H-Ser(tBu)-CTC resin carries out linked reaction and obtains Fmoc-Thr(tBu)-Ser(tBu)-CTC resin;
Step C3, according to the order that aminoacid sequence C shown in SEQ ID NO:3 holds N to hold, successively one by one by Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid according to step C2 coupling mode and extend coupling, obtain Fmoc-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, remove after N holds Fmoc protecting group and obtain H-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, then carry out acetylization reaction at N end and obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, add lysate and remove 2-CTC resin, obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-OH, be polypeptide fragment 2,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, tBu are amino acid side chain protecting group.
In the preferred version of solid-phase synthetic peptide fragment 2, further preferably, described coupling system is HOBt/DIC double reagent coupling system, PyBOP/HOBt double reagent coupling system, HATU/HOAT double reagent coupling system or TBTU/HOBt double reagent coupling system, and the mol ratio between each coupling reagent of the coupling system adopted is preferably:
PyBOP:HOBt is 1:1, TBTU:HOBt be 1:1, HOBt:DIC be 1:1, HATU:HOAT is 1:1.
In the preferred version of solid-phase synthetic peptide fragment 2, further preferably, described linked reaction is with one or more in DCM, NMP, DMF, DMSO for solvent, and being preferably volume ratio DCM:DMF is the mixed solvent of 1:1.
In the preferred version of solid-phase synthetic peptide fragment 2, further preferably, the mixed pyrolysis liquid of described lysate to be volume ratio TFE:DCM be 1:4.
In the preferred version of solid-phase synthetic peptide fragment 2, further preferably, described acetylization reaction carries out acetylization reaction with N Amino End Group in the basic conditions by diacetyl oxide.
In technical solution of the present invention, described 2-CTC resin substitution degree is preferably 0.2-1.0mmol/g.In the preferred synthesis step of step 1 of the present invention, except the protecting group that the present invention limits, other suitable protecting groups also can be adopted to be protected amino acid N end and side chain.
In the preferred synthesis step of step 1, the mol ratio extending the coupling agent in the single protected amino acid of coupling and coupling system is preferably 1:1-1.2.
As preferably, described in step 2, the mol ratio of polypeptide fragment 1 and polypeptide resin 1 is 2-5:1, and described in step 3, the mol ratio of polypeptide fragment 2 and polypeptide resin 2 is 2-5:1.
In the method for the invention, first according to the synthesis of Thymosin-Alpha1 peptide sequence 1-8,9-19 and 20-28 fragment, and then these 3 polypeptide fragment couplings are obtained Thymosin-Alpha1, the amino-acid sequence numbering of holding C to hold with Thymosin-Alpha1 main chain N, as shown in the formula:
Ac-Ser 1-Asp 2-Ala 3-Ala 4-Val 5-Asp 6-Thr 7-Ser 8-Ser 9-Glu 10-Ile 11-Thr 12-Thr 13-Lys 14-Asp 15-Leu 16-Lys 17-Glu 18-Lys 19-Lys 20-Glu 21-Val 22-Val 23-Glu 24-Glu 25-Ala 26-Glu 27-Asn 28-OH
Shown in SEQ ID NO:1, aminoacid sequence is the peptide sequence of numbering 20-28 in above formula.The polypeptide resin 1 of the present invention's solid phase synthesis is in step 1 on aminoacid sequence basis shown in SEQ ID NO:1, and on its Lys side chain, on Glu side chain, coupling has protecting group and holds coupling to have resin at C; Shown in SEQ ID NO:2, aminoacid sequence is the peptide sequence of numbering 9-19 in above formula, polypeptide fragment 1 is on aminoacid sequence basis shown in SEQ IDNO:2, and on its N end, Ser side chain, on Glu side chain, on Thr side chain, on Lys side chain, on Asp side chain, coupling has protecting group; Shown in SEQ ID NO:3, aminoacid sequence is the peptide sequence of numbering 1-8 in above formula, polypeptide fragment 2 is on aminoacid sequence basis shown in SEQ ID NO:3, its N hold coupling have ethanoyl, on Ser side chain, on Thr side chain, on Asp side chain coupling have protecting group; Can be synthesized by protected amino acid synthesis material in the art above.
