CN103980357A - Method used for synthesizing thymalfasin - Google Patents
Method used for synthesizing thymalfasin Download PDFInfo
- Publication number
- CN103980357A CN103980357A CN201310412012.0A CN201310412012A CN103980357A CN 103980357 A CN103980357 A CN 103980357A CN 201310412012 A CN201310412012 A CN 201310412012A CN 103980357 A CN103980357 A CN 103980357A
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- China
- Prior art keywords
- otbu
- glu
- asp
- thymosin
- resin
- Prior art date
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method used for solid-phase synthesis of thymalfasin, and relates to a novel technology used for preparing thymalfasin via Fmoc strategy solid-phase method. The method comprises following steps: (1) synthesis of Fmoc-Asp(RinkAmideMBHA resin)-OtBu is realized using Fmoc-Asp-OtBu and RinkAmideMBHA with an appropriate substitution degree; (2) Fmoc-Asp(RinkAmideMBHA resin)-OtBu is subjected to stepwise coupling or fragment coupling so as to obtain thymosin resin, wherein coupling of Glu18 and Lys19 is realized via fragment one-step peptide connection, and the rest amino acids are subjected to stepwise coupling, a mixed deprotection agent is used for deprotection in deprotection processes before coupling of Asp6, Thr12, Leu16, Lys17, Glu18-Lys19, and an special acylation reagent is used for terminal blocking of thymosin resin before Asp2 coupling; and (3) thymosin resin is subjected to cracking so as to obtain a crude peptide, and thymalfasin is obtained via RP-HPLC purification, salt transport, and freeze-drying. The invention provides the solid-phase synthesis method of thymalfasin with high product purity and convenient for purification.
Description
Technical field
The present invention relates to a kind of solid phase synthesis process of Thymosin-Alpha1.
Background technology
Thymosin-Alpha1 (Thymosin α 1 also claims Thymosin alpha 1) is the main bioactive ingredients of thymosin, is important immune regulator in body, is the polypeptide of acetylizad 28 amino acid composition of nitrogen end.Research shows, Thymosin-Alpha1 promotes that bone marrow stem cell Development And Differentiation is lymphoblast and prolymphocyte; Inducer T lymphocyte differentiation, with ripe, makes mature T cell further be divided into several different subgroups, as killer cell, memory cell, effector cell, guidance T lymphocyte etc., and produces various solubility media; Strengthen the reaction of lymphocyte to mitogen, and can increase the synthetic of adenoid protein and nucleic acid; Increase the generation of r-IFN, a-IFN, IL-2, IL-3 and lymphotoxin, strengthen antiviral and antineoplastic immune response; Suppression of autoimmune responses; Recover the function of suppressor T cell.Current Thymosin-Alpha1 has been widely used in clinical, and treatment various diseases, as the treatment for chronic hepatitis B; For the treatment of hepatitis C, hepatocellular carcinoma, malignant melanoma; Be used for the treatment of the treatment of infectious diseases, autoimmune disorder, malignant tumour etc.
US4517119 adopts that full liquid phase method is synthetic, purifying Thymosin-Alpha1.Amino acid nitrogen end adopts benzyl protection, goes protection all to adopt Pd shortening to carry out at every turn, operates extremely numerous and diversely, does not provide total recovery.US4504415 adopts that full liquid phase method is synthetic, reversed-phase HPLC purifying Thymosin-Alpha1.Calculate total recovery taking 18 peptide fragment (11-28 peptide section) as starting raw material as 12%.US5856440 is taking mbha resin as carrier, and adopting the protection of nitrogen end is Moz(4-methoxyl group benzyloxy formyl radical) amino acid carry out Thymosin-Alpha1 solid phase synthesis.Compare Boc method synthetic, Moz more easily sloughs than Boc.Therefore in the synthetic polypeptide process of Moz method, can reduce because repeatedly going protection to cause the loss of peptide chain from resin.Thereby improve total synthesis yield.Moz method is synthesized Thymosin-Alpha1 yield 35%, than 25% height of Boc method.CN101484467 adopts the synthetic Thymosin-Alpha1 of CTC resin.When synthetic, adopt pseudo proline dipeptides form to synthesize at the Ile-Thr at sequence 11-12 place.Final synthesis yield 40%.CN102199205 adopts the synthetic Thymosin-Alpha1 of Wang resin, and it adopts the progressively synthetic Thymosin-Alpha1 of method, and its thick peptide purity is 69.6%, and synthesis yield is 56.6%.If the Wang resin after adopting PEG derivative synthesizes, thick peptide purity is 57.2%, and synthesis yield is 64.4%.Do not report total recovery.CN1291031 adopts the synthetic Thymosin-Alpha1 of thorugh biologic engineering method, and has carried out active contrast with commodity Thymosin-Alpha1.
The total technological difficulties that these existing synthetic methods exist are: (1) liquid phase is synthesized Thymosin-Alpha1 complex operation, wastes time and energy, and is unfavorable for suitability for industrialized production; (2) how existing solid-phase synthesis, obtain Thymosin-Alpha1 peptide resin by coupling mode one by one.Because the existence of multiple difficulty (Leu16, Lys17, Glu18, Lys19 etc.) makes thick peptide purity lower, easily comprise the impurity close with Thymosin-Alpha1 retention properties and cause HPLC purifying difficulty increase, total recovery is not high.
