CN104109189B - The liquid-phase synthesis process of Thr-Pro-Pro-Thr tetrapeptide - Google Patents
The liquid-phase synthesis process of Thr-Pro-Pro-Thr tetrapeptide Download PDFInfo
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- CN104109189B CN104109189B CN201310134375.2A CN201310134375A CN104109189B CN 104109189 B CN104109189 B CN 104109189B CN 201310134375 A CN201310134375 A CN 201310134375A CN 104109189 B CN104109189 B CN 104109189B
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- 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
Abstract
The present invention relates to a kind of liquid-phase synthesis process of tetrapeptide, specifically, the carboxyl of the tetrapeptide C-terminal threonine has amide structure the present invention relates to the liquid-phase synthesis process of tetrapeptide Thr-Pro-Pro-Thr, N-terminal is free amino.
Description
Technical field
The present invention relates to a kind of liquid-phase synthesis process of tetrapeptide, which has the amino acid sequence of Thr-Pro-Pro-Thr
The carboxyl of column, C-terminal threonine has amide structure.
Background technique
Depression is the most common Nervous and mental diseases, seriously affects the physical and mental health of people.In China as life saves
The quickening played, the increase of social pressures, incidence of depression significantly increase.
Drug therapy is the main means for the treatment of depression.Primary treatment drug includes tricyclic antidepressant such as the third miaow
Piperazine, amitriptyline etc.；Selective serotonin reuptake inhibitor, such as Prozac, Sertraline etc.；Serotonin/go on first kidney
Parathyrine reuptake double inhibitor, such as venlafaxin, Duloxetine etc..But common drug works slow, generally requiring several weeks arrives
Time several months, and it is obvious there are inefficient and toxicity the disadvantages of.
GLYX-13 is a kind of newly-developed antidepressant, currently carries out the clinical research of II phase.It is by adjusting in brain
NMDA (N-methyl-D-aspartate) receptor and work, and do not have serious and limiter stage pair as ketamine
Effect, such as there is illusion and schizophrenia class etc..GLYX-13 can play strong, quick, lasting antidepressant effect,
Onset time less than 24 hours, and sustainable average 7 days.As peptide medicament, GLYX-13 tolerance is good, and use is safe.
GLYX-13 is a tetrapeptide, and the sequential structure with Thr-Pro-Pro-Thr, N-terminal is free amino, and C-terminal is
Amide structure.The synthetic method of GLYX-13 includes traditional Solid phase peptide synthesis and two kinds of liquid phase peptide symthesis method, due to its sequence
Short, synthesis in solid state amino acid dosage is big, at high cost, and is not easy to realize a large amount of preparations.Liquid phase method amino acid dosage is small, high income,
It can largely prepare.
Summary of the invention
The present invention provides the liquid-phase synthesis process of tetrapeptide Thr-Pro-Pro-Thr, and this method is condensed by two dipeptide fragments
It forms, reaction yield can be improved compared to the method for extending peptide chain one by one, use water to post-process solvent as peptide bond condensation reaction, keep away
Exempt from largely to use organic solvent, intermediate is not required to column chromatographic purifying, has the characteristics that low cost and environmental protection.
The liquid-phase synthesis process of tetrapeptide Thr-Pro-Pro-Thr provided by the invention, comprising the following steps:
1) threonine Carboxylamide structure H-Pro-NH2Synthesis: amino tertbutyloxycarbonyl (the Boc)/tertiary fourth of pendant hydroxyl group
The threonine of ether protection or amino 9-fluorenylmethyloxycarbonyl (Fmoc) protection is under the conditions of -15 DEG C, with isobutyl chlorocarbonate and N- first
The effect of base morpholine, forms mixed acid anhydride, then react with ammonia water, generates the protection threonine of carboxy amidation.Then with hydrochloric acid/
Organic solvent or trifluoroacetic acid (TFA) remove Boc protecting group, or with piperidines/n,N-Dimethylformamide (DMF) solution removal
Fmoc protecting group.