Protecting group of the present invention is the blocking group in order to the group of the interference synthesis such as amino, carboxyl, sulfydryl on protected amino acid main chain and side chain in Amino acid synthesis field; prevent amino, carboxyl etc. from reacting preparing in target product process, generate impurity.In the art, the group of protection, side-chain structure and how coupling protecting group is needed to be known to the skilled person for amino acid side chain.In the present invention, antithesis is associated with the amino acid representation of protecting group is also that representation is commonly used in this area; be well known to those skilled in the art; as Fmoc-Asp(OtBu)-OH; Fmoc is amino acid N end protecting group; OtBu in bracket is Asp Side chain protective group, and other protected amino acid synthesis materials of the present invention all can refer to this unless specifically indicated and explain.
Special situation; Fmoc-β-Asp(OtBu for the present invention adopts in synthesis)-OH protected amino acid; it has two carboxyls; i.e. α-the COOH of main chain and the β-COOH of side chain; Fmoc-β-Asp(OtBu) that OtBu protection is main chain α-COOH in-OH; and the β-COOH of side chain is used for becoming amido linkage with aminoresin coupling; during in the end cracking removing aminoresin; lysate is not excision amido linkage; but amino is cut from aminoresin; so Asp is just made to define Asn, consistent with Thymosin-Alpha1 the 28th amino acid.
Extension coupling of the present invention refers to after first amino acid and resin coupling, and remaining amino acid carries out coupling with the amino acid generation condensation reaction (condensation reaction of backbone amino and carboxyl) of previous coupling one by one according to the order of respective sequence.In extension coupling; because each amino acid N end has protecting group, therefore need first to remove N and hold the coupling again of Fmoc protecting group, this is common practise for a person skilled in the art; the present invention preferably with the DBLK(i.e. DMF solution of 20% piperidines, volume ratio) remove N hold protecting group.Owing to constantly having amino acid and resin coupling; synthesized polypeptide fragment resin is constantly change; as preferably, eachly treat that the protected amino acid synthetic materials of coupling is 2-3:1 with the mol ratio of the polypeptide fragment resin synthesized before, this preferred proportion is applicable in all schemes of the present invention.
As preferably, described RP-HPLC purifying is specially:
Be after the acetonitrile water ultrasonic dissolution of 10% by Thymosin-Alpha1 crude product percent by volume, adopt RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phase purifying, collects object peak cut and obtains Thymosin-Alpha1 sterling.
In clinical application, the less stable of Thymosin-Alpha1 own, needs to exist with acetate form, therefore the present invention also comprises Thymosin-Alpha1 sterling RP-HPLC system is turned salt step, is specially:
Thymosin-Alpha1 sterling solution is adopted RP-HPLC system, and chromatographic column is the anti-phase C18 post of 50 × 250mm, and 0.1% acetum/acetonitrile mobile phase turns salt, collects object peak cut, and rotary evaporation concentrates, and freeze-drying obtains Thymosin-Alpha1 acetate.
The Thymosin-Alpha1 synthesized by the method for the invention detects through RP-HPLC, and crude product purity is about 78.6%, and synthesis weight yield is 100%, and after purifying, sterling purity is greater than 99%, and total recovery is about 42%.And Thymosin-Alpha1 crude product purity is up to 69.6% in patent CN102199205, synthesis weight yield is up to 63.7%, is all starkly lower than the Thymosin-Alpha1 of the present invention's synthesis.In addition, the present invention only has suitable segment condense method can reach purity of the present invention and yield to verify further, carry out solid phase synthesis according to being different from segments of the present invention respectively, result shows, and the purity of the Thymosin-Alpha1 of its synthesis and total recovery are all lower than the present invention.
From above technical scheme, the present invention is according to the synthesis of Thymosin-Alpha1 peptide sequence 1-8,9-19 and 20-28 fragment, and then these 3 polypeptide fragment couplings are obtained Thymosin-Alpha1, the multiple segment condense of the present invention carries out simultaneously, and synthesis cycle decreases 2/3, the easy purifying of intermediate, cost is low, and the finished product purity is high, and by product is few, product yield is high, is beneficial to the scale operation of Thymosin-Alpha1.
