A kind of method for synthesizing thymalfasin
Technical field
The present invention relates to a kind of solid phase synthesis process of thymalfasin.
Background technology
Thymalfasin (Thymosin α 1, also referred to as Thymosin alpha 1) is thymosin extrasin primary bioactive components, is important in vivo
Immune regulator, is the polypeptide of 28 amino acid composition of nitrogen end acetylation.Research shows that thymalfasin promotes marrow stem thin
Born of the same parents' Development And Differentiation is lymphoblast and prolymphocyte;Inducer T lymphocyte breaks up and ripe, enters mature T cell
One step is divided into several different subgroups, such as kills cell, memory cell, effector cell, guidance T lymphocytes, and produce
Various soluble mediators;Reaction of the enhancing lymphocyte to mitogen, and the protein and nucleic acid of lymphoid tissue can be increased
Synthesis;Increase the generation of r-IFN, a-IFN, IL-2, IL-3 and lymphotoxin, strengthen antiviral and antineoplastic immune anti-
Should;Suppression of autoimmune responses;Recover the function of suppressor T lymphocyte.Current thymalfasin has been widely used in clinic, treats
Various diseases, are such as used for the treatment of chronic hepatitis B;For hepatitis C, hepatocellular carcinoma, malignant mela noma treatment;With
In the treatment for the treatment of infectious diseases, autoimmune disease, malignant tumour etc..
US4517119 is using the synthesis of full liquid phase method, purifying thymalfasin.Amino acid nitrogen end uses benzyl protection, goes every time
Protection is all carried out using Pd catalytic hydrogenations, and operation is extremely numerous and diverse, does not provide total recovery.US4504415 using full liquid phase method synthesis,
Reverse HPLC-purified thymalfasin.With 18 fragments of peptides(11-28 peptide fragments)It is 12% for initiation material calculates total recovery.US5856440
With mbha resin as carrier, it is Moz to use nitrogen end to protect(4- methoxyl group benzyloxy formoxyls)Amino acid carry out thymalfasin consolidate
It is combined to.Compared to the synthesis of Boc methods, Moz is easier to slough than Boc.Therefore during Moz method synthesis polypeptides, it is possible to decrease because repeatedly
Deprotection causes loss of the peptide chain from resin.So as to improve total synthesis yield.Moz methods synthesis thymalfasin yield 35%, than
The 25% of Boc methods is high.CN101484467 uses CTC resins synthesis thymalfasins.During synthesis, the Ile-Thr at sequence 11-12
Synthesized using pseudo proline dipeptides form.Final synthesis yield 40%.CN102199205 uses Wang resins synthesis thymus gland
Method is new, and it uses stepwise process to synthesize thymalfasin, and its thick peptide purity is 69.6%, and synthesis yield is 56.6%.Derive according to PEG
Wang resins afterwards are synthesized, then thick peptide purity is 57.2%, and synthesis yield is 64.4%.Total recovery is not reported.CN1291031
Thymalfasin is synthesized using thorugh biologic engineering method, and active contrast has been carried out with commodity thymalfasin.
The total technological difficulties that these existing synthetic methods are present are:(1)The technological operation of liquid phase synthesis thymalfasin is answered
It is miscellaneous, waste time and energy, it is unfavorable for industrialized production;(2)Existing solid-phase synthesis, obtain thymalfasin by coupling mode one by one more
Peptide resin.Due to multiple difficulties(Leu16, Lys17, Glu18, Lys19 etc.)Presence make thick peptide purity relatively low, easily include
The impurity close with thymalfasin retention properties and cause HPLC to purify difficulty and increase, total recovery is not high.
The content of the invention
It is an object of the invention to provide a kind of solid phase synthesis process of thymalfasin.The technical problem to be solved in the invention
It is:One suitable process route of selection, solves following technical problem:(1)Liquid phase synthesis thymalfasin complex operation, takes
When it is laborious, be unfavorable for industrialized production;(2)Existing solid-phase synthesis later-period purification difficulty is big, and product purity is low, yield is not high.
Preparation method of the invention is as shown in figure 1, first by the Fmoc-Asp-OtBu and Rink Amide of suitable substitution degree
MBHA resins start to synthesize Fmoc-Asp (Rink Amide mbha resins)-OtBu, then by Fmoc-Asp (Rink Amide
Mbha resin)-OtBu is synthesized by the way of being coupled one by one and obtains thymalfasin peptide resin, then carried out using acetic anhydride/pyridine
Nitrogen end acetylation;Finally thymalfasin peptide resin is cracked, thick peptide is obtained, high-purity thymus gland is obtained through RP-HPLC purifying
Method new soln, turn salt, freeze-drying through RP-HPLC again and obtain thymalfasin finished product.
