CN105001298A - Synthesis-separation and purification method for indissolvable polypeptide - Google Patents
Synthesis-separation and purification method for indissolvable polypeptide Download PDFInfo
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- CN105001298A CN105001298A CN201510466322.XA CN201510466322A CN105001298A CN 105001298 A CN105001298 A CN 105001298A CN 201510466322 A CN201510466322 A CN 201510466322A CN 105001298 A CN105001298 A CN 105001298A
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- Prior art keywords
- indissoluble
- polypeptide
- amino acid
- acid
- synthesis
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- BCIPGSZQUDLGSY-INIZCTEOSA-N tert-butyl (4s)-4-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 BCIPGSZQUDLGSY-INIZCTEOSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a synthesis-separation and purification method for indissolvable polypeptide. The synthesis-separation and purification method for the indissolvable polypeptide comprises the steps that coupling is conducted on indissolvable polypeptide chains and hydrophilic polypeptide chains through solid-phase synthesis firstly to achieve the dissolution of the indissolvable polypeptide in high performance liquid chromatography, then the indissolvable polypeptide chains and the hydrophilic polypeptide chains are disconnected by the utilization of LiOH saturated solution hydrolysis ester bonds to enable target peptide chains to be separated out directly, the synthesis-separation and purification method for the indissolvable polypeptide has the advantages that simplicity and high-efficiency are achieved, and the indissolvable polypeptide products obtained through the method can completely meet the required standards of customers.
Description
Technical field
The present invention relates to biotechnology and polypeptide production field, relate to a kind of synthesis-separation purification method of indissoluble polypeptide specifically.
Background technology
Solvability is that Study on Protein and polypeptide can run into important problem, the chemical property that each amino acid has it intrinsic.If leucine, Isoleucine, a word used in person's names propylhomoserin are hydrophobic, and Methionin, Histidine, arginine are hydrophilic.Indissoluble polypeptide mainly its indissoluble amino acid comprised causes, and in general, indissoluble amino acid accounts for polypeptide ratio will cause polypeptide indissoluble more than 75%.
Early stage in chemiluminescent polypeptide development, Peptide systhesis also mutually in carry out, although have the high advantage of output purity, the purification steps troublesome of intermediate, consuming time, too high for cost polypeptide manufacturing enterprise.And Peptide systhesis is completed on solid phase carrier, comparing liquid phase synthesis has significant superiority, but the purity of synthesis is inadequate, must be further purified, the main means of purification of polypeptide is RPLC, and it has good separating effect, the feature that resolving power high-recovery is high.RPLC is when carrying out separation and purification to polypeptide, and need polypeptide to be dissolved into completely in solvent, optimal Conventional solvents is H
2o, ACN, RPLC could be utilized like this to carry out effective separation and purification, but indissoluble polypeptide cannot be dissolved in water/acetonitrile solution, brings the deuce to pay to numerous scientific research personnel in this operation.
The method of existing solution indissoluble peptide purification selects excellent organic solvent (as: DMSO, DMF) to go to dissolve polypeptide, then carries out RPLC separation and purification.But DMSO is toxic, during use, suitable concentration must be considered; And DMF or DMSO is the secondary structure hydrotropy by destroying polypeptide, high performance liquid chromatography separating effect can be caused greatly to reduce, and limited to the dissolving power of peptide chain hard to tolerate.
Summary of the invention
Goal of the invention: the object of this invention is to provide the synthesis of a kind of indissoluble polypeptide liquid, separation purification method, for overcoming the problem of indissoluble polypeptide at liquid chromatography separation and purification insoluble.
Technical scheme: in order to realize foregoing invention object, the synthesis-separation purification method of a kind of indissoluble polypeptide of the present invention, comprises the steps:
(1) utilize Wang resin for initial resin, employing solid-phase synthesis one by one coupling has the hydrophilic amino acid of blocking group, after coupling protected hydrophilic amino acid, add DIC and HOBt and carry out condensation, wash after reacting completely, the next protected hydrophilic amino acid of coupling, obtains Wang resin-hydrophilic polypeptides chain again;
(2) connecting arm is added in the product obtained to step (1) complete to condensation reaction;
(3) the indissoluble amino acid with blocking group is added one by one, whenever coupling one has the indissoluble amino acid of blocking group, add DIC and HOBt and carry out condensation, wash after reacting completely, the next protected indissoluble amino acid of coupling again, until be coupled to last indissoluble amino acid;
(4) add cutting agent cracking resin, get filtrated stock and add ether precipitation, centrifugation obtains hydrophilic polypeptides-connecting arm-hydrophobic polypeptides crude product;
(5) the crude product polypeptide that previous step obtains is dissolved, utilize high performance liquid chromatography separating-purifying;
(6) the crude product polypeptide LiOH saturated solution after purifying is carried out ester linkage hydrolyzing, separate out indissoluble polypeptide.
