CN102690325A - Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl - Google Patents
Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl Download PDFInfo
- Publication number
- CN102690325A CN102690325A CN2012101894816A CN201210189481A CN102690325A CN 102690325 A CN102690325 A CN 102690325A CN 2012101894816 A CN2012101894816 A CN 2012101894816A CN 201210189481 A CN201210189481 A CN 201210189481A CN 102690325 A CN102690325 A CN 102690325A
- Authority
- CN
- China
- Prior art keywords
- pro
- compound method
- protecting group
- arg
- hcl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a synthesis method of developing a substrate PyroGlu-Pro-Arg-pNA.HCl, and belongs to the field of polypeptide synthesis. The synthesis method comprises the following steps of: condensing paranitroaniline and arginine of which guanidyl is protected by bis-Boc by using phosphorus oxychloride as a condensing agent; condensing the other two amino acids into dipeptide; and condensing the two synthesized peptide segments, and removing the protecting group bis-Boc of guanidyl by adopting HCl or a mixture of HCl and ethyl acetate to form the compound. The synthesis method has high yield, simple process and low preparation cost, and is suitable for preparation in batch.
Description
Technical field
The present invention relates to polypeptide synthetic field, the particularly compound method of a peptide species chromophoric substrate PyroGlu-Pro-Arg-pNAHCl.
Background technology
The structure of the polypeptide portion of PyroGlu-Pro-Arg-pNAHCl (S-2366) is that tripeptides adds chromophoric group; It is the chromophoric substrate of a kind of blood plasma activated protein C (being called for short APC) and plasma thromboplastin antecedent; This substrate can be used for the active detection by quantitative of blood plasma activated protein C, in scientific research with have a wide range of applications clinically.
Existing document is not seen relevant S-2366 synthetic report, and in the synthetic document of other relevant chromophoric substrates, methods such as azide method, active ester method, mixed anhydride method are used in the condensation of peptide bond usually, and operation is comparatively complicated, and yield is not high.
Arginic guanidine radicals has strong nucleophilicity and alkalescence, in polypeptide is synthetic, must protect, and guard method commonly used has nitro, carbobenzoxy protection and uses more benzene sulfonyl base class (like Pbf) protection etc. in the solid phase synthesis now.These blocking groups all need carry out the conversion of salt type to peptide substrate behind deprotection, change scientific research and the hydrochloride that uses clinically or the form of acetate into.Change at present salt and generally be through crossing the mode of ion exchange column, need expensive Ion Exchange Medium and very long spend the post time, especially when preparation amount all the more so greatly the time.
P-Nitroaniline (pNA), since the strong electrophilic effect of nitro, the non-constant of nucleophilicity, during with the l-arginine condensation, general amido linkage synthesizes the method poor, and yield is very low.
Summary of the invention
To the defective of prior art, the invention provides the compound method of a kind of PyroGlu-Pro-Arg-pNAHCl of high yield, this method technology is simple, cost is low, is fit to produce in enormous quantities.
For solving the problems of the technologies described above, the present invention has adopted following scheme:
The compound method of PyroGlu-Pro-Arg-pNAHCl may further comprise the steps:
(1) with the POCl3 is condensing agent, all has protection basic l-arginine and p-Nitroaniline condensation with guanidine radicals amino; Said arginic guanidine radicals protection base is two tertbutyloxycarbonyls;
(2) remove the amino protecting group of step (1) products therefrom, obtain midbody 1;
(3) with Pyrrolidonecarboxylic acid and the basic proline(Pro) condensation of carboxyl band protection;
(4) remove the carboxyl-protecting group of step (3) products therefrom, obtain midbody 2;
(5) said midbody 1 is carried out condensation with midbody 2;
(6) mixture with HCl or HCl and ETHYLE ACETATE is a deprotection base reagent, removes the guanidine radicals protection base of step (5) products therefrom, obtains PyroGlu-Pro-Arg-pNAHCl.
