CN102199205A - Synthetic method of polypeptide thymosin alpha1 - Google Patents
Synthetic method of polypeptide thymosin alpha1 Download PDFInfo
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- CN102199205A CN102199205A CN2011100698768A CN201110069876A CN102199205A CN 102199205 A CN102199205 A CN 102199205A CN 2011100698768 A CN2011100698768 A CN 2011100698768A CN 201110069876 A CN201110069876 A CN 201110069876A CN 102199205 A CN102199205 A CN 102199205A
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Abstract
The invention discloses a preparation method of thymosin alpha1 through solid phase polypeptide synthesis, comprising the steps of: (1) mixing Fmoc-Asn(Trt)-OH with hydroxyl functional resin, and performing esterification to the mixture so as to obtain Fmoc-Asn(Trt)- resin; (2) mixing Fmoc-Asn(Trt)- resin with a deprotection agent, thus obtaining Asn(Trt)- resin; (3) condensing Fmoc-Glu(OtBu)-OH and Asn(Trt)- resin, thus obtaining Fmoc-Glu(OtBu)-Asn(Trt)-resin; (4) according to solid phase synthesis method, repeating Fmoc removal in step (2) and condensation of amino acid and polypeptide on resin in step (3), and condensing amino acid from end C to end N in an order of Glu to Ser with polypeptide on resin, thus forming polypeptide resin as shown in formula II; and (5) separating the polypeptide on the polypeptide resin as shown in formula II and resin, thus obtaining thymosin alpha1 as shown in formula I.
Description
Technical field
The present invention relates to polypeptide solid phase synthesis field, relate in particular to the solid phase synthesis process of Thymosin-Alpha1.
Background technology
Zadaxin (having another name called thymosin, thymopeptide-5, Thymosin) is one group of polypeptide that the thymic tissue excretory has physiologically active.Can promote lymphocyte transformation, strengthen the macrophage phagocytic activity, can be used for treating panimmunity defective disease.
Single there are some researches show inside and outside in Thymosin-Alpha1 (Thymosin α 1) the treatment chronic viral hepatitis B, list is treated chronic viral hepatitis B with Thymosin-Alpha1, continue response rate (ALT again normal+DNA turn out cloudy+HBeAg turns out cloudy) about 37%,, but do not have the toxic side effect of Interferon, rabbit with single close with Interferon, rabbit.
The clinical study of using Thymosin-Alpha1 treatment chronic viral hepatitis B at TaiWan, China shows that Thymosin-Alpha1 has the treatment effect of delay.After the Thymosin-Alpha1 treatment finished, ALT had the property a crossed rising, and with the serum conversion of HBeAg, HBV DNA disappears simultaneously.
The result of study of HBV DNA negative conversion rate and serum conversion superiority shows: single have long-term efficacy with Thymosin-Alpha1 treatment chronic viral hepatitis B, and do not have obvious adverse reaction.For the patients with liver fibrosis in late period, the Thymosin-Alpha1 result of treatment of high dosage is better.
The chemosynthesis Zadaxin mainly contains solid-phase synthesis at present, as having reported the isolation and purification technical study of chemosynthesis Thymosin-Alpha1 in the 8th Chinese Chemical Society analytical chemistry annual meeting and the meeting of the 8th national atomic spectrum science, the synthetic Thymosin-Alpha1 of Fmoc solid phase method is analyzed
Though process for solid phase synthesis is very ripe, but the amino acid whose ultimate yield that is difficult to the synthetic sequence may very low (30%-45%), this may limit the amplification of technology from the angle of economy, can cause polypeptide purity deficiency from pharmaceutical industry to the requirement of polypeptide purity.
The acid residual position of Zadaxin, beta amino acids (Thr-Thr; Val-Val) continued presence and a large amount of protecting group of needs be protected polypeptide (20 side chain protected groups); in addition; it is reported; two fragments (1-10 and 11-28) of this peptide can form the beta sheet structure, and these all features show that it is very difficult synthesizing this polypeptide with the method for existing solid-phase synthetic peptide.
Therefore this area presses for the method that a kind of simple and effective synthetic Thymosin-Alpha1 is provided.