Wherein, in described step (1) and step (3), after adding the hydrophilic amino acid or indissoluble amino acid with blocking group, react 1 hour at 30 DEG C.
And in described step (3), synthesized Wang resin-hydrophilic polypeptides connect after-connecting arm after, adding first when there is the indissoluble amino acid of blocking group, except adding DIC and HOBt, also adding DMAP and condensation reaction 5 hours at 30 DEG C.
Described connecting arm is that Isosorbide-5-Nitrae position is respectively by benzene compound that hydroxyalkyl and carboxyalkyl replace.Specifically, connecting arm is from 4-HBA, to hydroxymethyl-benzoic acid, 4-(2-hydroxyethyl) phenylformic acid, 4-(3-hydroxypropyl) phenylformic acid, 4-(4-hydroxybutyl) phenylformic acid, p-hydroxyphenylaceticacid, 4-(methylol) toluylic acid, para hydroxybenzene propionic acid, 3-[4-(hydroxymethyl) phenyl] propionic acid, 4-(4-hydroxy phenyl) butyric acid, 2-(4-hydroxybenzene) propionic acid, 3-(4-hydroxy phenyl) butyric acid, 2-[(4-hydroxy phenyl) methyl] aminobutyric acid, 4-(1-hydroxyethyl)-benzoic any one, to be held with the N of hydrophilic polypeptides chain by conventional dehydration condensation and be connected.As preferred version of the present invention, described connecting arm be 4-HBA, to hydroxymethyl-benzoic acid, 4-(2-hydroxyethyl) phenylformic acid, p-hydroxyphenylaceticacid, 4-(methylol) toluylic acid any one.
Described hydrophilic polypeptides chain is preferably different hydrophilic amino acid dehydrating condensations and forms.Hydrophilic amino acid be arginine, Methionin, l-asparagine, aspartic acid, glutamine, L-glutamic acid, Histidine, proline(Pro) any one, these amino acid whose hydrophobic parameters are very little; And this for indissoluble polypeptide, can be understood as comprise indissoluble amino acid and in liquid chromatography the polypeptide chain of the very low or indissoluble of solubleness, indissoluble amino acid comprise L-Ala, methionine(Met), halfcystine, phenylalanine, leucine, α-amino-isovaleric acid, Isoleucine any one, these hydropathic amino acid parameters are large, all very low containing the general solubleness of these amino acid whose polypeptide chains.Hydrophilic amino acid and hydrophobic amino acid-specific are as shown in table 1, table 2:
The list of table 1 hydrophilic amino acid
| Amino acid name | Abbreviation | Code | Hydrophobic parameter |
| Arginine | Arg | R | -4.5 |
| Methionin | Lys | K | -3.9 |
| L-asparagine | Asn | N | -3.5 |
| Aspartic acid | Asp | D | -3.5 |
| Glutamine | Gln | Q | -3.5 |
| L-glutamic acid | Glu | E | -3.5 |
| Histidine | His | H | -3.2 |
| Proline(Pro) | Pro | P | -1.6 |
The list of table 2 indissoluble amino acid
| Amino acid name | Abbreviation | Code | Hydrophobic parameter |
| L-Ala | Ala | A | 1.8 |
| Methionine(Met) | Met | M | 1.9 |
| Halfcystine | Cys | C | 2.5 |
| Phenylalanine | Phe | F | 2.8 |
| Leucine | Leu | L | 3.8 |
| α-amino-isovaleric acid | Val | V | 4.2 |
| Isoleucine | Ile | I | 4.5 |
The present invention be based on solid phase synthesis send out coupling one by one or peptide chain, the amino acid added in step (1) and step (3) must be protected, and its blocking group is as shown in table 3:
Table 3 adds amino acid whose blocking group
| Amino acid | Protective group group | Alpha-amino group blocking group |
| Arg | Pbf | Fmoc |
| Lys | Boc | Fmoc |
| Asn | Trt | Fmoc |
| Asp | Otbu | Fmoc |
| Gln | Trt | Fmoc |
| Glu | Otbu | Fmoc |
| His | Trt | Fmoc |
| Pro | Nothing | Fmoc |
The present invention first becomes hydrophilic polypeptide chain by connecting arm coupling, carry out high performance liquid chromatography again and carry out separation and purification, the sterling obtained is hydrolyzed by LiOH saturated solution again, indissoluble polypeptide chain in polypeptide chain is directly separated out, and connecting arm-hydrophilic amino acid will ensure to be dissolved in preferably in hydrolysis environment.In order to reach this object, to select by the hydrophilic amino acid used to it when synthesis hydrophilic polypeptide chain.The preferred L-glutamic acid of hydrophilic amino acid in described hydrophilic polypeptides chain or aspartic acid, when L-glutamic acid and/or aspartic acid overall number account for 30% ~ 80% of total hydrophilic amino acid number, utilize X-Ph-Y-(B)
nseparation and purification in liquid chromatography, finally the effect of hydrolyse ester bond is best again.