Embodiment
The invention discloses a kind of method of synthesizing Thymosin-Alpha1, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope compound as herein described and preparation method are changed or suitably change with combination, realize and apply the technology of the present invention.
In the specific embodiment of the invention; all couplings by the amino acid of protecting group all by commercially available acquisition; protected amino acid in the present invention is purchased from the biochemical company limited of gill; resin used purchased from Tianjin Nankai with become company limited, the Chinese implication that in application documents, english abbreviation used is corresponding is in table 1.
The lexical or textual analysis of table 1 english abbreviation
In the process of improvement on synthesis fragment 1 of the present invention, polypeptide fragment 2, from filtrate, be settled out polypeptide fragment 1 and 2 normal hexane and ether sedimentation method can be adopted to realize, and Thymosin-Alpha1 crude product can go out by direct ether sedimentation.
Below in conjunction with embodiment, set forth the present invention further.
Embodiment 1: the preparation of polypeptide resin 1
1, Fmoc-β-Asp(OtBu)-Rink amide resin-made is standby
Get the Rink amide resin (20g of substitution degree sub=0.8mmol/g; 16mmol); join in reaction column; with DMF swelling more than 30 minutes, drain DMF, with DBLK(containing the DMF solution of 20% piperidines) the amino protecting group Fmoc of deresinate; remove reaction 5 minutes at every turn; totally twice, wash 6 times with DMF after removing, drain and wait to feed intake.
Take Fmoc-β-Asp(OtBu)-OH (6.6g, 16mmol), HOBt(2.6g, 19.2mmol) with DMF(40ml) and add DIC(2.4g under condition of ice bath after DCM(40ml) dissolving, 19.2mmol), activate and join in reaction column after 5 minutes, after 60 minutes, reaction terminates, resin washs three times with DMF respectively, add confining liquid (pyridine (20ml) and diacetyl oxide (22ml)), react and drain after 2 hours, DMF washs 6 times.Again with adding appropriate methanol wash 3 times, each 10 minutes, drying under reduced pressure obtained Fmoc-β-Asp(OtBu)-Rink amide Resin(22.5g), its substitution degree is 0.18mmol/g.
2, the preparation of polypeptide resin 1
Take the Fmoc-β-Asp(OtBu that substitution degree is 0.18mmol/g)-Rink amide resin (22.5g, 4mmol), add in solid state reaction post, wash 2 times with DMF, with DMF after swelling 30 minutes, remove Fmoc protection with DBLK, then wash 6 times with DMF.By Fmoc-Glu(OtBu)-OH(5.1g, 12mmol), HOBt(1.8g, 13.2mmol), be dissolved in the DCM(30ml that volume ratio is 1:1) and DMF(30ml) mixing solutions, DIC(1.7g, 13.2mmol is added under condition of ice bath), activate and solution is added in solid state reaction post after 5 minutes, room temperature reaction 2h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, represent that reaction not exclusively, needs linked reaction 1h again).Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to the order that aminoacid sequence C shown in SEQ ID NO:1 holds N to hold, successively one by one by Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH carries out amino acid and extends coupling, finally remove Fmoc protection with DBLK, obtain H-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-β-Asp(OtBu)-aminoresin, i.e. polypeptide resin 1.
Embodiment 2: the preparation of polypeptide fragment 1
Take the 2-CTC resin (40g that substitution degree is 0.5mmol/g, 20mmol), join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get Fmoc-Lys (Boc)-OH (28.1g, 60mmol) be dissolved in the DCM(30ml that volume ratio is 1:1) and DMF(30ml) mixing solutions, DIPEA(15.5g is added under condition of ice bath, 120mmol), activate and solution is added in solid state reaction post after 5 minutes, room temperature reaction 2h, reaction terminates rear confining liquid (DIPEA: methyl alcohol: DCM=1:2:17v:v) and closes three times, each 3min.Sealing liquid volume is pressed 4.0ml/ gram of resin and is calculated.Again with adding appropriate methanol wash 3 times, each 10 minutes, wash 6 times with DMF.Remove Fmoc protection with DBLK, then wash 6 times with DMF.