In the above-mentioned compound method, said arginic amino protecting group is a 9-fluorenylmethyloxycarbonyl; When being amino protecting group with the 9-fluorenylmethyloxycarbonyl, the reagent that removes of the said amino protecting group of step (2) is alkali.
The carboxyl-protecting group of said proline(Pro) is an ester, the preferred tertiary butyl ester; When being the carboxyl-protecting group of proline(Pro) with ester (comprising the tert-butyl ester), the reagent that removes of the said carboxyl-protecting group of step (4) is acid.
In the above-mentioned compound method, the temperature of reaction of step (1) is-10 ℃～0 ℃, preferred-5 ℃.
The reaction solvent of step (1) is an alkali organic solvent, and the preferred reaction solvent is a pyridine.
In the above-mentioned compound method; It is condensing agent that carbodiimide is adopted in the said condensation reaction of step (3) and step (5); Said carbodiimide is selected from N; N'-NSC 57182, N, N-DIC, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, preferred 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate.
The 1-ethyl-when (3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate was condensing agent, adding HOBt helped to reduce the generation of side reaction when reaction, effectively improves condensation efficiency when adopting.
Preferred implementation route of the present invention is following:
Compound method according to the invention has following characteristics:
(1) condensation efficiency of l-arginine and chromophoric group (p-Nitroaniline) is high
P-Nitroaniline, since the strong electrophilic effect of nitro, the non-constant of nucleophilicity, and the synthetic method of general amido linkage is difficult to the condensation success.The present invention adopts POCl3 as condensing agent, and activating essence propylhomoserin carboxyl with p-Nitroaniline generation ammonolysis reaction, can react completely in 15-30 minute then at low temperatures, and yield is more than 80%.The solvent pyridine can react by the catalysis nucleophilic attack, improves reaction efficiency, can protect acid-unstable group (like Boc) simultaneously.
(2) the peptide bond condensation efficiency is high
The preferred carbodiimide type of peptide bond condensation of the present invention condensation reagent has reaction conditions gentleness, aftertreatment is simple, yield is higher characteristics.More preferably 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is a condensation reagent, and it is water-soluble, when reaction, adds HOBt, can side reaction be controlled at very low scope, effectively improves condensation efficiency.
(3) the deprotection technology of l-arginine guanidine radicals protection base is simple, and cost is low
It is arginic guanidine radicals blocking group that the present invention adopts two tertbutyloxycarbonyls (bis-Boc); In the final step of reaction, use the mixture of HCl or HCl and ETHYLE ACETATE to carry out deprotection, the deprotection reaction temperature is lower; Chromophoric group is unaffected; Product directly forms the hydrochloride of peptide substrate, saves and changes the salt step, has saved preparation time and cost.
Compared with prior art, reaction conditions of the present invention is gentle, and aftertreatment is simple, and yield is high, and the finished product yield is greater than 50%, and preparation cost is low, is fit to preparation in enormous quantities.
Embodiment
The invention discloses the compound method of a kind of chromophoric substrate PyroGlu-Pro-Arg-pNAHCl, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention is described through preferred embodiment, and the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
Embodiment PyroGlu-Pro-Arg-pNAHCl's is synthetic
(1) Fmoc-Arg (Boc)
2-pNA's is synthetic
In the 250mL there-necked flask, add 25g (41.9mmol) Fmoc-Arg (Boc)
2-OH, 5.8g (41.9mmol) p-Nitroaniline and 125mL pyridine are cooled to-5 ℃ after the dissolving.Controlled temperature T≤0 ℃ drips 7.1g (46.3mmol) POCl
3, add continued reaction 15～30min.Reaction solution is transferred in the 500mL frozen water, adds dichloromethane extraction, organic layer is water, 10% Hydrocerol A, saturated sodium bicarbonate, saturated common salt water washing successively, anhydrous Na
2SO
4Drying, revolve dried, the silicagel column purifying, little yellow solid 25.5g, yield 85%.
The yield calculation formula:
Yield=product Fmoc-Arg (Boc)
2-pNA mole number/raw material Fmoc-Arg (Boc)
2-OH mole number * 100%.