In described step (4), cutting agent is TFA, TIS, EDT, and volume ratio is 95:3:2.
In the present invention some conventional abbreviations and implication as follows:
HMBA 4-hydroxymethyl-benzoic acid, connecting arm;
DIC N, N'-DIC, condensing agent;
DMAP DMAP, as superpower nucleophilic catalyst, increases hydroxyl and carboxyl link efficiency;
HOBT I-hydroxybenzotriazole, for preventing racemization, reduces side reaction;
DMF dimethyl formamide, reaction solvent;
TFA trifluoroacetic acid, for cracking polypeptide and resin;
TIS tri isopropyl silane, for removing amino acid protective group better;
EDT dithioglycol, for removing amino acid protective group better;
ACN acetonitrile, increases the solvent degree of polypeptide at the aqueous solution;
Fmoc N-fluorenylmethyloxycarbonyl, protective group group;
Pbf [(2,3-dihydro-2,2,4,6,7-pentamethyl-cumarone-5-base) alkylsulfonyl, alpha-amino group blocking group;
Boc tertbutyloxycarbonyl, alpha-amino group blocking group;
Trt trityl, alpha-amino group blocking group;
The OtBu tert-butyl ester, alpha-amino group blocking group
Beneficial effect: the separation purification method that the invention provides a kind of indissoluble polypeptide, first by solid phase synthesis, indissoluble polypeptide chain and hydrophilic polypeptides are connected coupling, realize the dissolving of indissoluble polypeptide in high performance liquid chromatography, recycling LiOH saturated solution hydrolyse ester bond disconnects indissoluble polypeptide chain and hydrophilic polypeptides chain, target peptide chain is directly separated out, there is simple and easy, efficient feature, customer requirement standard can be reached completely with the indissoluble polypeptide products that the method obtains.
Accompanying drawing explanation
Fig. 1 is the Peptide systhesis steps flow chart schematic diagram of the embodiment of the present invention 1;
Fig. 2 is the Peptide systhesis steps flow chart schematic diagram of the embodiment of the present invention 2.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1
The present embodiment carries out separation and purification for indissoluble polypeptide ILVLLIII, and connecting arm selects 4-hydroxymethyl-benzoic acid (HMBA), connects DDDDDEEKEEEE hydrophilic polypeptides chain by connecting arm.Wherein, D represents aspartic acid, hydrophobic parameter-3.5, utilizes OtBu to protect functional group; E represents L-glutamic acid, hydrophobic parameter-3.5, and utilize OtBu to protect functional group, K represents Methionin, hydrophobic parameter-3.9, utilizes Boc to protect functional group; These amino acid all adopt Fmoc to protect alpha-amino group to carry out solid phase synthesis.As shown in Figure 1, concrete synthesis step is as follows:
Take 1g Fmoc-Glu (OtBu)-Wang resin resin; perform [operation A]; that is: piperidines is added: DMF=1:4 (volume ratio) removes the Fmoc blocking group of N end; temperature controls at 30 DEG C; react 20 minutes; utilize DMF/ methyl alcohol respectively to wash 3 times after reaction, utilize triketohydrindene hydrate detection reagent to detect, color reacts completely for blueness represents.