Some conventional abbreviations have following meanings in the present invention;
Ac2O:Acetic anhydride
Pyridine:Pyridine
Boc:Tertbutyloxycarbonyl
Fmoc :9-fluorenylmethyloxycarbonyl
tBu :The tert-butyl group
Fmoc-AA-OH :The amino acid of 9-fluorenylmethyloxycarbonyl protection
Cl-HOBt :The chloro- I-hydroxybenzotriazoles of 6-
DIC :N, N '-Diisopropylcarbodiimide
Asp:Aspartic acid
Glu:Glutamic acid
Ala:Alanine
Val:Valine
Lys:Lysine
Leu:Leucine
Thr:Threonine
Ser:Serine
Ile:Isoleucine
DMF :N, N '-dimethylformamide
Piperidine :Hexahydropyridine
TFA :Trifluoracetic acid
TIS:Tri isopropyl silane
MeOH :Methyl alcohol
DCM:Dichloromethane
Kniser test detection reagents are:Ninhydrin
HOAc:Acetic acid
ACN:Acetonitrile
Therefore, the present invention provides a kind of method of solid cyclization into thymalfasin, methods described step is as follows:
Step 1, using Fmoc/tBu methods, with synthesis in solid state thymalfasin peptide resin on resin;
Step 2, two amino acid residues of Glu18, Lys19 are introduced in dipeptide fragment form;
Step 3, the deprotection before the coupling such as Asp6, Thr12, Leu16, Lys17, Glu18-Lys19 is using mixing
Deprotection reagent D BU/piperidines/DMF solution carries out 2 protective reactions, and the protective reaction time is first time 15min, the
Secondary protective reaction 30min;
Step 4, after being coupled Asp2 and having washed, special acylating reagent propionyl chloride etc. is carried out to peptide resin and is blocked
Operation.
Step 5, cracks to thymalfasin peptide resin, obtains thick peptide, through RP-HPLC purifying, turns salt, lyophilized obtains chest
Gland method is new.
Wherein, thymalfasin 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, dipeptide fragment form described in step 2 passes through Fmoc-Glu (OtBu)- Lys (Boc)-OH is coupled;
Wherein, described in step 3 mix deprotection reagent be as piperidines/DMF solution, DBU/DMF solution or DBU/piperidines/
DMF solution etc., preferably DBU/piperidines/DMF=1/5/20 (volume ratio).And deprotection is carried out using 2 times, first set reaction
15min, 25 DEG C of temperature;Second secondary response 30min, 25 DEG C of temperature;
Wherein, acylating reagent described in step 4 is propionyl chloride, butyl chloride, paratoluensulfonyl chloride, preferably propionyl chloride.Reaction bar
Part is fed intake for acyl chlorides using 10eq, and 15min is reacted at 20 DEG C.
Wherein, cracking uses decomposition agent described in step 5:TIS and TFA, its allocation ratio is TIS/TFA=5/95, and is cracked
The volume number of agent is 10 times of thymalfasin peptide resin mass number.Reaction time is 1 hour, and reaction temperature is 25 DEG C.Decomposition agent
Compound method is:5 volume TIS are added in the TFA of 95 volumes.
The present invention obtains the combination of specific synthesis mode by screening, will fragment method, reinforcement protective reaction condition, spy
The conditional joints such as different reagent end-blocking are used, and obtain a kind of in high yield and being easy to the thymalfasin synthetic method of purifying.
Beneficial effects of the present invention are illustrated below by way of experimental data:
Stepwise process is screened with fragment method:By Fmoc-Asp-OtBu and the Rink Amide MBHA resins of suitable substitution degree
Start to synthesize Fmoc-Asp (Rink Amide mbha resins)-OtBu, then by Fmoc-Asp (Rink Amide MBHA trees
Fat)-OtBu is synthesized by the way of being coupled one by one and obtains thymalfasin peptide resin, then nitrogen end second is carried out using acetic anhydride/pyridine
It is acylated;Finally thymalfasin peptide resin is cracked, the thick peptide of thymalfasin is obtained.Detection is sampled to thick peptide.Wherein exist
18th, following two modes are employed respectively when 19 two position residues are coupled:Fmoc-Lys (Boc)-OH is first coupled, then is coupled
The stepwise process of Fmoc-Glu (OtBu)-OH;And Fmoc- Glu (OtBu)-Lys (Boc)-OH fragment methods.
Purpose peptide is prepared using above-mentioned condition, the thymalfasin of different yields and purity is respectively obtained, result data is as follows:
Deprotecting regent (volume ratio) | Thick peptide purity | Synthetic peptide yield |
Stepwise process | 42.36% | 24% |
Fragment method | 51.22% | 33% |
General deprotection and the de-protected screening of reinforcement:
Start to synthesize Fmoc-Asp by the Rink Amide MBHA resins of Fmoc-Asp-OtBu and suitable substitution degree
(Rink Amide mbha resins)-OtBu, then by Fmoc-Asp (Rink Amide mbha resins)-OtBu using even one by one
The mode of connection synthesizes and obtains thymalfasin peptide resin, then carries out nitrogen end acetylation using acetic anhydride/pyridine;Finally to thymalfasin
Peptide resin is cracked, and obtains the thick peptide of thymalfasin.Detection is sampled to thick peptide.But Asp6, Thr12, Leu16,
Deprotection before the coupling such as Lys17, Glu18-Lys19 is respectively adopted general deprotection method and strengthens deprotection method.It is logical
Spending Protection Code is:Using deprotection reagent piperidines/DMF=1/4(Volume ratio)2 protective reactions are carried out, during protective reaction
Between be first time 5min, second protective reaction 10min, reaction temperature all be 25 DEG C.Strengthening deprotection method is:Using mixing
It is that deprotection reagent D BU/piperidines/DMF=1/5/20 (volume ratio) carries out 2 protective reactions, and protective reaction time
15min, second protective reaction 30min, reaction temperature are all 25 DEG C.