Summary of the invention
The object of this invention is to provide a kind of solid phase synthesis process of Thymosin-Alpha1.The technical issues that need to address of the present invention are: select a suitable operational path, solve following technical problem: (1) liquid phase is synthesized Thymosin-Alpha1 complex operation, wastes time and energy, and is unfavorable for suitability for industrialized production; (2) existing solid-phase synthesis later stage purifying difficulty is large, and product purity is low, yield is not high.
Preparation method of the present invention as shown in Figure 1, first start synthetic Fmoc-Asp (Rink Amide mbha resin)-OtBu by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and suitable substitution degree, then Fmoc-Asp (Rink Amide mbha resin)-OtBu is adopted to the mode of coupling is one by one synthetic obtains Thymosin-Alpha1 peptide resin, then adopt acetic anhydride/pyridine to carry out the acetylize of nitrogen end; Finally Thymosin-Alpha1 peptide resin is carried out to cracking, obtains thick peptide, through RP-HPLC purifying obtain high purity Thymosin-Alpha1 solution, again through RP-HPLC turn salt, lyophilize obtains Thymosin-Alpha1 finished product.
In the present invention, some conventional abbreviations have following implication;
Ac2O: acetic anhydride
Pyridine: pyridine
Boc: tertbutyloxycarbonyl
Fmoc: 9-fluorenylmethyloxycarbonyl
TBu: the tertiary butyl
Fmoc-AA-OH: the amino acid of 9-fluorenylmethyloxycarbonyl protection
The chloro-I-hydroxybenzotriazole of Cl-HOBt: 6-
DIC: N, N '-di-isopropyl carbodiimide
Asp: aspartic acid
Glu: L-glutamic acid
Ala: L-Ala
Val: α-amino-isovaleric acid
Lys: Methionin
Leu: leucine
Thr: Threonine
Ser: Serine
Ile: Isoleucine
DMF: N, N '-dimethyl formamide
Piperidine: hexahydropyridine
TFA: trifluoracetic acid
TIS: tri isopropyl silane
MeOH: methyl alcohol
DCM: methylene dichloride
Kniser test detection reagent is: triketohydrindene hydrate
HOAc: acetic acid
ACN: acetonitrile
For this reason, the invention provides a kind of solid cyclization and become the method for Thymosin-Alpha1, described method steps is as follows:
Step 1, application Fmoc/tBu method, on resin with solid phase synthesis Thymosin-Alpha1 peptide resin;
Step 2, introduces Glu18, two amino-acid residues of Lys19 with dipeptides pieces;
Step 3, the protection of going before the couplings such as Asp6, Thr12, Leu16, Lys17, Glu18-Lys19 adopts mixing to go to protect reagent D BU/piperidines/DMF solution to carry out protective reaction 2 times, and the protective reaction time is 15min, for the second time protective reaction 30min for the first time;
Step 4, after the complete Asp2 of coupling washing, carries out special acylating reagent propionyl chloride etc. to peptide resin and carries out end-blocking operation.
Step 5, carries out cracking to Thymosin-Alpha1 peptide resin, obtains thick peptide, through RP-HPLC purifying, turn salt, freeze-drying obtains Thymosin-Alpha1.
Wherein, Thymosin-Alpha1 resin described in step 1, structure is as follows:
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asp(NH-Resin)-OtBu
Wherein, described in step 2, dipeptides pieces is carried out coupling by Fmoc-Glu (OtBu)-Lys (Boc)-OH;
Wherein, mix that to remove to protect reagent be as piperidines/DMF solution, DBU/DMF solution or DBU/piperidines/DMF solution etc. described in step 3, preferably DBU/piperidines/DMF=1/5/20 (volume ratio).And go protection to adopt and carry out for 2 times, first set reaction 15min, 25 DEG C of temperature; React for the second time 30min, 25 DEG C of temperature;
Wherein, acylating reagent is propionyl chloride, butyryl chloride, Tosyl chloride described in step 4, preferably propionyl chloride.Reaction conditions is that acyl chlorides adopts 10eq to feed intake, and reacts 15min at 20 DEG C.
Wherein, cracking adopts cracking agent: TIS and TFA described in step 5, and its allocation ratio is TIS/TFA=5/95, and the volume number of cracking agent is 10 times of Thymosin-Alpha1 peptide resin total mass number.Reaction times is 1 hour, and temperature of reaction is 25 DEG C.Cracking agent compound method is: 5 volume TIS are joined in the TFA of 95 volumes.
The present invention obtains the combination of specific synthesis mode by screening, combine use by fragment method, the conditions such as protective reaction condition, special reagent end-blocking of strengthening, and obtains a kind of high yield and is easy to the Thymosin-Alpha1 synthetic method of purifying.
Below data declaration beneficial effect of the present invention by experiment:
Progressively method and the screening of fragment method: start synthetic Fmoc-Asp (Rink Amide mbha resin)-OtBu by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and suitable substitution degree, then Fmoc-Asp (Rink Amide mbha resin)-OtBu is adopted to the mode of coupling is one by one synthetic obtains Thymosin-Alpha1 peptide resin, then adopt acetic anhydride/pyridine to carry out the acetylize of nitrogen end; Finally Thymosin-Alpha1 peptide resin is carried out to cracking, obtain the thick peptide of Thymosin-Alpha1.Thick peptide is sampled to detection.Two kinds of modes below wherein having adopted respectively in the time of 18,19 two position residue couplings: first coupling Fmoc-Lys (Boc)-OH, then the progressively method of coupling Fmoc-Glu (OtBu)-OH; And Fmoc-Glu (OtBu)-Lys (Boc)-OH fragment method.