Summary of the invention
The present invention aims to provide the preparation method of a peptide species solid phase synthesis Thymosin-Alpha1.
The invention provides a kind of solid phase synthesis preparation method thereof suc as formula the Thymosin-Alpha1 shown in the I, described method comprises step:
(1) Fmoc-Asn (Trt)-OH and hydroxyl functional resin are mixed, obtain Fmoc-Asn (Trt)-resin through esterification;
(2), obtain Asn (Trt)-resin with Fmoc-Asn (Trt)-resin with go protective material to mix;
(3), obtain Fmoc-Glu (OtBu)-Asn (Trt)-resin with Fmoc-Glu (OtBu)-OH and Asn (Trt)-resin condensation;
(4) according to the method repeating step (2) of solid phase synthesis and (3) take off Fmoc and with the step of amino acid with the polypeptide condensation on the resin, with amino acid from C end to the N end according to the order of Glu to Ser successively with the polypeptide condensation on the resin, form suc as formula the polypeptide resin shown in the II; With
(5) make polypeptide on the polypeptide resin and resin isolation shown in the formula II, obtain suc as formula the Thymosin-Alpha1 shown in the I;
In above-mentioned preparation method, described esterification is with Fmoc-Asn (Trt)-OH, hydroxyl functional resin and 2, and the 6-dichlorobenzoyl chloride mixes in dinethylformamide (DMF) solution and carries out at N.
In above-mentioned preparation method, the described protective material that goes wherein contains the 3%-20% piperidines in its cumulative volume, 0.5%-10% bicyclic amidine (DBU) and 01M-0.5M1-hydroxybenzotriazole (HOBt); Described another kind removes protective material, in its cumulative volume, wherein contains 1%-10% piperazine and 0.1M-0.5M1-hydroxybenzotriazole (HOBt).
In above-mentioned preparation method, the condensation of described amino acid and resin be selected from one or more following condensing agents in the presence of carry out: N, N '-DIC (DIC), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU), TriMethylolPropane(TMP) (TMP), diisopropylethylamine (DIPEA), I-hydroxybenzotriazole (HOBt); Preferably, contain N in the described condensing agent, N '-DIC (DIC), I-hydroxybenzotriazole (HOBt), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU) and TriMethylolPropane(TMP) TMP); Preferably, contain N in the described condensing agent, N '-DIC (DIC), I-hydroxybenzotriazole (HOBt), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU) and diisopropylethylamine (DIPEA).
In above-mentioned preparation method, step (5) is 92-97 trifluoroacetic acid (TFA) in each material proportioning: 1.5-4 tri isopropyl silane (TIS): carry out in the presence of the cutting agent of 1.5-4 water.
In above-mentioned preparation method, described hydroxyl functional resin be selected from PEG-king's resin,
200 PHB,
400 PHB, NovaPEG king's resin.
In view of the above, the invention provides a kind of method of simple and effective synthetic Thymosin-Alpha1.
Description of drawings
Fig. 1 has shown that the present invention prepares the flow process of Fmoc-Asn (Trt)-PEG-king's resin.
Fig. 2 has shown the flow process of solid phase synthesis Thymosin-Alpha1 of the present invention.
Embodiment
The contriver has been surprised to find that the method for a simple and clear solid phase synthesis Thymosin-Alpha1 through extensive and deep research, the one, and to hydroxyl functional resin's esterification, productive rate is higher and kept stereochemical integrity like this; The 2nd, having selected with PEG is that solid carrier and DIC/HOBT are that main activator, TBTU/TMP or TBTU/DIPEA are secondary activation agent, DMF/DCM is as solvent, and the DMF solution of Piperidine/DBU/HOBt or piperazine/HOBt/DMF are as deprotection agent; The 3rd, use TFA/TIS/H
2O is as cutting liquid.