Purpose peptide is prepared using above-mentioned condition, the thymalfasin of different yields and purity is respectively obtained, result data is as follows:
Deprotection mode | Thick peptide purity | Synthetic peptide yield |
General method | 42.36% | 24% |
Reinforcement | 57.01% | 37% |
The screening of special capping reagent:By Fmoc-Asp-OtBu and the Rink Amide MBHA resins of suitable substitution degree
Start to synthesize Fmoc-Asp (Rink Amide mbha resins)-OtBu, then by Fmoc-Asp (Rink Amide MBHA trees
Fat)-OtBu is synthesized by the way of being coupled one by one and obtains thymalfasin peptide resin, then nitrogen end second is carried out using acetic anhydride/pyridine
It is acylated;Finally thymalfasin peptide resin is cracked, the thick peptide of thymalfasin is obtained, high-purity chest is obtained through RP-HPLC purifying
Gland method new soln, turn salt, freeze-drying through RP-HPLC again and obtain thymalfasin finished product.It is right after being coupled Asp2 and having washed
Peptide resin carries out two kinds of processing methods.Respectively directly carry out next amino acid couplings;It is laggard with being blocked using acylating reagent
The next amino acid couplings of row.
Purpose peptide is prepared using above-mentioned condition, the thymalfasin of different yields and purity is respectively obtained, result data is as follows:
Deprotection mode | Thick peptide purity | Synthesis yield | Fine peptide purity | Total recovery |
It is uncapped | 42.36% | 24% | 96.35% | 11% |
Propionyl chloride is blocked | 42.10% | 24% | 98.06% | 17% |
Compared to the prior art the method for the present invention has obvious advantage, and relevant contrast experiment is as follows:
First, thick peptide purity, synthesis yield and total recovery compare:
Yield calculating is carried out with the method for the inventive method and prior art, it is as a result as follows:
Method for detecting purity therein is as follows:
HPLC detection methods are as follows:
Chromatographic column:5 μm of C18 (250mm × 4.6mm) chromatographic columns of Waters X-bridge or suitable post;Mobile phase A:
0.1% triethylamine phosphoric acid adjusts pH2.0;Mobile phase B:Acetonitrile;Elution flow rate:1ml/min;Detection wavelength:210nm;Gradient:
Time(min) | Mobile phase A(%) | Mobile phase B(%) |
0 | 86 | 14 |
32 | 80 | 20 |
33 | 86 | 14 |
45 | 86 | 14 |
The beneficial effects of the invention are as follows:The combination of specific synthesis mode is obtained by screening, will fragment method, reinforcement go to protect
Shield reaction condition, special reagent end-blocking etc. conditional joint use, obtain it is a kind of in high yield and be easy to purifying thymalfasin synthesis
Method.
Brief description of the drawings
Fig. 1 is synthetic route chart of the present invention;
Fig. 2 is the thick peptide HPLC chromatogram of the thymalfasin of embodiment 5;
Fig. 3 is the HPLC chromatogram of thymalfasin reference substance;
Fig. 4 is the thymalfasin fine peptide HPLC chromatogram of embodiment 5.
Specific embodiment
The present invention is further illustrated by the following examples.
