Preparation method of amino acid capable of protecting tetrapeptide
By adopting multiple efficient chemical steps in polypeptide synthesis, including base-catalyzed methylation, deprotection, acylation and saponification, the problems of low reaction selectivity and insufficient product purity are solved, and the Boc with high purity and high yield is achieved -The synthesis of -Gly-Gly-Phe-Gly-OH tetrapeptide simplifies the purification process and reduces costs.
Patent Information
- Application Number
- CN202411434944.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2024-12-24
AI Technical Summary
In the existing polypeptide synthesis technology, there are problems such as low reaction selectivity, insufficient product purity and yield in the synthesis process of tetrapeptides, especially in the step of producing Phe-Gly-Ome-HCl, it is difficult to effectively improve the selectivity and product of the reaction. purity.
A preparation method comprising multiple efficient chemical steps, the specific steps include alkali-catalyzed addition of dimethyl sulfate in N,N-dimethylformamide to produce BOC-Phe-Gly-OMe, followed by HCl/Bacetic acid Remove BOC protection from the ester solution to obtain Phe-Gly-OMe.HCl, then add oxalyl chloride and triethylamine dropwise to dichloromethane for acylation, and finally saponified in a mixture of tetrahydrofuran and methanol to obtain high purity Boc -Gly-Gly-Phe-Gly-OH.
This method can synthesize high-purity Boc-Gly-Gly-Phe-Gly-OH tetrapeptide in a short time, optimize the reaction conditions and time, improve the reaction yield, and reduce the time and cost during the synthesis process. The purification process is simplified.
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Figure CN119176852A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for preparing an amino acid with protected tetrapeptide. Background Art
[0002] BOC-Gly-Gly-Phe-Gly-OH is a self-assembled N-terminal and C-terminal protected tetrapeptide with excellent biocompatibility and low immunogenicity. This self-assembly property makes it easier to be recognized and processed in the body, which can effectively improve the efficacy of ADC. At the same time, the tetrapeptide can be used as an intermediate to participate in the synthesis of ADC dual-drug linker, providing a good synthetic basis for complex ADC structures.
[0003] During the synthesis process, the derivatization reaction of BOC-Phe-Gly generates Phe-Gly-Ome-HCl, which is an important step in the synthesis of ADC dual-drug connection assembly units. The introduction of Phe-Gly-Ome-HCl can not only effectively improve the selectivity of the reaction and the purity of the product, but also can be connected to the peptide in the organic phase to remove the BOC-Gly-Gly generated during the decomposition of BOC-Gly-Gly-Cl, thereby further improving the purity and yield of the final product. Summary of the invention
[0004] The present application designs a synthesis method with simple operation, easy-to-control conditions, stable quality, stable yield, and superior overall cost and benefits, ensuring that a polypeptide that meets polypeptide requirements is synthesized.
[0005] In order to achieve the above-mentioned purpose, the technical solution adopted in the present application is: a method for preparing an amino acid with a protected tetrapeptide, wherein the amino acid is Boc-Gly-Gly-Phe-Gly-OH, and the preparation method comprises the following steps:
[0006] Step 1: Dissolve Boc-Phe-Gly-OH in N,N-dimethylformamide, then add dimethyl sulfate dropwise under base catalysis to obtain BOC-Phe-Gly-OMe.
[0007] Step 2: Deprotect BOC from BOC-Phe-Gly-OMe in HCl / ethyl acetate solution to obtain Phe-Gly-OMe.HCl.
[0008] Step 3: Dissolve Boc-Gly-Gly-OH in dichloromethane, and quickly add a dichloromethane solution of oxalyl chloride dropwise under reflux. After the addition is complete and the reaction is continued for a period of time, a Boc-Gly-Gly-Cl acyl chloride product is obtained.
[0009] Step 4: Dissolve Phe-Gly-OMe.HCl in dichloromethane, add triethylamine as an acid-binding agent, cool with ice water, add the reaction solution of step 3 to react, and after the addition is complete, naturally warm to room temperature to react for one hour, and then treat to obtain pure BOC-Gly-Gly-Phe-Gly-OMe.
[0010] Step 5: Dissolve the pure BOC-Gly-Gly-Phe-Gly-OMe in a mixture of tetrahydrofuran and methanol, saponify with liquid alkali, and obtain BOC-Gly-Gly-Phe-Gly-OH.
