CN102690325B - Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl - Google Patents

Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl Download PDF

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CN102690325B
CN102690325B CN201210189481.6A CN201210189481A CN102690325B CN 102690325 B CN102690325 B CN 102690325B CN 201210189481 A CN201210189481 A CN 201210189481A CN 102690325 B CN102690325 B CN 102690325B
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protecting group
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CN102690325A (en
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谢永华
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SHANGHAI SUN BIO-TECH Co Ltd
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SHANGHAI SUN BIO-TECH Co Ltd
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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

Polypeptide PyroGlu-Pro-Arg-pNAHCl's is synthetic
Technical field
The present invention relates to Peptides Synthesis, particularly the synthetic method of a peptide species chromophoric substrate PyroGlu-Pro-Arg-pNAHCl.
Background technology
The structure of polypeptide portion 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 detection by quantitative of blood plasma activated protein C activity, in scientific research with have a wide range of applications clinically.
Existing document has no the synthetic report of relevant S-2366, and in the synthetic document of other relevant chromophoric substrates, the methods such as azide method, active ester method, mixed anhydride method are used in the condensation of peptide bond conventionally, and operation is comparatively complicated, and yield is not high.
Arginic guanidine radicals has strong nucleophilicity and alkalescence, in polypeptide is synthetic, must be protected, and conventional guard method has nitro, carbobenzoxy protection and in solid phase synthesis, uses more benzene sulfonyl base class (as Pbf) protection etc. now.These blocking groups all need peptide substrate to carry out the conversion of salt type after deprotection, change scientific research and the hydrochloride using clinically or the form of acetate into.Turning at present salt is generally by the mode of mistake ion exchange column, needs expensive Ion Exchange Medium and very long post time excessively, especially all the more so when preparation amount is larger.
P-Nitroaniline (pNA), due to the strong electron attraction of nitro, the non-constant of nucleophilicity, during with arginine condensation, the synthetic method poor effect of general amido linkage, yield is very low.
Summary of the invention
For the defect of prior art, the invention provides the synthetic method of a kind of PyroGlu-Pro-Arg-pNAHCl of high yield, the method technique is simple, cost is low, is applicable to producing in enormous quantities.
For solving the problems of the technologies described above, the present invention has adopted following scheme:
The synthetic method of PyroGlu-Pro-Arg-pNAHCl, comprises the following steps:
(1) take phosphorus oxychloride as condensing agent, by amino and guanidine radicals all with arginine and the p-Nitroaniline condensation of protecting group; Described arginic guanidine radicals protecting group is two tertbutyloxycarbonyls;
(2) remove the amino protecting group of step (1) products therefrom, obtain intermediate 1;
(3) by the proline(Pro) condensation of Pyrrolidonecarboxylic acid and carboxyl band protecting group;
(4) remove the carboxyl-protecting group of step (3) products therefrom, obtain intermediate 2;
(5) described intermediate 1 and intermediate 2 are carried out to condensation;
(6) take the mixture of HCl or HCl and ethyl acetate is Deprotection reagent, removes the guanidine radicals protecting group of step (5) products therefrom, obtains PyroGlu-Pro-Arg-pNAHCl.
In above-mentioned synthetic method, described arginic amino protecting group is 9-fluorenylmethyloxycarbonyl; When take 9-fluorenylmethyloxycarbonyl during as amino protecting group, the reagent that removes of the described amino protecting group of step (2) is alkali.
The carboxyl-protecting group of described proline(Pro) is ester, preferred tertiary butyl ester; When take the carboxyl-protecting group that ester (comprising the tert-butyl ester) is proline(Pro), the described carboxyl-protecting group of step (4) remove reagent for acid.
In above-mentioned synthetic method, the temperature of reaction of step (1) is-10 ℃～0 ℃, preferably-5 ℃.
The reaction solvent of step (1) is alkali organic solvent, and preferred reaction solvent is pyridine.
In above-mentioned synthetic method, it is condensing agent that the described condensation reaction of step (3) and step (5) adopts carbodiimide, described carbodiimide is selected from N, N'-dicyclohexylcarbodiimide, N, N-DIC, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, preferably 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate.
When adopting 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate to be condensing agent, when reaction, add HOBt, contribute to reduce the generation of side reaction, effectively improve condensation efficiency.
Preferred implementation route of the present invention is as follows:
Figure BDA00001744403300021
Synthetic method of the present invention, has following characteristics:
(1) condensation efficiency of arginine and chromophoric group (p-Nitroaniline) is high