Then perform [operation B]; that is: DIC and HOBT of initial resin mole number twice is added; and the amino acid with blocking group of doubling dose reacts 1 hour; temperature controls at 30 DEG C; reaction terminates after rear DMF washs 3 times; it is colourless for detecting color with triketohydrindene hydrate, then illustrate and react completely.
Follow-uply hocket with [operation A], [operation B], the pro amino acid just added in [operation B] is changed along with the carrying out of synthesis order.Wherein there is the L-glutamic acid of blocking group, aspartic acid and Methionin and be respectively Fmoc-Glu (OtBu)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH.Reaction like this is until connected HMBA, and testing look with ninhydrin solution is that colourless i.e. explanation reaction completes.More than react and namely obtain X-Ph-Y-(B)
nhydrophilic coupling peptide chain.
Based on the peptide chain of above-mentioned acquisition, directly carry out [operation B], when operating, first add DIC and HOBT herein, then add DMAP, and the add-on of DMAP is only and adds 1/10th of amino acid mol times, at 30 DEG C, the reaction times extends to 5 hours.And replace at subsequent step and [operating A], until obtain Wang resin-indissoluble polypeptide chain-connecting arm-hydrophilic polypeptides chain.
Then 10ml TFA:TIS:EDT=95%:3%:2% cracking resin 2 hours are used, by coupling peptide chain deprotection group.Then resin filtering is left mother liquor, add 100ml ether and separate out polypeptide.With 3000 revolutions per seconds of centrifugal 2min, precipitation obtains crude product polypeptide, dry in Vacuumdrier after repeated washing is centrifugal, obtains indissoluble polypeptide chain-connecting arm-hydrophilic polypeptides chain crude product.
The mixing liquid of above-mentioned crude product water/acetonitrile is dissolved, carries out separating-purifying, process H in high performance liquid chromatography dress sample
2o/0.1TFA% does aqueous phase, and ACN/0.1%TFA does the gradient elution chromatography system separating-purifying of organic phase, collects target peak.The analytical high performance liquid chromatography of the target peak collected is detected purity.Qualified sample Rotary Evaporators concentrates, and puts refrigerator and is frozen into solid in advance.Finally put into vacuum freeze drier freeze-drying, obtain sterling ILVLLIII-HMBA-DDDDDEEKEEEE.
Above-mentioned sterling LiOH saturated solution is carried out ester linkage hydrolyzing, react under normal temperature after 3 hours, target polypeptides chain ILVLLIII separates out, and the sequence HMBA-DDDDDEEKEEEE increased is because the strong and whole polypeptide of wetting ability is in acid, to be dissolved in LiOH solution, then the target polypeptides of precipitation can be obtained finished product through filtering freeze-drying.
In above-mentioned reaction, Fmoc amino acid is purchased from gill biochemistry, and product batch number GLS141015-4071, DIC HobtDMAP is purchased from Suzhou sky sail, HMBA is purchased from gill biochemistry, and it is fine for evening that TFA, TIS, EDT, ether, piperidines, DMF are all purchased from Nanjing.
Embodiment 2
The present embodiment carries out separation and purification for indissoluble polypeptide ILVLLIII, and connecting arm selects 4-(2-hydroxyethyl) phenylformic acid (HPA), connects RRRRREDKKKKK hydrophilic polypeptides chain by connecting arm.Wherein, D represents aspartic acid, hydrophobic parameter-3.5, utilizes OtBu to protect functional group; E represents L-glutamic acid, hydrophobic parameter-3.5, utilizes OtBu to protect functional group; K represents Methionin, hydrophobic parameter-3.9, and utilize Boc to protect functional group, R represents arginine, hydrophobic parameter-4.5, utilizes Pbf to protect functional group; These amino acid all adopt Fmoc to protect alpha-amino group to carry out solid phase synthesis.As shown in Figure 2, concrete synthesis step is as follows:
Take 1g Fmoc-Lys (Boc)-Wang resin resin; perform [operation A]; that is: piperidines is added: DMF=1:4 (volume ratio) removes the Fmoc blocking group of N end; temperature controls at 30 DEG C; react 20 minutes; utilize DMF/ methyl alcohol respectively to wash 3 times after reaction, utilize triketohydrindene hydrate detection reagent to detect, color reacts completely for blueness represents.
Then perform [operation B], that is: add DIC and HOBT of initial resin mole number twice, and the amino acid with blocking group of doubling dose reacts 1 hour; temperature controls at 30 DEG C; reaction terminates after rear DMF washs 3 times, and it is colourless for detecting color with triketohydrindene hydrate, then illustrate and react completely.
