CN116813504A - Process for preparing Fmoc amino acid based on two-phase circulation method - Google Patents
Process for preparing Fmoc amino acid based on two-phase circulation method Download PDFInfo
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- CN116813504A CN116813504A CN202310837896.8A CN202310837896A CN116813504A CN 116813504 A CN116813504 A CN 116813504A CN 202310837896 A CN202310837896 A CN 202310837896A CN 116813504 A CN116813504 A CN 116813504A
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- -1 Fmoc amino acid Chemical class 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 107
- 238000006243 chemical reaction Methods 0.000 claims abstract description 89
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 claims abstract description 50
- 150000001413 amino acids Chemical class 0.000 claims abstract description 28
- 239000003208 petroleum Substances 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 125000004122 cyclic group Chemical group 0.000 claims abstract description 5
- 239000012071 phase Substances 0.000 claims description 89
- 238000003756 stirring Methods 0.000 claims description 58
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 56
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- 239000008346 aqueous phase Substances 0.000 claims description 42
- 239000003960 organic solvent Substances 0.000 claims description 26
- 239000000047 product Substances 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- 239000002244 precipitate Substances 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000013078 crystal Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 9
- 239000005457 ice water Substances 0.000 claims description 9
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000002390 rotary evaporation Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 5
- 239000002585 base Substances 0.000 claims description 5
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- KVNYFPKFSJIPBJ-UHFFFAOYSA-N ortho-diethylbenzene Natural products CCC1=CC=CC=C1CC KVNYFPKFSJIPBJ-UHFFFAOYSA-N 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 239000000376 reactant Substances 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- 230000003113 alkalizing effect Effects 0.000 abstract 2
- 239000012535 impurity Substances 0.000 abstract 1
- 238000004064 recycling Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 86
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000007086 side reaction Methods 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 6
- 239000012456 homogeneous solution Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 3
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 3
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 2
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 2
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000003875 Wang resin Substances 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of organic synthesis, and discloses a process for preparing Fmoc amino acid based on a two-phase circulation method. Comprises the steps of alkalizing amino acid raw materials, preparing Fmoc-Cl solution, adding the Fmoc-Cl solution in four times, extracting, adding petroleum ether, removing impurities, alkalizing and recycling. The invention uses two-phase reaction, the system is simple, and the post-treatment is easy; the cyclic reaction is used, so that the utilization rate of reactants is improved, and the cost is reduced.
Description
Technical Field
The invention belongs to the technical field of organic synthesis, and particularly relates to a process for preparing Fmoc amino acid based on a two-phase circulation method.
Background
Fmoc-protected amino acid or other amino protection is of great importance in organic synthesis, especially in peptide synthesis, especially solid phase peptide synthesis using Wang-resin, where Fmoc-protected amino acids are commonly used. The conventional Fmoc-Cl reagents are insoluble in water and amino acids are insoluble in organic solvents, so that in the synthesis, the conventional methods use expensive or water-miscible reagents which react side-reactions with amino groups to cause Fmoc-reaction of amino acids and Fmoc-Cl in a homogeneous phase. At least three disadvantages are caused by the fact that expensive organic solvents are used, cost is increased, post-treatment difficulty is increased, and yield is reduced; secondly, the homogeneous reaction can not neutralize the acid generated by the reaction, and the yield can be reduced, thirdly, side reactions are easy to occur, and in order to avoid the side reactions, the reaction activity of Fmoc-Cl needs to be reduced by an ice bath.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a process for preparing Fmoc amino acid based on a two-phase circulation method, which uses a two-phase reaction, has a simple system and is easy to post-treat; the cyclic reaction is used, so that the utilization rate of reactants is improved, and the cost is reduced.