P-Nitroaniline, due to the strong electron attraction of nitro, the non-constant of nucleophilicity, the synthetic method of general amido linkage is difficult to condensation success.The present invention adopts phosphorus oxychloride as condensing agent, and activating essence propylhomoserin carboxyl, then, with p-Nitroaniline generation ammonolysis reaction, can react completely in 15-30 minute at low temperatures, and yield is more than 80%.Solvent pyridine can react by catalysis nucleophilic attack, improves reaction efficiency, can protect acid-unstable group (as Boc) simultaneously.
(2) peptide bond condensation efficiency is high
The preferred carbodiimide type of peptide bond condensation of the present invention condensation reagent, has the advantages that reaction conditions gentleness, aftertreatment are simple, yield is higher.More preferably 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is condensation reagent, and it is water-soluble, when reaction, adds HOBt, side reaction can be controlled to very low scope, effectively improves condensation efficiency.
(3) the deprotection technique of arginine guanidine radicals protecting group is simple, and cost is low
It is arginic guanidine radicals blocking group that the present invention adopts two tertbutyloxycarbonyls (bis-Boc); final step in reaction; use the mixture of HCl or HCl and ethyl acetate to carry out deprotection; deprotection reaction temperature is lower; chromophoric group is unaffected; product directly forms the hydrochloride of peptide substrate, saves and turns 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 finished product yield is greater than 50%, and preparation cost is low, is applicable to preparation in enormous quantities.
Embodiment
The invention discloses the synthetic method of a kind of chromophoric substrate PyroGlu-Pro-Arg-pNAHCl, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, and related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment PyroGlu-Pro-Arg-pNAHCl's is synthetic
(1) Fmoc-Arg (Boc) 2-pNA's is synthetic
In 250mL there-necked flask, add 25g(41.9mmol) Fmoc-Arg (Boc) 2-OH, 5.8g(41.9mmol) p-Nitroaniline and 125mL pyridine, after dissolving, be cooled to-5 ℃.Control temperature T≤0 ℃, drip 7.1g(46.3mmol) POCl 3, add rear continuation reaction 15～30min.Reaction solution is transferred in 500mL frozen water, adds dichloromethane extraction, organic layer is water, 10% citric acid, saturated sodium bicarbonate, saturated common salt water washing successively, anhydrous Na 2sO 4dry, be spin-dried for, silicagel column purifying, obtains micro-yellow solid 25.5g, yield 85%.
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 250mL flask, add 25.5g Fmoc-Arg (Boc) 2-pNA and 300mL DMF, add 25mL piperidines after dissolving, under room temperature, react 20～30min.Toward reaction solution, add 500mL water, then use dichloromethane extraction.Organic layer washing, is cleaned anhydrous Na to piperidines 2sO 4dry, be spin-dried for, add 150mL petroleum ether and stirring 2～3h, filter, solid is pulled an oar once with sherwood oil again, filters, and obtains off-white color solid 15.4g, yield 88%.
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 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, after adding, at-5～0 ℃ of reaction 1h, then naturally rise to room temperature, reaction is spent the night.Toward reaction solution, add saturated aqueous common salt, then use dichloromethane extraction.Organic layer is used 10% citric acid, saturated sodium bicarbonate, saturated common salt water washing successively.Anhydrous Na 2sO 4dry, be spin-dried for, obtain off-white color waxy solid 16g, yield 97%.
Yield calculation formula:
Yield=product P yroGlu-Pro-OtBu mole number/raw material Pro-OtBu mole number * 100%.
（4）PyroGlu-Pro-OH
In 250mL flask, add 16g PyroGlu-Pro-OtBu and 40mL methylene dichloride, after dissolving, add 40mL trifluoroacetic acid, under room temperature, react 1h.Reaction solution is spin-dried for, and obtains oily matter, adds 100mL methyl tertiary butyl ether that oily matter is solidified, and smashs rear stirring 2～3h to pieces, filters, and solid is pulled an oar once with methyl tertiary butyl ether again, filters, and obtains white solid 11.2g, yield 87%.
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 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, be cooled to-5 ℃.Add 8.9g(46.4mmol) EDC.HCl, after adding, at-5～0 ℃ of reaction 1h, then naturally rise to room temperature, reaction is spent the night.Toward reaction solution, add water, then use dichloromethane extraction.Organic layer is used 10% citric acid, saturated sodium bicarbonate, saturated common salt water washing successively.Anhydrous Na 2sO 4dry, be spin-dried for, silicagel column purifying, obtains off-white color solid 18.4g, yield 84%.
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/ ethyl acetate, react 2～3h under room temperature.Solvent is spin-dried for, filters after adding 200mL ethyl acetate, obtain thick peptide 14g.Thick peptide, by Sephadex G-15 purifying, with 1% acetum wash-out, is collected component freeze-drying, obtains white solid 11.7g, yield 83%, purity 96%.
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 is only the preferred embodiment of the present invention; it should be pointed out 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 (11)

  1. The synthetic method of 1.PyroGlu-Pro-Arg-pNAHCl, comprises the following steps:
    (1) take phosphorus oxychloride as condensing agent, by amino and guanidine radicals all with arginine and the p-Nitroaniline condensation of protecting group; Described arginic guanidine radicals protecting group is two tertbutyloxycarbonyls; Described arginic amino protecting group is 9-fluorenylmethyloxycarbonyl;
    (2) remove the amino protecting group of step (1) products therefrom, obtain intermediate 1;
    (3) by the proline(Pro) condensation of Pyrrolidonecarboxylic acid and carboxyl band protecting group;
    (4) remove the carboxyl-protecting group of step (3) products therefrom, obtain intermediate 2;
    (5) described intermediate 1 and intermediate 2 are carried out to condensation;
    (6) take the mixture of HCl or HCl and ethyl acetate is Deprotection reagent, removes the guanidine radicals protecting group of step (5) products therefrom, obtains PyroGlu-Pro-Arg-pNAHCl.
  2. 2. synthetic method as claimed in claim 1, is characterized in that, the reagent that removes of the described amino protecting group of step (2) is alkali.
  3. 3. synthetic method as claimed in claim 1, is characterized in that, the carboxyl-protecting group of described proline(Pro) is ester.
  4. 4. synthetic method as claimed in claim 1, is characterized in that, the carboxyl-protecting group of described proline(Pro) is the tert-butyl ester.
  5. 5. the synthetic method as described in claim 3 or 4, is characterized in that, the reagent that removes of the described carboxyl-protecting group of step (4) is acid.
  6. 6. synthetic method as claimed in claim 1, is characterized in that, the temperature of reaction of step (1) is-10 ℃～0 ℃.
  7. 7. synthetic method as claimed in claim 1, is characterized in that, the temperature of reaction of step (1) is-5 ℃.
  8. 8. synthetic method as claimed in claim 1, is characterized in that, the reaction solvent of step (1) is alkali organic solvent.
  9. 9. synthetic method as claimed in claim 1, is characterized in that, the reaction solvent of step (1) is pyridine.
  10. 10. synthetic method as claimed in claim 1, is characterized in that, it is condensing agent that the described condensation reaction of step (3) and step (5) adopts carbodiimide.
  11. 11. synthetic methods as claimed in claim 10, is characterized in that, described carbodiimide is selected from N, N'-dicyclohexylcarbodiimide, N, N-DIC, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate.
CN201210189481.6A 2012-06-08 2012-06-08 Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl Active CN102690325B (en)

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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
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

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