The implication of employed abbreviation or English full name is listed in the table below among the present invention:
| PEG | Polyoxyethylene glycol |
| Fmoc | 9 fluorenylmethyloxycarbonyls |
| DMF | N, dinethylformamide |
| DBU | 1,8-diazabicylo [5,4,0] 11 carbon-7-alkene |
| HOBt | I-hydroxybenzotriazole |
| DIC | N, N '-DIC |
| TBTU | Benzotriazole tetramethyl-urea Tetrafluoroboric acid |
| DIPEA | Diisopropylethylamine |
| TMP | TriMethylolPropane(TMP) |
| DCM | Methylene dichloride |
| Piperidine | Piperidines |
| Piperazine | Piperazine |
| Pyridine | Pyridine |
| Ac2O | Diacetyl oxide |
| TFA | Trifluoroacetic acid |
| TIS | Tri isopropyl silane |
| MTBE | Methyl tertiary butyl ether |
| Boc | Tertbutyloxycarbonyl |
| tBu | The tertiary butyl ,-C (CH 3) 3 |
| OtBu | -O-C(CH 3) 3 |
| 2，6-dichlorobenzoylchloride | 2, the 6-dichlorobenzoyl chloride |
As used herein, " solid phase synthesis " or " polypeptide solid phase synthesis (solid phase peptide synthesis) " is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: with the protected amino acid of amino covalently bound (bonding) on solid phase carrier; Going in the presence of the protective material, the protecting group of desamidizate is received on the solid phase carrier first amino acid; Then amino be closed (protection) second amino acid whose carboxyl by activation, second amino acid that carboxyl is activated forms peptide bond with first the amino acid whose amino reaction (condensation) that is connected on solid phase carrier again, has just generated a dipeptides that has protecting group like this on solid phase carrier; Repeat above-mentioned peptide bond and form reaction, peptide chain is grown, from the C end to the N end until reaching needed peptide chain length; The protecting group of last deaminize, the ester bond between hydrolysis peptide chain and the solid phase carrier (cutting) obtains synthetic good peptide.
As used herein, " removing protective material " or " deprotection agent " can exchange use, all is meant the chemical reagent that the amino protecting agent that is connected on the amino acid can be removed, described amino protecting agent can make well known in the art, such as but not limited to, Fmoc, Boc; Preferably; the protective material that goes of the present invention contains the 3%-20% piperidines in its cumulative volume, 0.5%-10% bicyclic amidine (DBU); with 0.1M-0.5M1-hydroxybenzotriazole (HOBt), or contain 1%-10% piperazine and 0.1M-0.5M1-hydroxybenzotriazole (HOBt).
As used herein, " condensing agent ", " activator " or " condensation activator " can exchange use, all be to instigate an amino acid whose amino and another amino acid whose carboxyl condensation to form the chemical reagent of peptide bond, can make well known in the art, such as but not limited to, carbodiimide, ByPOB, HATU, TBTU, TMP, DIPEA.
As used herein, " cutting agent " be meant with the polypeptide of resin-bonded and the chemical reagent of resin isolation, can make well known in the art, such as but not limited to, contain weakly acidic solution, the HCl solution of TFA.
In an example of the present invention, the preparation method of polypeptide solid phase synthesis Thymosin-Alpha1 of the present invention may further comprise the steps:
The first step with Fmoc-Asn (Trt)-OH and resin-bonded, obtains Fmoc-Asn (Trt)-resin; Preferred use the hydroxyl functional resin, more preferably PEG-Wang resin, the substitution rate of described PEG-Wang resin is 0.2-0.8mmol/g;
Second step with protective material and Fmoc-Asn (the Trt)-mixed with resin of going of the present invention, removed the Fmoc group, obtained Asn (Trt)-resin;
In the 3rd step,, obtain Fmoc-Glu (OtBu)-Asn (Trt)-resin with Fmoc-Glu (OtBu)-OH and Asn (Trt)-resin condensation;
In the 4th step, use the protective material that goes of the present invention to remove the Fmoc group;
The 5th step, repeat above-mentioned peptide bond and form step, peptide chain is grown, until obtaining FmocSer (tBu) Asp (OtBu) AlaAlaValAsp (OtBu) Thr (tBu) Ser (tBu) Ser (tBu) Glu (OtBu) IleThr (tBu) Thr (tBu) Lys (Boc) Asp (OtBu) LeuLys (Boc) Glu (OtBu) Lys (Boc) Lys (Boc) Glu (OtBu) ValValGlu (OtBu) Glu (OtBu) AlaGlu (OtBu) Asn (Trt)-resin to the N end from the C end; (OtBu/tBu is a protecting group, can be removed at last)
In the 6th step, use the protective material that goes of the present invention to remove the Fmoc group;
The 7th the step, in the presence of cutting agent, make on the polypeptide resin suc as formula polypeptide shown in the I and resin isolation, obtain Thymosin-Alpha1; Contain TFA, TIS and water in the described cutting agent.