The above object of the present invention is achieved by the following technical solutions: the Fmoc amino acid preparation process based on the two-phase circulation method comprises the following steps:
1. adding an amino acid raw material with the purity of more than 98 percent and strong alkali with the same mole number as the amino group of the amino acid raw material into deionized water, and stirring to dissolve the amino acid raw material into a transparent uniform solution 1 with the pH value of not more than 11.5; fmoc-Cl in one third of the number of moles of amino groups of the amino acid starting material was taken and dissolved in an organic solvent which was capable of forming a two-phase system with water to form solution 2.
2. And (2) adding the solution 1 obtained in the step (1) into a glass reactor, starting strong stirring under ice water bath, dividing the solution 2 into four parts on average, adding one part of the solution into the solution 1 for reaction under vigorous stirring for half an hour, suspending the reaction, dividing the solution into two phases, detecting the pH value of the water phase, adding the second part when the pH value reaches 10.6+/-0.1 for reaction, stopping stirring for half an hour, dividing the reaction system into two phases, detecting the pH value of the water phase, adding the third part for reaction when the pH value reaches 10.3+/-0.1, stopping stirring for detecting the pH value of the water phase after half an hour, adding the fourth part of Fmoc-Cl for reaction, stopping stirring for detecting the pH value of the water phase after half an hour, stopping the reaction when the pH value reaches 9.6+/-0.1, and performing post-treatment.
3. Separating the solution into two phases, separating the two phases by a liquid separating device, extracting the water phase by ethyl acetate, adding 6N hydrochloric acid solution into the water phase, adjusting the pH value of the solution to be between 1.5 and 2, and extracting the solution by using an organic solvent with the same volume for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, adding 3-5 times of petroleum ether solution into the concentrated solution, and precipitating or crystallizing.
4. The solution forming crystals or precipitates in step 3 is filtered and the residual organic solvent in the crystals or precipitates is removed to obtain the Fmoc amino acid product in the form of crystals or powder.
5. Adding one third of the amino acid added in the step 1 into the water phase extracted in the step 3, wherein the amino acid reaches the initial amount in the step 1, and adding a concentrated alkali solution into the solution to enable the pH value of the solution to reach the pH value of the solution 1 in the step 1;
6. and (3) circulating the steps 1-5, wherein the circulating times are 3-5 times.
Further, the organic solvent used for dissolving Fmoc-Cl in the step 1 and capable of forming a two-phase system with water is specifically diethyl ether, benzene or dichloromethane.
In a further preferred aspect of the present invention, the organic solvent used in step 1 to dissolve Fmoc-Cl and capable of forming a two-phase system with water is in particular diethyl ether.
Further, the strong base in the step 1 is specifically sodium hydroxide or potassium hydroxide.
In a further preferred aspect of the present invention, the strong base in step 1 is sodium hydroxide.
In a further preferred aspect of the present invention, the step 2 is: and (2) adding the solution 1 obtained in the step (1) into a glass reactor, starting strong stirring under ice water bath, dividing the solution 2 into four parts on average, adding one part of the solution into the solution 1 for reaction under vigorous stirring for half an hour, suspending the reaction, dividing the solution into two phases, detecting the pH value of the water phase, adding the second part when the pH value reaches 10.6 for reaction, stopping stirring after half an hour, dividing the reaction system into two phases, detecting the pH value of the water phase, adding the third part when the pH value reaches 10.3 for reaction, stopping stirring for detecting the pH value of the water phase after half an hour, adding the fourth part of Fmoc-Cl for reaction when the pH value reaches 9.9, stopping stirring for detecting the pH value of the water phase after half an hour, stopping the reaction when the pH value reaches 9.6, and performing aftertreatment.
Further, after the pH is adjusted to 1.5-2 in the step 3, the organic solvent used for extracting the product is diethyl ether when the side chain is Fmoc amino acid of alkane, and ethyl acetate when the side chain is Fmoc amino acid of polar group.
In a further preferred aspect of the present invention, after adding 6N hydrochloric acid solution to the aqueous phase in step 3, the pH of the solution is adjusted to 2.