Use above-mentioned condition to prepare object peptide, obtain respectively the Thymosin-Alpha1 of different yields and purity, result data is as follows:
| Deprotecting regent (volume ratio) | Thick peptide purity | Synthetic peptide yield |
| Progressively method | 42.36% | 24% |
| Fragment method | 51.22% | 33% |
General go protection with strengthen de-protected screening:
Start synthetic Fmoc-Asp (Rink Amide mbha resin)-OtBu by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and suitable substitution degree, then Fmoc-Asp (Rink Amide mbha resin)-OtBu is adopted to the mode of coupling is one by one synthetic obtains Thymosin-Alpha1 peptide resin, then adopt acetic anhydride/pyridine to carry out the acetylize of nitrogen end; Finally Thymosin-Alpha1 peptide resin is carried out to cracking, obtain the thick peptide of Thymosin-Alpha1.Thick peptide is sampled to detection.But going protection to adopt respectively generally to go guard method and reinforcement to go guard method before the couplings such as Asp6, Thr12, Leu16, Lys17, Glu18-Lys19.The general protection method of going is: adopt and go to protect reagent piperidines/DMF=1/4(volume ratio) carry out protective reaction 2 times, the protective reaction time is 5min for the first time, protective reaction 10min for the second time, temperature of reaction is all 25 DEG C.Strengthen going protection method to be: adopt to mix and go to protect reagent D BU/piperidines/DMF=1/5/20 (volume ratio) to carry out protective reaction 2 times, and the protective reaction time be 15min for the first time, protective reaction 30min for the second time, temperature of reaction is all 25 DEG C.
Use above-mentioned condition to prepare object peptide, obtain respectively the Thymosin-Alpha1 of different yields and purity, result data is as follows:
| Go protected mode | Thick peptide purity | Synthetic peptide yield |
| General method | 42.36% | 24% |
| Reinforcement | 57.01% | 37% |
The screening of special capping reagent: start synthetic Fmoc-Asp (Rink Amide mbha resin)-OtBu by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and suitable substitution degree, then Fmoc-Asp (Rink Amide mbha resin)-OtBu is adopted to the mode of coupling is one by one synthetic obtains Thymosin-Alpha1 peptide resin, then adopt acetic anhydride/pyridine to carry out the acetylize of nitrogen end; Finally Thymosin-Alpha1 peptide resin is carried out to cracking, obtains the thick peptide of Thymosin-Alpha1, through RP-HPLC purifying obtain high purity Thymosin-Alpha1 solution, again through RP-HPLC turn salt, lyophilize obtains Thymosin-Alpha1 finished product.After the complete Asp2 of coupling washing, peptide resin is carried out to two kinds for the treatment of processs.Be respectively and directly carry out next amino acid coupling; With adopt acylating reagent end-blocking after carry out next amino acid coupling.
Use above-mentioned condition to prepare object peptide, obtain respectively the Thymosin-Alpha1 of different yields and purity, result data is as follows:
| Go protected mode | Thick peptide purity | Synthesis yield | Essence peptide purity | Total recovery |
| Not end-blocking | 42.36% | 24% | 96.35% | 11% |
| Propionyl chloride end-blocking | 42.10% | 24% | 98.06% | 17% |
Compared to the prior art method of the present invention has obvious advantage, and relevant contrast experiment is as follows:
One, thick peptide purity, synthesis yield and total recovery comparison:
Carry out yield calculating by the method for the inventive method and prior art, result is as follows:
Method for detecting purity is wherein as follows:
HPLC detection method is as follows:
Chromatographic column: Waters X-bridge 5 μ m C18 (250mm × 4.6mm) chromatographic columns or quite post; Mobile phase A: 0.1% triethylamine phosphoric acid is adjusted pH2.0; Mobile phase B: acetonitrile; Elution flow rate: 1ml/min; Detect wavelength: 210nm; Gradient:
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 86 | 14 |
| 32 | 80 | 20 |
| 33 | 86 | 14 |
| 45 | 86 | 14 |
The invention has the beneficial effects as follows: obtain the combination of specific synthesis mode by screening, combine use by fragment method, the conditions such as protective reaction condition, special reagent end-blocking of strengthening, obtain a kind of high yield and be easy to the Thymosin-Alpha1 synthetic method of purifying.
Brief description of the drawings
Fig. 1 is synthetic route chart of the present invention;
Fig. 2 is the thick peptide HPLC of embodiment 5 Thymosin-Alpha1 color atlas;
Fig. 3 is the HPLC color atlas of Thymosin-Alpha1 reference substance;
Fig. 4 is embodiment 5 Thymosin-Alpha1 essence peptide HPLC color atlass.
Embodiment
Further illustrate by the following examples the present invention.
Embodiment 1: adopt the synthetic Thymosin-Alpha1 of coupling method one by one
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu is synthetic
Take Rink Amide MBHA resin (substitution degree 0.45mmol/g) (2.67 g, 1.0 equiv) and add in solid phase synthesis pipe, add DCM(10mL), temperature control 10-30 DEG C is stirred swelling 30min, and suction filtration is removed liquid.Add again DMF(10mL × 2) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration is removed liquid.Again measure 20%Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration is removed liquid.Measure DMF(10 mL × 7) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Separately take Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4 equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, after stirring dissolving completely, bathe and be cooled to 0-10 DEG C with cryosel, add DIC(0.89 mL, 4.8 equiv), stir, temperature control 0-10 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects and is negative.