In a preference of the present invention, with Fmoc-Asn (Trt)-OH, resin, 2, the 6-dichlorobenzoyl chloride mixes in DMF solution in the first step, carries out esterification.
In a preference of the present invention, in the reaction that the 3rd and/or the 5th step peptide bond forms, with the 1.0-4.5 times of normal Fmoc-amino acid of resin (or polypeptide), with DMF/DCM (volume ratio 1: the 1) dissolving of the 2.0-6.0 times of normal HOBt of resin with the 3mL/g amount of resin, add the 1.0-6.0 times of normal DIC reaction of resin again and add the 1.0-6.0 times of DIC reaction that resin is normal after 45 minutes again, in entire reaction course, monitor with ninhydrin (Kaiser) test (Kaiser test).If reaction not exclusively, add amino acid whose DMF/DCM (volume ratio 1: the 1) solution that contains corresponding Fmoc protection in addition, and then add the 1.0-3.0 times of normal TBTU of resin and the 2.0-6.0 times of normal TMP of resin (or DIPEA) and make and react completely.
Relevant ninhydrin (Kaiser) method of testing, and monitoring method can be referring to document VIRENDER K.SARIN, et al. " Quantitative Monitoring of Solid-Phase Peptide Synthesis by the Ninhydrin Reaction " ANALYTICAL BIOCHEMISTRY 117,147-157 (1981), E.KAISER, et al. " Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides " SHORT COMMUNICATIONS 595-598 (Received October 28,1969), with THORKILD CHRISTENSEN " A Qualitative Test for Monitoring Coupling Completeness in Solid Phase Peptide Synthesis Using Chlo
By the Thymosin-Alpha1 that preparation method provided by the invention obtains, productive rate and purity are all comparatively desirable.In a preference of the present invention, the Thymosin-Alpha1 that the 7th step obtained can also be carried out sedimentation, be about to polypeptide and MTBE or ether mixing that the 7th step obtained suc as formula I, form the polypeptide precipitation, obtain the Thymosin-Alpha1 of purifying.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets is disclosed can with any composition forms and usefulness, each feature that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, the preparation method is easy.
2, yield and purity ideal.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Among the embodiment, the measuring method of Thymosin-Alpha1 crude product purity is:
Instrument Agilent1200, chromatographic column Kromasil 100-5C18
Ultraviolet wavelength: 215nm
Column temperature: 30 ℃
Flow rate of mobile phase: 1.0ml/min
Mobile phase A: 0.1%TFA/ water moving phase B:0.1%TFA/ACN
Gradient: 10%-30%B wash-out 40 minutes
PEG-king's resin among the embodiment (PEG grafted Wang resin (0.72mmol/g)) is available from Rapp Polymere GmbH (T ü bingen Germany)
Preparation Fmoc-Asn (Trt)-PEG-king's resin
15g PEG-king resin (substitution value is 0.5-0.7mmol/g) is immersed among the 150mlDMF 30 minutes, drain, with 3.0 equivalent (19.33g, 32.4mmole) Fmoc-Asn (Trt)-OH, 4.8 equivalent (7.41mL, 51.8mmol) 2,6-dichlorobenzoylchloride and 8.4 equivalent (7.4mL, 90.7mmol) mixing of DMF solution, esterification 3 hours.Resin washs with DMF then, resin is carried out end-blocking, 1 hour with 100mL10%pyridine/DMF and 100mL 10%benzoyl chloride (Benzoyl chloride)/DMF mixed solution.With DMF and methanol wash resin, vacuum drying, survey substitution value (substitution value 0.43mmol/g).See Fig. 1.