Step 5 of the invention is the key of the process method, by controlling the proportion of reactants, reducing side reaction, increasing yield, and each cycle, the utilization rate of Fmoc-Cl of one reactant reaches approximately 100%, the quantity of the first reaction is reached again after the amino acid reacted by Fmoc-Cl is supplemented to the remaining amino acid in the water phase, the reaction state is returned by adding alkali, and a new reaction can be started by adding specified Fmoc-Cl. Fmoc-Cl is fully utilized during each reaction, a new cycle is performed through the residual amino acids in the water phase, and through the cycle, both the amino acids and Fmoc-Cl are fully utilized.
In the step 2, since Fmoc-Cl is too active and side reaction is easy to occur, one third of moles of Fmoc-Cl is subdivided into four parts, after each part reacts with amino acid for a period of time, the pH value in the aqueous phase is detected, and after the requirement is met, the subsequent Fmoc-Cl is added for further reaction, so that the occurrence of side reaction is avoided. According to the calculation, the pH of the aqueous phase of the first Fmoc-Cl complete reaction (Fmoc-Cl is completely consumed) was about 10.6, the second about 10.3, the third about 9.9 and the fourth about 9.6. The reaction condition can be obtained by detecting the pH value of the water phase, and the monitoring is convenient.
Compared with the prior art, the invention has the beneficial effects that:
1) The two-phase reaction is used, the system is simple, and the post-treatment is easy; the cyclic reaction is used, so that the utilization rate of reactants is improved, and the cost is reduced.
2) For two-phase reasons, active reactants can be added in batches, the proportion of the reactants is controlled, side reactions are reduced, the reaction can be stopped in the middle, and the reaction progress is monitored by detecting the pH value of the water phase.
3) The yield and the quality of the product are greatly superior to those of the currently used method.
4) The raw materials and reagents used in the method are low in price, and the simple reaction conditions greatly reduce the production cost.
5) Reasonable process design results in the advantages described above and makes the process suitable for large-scale industrial production.
Detailed Description
The present invention is described in detail below by way of specific examples, but the scope of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and all experimental equipment, materials, reagents, etc. used can be obtained from commercial sources.
Example 1: preparation of Fmoc-Ala-OH
Step 1: L-Ala-OH2.67 g (30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 100mL distilled water was added, and about 1.2 g (30 mmol) was added under stirring to form a clear, homogeneous solution with a pH no greater than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask under ice water bath, then opening a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl), quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding a second part if the pH value reaches about 10.6, performing the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding a third part if the pH value reaches about 10.3, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding a fourth part of Fmoc-Cl, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, and stopping the reaction if the pH value reaches about 9.6 or unchanged, performing the post-treatment.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white precipitate.
Step four: the white precipitate formed in step three was filtered and the remaining organic solvent in the crystals or precipitate was removed, fmoc-Ala-OH2.99 g in powder form, yield 99%. And (3) purity detection: [ alpha ]] 20 D = -18 (DMF, C1.0), the optical purity of the product was 100%.
Step 5: and (3) adding 0.89 g of Ala (about 10 mmol) into the final aqueous phase in the step (3) to ensure that the Ala in the aqueous phase reaches about 30mmol, putting the aqueous solution into a 250mL flask, and performing the circulation of the steps (1) to (5) according to the subsequent steps of the step (1) to obtain Fmoc-Ala-OH products of the second batch and the subsequent batches, wherein the yield and the purity of the products are the same as those of the first batch.
Example 2: preparation of Fmoc-Gly-OH
Step 1: L-Gly-OH2.26 g (about 30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 100mL distilled water was added, and NaOH was added again with stirring to about 1.2 g (30 mmol) to form a clear and homogeneous solution with a pH no more than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask under ice water bath, then opening a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl), quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding a second part if the pH value reaches about 10.6, performing the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding a third part if the pH value reaches about 10.3, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding a fourth part of Fmoc-Cl, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, and stopping the reaction if the pH value reaches about 9.6 or unchanged, performing the post-treatment.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white precipitate.