[0011] Preferably, in step 1, the base is one of potassium carbonate, sodium carbonate and triethylamine, preferably sodium carbonate.
[0012] Preferably, the ratio of sodium carbonate to Boc-Phe-Gly-OH is 1:1.2-3.0, preferably 1:2.
[0013] Preferably, in the step 2, the BOC protection is removed by one of concentrated hydrochloric acid, TFA / DCM, HCL / EA, and HCl / MeOH, preferably HCL / EA.
[0014] Preferably, in step 3, the solvent used is one of DCM, THF and EA, preferably DCM.
[0015] Preferably, the acyl chloride reagent in step three is one of SOCl2, PCl3, (COCl)2, preferably (COCl)2.
[0016] Preferably, in the step 3, the temperature of the dichloromethane to which oxalyl chloride is added dropwise is 0°C to 40°C, preferably 40°C.
[0017] Preferably, in step 4, the system temperature must be maintained at 5-10° C. when the acyl chloride solution is added dropwise.
[0018] Preferably, in step 4, the acid binding agent is one of triethylamine, DIPEA and sodium carbonate, preferably triethylamine.
[0019] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:
[0020] 1. The synthesis method of the present invention can synthesize high-purity Boc-Gly-Gly-Phe-Gly-OH tetrapeptide in a short time through multiple efficient chemical steps, and the conditions of each reaction step are optimized, the reaction time is short, the reaction yield is high, and the time and cost in the synthesis process are reduced. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The synthetic route of the present invention is shown in FIG. DETAILED DESCRIPTION
[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.
[0023] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0024] Embodiment 1:
[0025] A method for preparing an amino acid with a protected tetrapeptide, wherein the amino acid is Boc-Gly-Gly-Phe-Gly-OH, and the preparation method comprises the following steps:
[0026] Step 1: Dissolve Boc-Phe-Gly-OH in N,N-dimethylformamide, then add dimethyl sulfate dropwise under base catalysis to obtain BOC-Phe-Gly-OMe.
[0027] Step 2: Deprotect BOC from BOC-Phe-Gly-OMe in HCl / ethyl acetate solution to obtain Phe-Gly-OMe.HCl.
[0028] Step 3: Dissolve Boc-Gly-Gly-OH in dichloromethane, and quickly add a dichloromethane solution of oxalyl chloride dropwise under reflux. After the addition is complete and the reaction is continued for a period of time, the Boc-Gly-Gly-Cl acyl chloride product is obtained.
[0029] Step 4: Dissolve Phe-Gly-OMe.HCl in dichloromethane, add triethylamine as an acid-binding agent, cool with ice water, and add the reaction solution of step 3 to react. After the addition is complete, naturally warm to room temperature to react for one hour, and then treat to obtain pure BOC-Gly-Gly-Phe-Gly-OMe.
[0030] Step 5: Dissolve the pure BOC-Gly-Gly-Phe-Gly-OMe in a mixture of tetrahydrofuran and methanol, and saponify with liquid alkali to obtain BOC-Gly-Gly-Phe-Gly-OH. The product is easy to precipitate as a jelly, which affects purification. Through experiments, a solvent system with a certain ratio is found to be beneficial to the purification of the product.
[0031] In the step 1, the base is one of potassium carbonate, sodium carbonate and triethylamine, preferably sodium carbonate.
[0032] In the step 1, the ratio of Boc-Phe-Gly-OH to sodium carbonate is 1:1.2-3.0, preferably 1:2.
[0033] In the step 2, the BOC protection is removed by one of concentrated hydrochloric acid, TFA / DCM, HCL / EA, and HCl / MeOH, preferably HCL / EA.
[0034] In the step 3, the solvent used is one of DCM, THF and EA, preferably DCM.
[0035] In the step 3, the acyl chloride reagent is one of SOCl2, PCl3, and (COCl)2, preferably (COCl)2.
[0036] In the step 3, the temperature of the dichloromethane to which oxalyl chloride is added dropwise is 0°C to 40°C, preferably 40°C.
[0037] In the step 4, the reaction liquid of the step 3 is added dropwise into the reaction system of the step 4 to carry out reaction.
[0038] In the step 4, the system temperature must be maintained at 5-10° C. when the acyl chloride solution is added dropwise.
[0039] In the step 4, the acid binding agent is one of triethylamine, DIPEA and sodium carbonate, preferably triethylamine.