Embodiment 1:Thymalfasin is synthesized using coupling method one by one
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu synthesizes
Weigh Rink Amide MBHA resins(Substitution degree 0.45mmol/g)(2.67 g, 1.0 equiv)Add solid phase
In composite tube, DCM is added(10mL), 10-30 DEG C of swelling 30min of stirring of temperature control, suction filtration removing liquid.Add DMF(10mL×
2)Washing.Measure 20%(V/v, similarly hereinafter)Piperidine/DMF solution(9.0 mL), add in synthesis in solid state pipe, temperature control 20-30
DEG C stirring reaction 5min, suction filtration removes liquid.20%Piperidine/DMF solution is measured again(9.0 mL), add solid phase to close
Into in pipe, 20-30 DEG C of stirring reaction 10min of temperature control, suction filtration removes liquid.Measure DMF(10 mL×7)Add synthesis in solid state pipe
Suction filtration removes liquid after washing.Separately weigh Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4
equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to use ice salt bath after being completely dissolved
0-10 DEG C is cooled to, DIC is added(0.89 mL, 4.8 equiv), stirring, then 0-10 DEG C of reaction 5min of temperature control react this
Liquid is added in synthesis in solid state pipe, and suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Taken out after adding the washing of synthesis in solid state pipe
Filter liquid.Ninhydrin detection 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 synthesizes
Step 1 product amino acid resin is sloughed into Fmoc by the same way.Weigh Fmoc-AA-OH(4 equiv), Cl-
HOBt(4equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to be used after being completely dissolved
Ice salt bath is cooled to 5-10 DEG C, adds DIC(4.8 equiv), stirring, 5-25 DEG C of reaction 5min of temperature control, then by this reaction solution
It is added in synthesis in solid state pipe, suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Add suction filtration after the washing of synthesis in solid state pipe
Remove liquid.Ninhydrin detects that such as result is the positive, then repeat coupling once.After being coupled all amino acid successively, then
Carry out Fmoc to operate, and washed with DMF 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 synthesizes
Added toward step 2 products therefrom peptide resin and contain Ac2O(0.57mL, 5 equiv), pyridine (0.48mL, 5
Equiv DMF)(8mL), in 15-20 DEG C, suction filtration removes liquid after reaction 1h, then uses DMF successively(18mL×3)、MeOH
(18mL×2)、DCM(20mL×2)、MeOH(18mL×2)Washing resin.Thymalfasin peptide resin is taken out, after drying
7.20g。
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Synthesis
Measure TFA(68 mL)、TIS(4.0 mL)It is added sequentially in 100mL there-necked flasks, stir and evenly mix, it is cooled to 15-
20 DEG C, by step 3 gained peptide resin addition there-necked flask.Stop stirring after being warmed up to 25-30 DEG C of reaction 1h.By reaction solution mistake
Filter, and use TFA(5 mL×2)Washing resin, takes filtrate after merging, vacuum distillation removes major part TFA in putting Rotary Evaporators.
Gained Liquid Residue is added to and pre- is cooled to -10~-5 DEG C of methyl tertiary butyl ether(MTBE)s(120mL)In, settled.Gained suspension is passed through
After repeated centrifugation, methyl tertiary butyl ether(MTBE) wash 6 times, the thick peptide of pasty state is obtained, thick peptide 4.12g is obtained after drying under reduced pressure.HPLC purity(Face
Product normalization method)42.36%.0.90g containing thymalfasin, synthesis yield in thick peptide are calculated through quantified by external standard method:24%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)RP-HPLC is purified
The thick peptide of step 4 gained is obtained into target product after C18 or C8 posts are purified for 3 times, turn salt, freeze-drying.First
Secondary purification condition:Mobile phase is:A phases: 0.1%TFA;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Second pure
Change condition:Mobile phase is:A phases:0.3% acetic acid;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Third time is purified
Condition:Mobile phase is:A phases:0.1%TFA ;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Turn salt condition:Flowing
Phase:A phases:20mM ammonium acetates-the aqueous solution;B phases:Acetonitrile;Detection wavelength 220nm.Flow velocity 80mL/min.Freeze-drying condition is:-
27℃ 16h;-5℃ 10h;5℃ 2h; 30℃ 16h.Obtain target product 0.41g, HPLC purity(Area normalization method)
96.35%, total recovery 11%.
Embodiment 2:Thymalfasin is synthesized using fragment coupling method
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Prepare
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu synthesizes
Weigh Rink Amide MBHA resins(Substitution degree 0.45mmol/g)(2.67 g, 1.0 equiv)Add solid phase
In composite tube, DCM is added(10mL), 10-30 DEG C of swelling 30min of stirring of temperature control, suction filtration removing liquid.Add DMF(10mL×
2)Washing.Measure 20%(V/v, similarly hereinafter)Piperidine/DMF solution(9.0 mL), add in synthesis in solid state pipe, temperature control 20-30
DEG C stirring reaction 5min, suction filtration removes liquid.20%Piperidine/DMF solution is measured again(9.0 mL), add solid phase to close
Into in pipe, 20-30 DEG C of stirring reaction 10min of temperature control, suction filtration removes liquid.Measure DMF(10 mL×7)Add synthesis in solid state pipe
Suction filtration removes liquid after washing.Separately weigh Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4
equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to use ice salt bath after being completely dissolved
0-10 DEG C is cooled to, DIC is added(0.89 mL, 4.8 equiv), stirring, then 0-10 DEG C of reaction 5min of temperature control react this
Liquid is added in synthesis in solid state pipe, and suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Taken out after adding the washing of synthesis in solid state pipe
Filter liquid.Ninhydrin detection 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 synthesizes
Step 1 product amino acid resin is sloughed into Fmoc by the same way.Weigh Fmoc-AA-OH(4 equiv), Cl-
HOBt(4equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to be used after being completely dissolved
Ice salt bath is cooled to 5-10 DEG C, adds DIC(4.8 equiv), stirring, 5-25 DEG C of reaction 5min of temperature control, then by this reaction solution
It is added in synthesis in solid state pipe, suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Add suction filtration after the washing of synthesis in solid state pipe
Remove liquid.Ninhydrin detects that such as result is the positive, then repeat coupling once.Carrying out two amino of Glu18, Lys19
During acid coupling, with dipeptide fragment Fmoc-Glu-Lys-OH as raw material, step coupling completes Glu18, Lys19 amino acid couplings, its
Remaining amino acid couplings condition is same as Example 1.After being coupled all amino acid successively, then carry out Fmoc and operate, and use 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 synthesizes
Added toward step 2 products therefrom peptide resin and contain Ac2O(0.57mL, 5 equiv), pyridine (0.48mL, 5
Equiv DMF)(8mL), in 15-20 DEG C, suction filtration removes liquid after reaction 1h, then uses DMF successively(18mL×3)、MeOH
(18mL×2)、DCM(20mL×2)、MeOH(18mL×2)Washing resin.Thymalfasin peptide resin is taken out, after drying
7.06g。
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Synthesis
Measure TFA(68 mL)、TIS(4.0 mL)It is added sequentially in 100mL there-necked flasks, stir and evenly mix, it is cooled to 15-
20 DEG C, by step 3 gained peptide resin addition there-necked flask.Stop stirring after being warmed up to 25-30 DEG C of reaction 1h.By reaction solution mistake
Filter, and use TFA(5 mL×2)Washing resin, takes filtrate after merging, vacuum distillation removes major part TFA in putting Rotary Evaporators.