Step four: the white precipitate formed in step three was filtered and the crystals or residual organic solvent from the precipitate was removed to give Fmoc-Gly-OH2.70 g as a powder in 95% yield.
Step 5: and (3) adding 0.75 g Gly (about 10 mmol) into the final water phase in the step (3) to ensure that the Gly in the water phase reaches about 30mmol, putting the water solution into a 250mL flask, and carrying out the circulation of the steps (1) to (5) according to the subsequent steps of the step (1), thus obtaining Fmoc-Gly-OH products of the second batch and the subsequent batches, wherein the purity and the yield of the products are the same as those of the first batch.
Example 3: preparation of Fmoc-Val-OH
Step 1: L-Val-OH3.51 g (about 30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 50mL distilled water was added, and about 1.2 g (30 mmol) was added under stirring to form a clear, homogeneous solution with a pH no greater than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask under ice water bath, then opening a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl), quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding a second part if the pH value reaches about 10.6, performing the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding a third part if the pH value reaches about 10.3, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding a fourth part of Fmoc-Cl, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, and stopping the reaction if the pH value reaches about 9.6 or unchanged, performing the post-treatment.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white crystal.
Step four: the white crystals formed in step three were filtered and the residual organic solvent in the crystals was removed to give Fmoc-Val-OH2.95 g as crystals in 87% yield. And (3) purity detection: [ alpha ]] 20 D = -16.5 ° (DMF, C1.0), product optical purity 96%.
Step 5: and (3) adding 1.17 g of L-Val (about 10 mmol) into the final water phase in the step (3) to ensure that the L-Val in the water phase reaches about 30mmol, putting the water solution into a 250mL flask, and performing the circulation of the steps (1) to (5) according to the subsequent steps of the step (1) to obtain Fmoc-Val-OH products of the second batch and the subsequent batches, wherein the yield and the purity are the same as those of the first batch.
Example 4: preparation of Fmoc-Leu-OH
Step 1: L-Leu-OH3.93 g (about 30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 50mL distilled water was added, and about 1.2 g (30 mmol) was added under stirring to form a clear, homogeneous solution with a pH no greater than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask under ice water bath, then opening a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl), quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding a second part if the pH value reaches about 10.6, performing the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding a third part if the pH value reaches about 10.3, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding a fourth part of Fmoc-Cl, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, and stopping the reaction if the pH value reaches about 9.6 or unchanged, performing the post-treatment.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white precipitate.
Step four: the white precipitate formed in step three was filtered and the remaining organic solvent in the crystals or precipitate was removed, 3.17 g Fmoc-Leu-OH was powdered in 90% yield. And (3) purity detection: [ alpha ]] 20 D = -25 ° (DMF, C1.0), product optical purity 100%.
Step 5: and (3) adding 1.32 g of Leu (about 10 mmol) into the final water phase in the step (3), enabling the Leu in the water phase to reach about 30mmol, putting the water solution into a 250mL flask, and performing the circulation of the steps (1) to (5) according to the subsequent steps of the step (1), thus obtaining Fmoc-Leu-OH products of the second batch and the subsequent batches, wherein the yield and the purity of the products are the same as those of the first batch.
Example 5: preparation of Fmoc-Ile-OH
Step 1: L-Ile-OH3.93 g (30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 50mL distilled water was added, and about 1.2 g (30 mmol) was added under stirring to form a clear, homogeneous solution with a pH no greater than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask under ice water bath, then opening a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl), quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding a second part if the pH value reaches about 10.6, performing the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding a third part if the pH value reaches about 10.3, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding a fourth part of Fmoc-Cl, performing the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, and stopping the reaction if the pH value reaches about 9.6 or unchanged, performing the post-treatment.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white precipitate.
Step four: the white precipitate formed in step three was filtered and the remaining organic solvent in the crystals or precipitate was removed, 3.17 g Fmoc-Ile-OH in powder form, 90% yield. And (3) purity detection: [ alpha ]] 20 D = -12 (DMF, C1.0), the optical purity of the product was 100%.