By Fmoc-Glu(OtBu)-OH(25.5g, 60mmol), HOBt(8g, 60mmol), be dissolved in the DCM(60ml that volume ratio is 1:1) and DMF(60ml) mixing solutions, add DIC(7.6g, 60mmol under condition of ice bath), activate and solution is added in solid state reaction post after 5 minutes, room temperature reaction 2h, (reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again).Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to the order that aminoacid sequence C shown in SEQ ID NO:2 holds N to hold, successively one by one by Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid and extend coupling, shrink with methyl alcohol after end, resin vacuum dried overnight, weigh and obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-CTC resin 95.6g,
By above-mentioned Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-CTC resin, be added in 2L flask.Configuration lytic reagent 1000ml(volume ratio, TFE:DCM=1:4), lytic reagent is poured in flask, room temperature reaction 3h.Reaction terminates, and filters resin, collects filtrate.Filtrate volume is revolved and steams to about 400ml, drop to (volume ratio in 4000ml precipitation reagent, normal hexane: ether=1:4), centrifugal, anhydrous diethyl ether washs, and vacuum-drying, obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-OH, be polypeptide fragment 1, by water-soluble for crude product rear freeze-drying, obtain 42.0g polypeptide fragment 1, purity 90%.
Embodiment 3: the preparation of polypeptide fragment 2
Take the 2-CTC resin (40g that substitution degree is 0.5mmol/g, 20mmol), join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get Fmoc-Ser(tBu)-OH (23.0g, 60mmol) be dissolved in the DCM(30ml that volume ratio is 1:1) and DMF(30ml) mixing solutions, DIPEA(15.5g is added under condition of ice bath, 120mmol), activate and solution is added in solid state reaction post after 5 minutes, room temperature reaction 2h, reaction terminates rear confining liquid (DIPEA: methyl alcohol: DCM=1:2:17v:v) and closes three times, each 3min.Sealing liquid volume is pressed 4.0ml/ gram of resin and is calculated.Again with adding appropriate methanol wash 3 times, each 10 minutes, wash 6 times with DMF.Remove Fmoc protection with DBLK, then wash 6 times with DMF.
By Fmoc-Thr(tBu)-OH(23.8g, 60mmol), HOBt(8g, 60mmol), be dissolved in the DCM(60ml that volume ratio is 1:1) and DMF(60ml) mixing solutions, add DIC(7.6g, 60mmol under condition of ice bath), activate and solution is added in solid state reaction post after 5 minutes, room temperature reaction 2h, (reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again).Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to the order that aminoacid sequence C shown in SEQ ID NO:3 holds N to hold, successively one by one by Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid and extend coupling, after coupling, DBLK removes Fmoc protecting group, add pyridine (23.7g, 200mmol) with diacetyl oxide (20.4g, 200mmol), acetylization reaction 3 hours, after reaction terminates, DMF washs 6 times, shrink with methyl alcohol, resin vacuum dried overnight, weigh and obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin 65.5g.
Take above-mentioned Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin 65.5g, be added in 1L flask.Configuration lytic reagent 700ml(volume ratio, TFE:DCM=1:4), lytic reagent is poured in flask, room temperature reaction 3h.Reaction terminates, and filters resin, collects filtrate.Filtrate volume is revolved and steams to about 200ml, drop to (volume ratio in 2000ml precipitation reagent, normal hexane: ether=1:4), centrifugal, anhydrous diethyl ether washs, and vacuum-drying, obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-OH, be polypeptide fragment 2, by water-soluble for crude product rear freeze-drying, obtain 23.1g polypeptide fragment 2, purity 92%.
Embodiment 4: the preparation of polypeptide resin 2
Take the polypeptide fragment 1 (42g of the 2-in-1 one-tenth of embodiment, 20mmol), HBTU(7.6g, 20mmol), HOBt(2.7g, 20mmol), DIPEA(5.2g is added under adding condition of ice bath after dissolving with the mixed solution that DMF and DMSO is 1:1,40mmol), mixed solution adds in the solid state reaction post of the polypeptide resin 1 that embodiment 1 is synthesized (4mmol), room temperature reaction 3h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, represent that reaction is not exclusively, needs linked reaction 1h again) obtain polypeptide resin 2.