Follow-uply hocket with [operation A], [operation B], the pro amino acid just added in [operation B] is changed along with the carrying out of synthesis order.Wherein there is the Methionin of blocking group, aspartic acid, L-glutamic acid, arginine be respectively Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Arg (Pbf)-OH; reaction like this is until connected HPA, and testing look with ninhydrin solution is that colourless i.e. explanation reaction completes.More than react and namely obtain X-Ph-Y-(B)
nhydrophilic coupling peptide chain.
Based on the peptide chain of above-mentioned acquisition, directly carry out [operation B], when operating, first add DIC and HOBT herein, then add DMAP, and the add-on of DMAP is only and adds 1/10th of amino acid mol times, at 30 DEG C, the reaction times extends to 5 hours.And replace at subsequent step and [operating A], until obtain Wang resin-indissoluble polypeptide chain-connecting arm-hydrophilic polypeptides chain.
Then 10ml TFA:TIS:EDT=95%:3%:2% cracking resin 2 hours are used, by coupling peptide chain deprotection group.Then resin filtering is left mother liquor, add 100ml ether and separate out polypeptide.With 3000 revolutions per seconds of centrifugal 2min, precipitation obtains crude product polypeptide, dry in Vacuumdrier after repeated washing is centrifugal, obtains indissoluble polypeptide chain-connecting arm-hydrophilic polypeptides chain crude product.
The mixing liquid of above-mentioned crude product water/acetonitrile is dissolved, carries out separating-purifying, process H in high performance liquid chromatography dress sample
2o/0.1TFA% does aqueous phase, and ACN/0.1%TFA does the gradient elution chromatography system separating-purifying of organic phase, collects target peak.The analytical high performance liquid chromatography of the target peak collected is detected purity.Qualified sample Rotary Evaporators concentrates, and puts refrigerator and is frozen into solid in advance.Finally put into vacuum freeze drier freeze-drying, obtain sterling ILVLLIII-HPA-RRRRREDKKKKK.
Above-mentioned sterling LiOH saturated solution is carried out ester linkage hydrolyzing, react under normal temperature after 3 hours, target polypeptides chain ILVLLIII separates out, and the sequence HPA-RRRRREDKKKKK increased is because the strong and whole polypeptide of wetting ability is in acid, to be dissolved in LiOH solution, then the target polypeptides of precipitation can be obtained finished product through filtering freeze-drying.
Test example
This test passes judgment on its ACN:H in DMF solution, DMSO solution, HPLC respectively for the peptide chain of each group
2under O (1:3) solution and LiOH saturated solution, the dissolving power of 2mg/mL.Indissoluble polypeptide chain-connecting arm-hydrophilic polypeptides chain in above-described embodiment 1,2 is respectively as group 1, group 2, and independent indissoluble polypeptide chain ILVLLIII as a control group.As shown in table 3:
Table 3 indissoluble polypeptide connects the dissolving power evaluation after process
The sequence of target peptide chain is: ILVLLIII, at DMF, DMSO and conventional HPLC purification condition ACN:H
2o=1:3 is very muddy and have solid to separate out after dissolving.ILVLLIII4-HMBA-DDDDDEEKEEEE is at DMF, DMSO and conventional HPLC purification condition ACN:H
2o (1:3) is all very limpid and separate out without solid after dissolving.And ILVLLIII-HPA-RRRRREDKKKKK is at ACN:H
2dissolve completely in O (1:3), but in DMF and DMSO, still have solid substance to separate out.