Step 5: and (3) adding 1.32 g of Ile (about 10 mmol) into the final water phase in the step (3), enabling the Ile in the water phase to reach about 30mmol, putting the water solution into a 250mL flask, and performing the circulation of the steps (1) to (5) according to the subsequent steps of the step (1), thus obtaining Fmoc-Ile-OH products of the second batch and the subsequent batches, wherein the yield and the purity of the products are the same as those of the first batch.
Example 6: preparation of Fmoc-Phe-OH
Step 1: L-Phe-OH4.95 g (30 mmol) with 98% purity was taken and added to a 250mL round bottom flask, about 50mL distilled water was added, and about 1.2 g (30 mmol) was added under stirring to form a clear, homogeneous solution with a pH no greater than 11.5. Fmoc-Cl2.60 g (about 10 mmol) was additionally taken in 40mL dry diethyl ether and placed in a 100mL dropping funnel.
Step 2: placing a dropping funnel on a flask, starting stirring of the flask, then starting a knob of the dropping funnel, quickly dripping 10mL of Fmoc-Cl diethyl ether solution (about 2.5 mmoles of Fmoc-Cl) into the flask, quickly stirring to enable the Fmoc-Cl diethyl ether solution to be dispersed in an amino acid aqueous solution, suspending the reaction after half an hour, separating the solution into two phases, detecting the pH value of the aqueous phase, adding the second part if the pH value reaches about 10.6, carrying out the reaction after half an hour, stopping stirring, separating the reaction system into two phases, detecting the pH value of the aqueous phase, adding the third part if the pH value reaches about 10.3, carrying out the reaction after half an hour, stopping stirring to detect the pH value of the aqueous phase, adding the fourth part of Fmoc-Cl to carry out the reaction if the pH value reaches about 9.9, stopping the stirring to detect the pH value of the aqueous phase after half an hour, and carrying out the post-treatment if the pH value reaches about 9.6 or is unchanged.
Step 3: step three: after stopping the reaction, separating the solution into two phases, separating the two phases by a liquid separation device, extracting the aqueous phase by ethyl acetate, then adding 6N hydrochloric acid solution into the aqueous phase, adjusting the pH value of the solution to about pH2, and extracting the solution by using an equal volume of organic solvent (diethyl ether or ethyl acetate) for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, and adding petroleum ether solution with certain volume ratio to obtain white precipitate.
Step four: the white precipitate formed in step three was filtered and the remaining organic solvent in the crystals or precipitate was removed, fmoc-Phe-OH3.48 g in powder form, 90% yield. And (3) purity detection: [ alpha ]] 20 D = -38 (DMF, C1.0), the optical purity of the product was 98%.
Step 5: and (3) adding 1.65 g of Phe (about 10 mmol) into the final water phase in the step (3) to ensure that the Phe in the water phase reaches about 30mmol, putting the water solution into a 250mL flask, and performing the circulation of the steps (1) to (5) according to the subsequent steps of the step (1) to obtain Fmoc-Phe-OH products of the second batch and the subsequent batches, wherein the yield and the purity of the products are the same as those of the first batch.