[0040] Specifically, DMF (N,N-dimethylformamide) is an excellent polar solvent that can effectively dissolve the target compound and help the subsequent reaction proceed smoothly; sodium carbonate, as a weak base, can deprotonate carboxylic acids or amino groups, improve the reactivity of the reactants, and promote esterification reactions; dimethyl sulfate, as a methylating agent, can methylate the carboxylic acids or amino groups in the target molecules, increasing the hydrophobicity and stability of the molecules; ethyl acetate, as an extractant, helps to extract the desired organic product from the reaction mixture, and the addition of water can help stratification; brine washing helps to remove polar impurities and can adjust the pH to neutral, promoting the subsequent separation process.
[0041] Add appropriate amount of HCl / AcOEt to a dry and clean reaction bottle, and then add BOC-Phe-Gly-OMe in batches. After the addition, react at room temperature for about 1 hour, filter, wash the solid with EA, filter dry, and dry to obtain Phe-Gly-OMe.HCl.
[0042] HCl removes the protecting group of BOC-Phe-Gly-OMe and converts it into an active amino compound, which is convenient for subsequent reactions.
[0043] In the reaction bottle, add BOC-Gly-Gly-OH and an appropriate amount of DCM, then slowly add oxalyl chloride dropwise under reflux to absorb the tail gas. After the reaction is complete, appropriately concentrate the DCM and then seal it for later use.
[0044] Oxalyl chloride, as a carboxylic acid acylating agent, can effectively convert BOC-Gly-Gly-OH into acyl chloride, improve the reaction activity, and promote the subsequent acylation reaction.
[0045] Add DCM and Phe-Gly-OMe.HCl to the reaction flask, then cool it with ice water, and drop triethylamine and BOC-Gly-Gly-Cl in dichloromethane. After the dropwise addition, warm the temperature to room temperature and react for 1 hour. Wash the reaction solution with a large amount of water and dry it. Filter and concentrate the filtrate to obtain BOC-Gly-Gly-Phe-Gly-OMe.
[0046] Triethylamine, as an alkaline catalyst, can deprotonate the reactants, promote the acylation reaction, and make the reaction more efficient; water washing can remove unreacted compounds and by-products and improve the purity.
[0047] Add an appropriate amount of THF and a small amount of water to the reaction flask, add BOC-Gly-Gly-Phe-Gly-OMe and stir to dissolve, then cool with tap water, and add liquid alkali for saponification. After the saponification is complete, add a large amount of water to dilute, extract impurities with petroleum ether, and separate the layers. The water layer is then extracted with EA / Pet until there is no impurity, the aqueous phase is acidified and the product is extracted with EA, washed, and dried. Filter, concentrate the filtrate to a small volume, add MTBE and petroleum ether for crystallization, and obtain the target product.
[0048] The saponification reaction removes the protecting groups, making the final product a more active amino acid form and increasing the reaction activity; petroleum ether is a good non-polar solvent that can effectively remove polar impurities and improve the purity of the final product; acidification of the aqueous phase can help transfer the target compound from the aqueous phase to the organic phase and improve the product yield.
[0049] Through this preparation method, the cumbersome processing steps in liquid phase synthesis of peptides can be effectively reduced, the purification process of such peptide products can be greatly simplified, the quality of the products can be guaranteed, and thus the later use of the products can be guaranteed.
[0050] Embodiment 2:
[0051] In a dry and clean 1L three-necked flask, add 32.2g (0.1mol) of BOC-Phe-Gly, 200ml of DMF, and 21.2g of sodium carbonate, and stir. Add 15.1g of dimethyl sulfate dropwise at room temperature, and react for 2 hours. TLC (DCM:MeOH=5:1) shows no raw material, and the mixture is treated.
[0052] Add 200ml EA and 300ml water to the reaction flask, and dissolve the solid with 2N hydrochloric acid, while ensuring that the pH value of the system is 8. Separate the water layer, and discard the water layer without product. Wash the EA layer with brine until there is no large polar impurities and the pH value of the water layer is neutral, use 30ml of brine each time, and wash about 5 times. Remove all the water, dry the EA layer with anhydrous sodium sulfate, filter, and concentrate the filtrate at 65℃ in a vacuum to obtain BOC-Phe-Gly-OMe oil, the oil weight is 31.2g. Y: 93%
[0053] In a 500ml dry three-necked flask, add 100ml of 3N HCl / EA, and then slowly add 31.2g of BOC-Phe-Gly-OMe oil. After the addition is complete, react at room temperature for 1 hour. TLC (EA:Pet=1:1) shows that the reaction is basically complete and a large amount of solid is precipitated in the system.