Step 2
H-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Step 1 product amino-acid resin is sloughed to Fmoc by the same way.Take Fmoc-AA-OH(4 equiv), Cl-HOBt(4equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, stirring is bathed and is cooled to 5-10 DEG C with cryosel after dissolving completely, adds DIC(4.8 equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects, as positive in result, repeats coupling once.Successively after the complete all amino acid of coupling, then go Fmoc operation, and with DMF washing 7 times.
Step 3
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Contain Ac2O(0.57mL toward adding in step 2 products therefrom peptide resin, 5 equiv), pyridine (0.48mL, 5 equiv) DMF(8mL), in 15-20 DEG C, after reaction 1h, suction filtration is removed liquid, then use DMF(18mL successively × 3), MeOH(18mL × 2), DCM(20mL × 2), MeOH(18mL × 2) washing resin.Take out Thymosin-Alpha1 peptide resin, after being dried, obtain 7.20g.
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) synthetic
Measure TFA(68 mL), TIS(4.0 mL) join successively in 100mL there-necked flask,, stirring and evenly mixing, is cooled to 15-20 DEG C, and step 3 gained peptide resin is added in there-necked flask.After being warmed up to 25-30 DEG C of reaction 1h, stop stirring.By reacting liquid filtering, and with TFA(5 mL × 2) washing resin, after merging, get filtrate, put underpressure distillation in Rotary Evaporators and remove most of TFA.Gained debris is joined in advance and is chilled in-10～-5 DEG C of methyl tertiary butyl ethers (120mL), carry out sedimentation.Gained suspension liquid washs after 6 times through repeated centrifugation, methyl tertiary butyl ether, obtains the thick peptide of pasty state, obtains thick peptide 4.12g after drying under reduced pressure.HPLC purity (area normalization method) 42.36%.Obtain in thick peptide containing Thymosin-Alpha1 0.90g synthesis yield: 24% through external standard method quantitative Analysis.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) RP-HPLC purifying
By thick step 4 gained peptide by C18 or 3 purifying of C8 post, obtain target product after turning salt, lyophilize.Purification condition for the first time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the second time: moving phase is: A phase: 0.3% acetic acid; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the third time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Turn salt condition: moving phase: A phase: 20mM ammonium acetate-aqueous solution; B phase: acetonitrile; Detect wavelength 220nm.Flow velocity 80mL/min.Lyophilize condition is :-27 DEG C of 16h;-5 DEG C of 10h; 5 DEG C of 2h; 30 DEG C of 16h.Obtain target product 0.41g, HPLC purity (area normalization method) 96.35%, total recovery 11%.
Embodiment 2: adopt the synthetic Thymosin-Alpha1 of fragment coupling method
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) preparation
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu is synthetic
Take Rink Amide MBHA resin (substitution degree 0.45mmol/g) (2.67 g, 1.0 equiv) and add in solid phase synthesis pipe, add DCM(10mL), temperature control 10-30 DEG C is stirred swelling 30min, and suction filtration is removed liquid.Add again DMF(10mL × 2) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration is removed liquid.Again measure 20%Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration is removed liquid.Measure DMF(10 mL × 7) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Separately take Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4 equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, after stirring dissolving completely, bathe and be cooled to 0-10 DEG C with cryosel, add DIC(0.89 mL, 4.8 equiv), stir, temperature control 0-10 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects and is negative.
Step 2
H-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Step 1 product amino-acid resin is sloughed to Fmoc by the same way.Take Fmoc-AA-OH(4 equiv), Cl-HOBt(4equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, stirring is bathed and is cooled to 5-10 DEG C with cryosel after dissolving completely, adds DIC(4.8 equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects, as positive in result, repeats coupling once.In the time carrying out Glu18, two amino acid couplings of Lys19, taking two peptide fragment Fmoc-Glu-Lys-OH as raw material, a step coupling completes Glu18, the coupling of Lys19 amino acid, and all the other amino acid coupling conditions are identical with embodiment 1.Successively after the complete all amino acid of coupling, then go Fmoc operation, and with DMF washing 7 times.
Step 3
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Contain Ac2O(0.57mL toward adding in step 2 products therefrom peptide resin, 5 equiv), pyridine (0.48mL, 5 equiv) DMF(8mL), in 15-20 DEG C, after reaction 1h, suction filtration is removed liquid, then use DMF(18mL successively × 3), MeOH(18mL × 2), DCM(20mL × 2), MeOH(18mL × 2) washing resin.Take out Thymosin-Alpha1 peptide resin, after being dried, obtain 7.06g.
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) synthetic
Measure TFA(68 mL), TIS(4.0 mL) join successively in 100mL there-necked flask,, stirring and evenly mixing, is cooled to 15-20 DEG C, and step 3 gained peptide resin is added in there-necked flask.After being warmed up to 25-30 DEG C of reaction 1h, stop stirring.By reacting liquid filtering, and with TFA(5 mL × 2) washing resin, after merging, get filtrate, put underpressure distillation in Rotary Evaporators and remove most of TFA.Gained debris is joined in advance and is chilled in-10～-5 DEG C of methyl tertiary butyl ethers (120mL), carry out sedimentation.Gained suspension liquid washs after 6 times through repeated centrifugation, methyl tertiary butyl ether, obtains the thick peptide of pasty state, obtains thick peptide 4.23g after drying under reduced pressure.HPLC purity (area normalization method) 51.22%.Obtain in thick peptide containing Thymosin-Alpha1 1.22g synthesis yield: 33% through external standard method quantitative Analysis.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) RP-HPLC purifying
By thick step 4 gained peptide by C18 or 3 purifying of C8 post, obtain target product after turning salt, lyophilize.Purification condition for the first time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the second time: moving phase is: A phase: 0.3% acetic acid; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the third time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Turn salt condition: moving phase: A phase: 20mM ammonium acetate-aqueous solution; B phase: acetonitrile; Detect wavelength 220nm.Flow velocity 80mL/min.Lyophilize condition is :-27 DEG C of 16h;-5 DEG C of 10h; 5 DEG C of 2h; 30 DEG C of 16h.Obtain target product 0.75g, HPLC purity (area normalization method) 97.34%, total recovery 20%.