（2）H-Arg(Boc)
2-pNA
In the 250mL flask, add 25.5g Fmoc-Arg (Boc)
2-pNA and 300mL DMF, the dissolving back adds the 25mL piperidines, reacts 20～30min under the room temperature.Add 500mL water toward reaction solution, use dichloromethane extraction then.The organic layer washing, clean to the piperidines quilt, anhydrous Na
2SO
4Drying is revolved driedly, adds 150mL petroleum ether and stirring 2～3h, filters, and solid is pulled an oar once with sherwood oil again, filter, off-white color solid 15.4g, yield 88%.
The yield calculation formula:
Yield=product H-Arg (Boc)
2-pNA mole number/raw material Fmoc-Arg (Boc)
2-pNA mole number * 100%.
（3）PyroGlu-Pro-OtBu
In the 250mL there-necked flask, add 10g (58.4mmol) Pro-OtBu, 16.0g (123.9mmol) PyroGlu-OH, 8.7g (64.3mmol) HOBt, 100mL methylene dichloride and 11.3g (87.4mmol) DIEA, be cooled to-5 ℃.Add 15.6g (81.4mmol) EDC.HCl, add the back at-5～0 ℃ of reaction 1h, rise to room temperature then naturally, reaction is spent the night.Add saturated aqueous common salt toward reaction solution, use dichloromethane extraction then.Organic layer is used 10% Hydrocerol A, saturated sodium bicarbonate, saturated common salt water washing successively.Anhydrous Na
2SO
4Drying, revolve dried, off-white color waxy solid 16g, yield 97%.
The yield calculation formula:
Yield=product P yroGlu-Pro-OtBu mole number/raw material Pro-OtBu mole number * 100%.
（4）PyroGlu-Pro-OH
In the 250mL flask, add 16g PyroGlu-Pro-OtBu and 40mL methylene dichloride, the dissolving back adds the 40mL trifluoroacetic acid, reacts 1h under the room temperature.Reaction solution revolves dried, gets oily matter, adds the 100mL MTBE and makes oily matter curing, and smash the back to pieces and stir 2～3h, filtration, solid is pulled an oar once with MTBE again, filters, and gets white solid 11.2g, yield 87%.
The yield calculation formula:
Yield=product P yroGlu-Pro-OH mole number/raw material PyroGlu-Pro-OtBu mole number * 100%.
（5）PyroGlu-Pro-Arg(Boc)
2-pNA
In the 250mL there-necked flask, add 15.4g (31.1mmol) H-Arg (Boc)
2-pNA, 10.5g (46.4mmol) PyroGlu-Pro-OH, 6.3g (46.6mmol) HOBt, 150mL methylene dichloride and 6g (46.4mmol) DIEA are cooled to-5 ℃.Add 8.9g (46.4mmol) EDC.HCl, add the back at-5～0 ℃ of reaction 1h, rise to room temperature then naturally, reaction is spent the night.Add water toward reaction solution, use dichloromethane extraction then.Organic layer is used 10% Hydrocerol A, saturated sodium bicarbonate, saturated common salt water washing successively.Anhydrous Na
2SO
4Drying, revolve dried, the silicagel column purifying, off-white color solid 18.4g, yield 84%.
The yield calculation formula:
Yield=product P yroGlu-Pro-Arg (Boc)
2-pNA mole number/raw material H-Arg (Boc)
2-pNA mole number * 100%.
（6）PyroGlu-Pro-Arg-pNA.HCl
In 500mL single port flask, add 18.4g PyroGlu-Pro-Arg (Boc)
2-pNA and 200mL5～6M HCl/ ETHYLE ACETATE reacts 2～3h under the room temperature.Solvent is revolved dried, adds 200mL ETHYLE ACETATE after-filtration, thick peptide 14g.Thick peptide with 1% acetum wash-out, is collected the component freeze-drying through Sephadex G-15 purifying, gets white solid 11.7g, yield 83%, purity 96%.