2)H-Pro-Thr-NH2The synthesis of dipeptide fragment: Boc or fmoc-protected proline are under the conditions of -15 DEG C, with chlorine
Iso-butyl formate and the effect of N- methylmorpholine form mixed acid anhydride, then contract with the threonine of the carboxy amidation of upper step preparation
Reaction is closed, the dipeptides of protection is generated.Then hydrochloric acid/organic solvent or trifluoroacetic acid (TFA) deprotection base are used, or uses piperazine
Pyridine/n,N-Dimethylformamide (DMF) solution removal Fmoc protecting group.Reaction solution is poured into pure water, weak base or weak acid are passed through
Adjust the pH value of solution, the dipeptides after making deprotection base is precipitated from water, filters, and washs, vacuum drying, obtain amino from
By dipeptide fragment H-Pro-Thr-NH2。
3) synthesis of Boc-Thr (tBu)-Pro-OH dipeptide fragment: amino Boc or Fmoc protection, the tertiary butyl ether of pendant hydroxyl group
Protection or the unshielded proline of side chain act under the conditions of -15 DEG C with isobutyl chlorocarbonate and N- methylmorpholine, are formed
Mixed acid anhydride, then reacted with the proline H-Pro-OMe of carboxyl ester protection, generate the dipeptides of protection.Then it is saponified with NaOH
Reaction removing methyl esters protecting group.Reaction solution is poured into pure water, the pH value of acid-conditioning solution is added, makes the free dipeptides of carboxyl from water
Middle precipitation is filtered, and is washed, vacuum drying, dipeptide fragment Boc-Thr (the tBu)-Pro-OH protected.
4) tetrapeptide H-Thr-Pro-Pro-Thr-NH2Synthesis: dipeptide fragment Boc-Thr (tBu)-Pro-OH of protection
Under the conditions of -15 DEG C, acted on isobutyl chlorocarbonate and N- methylmorpholine, form mixed acid anhydride, then with amino free two
Peptide fragment H-Pro-Thr-NH2Condensation, generates the tetrapeptide of protection.Then it is removed with hydrochloric acid/organic solvent or trifluoroacetic acid (TFA)
Protecting group.Reaction solution is poured into pure water, the pH value of solution is adjusted by weak base or weak acid, the tetrapeptide after making deprotection base
It is precipitated, filters from water, wash, vacuum drying obtains target compound tetrapeptide H-Thr-Pro-Pro-Thr-NH2。
Specific embodiment
The present invention can be further described by the following examples.
1 tetrapeptide H-Thr-Pro-Pro-Thr-NH of embodiment2Preparation
Amidation (the H-Thr-NH of 1.1 threonine carboxyls2)
Boc-Thr (tBu)-OH20g (0.073mol), anhydrous tetrahydro furan (THF) 150ml are added in 500ml there-necked flask,
Stirring dissolves solid.Ice salt bath cools to -10 DEG C~-15 DEG C, and N- methylmorpholine 8ml is added, and it is different that chloro-carbonic acid is then added dropwise
Butyl ester 10ml keeps temperature to be not higher than -10 DEG C, and low-temp reaction 10min is kept after being added dropwise, and ammonium hydroxide 20ml, ice is then added
Bath reaction 30min, then reacts at room temperature 8h.Stop reaction, add water 300ml, ethyl acetate 200ml is added to extract precipitate, washing 3
Time.Anhydrous sodium sulfate dries 6h.Filtering, then evaporating solvent under reduced pressure, obtains white solid 16.6g, yield 83%.
Above-mentioned product is dissolved in 50ml trifluoroacetic acid or 2N hydrochloric acid/ethyl acetate solution, 1h is reacted at room temperature, after solvent is evaporated off
Obtain white solid, i.e. threonine trifluoroacetic acid/hydrochloride H-Thr-NH of carboxy amidation2.HCl。
1.2 proline-threonine dipeptide fragment H-Pro-Thr-NH2Preparation
Boc-Pro-OH20g (0.093mol) is added in 500ml there-necked flask, anhydrous tetrahydro furan (TH F) 200ml, stirring
Dissolve solid, ice salt bath cools to -10 DEG C~-15 DEG C, and N- methylmorpholine 11ml is added, isobutyl chloroformate is then added dropwise
Ester 13ml keeps temperature to be not higher than -10 DEG C, and low-temp reaction 10min is kept after being added dropwise.H-Thr-NH2.HCl14.5g molten
In 50ml tetrahydrofuran, N- methylmorpholine 11ml is added.Above-mentioned solution is added in reaction solution, low-temp reaction 30min,
Then 8h is reacted at room temperature.Stop reaction, add water 300ml, ethyl acetate 200ml is added to extract precipitate, washes 3 times.Anhydrous slufuric acid
Sodium dries 6h.Filtering, then evaporating solvent under reduced pressure, obtains white solid 25.7g, yield 82%.