The Fmoc deprotection
Remove Fmoc with 0.2MHOBt/6%piperazine/DMF, double deprotection, the time is respectively 10min and 20min.Drain deprotection solution, use DMF and methanol wash respectively.Thoroughly draining back Kaiser test detection assessment Fmoc removes.
The Fmoc amino acid condensation
DMF/DCM (volume ratio is 1: the 1) solution (3ml/g resin) that in reactor, adds Fmoc-AA-OH/HOBt (1.5 equivalents/2 equivalents), add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) subsequently, add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) after 45 minutes again.Monitored with Kaiser test in the process in three hours in reaction.The peptide chain resin that increases is thoroughly drained after with methanol wash.If reaction not exclusively, the DMF solution that adds Fmoc-AA-OH (0.5 equivalent) so in addition, reaction is more than 10 hours, with Kaiser test monitoring, if reaction not exclusively, drain solution, with methyl alcohol/DMF washing, add DMF/DIC (volume ratio is 1: the 1) solution (4ml/g resin) of Fmoc-AA-OH (1.5 equivalent), add 2.0 equivalent TBTU then, 4.0 equivalent TMP, back methanol wash resin reacts completely.
If α after the condensation-end amino does not also have condensation intact again, then carry out end-blocking with diacetyl oxide, diacetyl oxide/pyridine/DMF (volume ratio is 6: 5: 50), with DMF and MTBE washing, drying is weighed.
Cutting
The configuration of cutting liquid
In the container of suitable size, mix TFA: TIS by volume: water (92v: 4v: 4v), with refrigeration agent or frozen water cooling cutting liquid.
Cutting
In refrigerative cutting liquid, add the polypeptide tree slowly.Stirring is risen again 2-3 hour to 25 ℃ ± 5 ℃.Filtration or centrifugal removal resin are also used twice of trifluoroacetic acid washing resin.Collect the filtrate packaging resin.
Sedimentation
Concentrate good filtrate (every approximately 1ml filtrate 10ml MTBE) and pour the good methyl tert-butyl ether (MTBE) of cooling, (0-8 ℃) lining sedimentation into.Cooling was left standstill crystallization 0.5-1.5 hour.Filter or centrifugally must consider cake, thoroughly wash the worry cake three times with refrigerated MTBE.
The crude product drying
The crude product polypeptide is transferred to moisture eliminator, and drying is at least 12 hours under the vacuum.
Synthetic crude product purity is 52.6%, and synthetic yield is 55.8%.
Flow process can be referring to Fig. 2.
Embodiment 2
Preparation Fmoc-Asn (Trt)-PEG-king's resin
15g PEG-king resin (substitution value is 0.5-0.75mmol/g) is immersed among the 150mlDMF 30 minutes, drain, with 3.0 equivalent (19.33g, 32.4mmole) Fmoc-Asn (Trt)-OH, 4.8 equivalent (7.41mL, 51.8mmol) 2,6-dichlorobenzoylchloride and 8.4 equivalent (7.4mL, 90.7mmol) mixing of DMF solution, esterification 3 hours.Resin washs with DMF then, resin is carried out end-blocking, 1 hour with 100mL10%pyridine/DMF and 100mL 10%benzoyl chloride/DMF mixed solution.With DMF and methanol wash resin, vacuum drying, survey substitution value.
The Fmoc deprotection
Remove Fmoc with 0.1MHOBt/10%piperazine/DMF, double deprotection, the time is respectively 10min and 20min.Drain deprotection solution, use DMF and methanol wash respectively.Thoroughly draining back Kaiser test detection assessment Fmoc removes.