Embodiment 5: the preparation of polypeptide resin 3
The N removing polypeptide resin 2 with DBLK holds Fmoc protecting group, then washs 6 times with DMF;
Take the polypeptide fragment 2 (23.1g that embodiment 3 is synthesized, 20mmol), PyBOP(10.4), HOBt(2.7g, 20mmol), DIPEA(5.2g is added under adding condition of ice bath after dissolving with the mixed solution that DMF and DMSO is 1:1,40mmol), mixed solution adds in the solid state reaction post of the polypeptide resin 2 that embodiment 4 is synthesized, room temperature reaction 3h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again).Then wash 6 times with DMF, methyl alcohol contracting is dry obtains 42.5g polypeptide resin 3.
Embodiment 6: polypeptide resin 3 cracking obtains Thymosin-Alpha1 crude product
42.5g polypeptide resin 3 in embodiment 5 is joined in 1000ml flask, configuration lytic reagent 500ml(volume ratio, TFA:DET:PHSH:PHOH:H 2o=80:5:5:5:5), lytic reagent is poured in flask, room temperature reaction 3 hours.Reaction terminates, and filters resin, collects filtrate.Drop in 5000ml ether reagent, centrifugal, anhydrous diethyl ether washs, and vacuum-drying, obtain 14.2g crude product Thymosin-Alpha1, purity 78.6%, synthesis weight yield 100%.
Embodiment 7: crude product Thymosin-Alpha1 is purified obtains product Thymosin-Alpha1
By the Thymosin-Alpha1 crude product aqueous solution 0.45um filtering with microporous membrane in embodiment 6.Adopt Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phase purifying, collects object peak cut, obtains purity and be greater than 99% sterling.
Thymosin-Alpha1 sterling solution is adopted RP-HPLC system, and chromatographic column is the anti-phase C18 post of 50 × 250mm, and 0.1% acetum/acetonitrile mobile phase turns salt, collect object peak cut, rotary evaporation concentrates, and freeze-drying obtains Thymosin-Alpha1 acetate 5.2g, HPLC purity 99.4%, total recovery 42.0%.
Embodiment 8: different fragments synthetic test
In order to checking only has suitable segment condense method can reach purity of the present invention and yield further, carry out solid phase synthesis according to being different from segments of the present invention respectively, synthetic method is identical with the present invention, the results are shown in Table 2.
The Thymosin-Alpha1 purity of the different synthetic schemes of table 2 and yield
As shown in Table 2, not according to the Thymosin-Alpha1 (method 1-3) of synthetic schemes synthesis of the present invention, its crude product purity and total recovery are all not as synthetic schemes of the present invention (method 4), show thus, carry out synthesizing according to different fragments number and the purity of Thymosin-Alpha1 and yield tool are had a significant impact, only can reach object of the present invention according to suitable synthetic schemes.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. synthesize a method for Thymosin-Alpha1, it is characterized in that, comprise the following steps:
The aminoacid sequence coupling on Lys side chain, on Glu side chain shown in SEQ ID NO:1 of step 1, solid phase synthesis has protecting group and holds coupling to have the polypeptide resin 1 of resin at C;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:2, Ser side chain, on Glu side chain, on Thr side chain, on Lys side chain, on Asp side chain has the polypeptide fragment 1 of protecting group;
Solid phase synthesis aminoacid sequence N shown in SEQ ID NO:3 holds the polypeptide fragment 2 that coupling has ethanoyl, coupling has protecting group on Ser side chain, on Thr side chain, on Asp side chain;
Step 2, the C section of polypeptide fragment 1 and the N of polypeptide resin 1 held and carries out linked reaction and obtain polypeptide resin 2;
Step 3, the N sloughing polypeptide resin 2 hold protecting group and carry out linked reaction with the C section of polypeptide fragment 2 and obtains polypeptide resin 3, and the resin and all protecting groups that add lysate removal polypeptide resin 3 obtain Thymosin-Alpha1 crude product;
Thymosin-Alpha1 sterling is obtained after step 4, Thymosin-Alpha1 crude product micro-filtration, RP-HPLC purifying.