Gained Liquid Residue is added to and pre- is cooled to -10~-5 DEG C of methyl tertiary butyl ether(MTBE)s(120mL)In, settled.Gained suspension is passed through
After repeated centrifugation, methyl tertiary butyl ether(MTBE) wash 6 times, the thick peptide of pasty state is obtained, thick peptide 4.23g is obtained after drying under reduced pressure.HPLC purity(Face
Product normalization method)51.22%.1.22g containing thymalfasin, synthesis yield in thick peptide are calculated through quantified by external standard method:33%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)RP-HPLC is purified
The thick peptide of step 4 gained is obtained into target product after C18 or C8 posts are purified for 3 times, turn salt, freeze-drying.First
Secondary purification condition:Mobile phase is:A phases: 0.1%TFA;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Second pure
Change condition:Mobile phase is:A phases:0.3% acetic acid;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Third time is purified
Condition:Mobile phase is:A phases:0.1%TFA ;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Turn salt condition:Flowing
Phase:A phases:20mM ammonium acetates-the aqueous solution;B phases:Acetonitrile;Detection wavelength 220nm.Flow velocity 80mL/min.Freeze-drying condition is:-
27℃ 16h;-5℃ 10h;5℃ 2h; 30℃ 16h.Obtain target product 0.75g, HPLC purity(Area normalization method)
97.34%, total recovery 20%.
Embodiment 3:Using special reagent end-blocking synthesis thymalfasin
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Prepare
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu synthesizes
Weigh Rink Amide MBHA resins(Substitution degree 0.45mmol/g)(2.67 g, 1.0 equiv)Add solid phase
In composite tube, DCM is added(10mL), 10-30 DEG C of swelling 30min of stirring of temperature control, suction filtration removing liquid.Add DMF(10mL×
2)Washing.Measure 20%(V/v, similarly hereinafter)Piperidine/DMF solution(9.0 mL), add in synthesis in solid state pipe, temperature control 20-30
DEG C stirring reaction 5min, suction filtration removes liquid.20%Piperidine/DMF solution is measured again(9.0 mL), add solid phase to close
Into in pipe, 20-30 DEG C of stirring reaction 10min of temperature control, suction filtration removes liquid.Measure DMF(10 mL×7)Add synthesis in solid state pipe
Suction filtration removes liquid after washing.Separately weigh Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4
equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to use ice salt bath after being completely dissolved
0-10 DEG C is cooled to, DIC is added(0.89 mL, 4.8 equiv), stirring, then 0-10 DEG C of reaction 5min of temperature control react this
Liquid is added in synthesis in solid state pipe, and suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Taken out after adding the washing of synthesis in solid state pipe
Filter liquid.Ninhydrin detection 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 synthesizes
Step 1 product amino acid resin is sloughed into Fmoc by the same way.Weigh Fmoc-AA-OH(4 equiv), Cl-
HOBt(4equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to be used after being completely dissolved
Ice salt bath is cooled to 5-10 DEG C, adds DIC(4.8 equiv), stirring, 5-25 DEG C of reaction 5min of temperature control, then by this reaction solution
It is added in synthesis in solid state pipe, suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Add suction filtration after the washing of synthesis in solid state pipe
Remove liquid.Ninhydrin detects that such as result is the positive, then repeat coupling once.But it is being coupled Asp2 and is being washed with DMF
After washing 3 times, propionyl chloride is contained toward interior addition of synthesis in solid state pipe(5 equiv)、DIEA(5 equiv)DMF solution(15mL), and
After 15-20 DEG C of reaction 30min of control temperature, suction filtration removes liquid.Measure DMF(12 mL×3)After adding the washing of synthesis in solid state pipe
Suction filtration removes liquid.It is further continued for carrying out the coupling of last amino acid Ser1, after all amino acid couplings terminate, then is gone
Fmoc is operated, and is washed with DMF 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 synthesizes
Added toward step 2 products therefrom peptide resin and contain Ac2O(0.57mL, 5 equiv), pyridine (0.48mL, 5
Equiv DMF)(8mL), in 15-20 DEG C, suction filtration removes liquid after reaction 1h, then uses DMF successively(18mL×3)、MeOH
(18mL×2)、DCM(20mL×2)、MeOH(18mL×2)Washing resin.Thymalfasin peptide resin is taken out, after drying
7.25g。
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Synthesis
Measure TFA(68 mL)、TIS(4.0 mL)It is added sequentially in 100mL there-necked flasks, stir and evenly mix, it is cooled to 15-
20 DEG C, by step 3 gained peptide resin addition there-necked flask.Stop stirring after being warmed up to 25-30 DEG C of reaction 1h.By reaction solution mistake
Filter, and use TFA(5 mL×2)Washing resin, takes filtrate after merging, vacuum distillation removes major part TFA in putting Rotary Evaporators.