The Fmoc amino acid condensation
DMF/DCM (volume ratio is 1: the 1) solution (3ml/g resin) that in reactor, adds Fmoc-AA-OH/HOBt (2 equivalents/3 equivalents), add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) subsequently, add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) after 45 minutes again.Monitored with Kaiser test in the process in three hours in reaction.The peptide chain resin that increases is thoroughly drained after with methanol wash.If reaction not exclusively, the DMF solution that adds Fmoc-AA-OH (0.5 equivalent) so in addition, reaction is more than 10 hours, with Kaiser test monitoring, if reaction not exclusively, drain solution, with methyl alcohol/DMF washing, add DMF/DIC (volume ratio is 1: the 1) solution (4ml/g resin) of Fmoc-AA-OH (1.5 equivalent), add 1.45 equivalent TBTU then, 3.0 equivalent DIPEA, back methanol wash resin reacts completely.
If α after the condensation-end amino does not also have condensation intact again, then carry out end-blocking with diacetyl oxide, diacetyl oxide/pyridine/DMF (volume ratio is 6: 5: 50), with DMF and MTBE washing, drying is weighed.
Cutting
The configuration of cutting liquid
In the container of suitable size, mix TFA: TIS by volume: water (94v: 3v: 3v), with refrigeration agent or frozen water cooling cutting liquid.
Cutting
In refrigerative cutting liquid, add the polypeptide tree slowly.Stirring is risen again 2-3 hour to 25 ℃ ± 5 ℃.Filtration or centrifugal removal resin are also used twice of trifluoroacetic acid washing resin.Collect the filtrate packaging resin.
Sedimentation
Concentrate good filtrate (every approximately 1ml filtrate 10ml MTBE) and pour the good methyl tert-butyl ether (MTBE) of cooling, (0-8 ℃) lining sedimentation into.Cooling was left standstill crystallization 0.5-1.5 hour.Filter or centrifugally must consider cake, thoroughly wash the worry cake three times with refrigerated MTBE.
The crude product drying
The crude product polypeptide is transferred to moisture eliminator, and drying is at least 12 hours under the vacuum.
Synthetic crude product purity is 69.6%, and synthetic yield is 56.6%.
Preparation Fmoc-Asn (Trt)-PEG-king's resin
15g PEG-king resin (substitution value is 0.4-0.8mmol/g) is immersed among the 150mlDMF 30 minutes, drain, with 3.0 equivalent (19.33g, 32.4mmole) Fmoc-Asn (Trt)-OH, 4.8 equivalent (7.41mL, 51.8mmol) 2,6-dichlorobenzoylchloride and 8.4 equivalent (7.4mL, 90.7mmol) mixing of DMF solution, esterification 3 hours.Resin washs with DMF then, resin is carried out end-blocking, 1 hour with 100mL10%pyridine/DMF and 100mL 10%benzoyl chloride/DMF mixed solution.With DMF and methanol wash resin, vacuum drying, survey substitution value.
The Fmoc deprotection
Remove Fmoc with 1%HOBt/3-20%piperidine/0.5-10%DBU/DMF, double deprotection, the time is respectively 10min and 20min.Drain deprotection solution, use DMF and methanol wash respectively.Thoroughly draining back Kaiser test detection assessment Fmoc removes.
The Fmoc amino acid condensation
DMF/DCM (volume ratio is 1: the 1) solution (3ml/g resin) that in reactor, adds Fmoc-AA-OH/HOBt (1.0 equivalents/3 equivalents), add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) subsequently, add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) after 45 minutes again.Monitored with Kaiser test in the process in three hours in reaction.The peptide chain resin that increases is thoroughly drained after with methanol wash.If reaction not exclusively, the DMF solution that adds Fmoc-AA-OH (0.5 equivalent) so in addition, reaction is more than 10 hours, with Kaiser test monitoring, if reaction not exclusively, drain solution, with methyl alcohol/DMF washing, add DMF/DIC (volume ratio is 1: the 1) solution (4ml/g resin) of Fmoc-AA-OH (1.5 equivalent), add 1.45 equivalent TBTU then, 3.0 equivalent TMP, back methanol wash resin reacts completely.
If α after the condensation-end amino does not also have condensation intact again, then carry out end-blocking with diacetyl oxide, diacetyl oxide/pyridine/DMF (volume ratio is 6: 5: 50), with DMF and MTBE washing, drying is weighed.