2. method according to claim 1, it is characterized in that, described in step 1, solid-phase synthetic peptide resin 1 is:
Steps A 1, Fmoc-β-Asp(OtBu)-OH carries out linked reaction and obtains Fmoc-β-Asp(OtBu under coupling system effect with aminoresin)-aminoresin;
Steps A 2, remove Fmoc protecting group and obtain H-β-Asp(OtBu)-aminoresin, Fmoc-Glu(OtBu)-OH under coupling system effect with H-β-Asp(OtBu)-aminoresin carries out linked reaction and obtains Fmoc-Glu(OtBu)-β-Asp(OtBu)-aminoresin;
Steps A 3, according to the order that aminoacid sequence C shown in SEQ ID NO:1 holds N to hold, successively one by one by Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH carries out amino acid according to steps A 2 coupling mode and extends coupling, finally removing N holds Fmoc protecting group to obtain H-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-β-Asp(OtBu)-aminoresin, i.e. polypeptide resin 1,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, Boc are amino acid side chain protecting group.
3. method according to claim 2, is characterized in that, described aminoresin is the Rink amide resin of 0.2-1.0mmol/g, Rink amide AM resin or Rink amide mbha resin.
4. method according to claim 1, it is characterized in that, described in step 1, solid-phase synthetic peptide fragment 1 is:
Step B1, Fmoc-Lys(Boc)-OH carries out linked reaction with 2-CTC resin and obtains Fmoc-Lys(Boc under DIPEA effect)-CTC resin;
Step B2, remove Fmoc protecting group and obtain H-Lys(Boc)-CTC resin, Fmoc-Glu(OtBu)-OH under coupling system effect with H-Lys(Boc)-CTC resin carries out linked reaction and obtains Fmoc-Glu(OtBu)-Lys(Boc)-CTC resin;
Step B3, according to the order that aminoacid sequence C shown in SEQ ID NO:2 holds N to hold, successively one by one by Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid according to step B2 coupling mode and extend coupling, obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-CTC resin, add lysate and remove 2-CTC resin, obtain Fmoc-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-OH, be polypeptide fragment 1,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, tBu, Boc are amino acid side chain protecting group.
5. method according to claim 1, it is characterized in that, described in step 1, solid-phase synthetic peptide fragment 2 is:
Step C1, Fmoc-Ser(tBu)-OH carries out linked reaction with 2-CTC resin and obtains Fmoc-Ser(tBu under DIPEA effect)-CTC resin;
Step C2, remove Fmoc protecting group and obtain H-Ser(tBu)-CTC resin, Fmoc-Thr(tBu)-OH under coupling system effect with H-Ser(tBu)-CTC resin carries out linked reaction and obtains Fmoc-Thr(tBu)-Ser(tBu)-CTC resin;
Step C3, according to the order that aminoacid sequence C shown in SEQ ID NO:3 holds N to hold, successively one by one by Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, carry out amino acid according to step C2 coupling mode and extend coupling, obtain Fmoc-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, remove after N holds Fmoc protecting group and obtain H-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, then carry out acetylization reaction at N end and obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-CTC resin, add lysate and remove 2-CTC resin, obtain Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-OH, be polypeptide fragment 2,
Wherein, described Fmoc is amino acid N end protecting group, and described OtBu, tBu are amino acid side chain protecting group.
6. method according to claim 2,4 or 5 any one, it is characterized in that, described coupling system is HOBt/DIC double reagent coupling system, PyBOP/HOBt double reagent coupling system, HATU/HOAT double reagent coupling system or TBTU/HOBt double reagent coupling system.
7. method according to claim 1,2,4 or 5 any one, is characterized in that, described linked reaction with one or more in DCM, NMP, DMF, DMSO for solvent.
8. method according to claim 4 or 5 any one, is characterized in that, the mixed pyrolysis liquid of described lysate to be volume ratio TFE:DCM be 1:4.
9. method according to claim 1, is characterized in that, described in step 2 and step 3, linked reaction carries out coupling with HOBt/DIC double reagent coupling system, PyBOP/HOBt/DIPEA tri-reagent coupling system or HBTU/HOBt/DIPEA tri-reagent coupling system.
10. method according to claim 1, it is characterized in that, lysate described in step 3 is volume ratio TFA: phenol: thioanisole: dithioglycol: water is the mixed pyrolysis of 80-85:1-5:1-5:1-5:1-5.
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