Gained Liquid Residue is added to and pre- is cooled to -10~-5 DEG C of methyl tertiary butyl ether(MTBE)s(120mL)In, settled.Gained suspension is passed through
After repeated centrifugation, methyl tertiary butyl ether(MTBE) wash 6 times, the thick peptide of pasty state is obtained, thick peptide 4.05g is obtained after drying under reduced pressure.HPLC purity(Face
Product normalization method)42.10%.1.10g containing thymalfasin, synthesis yield in thick peptide are calculated through quantified by external standard method:24%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)RP-HPLC is purified
The thick peptide of step 4 gained is obtained into target product after C18 or C8 posts are purified for 3 times, turn salt, freeze-drying.First
Secondary purification condition:Mobile phase is:A phases: 0.1%TFA;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Second pure
Change condition:Mobile phase is:A phases:0.3% acetic acid;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Third time is purified
Condition:Mobile phase is:A phases:0.1%TFA ;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Turn salt condition:Flowing
Phase:A phases:20mM ammonium acetates-the aqueous solution;B phases:Acetonitrile;Detection wavelength 220nm.Flow velocity 80mL/min.Freeze-drying condition is:-
27℃ 16h;-5℃ 10h;5℃ 2h; 30℃ 16h.Obtain target product 0.62g, HPLC purity(Area normalization method)
98.06%, total recovery 17%.
Embodiment 4:Synthesize thymalfasin using deprotection condition is strengthened
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Prepare
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu synthesizes
Weigh Rink Amide MBHA resins(Substitution degree 0.45mmol/g)(2.67 g, 1.0 equiv)Add solid phase
In composite tube, DCM is added(10mL), 10-30 DEG C of swelling 30min of stirring of temperature control, suction filtration removing liquid.Add DMF(10mL×
2)Washing.Measure 20%(V/v, similarly hereinafter)Piperidine/DMF solution(9.0 mL), add in synthesis in solid state pipe, temperature control 20-30
DEG C stirring reaction 5min, suction filtration removes liquid.20%Piperidine/DMF solution is measured again(9.0 mL), add solid phase to close
Into in pipe, 20-30 DEG C of stirring reaction 10min of temperature control, suction filtration removes liquid.Measure DMF(10 mL×7)Add synthesis in solid state pipe
Suction filtration removes liquid after washing.Separately weigh Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4
equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to use ice salt bath after being completely dissolved
0-10 DEG C is cooled to, DIC is added(0.89 mL, 4.8 equiv), stirring, then 0-10 DEG C of reaction 5min of temperature control react this
Liquid is added in synthesis in solid state pipe, and suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Taken out after adding the washing of synthesis in solid state pipe
Filter liquid.Ninhydrin detection 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 synthesizes
Step 1 product amino acid resin is sloughed into Fmoc by the same way.Weigh Fmoc-AA-OH(4 equiv), Cl-
HOBt(4equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to be used after being completely dissolved
Ice salt bath is cooled to 5-10 DEG C, adds DIC(4.8 equiv), stirring, 5-25 DEG C of reaction 5min of temperature control, then by this reaction solution
It is added in synthesis in solid state pipe, suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Add suction filtration after the washing of synthesis in solid state pipe
Remove liquid.Ninhydrin detects that such as result is the positive, then repeat coupling once.But Asp6, Thr12, Leu16,
Going in Fmoc reactions before the coupling such as Lys17, Glu18, Lys19, it is DBU/ piperidines/DMF=1/20/ to change deprotection reagent
80(Volume ratio)Carry out, the constancy of volume of deprotection reagent place, but the protective reaction time is respectively 15min and 30min.It is all
After amino acid couplings terminate, then carry out Fmoc and operate, and washed with DMF 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 synthesizes
Added toward step 2 products therefrom peptide resin and contain Ac2O(0.57mL, 5 equiv), pyridine (0.48mL, 5
Equiv DMF)(8mL), in 15-20 DEG C, suction filtration removes liquid after reaction 1h, then uses DMF successively(18mL×3)、MeOH
(18mL×2)、DCM(20mL×2)、MeOH(18mL×2)Washing resin.Thymalfasin peptide resin is taken out, after drying
7.44g。
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Synthesis
Measure TFA(68 mL)、TIS(4.0 mL)It is added sequentially in 100mL there-necked flasks, stir and evenly mix, it is cooled to 15-
20 DEG C, by step 3 gained peptide resin addition there-necked flask.Stop stirring after being warmed up to 25-30 DEG C of reaction 1h.By reaction solution mistake
Filter, and use TFA(5 mL×2)Washing resin, takes filtrate after merging, vacuum distillation removes major part TFA in putting Rotary Evaporators.