Above-mentioned product is dissolved in 100ml trifluoroacetic acid or 2N hydrochloric acid/ethyl acetate solution, 1h is reacted at room temperature, solvent is evaporated off
White solid, i.e. proline-threonine dipeptides hydrochloride H-Pro-Thr-NH are obtained afterwards2.HCl。
Above-mentioned product is dissolved in 100ml pure water, sodium carbonate liquor is added and adjusts pH value, white solid is precipitated, filters, very
Sky is dry, obtains target product proline-threonine dipeptide fragment H-Pro-Thr-NH223g。
The preparation of Thr-Pro dipeptide fragment Boc-Thr (tBu)-Pro-OH of 1.3 protections
Boc-Thr (tBu)-OH20g (0.073mol) is dissolved in anhydrous tetrahydro furan (THF) 150ml, and stirring keeps solid molten
Solution.Ice salt bath cools to -10 G~-15 DEG C, and N- methylmorpholine 8ml is added, and isobutyl chlorocarbonate 10ml is then added dropwise, and protects
Temperature is held not higher than -10 DEG C, low-temp reaction 10min is kept after being added dropwise.Proline methyl ester hydrochloride H-Pro-
OMe.HCl12g is dissolved in 30ml tetrahydrofuran, and N- methylmorpholine 8ml is added.Above-mentioned mixed solution is added to reaction solution
In, ice bath reacts 30min, then reacts at room temperature 8h.Stop reaction, add water 300ml, ethyl acetate 200ml is added to extract precipitate,
Washing 3 times.Anhydrous sodium sulfate dries 6h.Filtering, evaporating solvent under reduced pressure obtain white solid 24.9g, yield 81%.
Above-mentioned product is dissolved in 30ml n,N-Dimethylformamide (DMF) solution, adds people's 2N NaOH aqueous solution, room temperature
It is stirred to react 1h, adds water 200ml, pH value is adjusted to 3 with 5% citric acid, ethyl acetate 200ml is added to extract precipitate, organic phase
Washing 3 times.Anhydrous sodium sulfate dries 6h.Filtering, evaporating solvent under reduced pressure obtain white solid Boc-Thr (tBu)-Pro-
OH23.8g, yield 84%.
1.4 tetrapeptide H-Thr-Pro-Pro-Thr-NH2Preparation
Boc-Thr (tBu)-Pro-OH20g (0.054mol) is dissolved in anhydrous tetrahydro furan (THF) 150ml, and stirring makes solid
Dissolution.Ice salt bath cools to -10 DEG C~-15 DEG C, and N- methylmorpholine 6ml is added, and isobutyl chlorocarbonate 7ml is then added dropwise,
It keeps temperature to be not higher than -10 DEG C, low-temp reaction 10min is kept after being added dropwise.Proline-threonine dipeptides hydrochloride H-
Pro-Thr-NH2.HCl13.6g it is dissolved in 50ml tetrahydrofuran, N- methylmorpholine 6ml is added.Above-mentioned mixed solution is added
Into reaction solution, ice bath reacts 30min, then reacts at room temperature 8h.Stop reaction, adds water 300ml, ethyl acetate 200ml is added to mention
Precipitate is taken, is washed 3 times.Anhydrous sodium sulfate dries 6h.Filtering, evaporating solvent under reduced pressure obtain white solid 25.4g, yield 82%.