The yield calculation formula:
Yield=product P yroGlu-Pro-Arg-pNA.HCl mole number/raw material PyroGlu-Pro-Arg (Boc)
2-pNA mole number * 100%.
The calculation formula of total recovery:
Total recovery=end product PyroGlu-Pro-Arg-pNA.HCl mole number/starting raw material Fmoc-Arg (Boc)
2-OH mole number=51.8%.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (12)
1.PyroGlu-Pro-Arg-pNAHCl compound method, may further comprise the steps:
(1) with the POCl3 is condensing agent, all has protection basic l-arginine and p-Nitroaniline condensation with guanidine radicals amino; Said arginic guanidine radicals protection base is two tertbutyloxycarbonyls;
(2) remove the amino protecting group of step (1) products therefrom, obtain midbody 1;
(3) with Pyrrolidonecarboxylic acid and the basic proline(Pro) condensation of carboxyl band protection;
(4) remove the carboxyl-protecting group of step (3) products therefrom, obtain midbody 2;
(5) said midbody 1 is carried out condensation with midbody 2;
(6) mixture with HCl or HCl and ETHYLE ACETATE is a deprotection base reagent, removes the guanidine radicals protection base of step (5) products therefrom, obtains PyroGlu-Pro-Arg-pNAHCl.
2. compound method as claimed in claim 1 is characterized in that, said arginic amino protecting group is a 9-fluorenylmethyloxycarbonyl.
3. compound method as claimed in claim 2 is characterized in that, the reagent that removes of the said amino protecting group of step (2) is alkali.
4. compound method as claimed in claim 1 is characterized in that, the carboxyl-protecting group of said proline(Pro) is an ester.
5. compound method as claimed in claim 1 is characterized in that, the carboxyl-protecting group of said proline(Pro) is the tert-butyl ester.
6. like claim 4 or 5 described compound methods, it is characterized in that the reagent that removes of the said carboxyl-protecting group of step (4) is acid.
7. compound method as claimed in claim 1 is characterized in that, the temperature of reaction of step (1) is-10 ℃～0 ℃.
8. compound method as claimed in claim 1 is characterized in that, the temperature of reaction of step (1) is-5 ℃.
9. compound method as claimed in claim 1 is characterized in that, the reaction solvent of step (1) is an alkali organic solvent.
10. compound method as claimed in claim 1 is characterized in that, the reaction solvent of step (1) is a pyridine.
11. compound method as claimed in claim 1 is characterized in that, it is condensing agent that carbodiimide is adopted in the said condensation reaction of step (3) and step (5).
12. compound method as claimed in claim 11 is characterized in that, said carbodiimide is selected from N, N'-NSC 57182, N, N-DIC, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210189481.6A CN102690325B (en) | 2012-06-08 | 2012-06-08 | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210189481.6A CN102690325B (en) | 2012-06-08 | 2012-06-08 | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102690325A true CN102690325A (en) | 2012-09-26 |
| CN102690325B CN102690325B (en) | 2014-01-15 |
Family
ID=46856077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210189481.6A Active CN102690325B (en) | 2012-06-08 | 2012-06-08 | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102690325B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911253A (en) * | 2012-11-30 | 2013-02-06 | 北京华宇亿康生物工程技术有限公司 | New dipeptide derivative and salt for detecting enzyme activity |
| CN104109189A (en) * | 2013-04-18 | 2014-10-22 | 中国人民解放军军事医学科学院毒物药物研究所 | Liquid-phase synthetic method of Thr-Pro-Pro-Thr tetrapiptide |
| CN107474108A (en) * | 2017-07-18 | 2017-12-15 | 上海太阳生物技术有限公司 | Suc Ile Glu (γ Pip) Gly Arg pNAHCl preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405168A (en) * | 2001-09-07 | 2003-03-26 | 中国科学院上海药物研究所 | Tert-butoxy carbonyl dihydro artemisinin, preparation method and drug composition thereof |
| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
-
2012
- 2012-06-08 CN CN201210189481.6A patent/CN102690325B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405168A (en) * | 2001-09-07 | 2003-03-26 | 中国科学院上海药物研究所 | Tert-butoxy carbonyl dihydro artemisinin, preparation method and drug composition thereof |
| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
Non-Patent Citations (4)
| Title |
|---|
| RIJKERS D.T.等: "A convenient synthesis of amino acid p-nitroanilides; synthons in the synthesis of protease substrates", 《TETRAHEDRON》 * |
| RIJKERS D.T.等: "A generally applicable synthesis of amino acid p-nitroanilides as synthons", 《PEPTIDES》 * |
| RIJKERS D.T.等: "The use of phosphorus oxychlorides in the synthesis of amino acid p-nitroanilides", 《RECUEIL DES TRAVAUX CHIMIQUES DES PAYS-BAS》 * |
| WU YOULING等: "An efficient method for the preparation of w, w-bis-urethane protected arginine derivatives", 《SYNTHETIC COMMUNICATIONS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911253A (en) * | 2012-11-30 | 2013-02-06 | 北京华宇亿康生物工程技术有限公司 | New dipeptide derivative and salt for detecting enzyme activity |
| CN104109189A (en) * | 2013-04-18 | 2014-10-22 | 中国人民解放军军事医学科学院毒物药物研究所 | Liquid-phase synthetic method of Thr-Pro-Pro-Thr tetrapiptide |
| CN104109189B (en) * | 2013-04-18 | 2019-05-10 | 中国人民解放军军事医学科学院毒物药物研究所 | The liquid-phase synthesis process of Thr-Pro-Pro-Thr tetrapeptide |
| CN107474108A (en) * | 2017-07-18 | 2017-12-15 | 上海太阳生物技术有限公司 | Suc Ile Glu (γ Pip) Gly Arg pNAHCl preparation method |
| CN107474108B (en) * | 2017-07-18 | 2021-09-03 | 上海太阳生物技术有限公司 | Preparation method of Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA & HCl |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102690325B (en) | 2014-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107857797A (en) | The liquid-phase fragment synthetic method of one species snake venom tripeptides | |
| CN106632609A (en) | Method for preparing hexapeptide and product thereof | |
| CN102690325B (en) | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl | |
| CN103694316A (en) | Preparation method of argireline | |
| CN104086632A (en) | Method for preparing cetrorelix | |
| CN103992383A (en) | Method for preparing icatibant | |
| CN103012563A (en) | Solid-phase synthesis method of antibacterial peptide Iseganan | |
| CN105330726A (en) | Leuprorelin synthesis method | |
| CN102532267A (en) | Method for preparing icatibant | |
| CN103183716B (en) | Preparation method of tauro ursodesoxy cholic acid | |
| CN104211801A (en) | Method for preparing lixisenatide | |
| CN103897029B (en) | A kind of preparation method of romidepsin | |
| CN101362807B (en) | Process for preparation of cyclodextrin aminoacid derivates | |
| CN104817631B (en) | A kind of method for synthesizing sinapultide | |
| CN101519429B (en) | Solid phase method for synchronizing Argatroban | |
| CN101250172B (en) | Arginine double-protective preparation technique | |
| CN106046077A (en) | Tulathromycin A synthesis method | |
| CN108484735B (en) | Synthesis method of wide-activity cyclic heptapeptide Reniochalistatin A-D | |
| CN105418547A (en) | Preparation for sofosbuvir key intermediate | |
| CN103373940B (en) | Novel process for synthesizing N-FMOC-amino acid crude product of non-active side chain | |
| CN107474108B (en) | Preparation method of Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA & HCl | |
| CN110903249B (en) | Lopinavir prepared by one-pot method | |
| CN111018791B (en) | Novel method for preparing lopinavir | |
| CN107778350A (en) | A kind of method for synthesizing romidepsin | |
| CN103396475B (en) | A kind of method of pure solid-phase synthetic peptide class microbiotic Colistin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |