CN112110868A - Preparation method of Fmoc-beta-Ala-AA-OH - Google Patents

Preparation method of Fmoc-beta-Ala-AA-OH Download PDF

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CN112110868A
CN112110868A CN201910542465.2A CN201910542465A CN112110868A CN 112110868 A CN112110868 A CN 112110868A CN 201910542465 A CN201910542465 A CN 201910542465A CN 112110868 A CN112110868 A CN 112110868A
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ala
fmoc
beta
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preparing
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张东海
胡佳伟
郑征
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Chengdu Zhengyuan Biochemical Technology Co ltd
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Chengdu Zhengyuan Biochemical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/16—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C07D249/18—Benzotriazoles

Abstract

The invention relates to a preparation method of Fmoc-beta-Ala-AA-OH, belonging to the technical field of polypeptide synthesis. The preparation method of Fmoc-beta-Ala-AA-OH comprises the following steps: (1) mixing HBTA and thionyl chloride, and then adding Fmoc-beta-Ala-OH for reaction to generate Fmoc-beta-Ala-Bt; (2) dissolving AA in a buffer system, adding Fmoc-beta-Ala-Bt solution for reaction to prepare Fmoc-beta-Ala-AA-OH; wherein, the AA is an amino acid or an amino acid derivative with only one amino group; the buffer system is Na2CO3，NaHCO3，K2CO3，NaHCO3At least one of the solutions. The preparation method provided by the invention is simple to operate, effectively shortens the production steps, is low in cost, improves the production efficiency and improves the yield.

Description

Preparation method of Fmoc-beta-Ala-AA-OH
Technical Field
The invention relates to a preparation method of Fmoc-beta-Ala-AA-OH, belonging to the technical field of polypeptide synthesis.
Background
Fmoc-beta-Ala-AA-OH is a byproduct which cannot be avoided when Fmoc-OSu is used for preparing Fmoc-protected amino acid, is also a chemical intermediate, and has important significance in research of the protected amino acid industry. The Fmoc-beta-Ala-AA-OH is synthesized by a simple and efficient method which is required by the industry at present, and the existing synthesis method mainly comprises the following steps: firstly, condensing Fmoc-beta-Ala-OH and AA-OEt, saponifying-OEt and Fmoc-protecting groups, and then generating Fmoc-beta-Ala-AA-OH by Fmoc-protection; secondly, preparing the Fmoc-beta-Ala-OH into activated ester, such as Fmoc-beta-Ala-ONB, Fmoc-beta-Ala-OBt and the like, and reacting with AA to generate Fmoc-beta-Ala-AA-OH.
However, the first method has long steps and low production efficiency; the second method, Fmoc-beta-Ala-ONB and Fmoc-beta-Ala-OBt, did not give crystals and the intermediates could not be purified.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel preparation method of Fmoc-beta-Ala-AA-OH.
In order to solve the technical problem of the invention, the preparation method of Fmoc-beta-Ala-AA-OH comprises the following steps:
(1) mixing HBTA and thionyl chloride, and then adding Fmoc-beta-Ala-OH for reaction to generate Fmoc-beta-Ala-Bt;
(2) dissolving AA in a buffer system, adding Fmoc-beta-Ala-Bt solution for reaction to prepare Fmoc-beta-Ala-AA-OH;
wherein, the AA is an amino acid or an amino acid derivative with only one amino group;
the buffer system is Na2CO3，NaHCO3，K2CO3，NaHCO3At least one of the solutions.
Preferably, the molar ratio of Fmoc-beta-Ala-OH, thionyl chloride and HBTA in the step (1) is 5: 11-12: 20.
Preferably, the thionyl chloride in the step (1) is added in two times, HBTA and part of thionyl chloride are mixed, then Fmoc-beta-Ala-OH is added and mixed evenly, and then the rest of thionyl chloride is added; preferably, the Fmoc-beta-Ala-OH is dissolved in a solvent to form a Fmoc-beta-Ala-OH solution.
Preferably, the step (1) further comprises purifying Fmoc-beta-Ala-Bt generated in the reaction; the purification is preferably carried out by filtering the liquid after the reaction to obtain a filtrate, concentrating the filtrate to obtain a solid, and subjecting the solid obtained by concentration to THF/H2Recrystallizing with 1/3 of the solvent or concentrating the solid, dissolving the solid with dichloromethane, washing with water, and washing with anhydrous Na2SO4Drying, concentrating and crystallizing.
Preferably, the Fmoc-beta-Ala-Bt solution in step (2) is preferably Fmoc-beta-Ala-Bt in acetonitrile, tetrahydrofuran or DMF.
Preferably, the molar ratio of AA to Fmoc-beta-Ala-Bt in the step (2) is 1.1-1.6: 1.
Preferably, the addition amount of the buffer system in the step (2) is based on the maintenance of the pH value of the reaction to be 8-9.
Preferably, the reaction time in the step (2) is 4-5 hours.
Preferably, the reaction in the step (2) is further purified, wherein the purification is to extract impurities by using ethyl acetate, the pH of the extracted water phase is adjusted to 5-6, solids are separated out, and Fmoc-beta-Ala-AA-OH solids are obtained by filtering.
Has the advantages that:
1. the preparation method provided by the invention is simple to operate, effectively shortens the production steps, is low in cost and improves the production efficiency.
2. The intermediate is easy to purify by adopting the method of the invention.
Detailed Description
In order to solve the technical problem of the invention, the preparation method of Fmoc-beta-Ala-AA-OH comprises the following steps:
(1) mixing HBTA and thionyl chloride, and then adding Fmoc-beta-Ala-OH for reaction to generate Fmoc-beta-Ala-Bt;
(2) dissolving AA in a buffer system, adding Fmoc-beta-Ala-Bt solution for reaction to prepare Fmoc-beta-Ala-AA-OH;
wherein, the AA is an amino acid or an amino acid derivative with only one amino group;
the buffer system is Na2CO3，NaHCO3，K2CO3，NaHCO3At least one of the solutions.
The AA has only one amino group, and if more than two amino groups are present, it reacts to form a variety of impurities.
Preferably, the molar ratio of Fmoc-beta-Ala-OH, thionyl chloride and HBTA in the step (1) is 5: 11-12: 20.
Preferably, the thionyl chloride in the step (1) is added in two times, HBTA and part of thionyl chloride are mixed, then Fmoc-beta-Ala-OH is added and mixed evenly, and then the rest of thionyl chloride is added; preferably, the Fmoc-beta-Ala-OH is dissolved in a solvent to form a Fmoc-beta-Ala-OH solution.
Preferably, the step (1) further comprises purifying Fmoc-beta-Ala-Bt generated in the reaction; preferably, the purification is carried out by filtering the liquid after the reaction to obtain a filtrate, concentrating the filtrate to obtain a solid, dissolving the solid with dichloromethane, washing with water, and using anhydrous Na as an organic phase after washing2SO4Drying, concentrating and crystallizing.
Preferably, the Fmoc-beta-Ala-Bt solution in step (2) is preferably Fmoc-beta-Ala-Bt in acetonitrile, tetrahydrofuran or DMF.
Preferably, the molar ratio of AA to Fmoc-beta-Ala-Bt in the step (2) is 1.1-1.6: 1.
Preferably, the addition amount of the buffer system in the step (2) is based on the maintenance of the pH value of the reaction to be 8-9.
Preferably, the reaction time in the step (2) is 4-5 hours.
Preferably, the reaction in the step (2) is further purified, wherein the purification is to extract impurities by using ethyl acetate, the pH of the extracted water phase is adjusted to 5-6, solids are separated out, and Fmoc-beta-Ala-AA-OH solids are obtained by filtering.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Abbreviations for examples and comparative examples of the present invention
TABLE 1 abbreviations for the examples of the present invention
Figure BDA0002102992990000031
Example 1
23.83g HBTA was added to the reaction flask and SOCl was added dropwise with stirring2(thionyl chloride) 5mL, 15.5g Fmoc-. beta. -Ala-OH was dissolved in 100mL THF, and added to the above system, followed by addition of 4mL SOCl2The reaction was carried out overnight at room temperature. Reaction ofAfter completion of the reaction, the mixture was filtered, the filtrate was concentrated to dryness, the obtained solid was dissolved in 300ml of methylene chloride, washed with water 3 times (150 ml each), and the organic phase was washed with anhydrous Na2SO4Drying, concentrating, adding petroleum ether, and crystallizing to obtain Fmoc-beta-Ala-Bt.
5g H-L-Gly-OH and 5g Na2CO3Dissolving 17g of Fmoc-beta-Ala-Bt in 150ml of water, dissolving the Fmoc-beta-Ala-Bt in 50ml of acetonitrile, adding the mixture into the system, maintaining the pH value of the reaction to be 8-9, reacting at room temperature for 4-5 hours, extracting impurities for 3 times by using 300ml of ethyl acetate after the reaction is finished, using hydrochloric acid to adjust the pH value of a water phase to be 5-6, separating out solids, and filtering to obtain Fmoc-beta-Ala-Gly-OH.
The product purity was 99.8%, the single maximum impurity was 0.1%, and the yield was 78.5%.
Example 2
238.3g HBTA was added to the reaction flask and SOCl was added dropwise with stirring2(thionyl chloride) 45mL, 155g of Fmoc-. beta. -Ala-OH was dissolved in 800mL of THF, and added to the above system, and 35mL of SOCl was further added2The reaction was carried out overnight at room temperature. After the reaction, the mixed system was filtered, the filtrate was concentrated to dryness, the obtained solid was dissolved in 2L of methylene chloride, washed with water 4 times (2L each), and the organic phase was purified with anhydrous Na2SO4Drying, concentrating, adding petroleum ether, and crystallizing to obtain Fmoc-beta-Ala-Bt.
17.0g of 17.0g H-L-Ile-OH and 10.6g of Na2CO3Dissolving the mixture in 300ml of water, dissolving 41.2g of Fmoc-beta-Ala-Bt in 300ml of acetonitrile, adding the mixture into the system, maintaining the pH value of the reaction to be 8-9, reacting at room temperature for 4-5 hours, extracting impurities for 3 times by using 300ml of ethyl acetate after the reaction is finished, using hydrochloric acid to adjust the pH value of a water phase to be 5-6, separating out solids, and filtering to obtain Fmoc-beta-Ala-Ile-OH.
The product purity was 99.1%, the single maximum impurity was 0.39%, and the yield was 70%.
Example 3
238.3g HBTA was added to the reaction flask and SOCl was added dropwise with stirring2(thionyl chloride) 45mL, 0.5mol 155g Fmoc-. beta. -Ala-OH was dissolved in 800mL THF, added to the above system, and 35mL SOCl was added2The reaction was allowed to proceed overnight. Inverse directionAfter completion of the reaction, the mixed system was filtered, the filtrate was concentrated to dryness, the obtained solid was dissolved in 2L of methylene chloride, washed with water 4 times (2L each), and the organic phase was purified with anhydrous Na2SO4Drying, concentrating, adding petroleum ether, and crystallizing to obtain Fmoc-beta-Ala-Bt.
35.10g of 35.10g H-L-Val-OH and 24.4g of Na2CO3Dissolving 41.2g of Fmoc-beta-Ala-Bt in 600ml of tetrahydrofuran in 600ml of water, adding the mixture into the system, maintaining the pH value of the reaction at 8-9, reacting for 4-5 hours, extracting impurities for 3 times by using 500ml of ethyl acetate after the reaction is finished, using hydrochloric acid to adjust the pH value of a water phase to 5-6, separating out solids, and filtering to obtain Fmoc-beta-Ala-Val-OH.
The product purity was 99.0%, the single maximum impurity was 0.41%, and the yield was 72%.

Claims (9)

  1. A method of Fmoc- β -Ala-AA-OH preparation, comprising the steps of:
    (1) mixing HBTA and thionyl chloride, and then adding Fmoc-beta-Ala-OH for reaction to generate Fmoc-beta-Ala-Bt;
    (2) dissolving AA in a buffer system, adding Fmoc-beta-Ala-Bt solution, and reacting to prepare Fmoc-beta-Ala-AA-OH;
    wherein, the AA is an amino acid or an amino acid derivative with only one amino group;
    the buffer system is Na2CO3，NaHCO3，K2CO3，NaHCO3At least one of the solutions.
  2. 2. The method for preparing Fmoc- β -Ala-AA-OH of claim 1, wherein the molar ratio of Fmoc- β -Ala-OH, thionyl chloride and HBTA in step (1) is 5:11 to 12: 20.
  3. 3. The method for preparing Fmoc- β -Ala-AA-OH as claimed in claim 1 or 2, wherein the thionyl chloride of step (1) is added in two steps, HBTA is mixed with part of thionyl chloride, then Fmoc- β -Ala-OH is added and mixed uniformly, and then the rest of thionyl chloride is added; preferably, the Fmoc-beta-Ala-OH is dissolved in a solvent to form a Fmoc-beta-Ala-OH solution.
  4. 4. The method for preparing Fmoc- β -Ala-AA-OH according to any one of claims 1 to 3, wherein the step (1) further comprises purifying Fmoc- β -Ala-Bt produced by the reaction; the purification is preferably carried out by filtering the liquid after the reaction to obtain a filtrate, concentrating the filtrate to obtain a solid, and subjecting the solid obtained by concentration to THF/H2Recrystallizing with 1/3 of the solvent or concentrating the solid, dissolving the solid with dichloromethane, washing with water, and washing with anhydrous Na2SO4Drying, concentrating and crystallizing.
  5. 5. The method for preparing Fmoc- β -Ala-AA-OH as claimed in any one of claims 1 to 4, wherein the Fmoc- β -Ala-Bt solution of step (2) is Fmoc- β -Ala-Bt in acetonitrile, tetrahydrofuran or DMF.
  6. 6. The method for preparing Fmoc-beta-Ala-AA-OH as claimed in any one of claims 1 to 5, wherein the molar ratio of AA to Fmoc-beta-Ala-Bt in step (2) is 1.1 to 1.6: 1.
  7. 7. The method for preparing Fmoc- β -Ala-AA-OH as claimed in any one of claims 1 to 6, wherein the buffer system in step (2) is added in an amount to maintain the reaction pH of 8-9.
  8. 8. The method for preparing Fmoc- β -Ala-AA-OH according to any one of claims 1 to 7, wherein the reaction time in step (2) is 4 to 5 hours.
  9. 9. The method for preparing Fmoc- β -Ala-AA-OH as claimed in any one of claims 1 to 8, wherein the reaction of step (2) is followed by purification, wherein the purification comprises extracting impurities with ethyl acetate, adjusting pH of the extracted water phase to 5-6, precipitating solids, and filtering to obtain Fmoc- β -Ala-AA-OH solids.
CN201910542465.2A 2019-06-21 2019-06-21 Preparation method of Fmoc-beta-Ala-AA-OH Pending CN112110868A (en)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALAN R. KATRITZKY ET AL.: "Chemical Ligation of S-Scylated Cysteine Peptides to Form Native Peptides via 5-, 11-, and 14-Membered Cyclic Transition States", 《J. ORG. CHEM.》 *
ALAN R. KATRITZKY ET AL.: "Convenient and Efficient Preparations of Azodye-Labeled Peptides", 《CHEM BIOL DRUG DES》 *
MALTE MEPPEN ET AL.: "Cyclic phosphoramidates as prodrugs of 2’-C-methylcytidine", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 *
ROMAN MARTY ET AL.: "Two-Fold Odd-Even Effect in Self-Assembled Nanowires from Oligopeptide-Polymer-Substituted Perylene Bisimides", 《J. AM. CHEM. SOC.》 *
SRINIVASARAO KONDAPARLA ET AL.: "Synthesis and antimalarial activity of new 4-aminoquinolines active against drug resistant strains", 《RSC ADV.》 *

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