Embodiment 3: adopt the synthetic Thymosin-Alpha1 of special reagent end-blocking
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) preparation
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu is synthetic
Take Rink Amide MBHA resin (substitution degree 0.45mmol/g) (2.67 g, 1.0 equiv) and add in solid phase synthesis pipe, add DCM(10mL), temperature control 10-30 DEG C is stirred swelling 30min, and suction filtration is removed liquid.Add again DMF(10mL × 2) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration is removed liquid.Again measure 20%Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration is removed liquid.Measure DMF(10 mL × 7) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Separately take Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4 equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, after stirring dissolving completely, bathe and be cooled to 0-10 DEG C with cryosel, add DIC(0.89 mL, 4.8 equiv), stir, temperature control 0-10 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects and is negative.
Step 2
H-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Step 1 product amino-acid resin is sloughed to Fmoc by the same way.Take Fmoc-AA-OH(4 equiv), Cl-HOBt(4equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, stirring is bathed and is cooled to 5-10 DEG C with cryosel after dissolving completely, adds DIC(4.8 equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects, as positive in result, repeats coupling once.But at the complete Asp2 of coupling and with after DMF washing 3 times, contain propionyl chloride (5 equiv), DIEA(5 equiv toward adding in solid phase synthesis pipe) DMF solution (15mL), and control temperature 15-20 DEG C and react after 30min, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Proceed again the coupling of last amino acid Ser1, after all amino acid couplings finish, then go Fmoc operation, and with DMF washing 7 times.
Step 3
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Contain Ac2O(0.57mL toward adding in step 2 products therefrom peptide resin, 5 equiv), pyridine (0.48mL, 5 equiv) DMF(8mL), in 15-20 DEG C, after reaction 1h, suction filtration is removed liquid, then use DMF(18mL successively × 3), MeOH(18mL × 2), DCM(20mL × 2), MeOH(18mL × 2) washing resin.Take out Thymosin-Alpha1 peptide resin, after being dried, obtain 7.25g.
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) synthetic
Measure TFA(68 mL), TIS(4.0 mL) join successively in 100mL there-necked flask,, stirring and evenly mixing, is cooled to 15-20 DEG C, and step 3 gained peptide resin is added in there-necked flask.After being warmed up to 25-30 DEG C of reaction 1h, stop stirring.By reacting liquid filtering, and with TFA(5 mL × 2) washing resin, after merging, get filtrate, put underpressure distillation in Rotary Evaporators and remove most of TFA.Gained debris is joined in advance and is chilled in-10～-5 DEG C of methyl tertiary butyl ethers (120mL), carry out sedimentation.Gained suspension liquid washs after 6 times through repeated centrifugation, methyl tertiary butyl ether, obtains the thick peptide of pasty state, obtains thick peptide 4.05g after drying under reduced pressure.HPLC purity (area normalization method) 42.10%.Obtain in thick peptide containing Thymosin-Alpha1 1.10g synthesis yield: 24% through external standard method quantitative Analysis.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) RP-HPLC purifying
By thick step 4 gained peptide by C18 or 3 purifying of C8 post, obtain target product after turning salt, lyophilize.Purification condition for the first time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the second time: moving phase is: A phase: 0.3% acetic acid; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the third time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Turn salt condition: moving phase: A phase: 20mM ammonium acetate-aqueous solution; B phase: acetonitrile; Detect wavelength 220nm.Flow velocity 80mL/min.Lyophilize condition is :-27 DEG C of 16h;-5 DEG C of 10h; 5 DEG C of 2h; 30 DEG C of 16h.Obtain target product 0.62g, HPLC purity (area normalization method) 98.06%, total recovery 17%.
Embodiment 4: adopt and strengthen going protective condition to synthesize Thymosin-Alpha1
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) preparation
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu is synthetic
Take Rink Amide MBHA resin (substitution degree 0.45mmol/g) (2.67 g, 1.0 equiv) and add in solid phase synthesis pipe, add DCM(10mL), temperature control 10-30 DEG C is stirred swelling 30min, and suction filtration is removed liquid.Add again DMF(10mL × 2) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration is removed liquid.Again measure 20%Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration is removed liquid.Measure DMF(10 mL × 7) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Separately take Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4 equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, after stirring dissolving completely, bathe and be cooled to 0-10 DEG C with cryosel, add DIC(0.89 mL, 4.8 equiv), stir, temperature control 0-10 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects and is negative.
Step 2
H-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Step 1 product amino-acid resin is sloughed to Fmoc by the same way.Take Fmoc-AA-OH(4 equiv), Cl-HOBt(4equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, stirring is bathed and is cooled to 5-10 DEG C with cryosel after dissolving completely, adds DIC(4.8 equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects, as positive in result, repeats coupling once.But going in Fmoc reaction before the couplings such as Asp6, Thr12, Leu16, Lys17, Glu18, Lys19; it is DBU/ piperidines/DMF=1/20/80(volume ratio that replacing removes to protect reagent) carry out; go to protect reagent place's constancy of volume, but the protective reaction time is respectively 15min and 30min.After all amino acid couplings finish, then go Fmoc operation, and with DMF washing 7 times.