Cutting
The configuration of cutting liquid
In the container of suitable size, mix TFA: TIS by volume: water (96v: 2v: 2v), with refrigeration agent or frozen water cooling cutting liquid.
Cutting
In refrigerative cutting liquid, add the polypeptide tree slowly.Stirring is risen again 2-3 hour to 25 ℃ ± 5 ℃.Filtration or centrifugal removal resin are also used twice of trifluoroacetic acid washing resin.Collect the filtrate packaging resin.
Sedimentation
Concentrate good filtrate (every approximately 1ml filtrate 10ml MTBE) and pour the good methyl tert-butyl ether (MTBE) of cooling, (0-8 ℃) lining sedimentation into.Cooling was left standstill crystallization 0.5-1.5 hour.Filter or centrifugally must consider cake, thoroughly wash the worry cake three times with refrigerated MTBE.
The crude product drying
The crude product polypeptide is transferred to moisture eliminator, and drying is at least 12 hours under the vacuum.
Synthetic crude product purity is 65.4%, and synthetic yield is 63.7%.
Embodiment 4
Preparation Fmoc-Asn (Trt)-PEG-king's resin
15g PEG-king resin (substitution value is 0.2-0.7mmol/g) is immersed among the 150mlDMF 30 minutes, drain, with 3.0 equivalent (19.33g, 32.4mmole) Fmoc-Asn (Trt)-OH, 4.8 equivalent (7.41mL, 51.8mmol) 2,6-dichlorobenzoylchloride and 8.4 equivalent (7.4mL, 90.7mmol) mixing of DMF solution, esterification 3 hours.Resin washs with DMF then, resin is carried out end-blocking, 1 hour with 100mL10%pyridine/DMF and 100mL 10%benzoyl chloride/DMF mixed solution.With DMF and methanol wash resin, vacuum drying, survey substitution value.
The Fmoc deprotection
Remove Fmoc with 0.1MHOBt/3%piperazine/DMF, double deprotection, the time is respectively 10min and 20min.Drain deprotection solution, use DMF and methanol wash respectively.Thoroughly draining back Kaisertest detection assessment Fmoc removes.
The Fmoc amino acid condensation
DMF/DCM (volume ratio is 1: the 1) solution (3ml/g resin) that in reactor, adds Fmoc-AA-OH/HOBt (1.5 equivalents/3 equivalents), add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) subsequently, add DIC (the 1.0-3.0 equivalent is with respect to Fmoc-Asn (Trt)-PEG-king's resin) after 45 minutes again.Monitored with Kaiser test in the process in three hours in reaction.The peptide chain resin that increases is thoroughly drained after with methanol wash.If reaction not exclusively, the DMF solution that adds Fmoc-AA-OH (0.5 equivalent) so in addition, reaction is more than 10 hours, with Kaiser test monitoring, if reaction not exclusively, drain solution, with methyl alcohol/DMF washing, add DMF/DIC (volume ratio is 1: the 1) solution (4ml/g resin) of Fmoc-AA-OH (1.5 equivalent), add 1.45 equivalent TBTU then, 3.0 equivalent TMP, back methanol wash resin reacts completely.
If α after the condensation-end amino does not also have condensation intact again, then carry out end-blocking with diacetyl oxide, diacetyl oxide/pyridine/DMF (volume ratio is 6: 5: 50), with DMF and MTBE washing, drying is weighed.
Cutting
The configuration of cutting liquid
In the container of suitable size, mix TFA: TIS by volume: water (95v: 2.5v: 2.5v), with refrigeration agent or frozen water cooling cutting liquid.
Cutting
In refrigerative cutting liquid, add the polypeptide tree slowly.Stirring is risen again 2-3 hour to 25 ℃ ± 5 ℃.Filtration or centrifugal removal resin are also used twice of trifluoroacetic acid washing resin.Collect the filtrate packaging resin.
Sedimentation
Concentrate good filtrate (every approximately 1ml filtrate 10ml MTBE) and pour the good methyl tert-butyl ether (MTBE) of cooling, (0-8 ℃) lining sedimentation into.Cooling was left standstill crystallization 0.5-1.5 hour.Filter or centrifugally must consider cake, thoroughly wash the worry cake three times with refrigerated MTBE.