Gained Liquid Residue is added to and pre- is cooled to -10~-5 DEG C of methyl tertiary butyl ether(MTBE)s(120mL)In, settled.Gained suspension is passed through
After repeated centrifugation, methyl tertiary butyl ether(MTBE) wash 6 times, the thick peptide of pasty state is obtained, thick peptide 4.39g is obtained after drying under reduced pressure.HPLC purity(Face
Product normalization method)57.01%.1.38g containing thymalfasin, synthesis yield in thick peptide are calculated through quantified by external standard method:37%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)RP-HPLC is purified
The thick peptide of step 4 gained is obtained into target product after C18 or C8 posts are purified for 3 times, turn salt, freeze-drying.First
Secondary purification condition:Mobile phase is:A phases: 0.1%TFA;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Second pure
Change condition:Mobile phase is:A phases:0.3% acetic acid;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Third time is purified
Condition:Mobile phase is:A phases:0.1%TFA ;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Turn salt condition:Flowing
Phase:A phases:20mM ammonium acetates-the aqueous solution;B phases:Acetonitrile;Detection wavelength 220nm.Flow velocity 80mL/min.Freeze-drying condition is:-
27℃ 16h;-5℃ 10h;5℃ 2h; 30℃ 16h.Obtain target product 0.73g, HPLC purity(Area normalization method)
97.89%, total recovery 20%.
Embodiment 5:Joint is using the technique synthesis thymus gland method such as fragment coupling method, reinforcement deprotection condition and propionyl chloride end-blocking
Newly.
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Prepare
Step 1
Fmoc-Asp (RinkAmide-MBHA-Resin)-OtBu synthesizes
Weigh Rink Amide MBHA resins(Substitution degree 0.45mmol/g)(2.67 g, 1.0 equiv)Add solid phase
In composite tube, DCM is added(10mL), 10-30 DEG C of swelling 30min of stirring of temperature control, suction filtration removing liquid.Add DMF(10mL×
2)Washing.Measure 20%(V/v, similarly hereinafter)Piperidine/DMF solution(9.0 mL), add in synthesis in solid state pipe, temperature control 20-30
DEG C stirring reaction 5min, suction filtration removes liquid.20%Piperidine/DMF solution is measured again(9.0 mL), add solid phase to close
Into in pipe, 20-30 DEG C of stirring reaction 10min of temperature control, suction filtration removes liquid.Measure DMF(10 mL×7)Add synthesis in solid state pipe
Suction filtration removes liquid after washing.Separately weigh Fmoc-Asp-OtBu(1.97 g, 4 equiv), Cl-HOBt(0.82 g, 4
equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to use ice salt bath after being completely dissolved
0-10 DEG C is cooled to, DIC is added(0.89 mL, 4.8 equiv), stirring, then 0-10 DEG C of reaction 5min of temperature control react this
Liquid is added in synthesis in solid state pipe, and suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Taken out after adding the washing of synthesis in solid state pipe
Filter liquid.Ninhydrin detection 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 synthesizes
Step 1 product amino acid resin is sloughed into Fmoc by the same way.Weigh Fmoc-AA-OH(4 equiv), Cl-
HOBt(4equiv)Add beaker.Measure DMF/DCM=1/1 mixed solvents(6.5 mL)Beaker, stirring is added to be used after being completely dissolved
Ice salt bath is cooled to 5-10 DEG C, adds DIC(4.8 equiv), stirring, 5-25 DEG C of reaction 5min of temperature control, then by this reaction solution
It is added in synthesis in solid state pipe, suction filtration removes liquid after 3h.Measure DMF(12 mL×3)Add suction filtration after the washing of synthesis in solid state pipe
Remove liquid.Ninhydrin detects that such as result is the positive, then repeat coupling once.But Asp6, Thr12, Leu16,
Going in Fmoc reactions before the coupling such as Lys17, Glu18-Lys19, it is DBU/ piperidines/DMF=1/20/ to change deprotection reagent
80(Volume ratio)Carry out, deprotection reagent place constancy of volume, the protective reaction time is respectively 15min and 30min.Exist simultaneously
When carrying out two amino acid couplings of Glu18, Lys19, with dipeptide fragment Fmoc-Glu-Lys-OH as raw material, step coupling is completed
Glu18, Lys19 amino acid couplings, remaining amino acid couplings condition are same as Example 1.And it is being coupled Asp2 and is using DMF
After washing 3 times, propionyl chloride is contained toward interior addition of synthesis in solid state pipe(5 equiv)、DIEA(5 equiv)DMF solution(15mL),
And after controlling 15-20 DEG C of reaction 30min of temperature, suction filtration removes liquid.Measure DMF(12 mL×3)Add the washing of synthesis in solid state pipe
Suction filtration removes liquid afterwards.It is further continued for carrying out the coupling of last amino acid Ser1.After all amino acid couplings terminate, then enter
Row goes Fmoc to operate, and is washed with DMF 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 synthesizes
Added toward step 2 products therefrom peptide resin and contain Ac2O(0.57mL, 5 equiv), pyridine (0.48mL, 5
Equiv DMF)(8mL), in 15-20 DEG C, suction filtration removes liquid after reaction 1h, then uses DMF successively(18mL×3)、MeOH
(18mL×2)、DCM(20mL×2)、MeOH(18mL×2)Washing resin.Thymalfasin peptide resin is taken out, after drying
7.83g。
Step 4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)Synthesis
Measure TFA(68 mL)、TIS(4.0 mL)It is added sequentially in 100mL there-necked flasks, stir and evenly mix, it is cooled to 15-
20 DEG C, by step 3 gained peptide resin addition there-necked flask.Stop stirring after being warmed up to 25-30 DEG C of reaction 1h.By reaction solution mistake
Filter, and use TFA(5 mL×2)Washing resin, takes filtrate after merging, vacuum distillation removes major part TFA in putting Rotary Evaporators.