Above-mentioned product is dissolved in 150ml trifluoroacetic acid or 2N hydrochloric acid/ethyl acetate solution, 1h is reacted at room temperature, solvent is evaporated off
White solid, i.e. the tetrapeptide hydrochloride H-Thr-Pro-Pro-Thr-NH of target compound carboxy amidation are obtained afterwards2.HCl。
Above compound H-Thr-Pro-Pro-Thr-NH2.HCl20g it is dissolved in 100ml methanol, 5% sodium bicarbonate is added
Solution 100ml, water 300ml filter precipitate, are washed to PH neutrality, vacuum drying.Obtain white solid 15.8g, i.e. target chemical combination
The tetrapeptide H-Thr-Pro-Pro-Thr-NH of object carboxy amidation2。
Claims (1)
1. tetrapeptide H-Thr-Pro-Pro-Thr-NH2Liquid-phase synthesis process, comprising the following steps:
1) threonine Carboxylamide structure H-Thr-NH2Synthesis: amino tertbutyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl
(Fmoc) threonine protected acts under the conditions of -15 DEG C with isobutyl chlorocarbonate and N- methylmorpholine, forms mixed acid
Acid anhydride, then react with ammonia water, generate the protection threonine of carboxy amidation；Then hydrochloric acid/organic solvent or trifluoroacetic acid (TFA) are used
Boc protecting group is removed, or with piperidines/n,N-Dimethylformamide (DMF) solution removal Fmoc protecting group；
2) dipeptide fragment H-Pro-Thr-NH2Synthesis: Boc or fmoc-protected proline are under the conditions of -15 DEG C, with chloro-carbonic acid
Isobutyl ester and the effect of N- methylmorpholine form mixed acid anhydride, then anti-with the threonine condensation of the carboxy amidation of upper step preparation
It answers, generates the dipeptides of amido protecting；Reaction solution is poured into pure water, precipitate is filtered out, it is dry；Then hydrochloric acid/organic solvent is used
Or trifluoroacetic acid (TFA) removes Boc protecting group, or is protected with piperidines/n,N-Dimethylformamide (DMF) solution removal Fmoc
Base；Reaction solution is poured into pure water, the pH value of solution is adjusted by weak base or weak acid, the dipeptides after making deprotection base is from water
Middle precipitation is filtered, and is washed, and vacuum drying obtains the free dipeptide fragment H-Pro-Thr-NH of amino2；
3) synthesis of Boc-Thr (tBu)-Pro-OH dipeptide fragment: amino Boc protection, the tertiary butyl ether protection of pendant hydroxyl group or side chain
Unshielded threonine acts under the conditions of -15 DEG C with isobutyl chlorocarbonate and N- methylmorpholine, forms mixed acid anhydride, then
It is reacted with the proline H-Pro-OMe of carboxyl ester protection, generates the dipeptides of protection；Then methyl esters is removed with NaOH saponification
Protecting group；Reaction solution is poured into pure water, the pH value of acid-conditioning solution is added, the free dipeptides of carboxyl is precipitated from water, is filtered,
Washing, vacuum drying, dipeptide fragment Boc-Thr (the tBu)-Pro-OH protected；
4) tetrapeptide H-Thr-Pro-Pro-Thr-NH2Synthesis: dipeptide fragment Boc-Thr (tBu)-Pro-OH of protection is at -15 DEG C
Under the conditions of, acted on isobutyl chlorocarbonate and N- methylmorpholine, form mixed acid anhydride, then with the free dipeptide fragment H- of amino
Pro-Thr-NH2Condensation, generates the tetrapeptide of protection；Then with hydrochloric acid/organic solvent or trifluoroacetic acid (TFA), disposably removing is protected
Protect base；Reaction solution is poured into pure water, the pH value of solution is adjusted by weak base, the tetrapeptide after making deprotection base is analysed from water
Out, it filters, washs, vacuum drying obtains target compound tetrapeptide H-Thr-Pro-Pro-Thr-NH2。
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| CN107141351A (en) * | 2017-05-15 | 2017-09-08 | 广州领晟医疗科技有限公司 | A kind of liquid-phase synthesis process of E75 peptides |
| CN107474107B (en) * | 2017-05-18 | 2023-01-17 | 江苏恩华药业股份有限公司 | Process for the preparation of GLYX-13 and compounds useful for the preparation of GLYX-13 |
| CN107857799B (en) * | 2017-11-20 | 2021-06-04 | 陕西慧康生物科技有限责任公司 | Liquid phase synthesis method of tetrapeptide-21 |
| CN113557028A (en) * | 2019-01-11 | 2021-10-26 | 诺雷克斯股份有限公司 | Salts and crystalline forms of rapatidine |
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| CN1715295A (en) * | 2004-07-03 | 2006-01-04 | 兰州大学 | Liquid phase synthetic method for endomorphine -1 and endomorphine -2 |
| CN102690325A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1715295A (en) * | 2004-07-03 | 2006-01-04 | 兰州大学 | Liquid phase synthetic method for endomorphine -1 and endomorphine -2 |
| CN104321071A (en) * | 2011-10-24 | 2015-01-28 | 西北大学 | Nmda receptor modulators and uses thereof |
| CN102690325A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Synthesis of polypeptide PyroGlu-Pro-Arg-pNA.HCl |
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