Step 3
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Contain Ac2O(0.57mL toward adding in step 2 products therefrom peptide resin, 5 equiv), pyridine (0.48mL, 5 equiv) DMF(8mL), in 15-20 DEG C, after reaction 1h, suction filtration is removed liquid, then use DMF(18mL successively × 3), MeOH(18mL × 2), DCM(20mL × 2), MeOH(18mL × 2) washing resin.Take out Thymosin-Alpha1 peptide resin, after being dried, obtain 7.44g.
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) synthetic
Measure TFA(68 mL), TIS(4.0 mL) join successively in 100mL there-necked flask,, stirring and evenly mixing, is cooled to 15-20 DEG C, and step 3 gained peptide resin is added in there-necked flask.After being warmed up to 25-30 DEG C of reaction 1h, stop stirring.By reacting liquid filtering, and with TFA(5 mL × 2) washing resin, after merging, get filtrate, put underpressure distillation in Rotary Evaporators and remove most of TFA.Gained debris is joined in advance and is chilled in-10～-5 DEG C of methyl tertiary butyl ethers (120mL), carry out sedimentation.Gained suspension liquid washs after 6 times through repeated centrifugation, methyl tertiary butyl ether, obtains the thick peptide of pasty state, obtains thick peptide 4.39g after drying under reduced pressure.HPLC purity (area normalization method) 57.01%.Obtain in thick peptide containing Thymosin-Alpha1 1.38g synthesis yield: 37% through external standard method quantitative Analysis.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) RP-HPLC purifying
By thick step 4 gained peptide by C18 or 3 purifying of C8 post, obtain target product after turning salt, lyophilize.Purification condition for the first time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the second time: moving phase is: A phase: 0.3% acetic acid; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the third time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Turn salt condition: moving phase: A phase: 20mM ammonium acetate-aqueous solution; B phase: acetonitrile; Detect wavelength 220nm.Flow velocity 80mL/min.Lyophilize condition is :-27 DEG C of 16h;-5 DEG C of 10h; 5 DEG C of 2h; 30 DEG C of 16h.Obtain target product 0.73g, HPLC purity (area normalization method) 97.89%, total recovery 20%.
Embodiment 5: combine and adopt fragment coupling method, reinforcement to go the technique such as protective condition and propionyl chloride end-blocking to synthesize Thymosin-Alpha1.
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) preparation
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu is synthetic
Take Rink Amide MBHA resin (substitution degree 0.45mmol/g) (2.67 g, 1.0 equiv) and add in solid phase synthesis pipe, add DCM(10mL), temperature control 10-30 DEG C is stirred swelling 30min, and suction filtration is removed liquid.Add again DMF(10mL × 2) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration is removed liquid.Again measure 20%Piperidine/DMF solution (9.0 mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration is removed liquid.Measure DMF(10 mL × 7) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Separately take Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4 equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, after stirring dissolving completely, bathe and be cooled to 0-10 DEG C with cryosel, add DIC(0.89 mL, 4.8 equiv), stir, temperature control 0-10 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects and is negative.
Step 2
H-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Step 1 product amino-acid resin is sloughed to Fmoc by the same way.Take Fmoc-AA-OH(4 equiv), Cl-HOBt(4equiv) add beaker.Measure DMF/DCM=1/1 mixed solvent (6.5 mL) and add beaker, stirring is bathed and is cooled to 5-10 DEG C with cryosel after dissolving completely, adds DIC(4.8 equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins this reaction solution in solid phase synthesis pipe, and after 3h, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Triketohydrindene hydrate detects, as positive in result, repeats coupling once.But going in Fmoc reaction before the couplings such as Asp6, Thr12, Leu16, Lys17, Glu18-Lys19; it is DBU/ piperidines/DMF=1/20/80(volume ratio that replacing removes to protect reagent) carry out; go to protect reagent place's constancy of volume, the protective reaction time is respectively 15min and 30min.In the time carrying out Glu18, two amino acid couplings of Lys19, taking two peptide fragment Fmoc-Glu-Lys-OH as raw material, a step coupling completes Glu18, the coupling of Lys19 amino acid simultaneously, and all the other amino acid coupling conditions are identical with embodiment 1.And at the complete Asp2 of coupling and with after DMF washing 3 times, contain propionyl chloride (5 equiv), DIEA(5 equiv toward adding in solid phase synthesis pipe) DMF solution (15mL), and control temperature 15-20 DEG C and react after 30min, suction filtration is removed liquid.Measure DMF(12 mL × 3) add the rear suction filtration of solid phase synthesis pipe washing to remove liquid.Proceed again the coupling of last amino acid Ser1.After all amino acid couplings finish, then go Fmoc operation, and with DMF washing 7 times.
Step 3
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (RinkAmide-MBHA-Resin)-OtBu is synthetic
Contain Ac2O(0.57mL toward adding in step 2 products therefrom peptide resin, 5 equiv), pyridine (0.48mL, 5 equiv) DMF(8mL), in 15-20 DEG C, after reaction 1h, suction filtration is removed liquid, then use DMF(18mL successively × 3), MeOH(18mL × 2), DCM(20mL × 2), MeOH(18mL × 2) washing resin.Take out Thymosin-Alpha1 peptide resin, after being dried, obtain 7.83g.