The above-described embodiments are only preferred embodiments of the invention, and not all embodiments of the invention are possible. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (8)
1. The process for preparing the Fmoc amino acid based on the two-phase circulation method is characterized by comprising the following steps of:
s1, adding an amino acid raw material with the purity of more than 98 percent and strong base with the same mole number as the amino group of the amino acid raw material into deionized water, and stirring to dissolve the amino acid raw material into a transparent uniform solution 1 with the pH value of not more than 11.5; taking Fmoc-Cl with one third mole number of amino groups of the amino acid raw material, and dissolving the Fmoc-Cl in an organic solvent which can form a two-phase system with water to form a solution 2;
s2, the solution 1 in the step S1 and the solution are added into a glass reactor, strong stirring is started under ice water bath, the solution 2 is divided into four parts which are averagely added, one part of the solution is added into the solution 1 for reaction under vigorous stirring, after half an hour, the reaction is stopped, the solution is divided into two phases, when the pH value reaches 10.6+/-0.1, the second part is added for reaction, after half an hour, the stirring is stopped, the reaction system is divided into two phases, the pH value of the water phase is detected, when the pH value reaches 10.3+/-0.1, the third part is added for reaction, after half an hour, the stirring is stopped, the pH value of the water phase is detected, when the pH value reaches 9.9+/-0.1, the fourth part of Fmoc-Cl is added for reaction, after half an hour, the stirring is stopped, the pH value of the water phase is detected, and when the pH value reaches 9.6+/-0.1, the reaction is stopped, and the post-treatment is performed;
s3, separating the solution into two phases, separating the two phases by a liquid separation device, extracting a water phase by ethyl acetate, adding a 6N hydrochloric acid solution into the water phase, adjusting the pH value of the solution to be between 1.5 and 2, and extracting the solution by using an organic solvent with the same volume for three times; mixing the extractive solutions, drying with anhydrous sodium sulfate, filtering, concentrating by rotary evaporation, adding petroleum ether solution 3-5 times of the concentrated solution, and precipitating or crystallizing;
s4, filtering the solution formed in the step S3 to form crystals or precipitates, and removing residual organic solvent in the crystals or precipitates to obtain crystalline or powdery Fmoc amino acid products;
s5, adding one third of the amino acid amount added in the step 1 into the water phase extracted in the step S3, wherein the amino acid amount reaches the initial amount in the step S1, and adding a concentrated alkali solution into the solution to enable the pH value of the solution to reach the pH value of the solution 1 in the step S1;
s6, circulating the steps S1 to S5, wherein the circulating times are 3 to 5.
2. The process for preparing Fmoc amino acids based on the two-phase cyclic method according to claim 1, wherein said step S1 is used for dissolving Fmoc-Cl, and the organic solvent capable of forming a two-phase system with water is diethyl ether, benzene or dichloromethane.
3. The process for preparing Fmoc amino acids based on the two-phase cyclic method according to claim 2, wherein said step S1 is for dissolving Fmoc-Cl, the organic solvent capable of forming a two-phase system with water being specifically diethyl ether.
4. The process for preparing Fmoc amino acids according to claim 1, wherein said strong base in step S1 is sodium hydroxide or potassium hydroxide.
5. The process for preparing Fmoc amino acids according to claim 4, wherein said strong base in step S1 is sodium hydroxide.
6. The process for preparing Fmoc amino acid based on two-phase circulation method of claim 1, wherein step S2 is: and (3) adding the solution 1 obtained in the step (S1) into a glass reactor, starting strong stirring under ice water bath, dividing the solution 2 into four parts on average, adding one part of the four parts into the solution 1 for reaction under vigorous stirring for half an hour, suspending the reaction, dividing the solution into two phases, detecting the pH value of the water phase, adding the second part when the pH value reaches 10.6 for reaction, stopping stirring after half an hour, dividing the reaction system into two phases, detecting the pH value of the water phase, adding the third part when the pH value reaches 10.3 for reaction, stopping stirring for detecting the pH value of the water phase after half an hour, adding the fourth part of Fmoc-Cl for reaction when the pH value reaches 9.9, stopping stirring for detecting the pH value of the water phase after half an hour, stopping the reaction when the pH value reaches 9.6, and performing post-treatment.
7. The process for preparing Fmoc amino acids according to claim 1, wherein after the pH is adjusted to 1.5-2 in the step S3, the organic solvent is ethyl ether when the side chain is an alkane Fmoc amino acid, and ethyl acetate when the side chain is a polar group Fmoc amino acid product.
8. The process for preparing Fmoc amino acids according to claim 1, wherein in step S3, after adding 6N hydrochloric acid solution to the aqueous phase, the pH of the solution is adjusted to 2.
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