Peptide chain for first group and second group is dissolved in LiOH saturated solution, requires dissolve completely when preferably adding, and then wait reaction after 3 hours, solid is separated out in the hydrolysis of indissoluble polypeptide chain.And the solute effect of second group obviously do not have first group good, product yield can be caused to decline.The amino acid structure of visible first group of hydrophilic polypeptides chain is more conducive to the precipitation of last indissoluble polypeptide chain.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. synthesis-separation purification method of indissoluble polypeptide, is characterized in that, comprise the steps:
(1) utilize Wang resin for initial resin, employing solid-phase synthesis one by one coupling has the hydrophilic amino acid of blocking group, after coupling protected hydrophilic amino acid, add DIC and HOBt and carry out condensation, wash after reacting completely, the next protected hydrophilic amino acid of coupling, obtains Wang resin-hydrophilic polypeptides chain again;
(2) connecting arm is added in the product obtained to step (1) complete to condensation reaction;
(3) the indissoluble amino acid with blocking group is added one by one, whenever coupling one has the indissoluble amino acid of blocking group, add DIC and HOBt and carry out condensation, wash after reacting completely, the next protected indissoluble amino acid of coupling again, until be coupled to last indissoluble amino acid;
(4) add cutting agent cracking resin, get filtrated stock and add ether precipitation, centrifugation obtains hydrophilic polypeptides-connecting arm-hydrophobic polypeptides crude product;
(5) the crude product polypeptide that previous step obtains is dissolved, utilize high performance liquid chromatography separating-purifying;
(6) the crude product polypeptide LiOH saturated solution after purifying is carried out ester linkage hydrolyzing, separate out indissoluble polypeptide.
2. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 1; it is characterized in that: in described step (1) and step (3); after adding the hydrophilic amino acid or indissoluble amino acid with blocking group, react 1 hour at 30 DEG C.
3. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 2; it is characterized in that: in described step (3); add first when there is the indissoluble amino acid of blocking group, also add DMAP and condensation reaction 5 hours at 30 DEG C.
4. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 1, is characterized in that: described connecting arm is that Isosorbide-5-Nitrae position is respectively by benzene compound that hydroxyalkyl and carboxyalkyl replace.
5. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 4, it is characterized in that: described connecting arm is from 4-HBA, to hydroxymethyl-benzoic acid, 4-(2-hydroxyethyl) phenylformic acid, 4-(3-hydroxypropyl) phenylformic acid, 4-(4-hydroxybutyl) phenylformic acid, p-hydroxyphenylaceticacid, 4-(methylol) toluylic acid, para hydroxybenzene propionic acid, 3-[4-(hydroxymethyl) phenyl] propionic acid, 4-(4-hydroxy phenyl) butyric acid, 2-(4-hydroxybenzene) propionic acid, 3-(4-hydroxy phenyl) butyric acid, 2-[(4-hydroxy phenyl) methyl] aminobutyric acid, 4-(1-hydroxyethyl)-benzoic any one.
6. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 1, is characterized in that: described hydrophilic polypeptides chain is that different hydrophilic amino acid dehydrating condensation forms.
7. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 6, is characterized in that: described hydrophilic amino acid be arginine, Methionin, l-asparagine, aspartic acid, glutamine, L-glutamic acid, Histidine, proline(Pro) any one.
8. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 7, is characterized in that: described L-glutamic acid and/or aspartic acid overall number account for 30% ~ 80% of total amino acid number.
9. synthesis-the separation purification method of a kind of indissoluble polypeptide according to claim 1, it is characterized in that: described cutting agent is TFA, Tis, EDT, volume ratio is 95:3:2.
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| WO2017020569A1 (en) * | 2015-07-31 | 2017-02-09 | 南京斯拜科生化实业有限公司 | Synthesis-separation purification method for indissolvable polypeptide |
| CN112110984A (en) * | 2020-09-27 | 2020-12-22 | 深圳瑞德林生物技术有限公司 | Process for producing polypeptide |
| CN113125594A (en) * | 2021-04-02 | 2021-07-16 | 宁波大学 | Peptide chain hydrolysis reagent and preparation method and application thereof |
| CN116162124A (en) * | 2023-04-21 | 2023-05-26 | 吉尔生化（上海）有限公司 | Preparation method of continuous glutamine polypeptide |
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| CN110386964B (en) * | 2018-04-21 | 2023-04-07 | 深圳市健元医药科技有限公司 | Solid-liquid synthesis method of leuprorelin |
| CN112526051B (en) * | 2020-12-18 | 2022-08-09 | 上海吉奉生物科技有限公司 | Fmoc-lysine high performance liquid chromatography determination method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017020569A1 (en) * | 2015-07-31 | 2017-02-09 | 南京斯拜科生化实业有限公司 | Synthesis-separation purification method for indissolvable polypeptide |
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| CN116162124A (en) * | 2023-04-21 | 2023-05-26 | 吉尔生化（上海）有限公司 | Preparation method of continuous glutamine polypeptide |
| CN116162124B (en) * | 2023-04-21 | 2023-06-30 | 吉尔生化（上海）有限公司 | Preparation method of continuous glutamine polypeptide |
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