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) synthetic
Measure TFA(68 mL), TIS(4.0 mL) join successively in 100mL there-necked flask,, stirring and evenly mixing, is cooled to 15-20 DEG C, and step 3 gained peptide resin is added in there-necked flask.After being warmed up to 25-30 DEG C of reaction 1h, stop stirring.By reacting liquid filtering, and with TFA(5 mL × 2) washing resin, after merging, get filtrate, put underpressure distillation in Rotary Evaporators and remove most of TFA.Gained debris is joined in advance and is chilled in-10～-5 DEG C of methyl tertiary butyl ethers (120mL), carry out sedimentation.Gained suspension liquid washs after 6 times through repeated centrifugation, methyl tertiary butyl ether, obtains the thick peptide of pasty state, obtains thick peptide 4.71g after drying under reduced pressure.HPLC purity (area normalization method) 78.04%, obtaining in thick peptide through external standard method quantitative Analysis is 53.32% containing Thymosin-Alpha1 content, containing object peptide 2.51g, object peptide synthesis yield: 67%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-OH(Thymosin-Alpha1) RP-HPLC purifying
By thick step 4 gained peptide by C18 or 3 purifying of C8 post, obtain target product after turning salt, lyophilize.Purification condition for the first time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the second time: moving phase is: A phase: 0.3% acetic acid; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Purification condition for the third time: moving phase is: A phase: 0.1%TFA; B phase: acetonitrile.Detect wavelength 220nm.Flow velocity 80mL/min.Turn salt condition: moving phase: A phase: 20mM ammonium acetate-aqueous solution; B phase: acetonitrile; Detect wavelength 220nm.Flow velocity 80mL/min.Lyophilize condition is :-27 DEG C of 16h;-5 DEG C of 10h; 5 DEG C of 2h; 30 DEG C of 16h.Obtain Thymosin-Alpha1 essence peptide 2.03g, HPLC purity (area normalization method) is 99.79%, taking dry product Thymosin-Alpha1 content as 99.78%, and total recovery 54%.
HPLC detection method is as follows:
Chromatographic column: Waters X-bridge 5 μ m C18 (250mm × 4.6mm) chromatographic columns or quite post;
Mobile phase A: 0.1% triethylamine phosphoric acid is adjusted pH2.0;
Mobile phase B: acetonitrile;
Elution flow rate: 1ml/min;
Detect wavelength: 210nm;
Gradient:
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 86 | 14 |
| 32 | 80 | 20 |
| 33 | 86 | 14 |
| 45 | 86 | 14 |
The thick peptide peak area of object peptide yield calculation formula=Thymosin-Alpha1 reference substance concentration × Thymosin-Alpha1 reference substance content × Thymosin-Alpha1 in thick peptide × thick peptide amount/(amount × Thymosin-Alpha1 molecular weight of the thick peptide concentration × synthetic of Thymosin-Alpha1 reference substance peak area × Thymosin-Alpha1) × 100%=8.98 × 86.31% × 922.469 × 4710/(854.044 × 15.78 × 1.2 × 3108) × 100%=67%
Note: the testing conditions of Thymosin-Alpha1 reference substance and the thick peptide of Thymosin-Alpha1 is consistent with sample size, is all: 25 μ l.
Thymosin-Alpha1 reference substance concentration: 8.98 mg/ml
Thymosin-Alpha1 reference substance peak area: 854.044 mAU*min (Fig. 3)
Thymosin-Alpha1 reference substance content: 86.31%
The thick peptide compound concentration of Thymosin-Alpha1: 15.78 mg/ml
The thick peptide main peak of Thymosin-Alpha1 area: 922.469 mAU*min(Fig. 2)
Thymosin-Alpha1 essence peptide yield calculation formula=Thymosin-Alpha1 reference substance concentration × Thymosin-Alpha1 reference substance content × Thymosin-Alpha1 essence peptide peak area × smart peptide amount/(amount × Thymosin-Alpha1 molecular weight of Thymosin-Alpha1 reference substance peak area × Thymosin-Alpha1 essence peptide concentration × synthetic) × 100%=8.98 × 86.31% × 1176.377 × 2030/(854.044 × 10.76 × 1.2 × 3108) × 100%=54%
Note: Thymosin-Alpha1 reference substance is consistent with sample size with the testing conditions of Thymosin-Alpha1 essence peptide, is all: 25 μ l.
Thymosin-Alpha1 reference substance concentration: 8.98 mg/ml
Thymosin-Alpha1 reference substance peak area: 854.044 mAU*min (Fig. 3)
Thymosin-Alpha1 reference substance content: 86.31%
Thymosin-Alpha1 essence peptide compound concentration: 10.76 mg/ml
Thymosin-Alpha1 essence peptide peak area: 1176.377 mAU*min(Fig. 4).
Claims (5)
1. a method for synthetic Thymosin-Alpha1, described method steps is as follows:
Step 1, starts synthetic Fmoc-Asp (Rink Amide mbha resin)-OtBu by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and suitable substitution degree;
Step 2, the order according to Thymosin-Alpha1 peptide order carbon teminal to nitrogen end adopts coupling mode one by one or fragment method to carry out all amino acid whose connections on resin Fmoc-Asp (Rink Amide mbha resin)-OtBu, obtains Thymosin-Alpha1 peptide resin;
Step 3, carries out cracking to Thymosin-Alpha1 peptide resin, obtains thick peptide, through RP-HPLC purifying, turn salt, freeze-drying obtains Thymosin-Alpha1.