Gained Liquid Residue is added to and pre- is cooled to -10~-5 DEG C of methyl tertiary butyl ether(MTBE)s(120mL)In, settled.Gained suspension is passed through
After repeated centrifugation, methyl tertiary butyl ether(MTBE) wash 6 times, the thick peptide of pasty state is obtained, thick peptide 4.71g is obtained after drying under reduced pressure.HPLC purity(Face
Product normalization method)78.04%, content containing thymalfasin is 53.32%, i.e. peptide containing purpose in calculating thick peptide through quantified by external standard method
2.51g, purpose peptide symthesis yield:67%.
Step 5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Thymalfasin)RP-HPLC is purified
The thick peptide of step 4 gained is obtained into target product after C18 or C8 posts are purified for 3 times, turn salt, freeze-drying.First
Secondary purification condition:Mobile phase is:A phases: 0.1%TFA;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Second pure
Change condition:Mobile phase is:A phases:0.3% acetic acid;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Third time is purified
Condition:Mobile phase is:A phases:0.1%TFA ;B phases:Acetonitrile.Detection wavelength 220nm.Flow velocity 80mL/min.Turn salt condition:Flowing
Phase:A phases:20mM ammonium acetates-the aqueous solution;B phases:Acetonitrile;Detection wavelength 220nm.Flow velocity 80mL/min.Freeze-drying condition is:-
27℃ 16h;-5℃ 10h;5℃ 2h; 30℃ 16h.Obtain thymalfasin fine peptide 2.03g, HPLC purity(Area normalization method)
It is 99.79%, it is 99.78% to count thymalfasin content with dry product, total recovery 54%.
HPLC detection methods are as follows:
Chromatographic column:5 μm of C18 (250mm × 4.6mm) chromatographic columns of Waters X-bridge or suitable post;
Mobile phase A:0.1% triethylamine phosphoric acid adjusts pH2.0;
Mobile phase B:Acetonitrile;
Elution flow rate:1ml/min;
Detection wavelength:210nm;
Gradient:
Time(min) | Mobile phase A(%) | Mobile phase B(%) |
0 | 86 | 14 |
32 | 80 | 20 |
33 | 86 | 14 |
45 | 86 | 14 |
Purpose peptide yield computing formula=thymalfasin reference substance concentration × thymalfasin reference substance content × thymus gland in thick peptide
The new thick peptide peak area of method × thick peptide amount/(The amount of the thick peptide concentration × synthetic of thymalfasin reference substance peak area × thymalfasin
× thymalfasin molecular weight)×100%=8.98×86.31%×922.469×4710/(854.044×15.78×1.2×
3108)×100%=67%
Note:Thymalfasin reference substance is consistent with sample size with the testing conditions of the thick peptide of thymalfasin, all for:25μl.
Thymalfasin reference substance concentration:8.98 mg/ml
Thymalfasin reference substance peak area:854.044 mAU*min (Fig. 3)
Thymalfasin reference substance content:86.31%
The thick peptide compound concentration of thymalfasin:15.78 mg/ml
The thick peptide main peak area of thymalfasin:922.469 mAU*min(Fig. 2)
Thymalfasin fine peptide yield computing formula=thymalfasin reference substance concentration × thymalfasin reference substance content × thymus gland
The new fine peptide peak area × fine peptide amount of method/(The amount of thymalfasin reference substance peak area × thymalfasin fine peptide concentration × synthetic
× thymalfasin molecular weight)×100%=8.98×86.31%×1176.377×2030/(854.044×10.76×1.2×
3108)×100%=54%
Note:Thymalfasin reference substance is consistent with sample size with the testing conditions of thymalfasin fine peptide, all for:25μl.
Thymalfasin reference substance concentration:8.98 mg/ml
Thymalfasin reference substance peak area:854.044 mAU*min (Fig. 3)
Thymalfasin reference substance content:86.31%
Thymalfasin fine peptide compound concentration:10.76 mg/ml
Thymalfasin fine peptide peak area:1176.377 mAU*min(Fig. 4).