The crude product drying
The crude product polypeptide is transferred to moisture eliminator, and drying is at least 12 hours under the vacuum.
Synthetic crude product purity is 57.2%, and synthetic yield is 64.4%.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (9)
1. the solid phase synthesis preparation method thereof suc as formula the Thymosin-Alpha1 shown in the I is characterized in that, described method comprises step:
(1) Fmoc-Asn (Trt)-OH and hydroxyl functional resin are mixed, obtain Fmoc-Asn (Trt)-resin through esterification;
(2), obtain Asn (Trt)-resin with Fmoc-Asn (Trt)-resin with go protective material to mix;
(3), obtain Fmoc-Glu (OtBu)-Asn (Trt)-resin with Fmoc-Glu (OtBu)-OH and Asn (Trt)-resin condensation;
(4) according to the method repeating step (2) of solid phase synthesis and (3) take off Fmoc and with the step of amino acid with the polypeptide condensation on the resin, with amino acid from C end to the N end according to the order of Glu to Ser successively with the polypeptide condensation on the resin, form suc as formula the polypeptide resin shown in the II; With
(5) make polypeptide on the polypeptide resin and resin isolation shown in the formula II, obtain suc as formula the Thymosin-Alpha1 shown in the I;
2. preparation method as claimed in claim 1 is characterized in that, described esterification is with Fmoc-Asn (Trt)-OH, hydroxyl functional resin and 2, and the 6-dichlorobenzoyl chloride mixes in dinethylformamide (DMF) solution and carries out at N.
3. preparation method as claimed in claim 1 is characterized in that, the described protective material that goes wherein contains the 3%-20% piperidines in its cumulative volume, 0.5%-10% bicyclic amidine (DBU) and 01M-0.5M1-hydroxybenzotriazole (HOBt).
4. preparation method as claimed in claim 1 is characterized in that, the described protective material that goes wherein contains 1%-10% piperazine and 0.1M-0.5M1-hydroxybenzotriazole (HOBt) in its cumulative volume.
5. preparation method as claimed in claim 1, it is characterized in that, the condensation of described amino acid and resin be selected from one or more following condensing agents in the presence of carry out: N, N '-DIC (DIC), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU), TriMethylolPropane(TMP) (TMP), diisopropylethylamine (DIPEA), I-hydroxybenzotriazole (HOBt).
6. preparation method as claimed in claim 5, it is characterized in that, contain N in the described condensing agent, N '-DIC (DIC), I-hydroxybenzotriazole (HOBt), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU) and TriMethylolPropane(TMP) TMP).
7. preparation method as claimed in claim 5, it is characterized in that, contain N in the described condensing agent, N '-DIC (DIC), I-hydroxybenzotriazole (HOBt), O-(benzotriazole-1-yl)-N, N, N, N-tetramethyl-urea Tetrafluoroboric acid (TBTU) and diisopropylethylamine (DIPEA).
8. preparation method as claimed in claim 1 is characterized in that, step (5) is 92-97 trifluoroacetic acid (TFA) in each material proportioning: 1.5-4 tri isopropyl silane (TIS): carry out in the presence of the cutting agent of 1.5-4 water.
9. as the described preparation method of claim 1-8 personnel, it is characterized in that, described hydroxyl functional resin be selected from PEG-king's resin,
200PHB,
400PHB, NovaPEG king's resin.
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| CN103265629A (en) * | 2013-05-28 | 2013-08-28 | 福建省闽东力捷迅药业有限公司 | Novel solid phase synthesis process for preparing thymalfasin |
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| CN103936848A (en) * | 2014-03-14 | 2014-07-23 | 深圳翰宇药业股份有限公司 | Synthesis method of thymosin alpha1 |
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| CN107857809A (en) * | 2017-12-12 | 2018-03-30 | 安徽省国平药业有限公司 | A kind of new method of synthesis in solid state Thymosin alpha 1 |
| CN108239147A (en) * | 2016-12-27 | 2018-07-03 | 江苏豪森药业集团有限公司 | The preparation method of thymosin α1 derivative |
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