2. according to the method described in claim 1, it is characterized in that:
Wherein, synthetic described in step 1, is characterized in that:
Fmoc-Asp (Rink Amide mbha resin)-OtBu is generated under DIC/Cl-HOBt effect by the Rink Amide MBHA resin of Fmoc-Asp-OtBu and substitution degree 0.40-0.50mmol/g
Wherein, Thymosin-Alpha1 peptide resin described in step 2, structure is as follows:
Ac-Ser, (tBu)-Asp, (OtBu)-Ala-Ala-Val-Asp, (OtBu)-Thr, (tBu)-Ser, (tBu)-Ser, (tBu)-Glu, (OtBu)-Ile-Thr, (tBu)-Thr, (tBu)-Lys, (Boc)-Asp, (OtBu)-Leu-Lys, (Boc)-Glu, (OtBu)-Lys, (Boc)-Lys, (Boc)-Glu, (OtBu)-Val-Val-Glu, (OtBu)-Glu, (OtBu)-Ala-Glu, (OtBu)-Asp, (Rink Amide mbha resin)-OtBu
The wherein coupling mode described in step 2, is characterized in that:
In the time of Glu18, two amino acid whose couplings of Lys19, adopt the one step coupling of fragment method to complete
The wherein coupling mode described in step 2, is characterized in that:
Before the couplings such as Asp6, Thr12, Leu16, Lys17, Glu18-Lys19 going protection to adopt to mix goes to protect reagent to carry out protective reaction 2 times as piperidines/DMF solution, DBU/DMF solution or DBU/piperidines/DMF solution, and the protective reaction time is 15min, for the second time protective reaction 30min for the first time;
The wherein coupling mode described in step 2, is characterized in that:
After the complete Asp2 of coupling washing, peptide resin is carried out to special acylating reagent and carry out end-blocking operation as propionyl chloride, butyryl chloride, Tosyl chloride etc.;
The wherein coupling mode described in step 2, is characterized in that:
The complete all amino acid of coupling obtains Thymosin-Alpha1 28 peptide resins, adopts acetic anhydride/pyridine to carry out the acetylize of nitrogen end
Wherein, fragmentation pattern described in step 3, is characterized in that:
The cracking agent using is selected from: tri isopropyl silane and trifluoroacetic acid, its allocation ratio is tri isopropyl silane: trifluoroacetic acid=5: 95(volume ratio), the reaction times is 1 hour, temperature of reaction is 25 DEG C;
Wherein, RP-HPLC purifying described in step 3, turn salt operation, it is characterized in that:
RP-HPLC purifying, turn salt and adopt anti-phase C18 filler preparative chromatography post, TFA/ acetonitrile, HOAc/ acetonitrile system carry out as moving phase.
3. according to the method described in claim 1, it is characterized in that: in the time of Glu18, two amino acid whose couplings of Lys19, adopt two peptide fragment method one step couplings to complete, available dipeptides is: Fmoc-Glu(OtBu)-Lys(Boc)-OH.
4. according to the method described in claim 1; it is characterized in that: after the complete Asp2 of coupling washing; peptide resin is carried out to special acylating reagent and carry out end-blocking, wherein acylating reagent is non-acetylizad other acylating reagent, as: propionyl chloride, butyryl chloride, Tosyl chloride etc.
5. according to the method described in claim 1, it is characterized in that: fragment method, the conditions such as protective reaction condition, special reagent end-blocking of strengthening are combined after use, significantly improved purity and yield.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104356221A (en) * | 2014-10-24 | 2015-02-18 | 杭州阿德莱诺泰制药技术有限公司 | Preparation method of pexiganan |
| 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 |
| CN107417786A (en) * | 2017-05-04 | 2017-12-01 | 苏州强耀生物科技有限公司 | A kind of preparation method of Thymosin alpha 1 |
| CN108218980A (en) * | 2016-12-21 | 2018-06-29 | 鲁南制药集团股份有限公司 | A kind of synthetic method of thymalfasin |
| CN109762057A (en) * | 2019-03-11 | 2019-05-17 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of thymalfasin peptide resin cleavage method |
| CN119735634A (en) * | 2024-09-24 | 2025-04-01 | 深圳瑞德林生物技术有限公司 | Method for preparing polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104356221A (en) * | 2014-10-24 | 2015-02-18 | 杭州阿德莱诺泰制药技术有限公司 | Preparation method of pexiganan |
| 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 |
| CN104987382B (en) * | 2015-06-30 | 2018-11-30 | 济南康和医药科技有限公司 | A kind of method that dipeptide fragment Liquid solid Bonding prepares thymalfasin |
| CN108218980A (en) * | 2016-12-21 | 2018-06-29 | 鲁南制药集团股份有限公司 | A kind of synthetic method of thymalfasin |
| CN108218980B (en) * | 2016-12-21 | 2020-12-08 | 鲁南制药集团股份有限公司 | Synthesis method of thymalfasin |
| CN107417786A (en) * | 2017-05-04 | 2017-12-01 | 苏州强耀生物科技有限公司 | A kind of preparation method of Thymosin alpha 1 |
| CN107417786B (en) * | 2017-05-04 | 2021-03-09 | 苏州强耀生物科技有限公司 | Preparation method of thymosin alpha 1 |
| CN109762057A (en) * | 2019-03-11 | 2019-05-17 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of thymalfasin peptide resin cleavage method |
| CN119735634A (en) * | 2024-09-24 | 2025-04-01 | 深圳瑞德林生物技术有限公司 | Method for preparing polypeptide |
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