[0054] The solid was filtered, washed with a mixture of 30 ml EA and 30 ml Pet, filtered and dried to obtain 24.25 g Phe-Gly-Ome.HCl Y: 89%
[0055] Connect a reflux device to a 500ml anhydrous clean three-necked flask, then add 23.2g of BOC-Gly-Gly and 150ml of DCM. Then slowly drop 15.5g of oxalyl chloride under reflux. It is important to slowly drop oxalyl chloride under reflux to facilitate the reaction. After dropping oxalyl chloride, reflux for 30 minutes. Because the amount is small, it does not need to be concentrated, and is directly sealed for the next step of reaction.
[0056] In a 1L three-necked flask, add 150ml DCM and 24.25g Phe-Gly-Ome.HCl, then cool to 5-10°C with ice water, add 20g triethylamine dropwise to adjust the pH to 8-9, and slowly add the reaction solution from step 3.1 dropwise. After the acid chloride solution is added, maintain the system pH at 8-9 and the temperature at 5-10°C for 30-60 minutes for post-treatment.
[0057] Add 200 ml of water to the reaction solution, separate the layers, and discard the water layer. Wash the DCM layer with 10% sodium carbonate brine 3 times, 50 ml each time, separate the water layer, and wash the DCM layer with 10% hydrochloric acid brine 3 times, 50 ml each time, and then wash the DCM layer with brine until neutral. Concentrate the DCM layer in vacuum to obtain 29.2 g of BOC-Gly-Gly-Phe-Gly-OMe oil.
[0058] 29.2 g of BOC-Gly-Gly-Phe-Gly-OMe oil was dissolved in 30 ml of methanol and diluted with 30 ml of THF, and then 20% liquid alkali was added dropwise at room temperature to adjust the pH to 10-11 for saponification. After saponification for about 1 hour, TLC (DCM:MeOH=10:1) showed no starting material.
[0059] Adjust the reaction solution to pH 8-9 with hydrochloric acid, then add 100 ml of water, then add 100 ml of petroleum ether and stir, separate the layers, discard the organic layer, and extract the water layer with 30% EA / Pet 30 ml × 3 for 3 times. Separate the organic layer, discard the organic layer, add 150 ml of DCM to the water layer, then acidify to pH 2-3 with concentrated hydrochloric acid, separate the layers, and extract the product with DCM 30 ml × 2 for 2 times. Combine the DCM layers, wash 3 times with brine 20 ml × 3, add 30 g of anhydrous sodium sulfate to the DCM layer, stir and dry.
[0060] Filter, concentrate the filtrate to a small volume, then add an appropriate amount of petroleum ether and stir, and pour out the supernatant. Add an appropriate amount of MTBE to the lower layer of oil and stir, then add petroleum ether and stir, pour out the supernatant, repeat this 3 times to obtain an oily solid that is slightly swollen. Add n-heptane to the oily solid and stir, pour out the supernatant, add n-heptane to the oily solid and stir, slowly solidify, beat, filter, and dry to obtain BOC-Gly-Gly-Phe-Gly24.5g, HPLC99.1%, yield 86.5%.
[0061] The yield of the product BOC-Phe-Gly-OMe in step 1 is 93%, the yield of Phe-Gly-OMe.HCl in step 2 is 89%, and the yield of the final product BOC-Gly-Gly-Phe-Gly is 86.5%. High yield means lower raw material waste and higher product output, which helps to improve overall production efficiency.
[0062] The HPLC purity of the final product BOC-Gly-Gly-Phe-Gly was 99.1%, which shows that the synthetic method can effectively remove impurities and obtain high-purity target molecules, which is especially important for drug development and fine chemical production.
[0063] Although the synthetic route of the present invention uses an organic solvent, multiple solvent exchanges and water washings reduce environmental pollution and waste generation. In some steps, desiccants such as anhydrous sodium sulfate are used to remove moisture, thereby avoiding unnecessary use of solvents.
[0064] The entire synthesis process has high yield, good purity, reasonable control of reaction conditions and effective post-processing steps, which are important advantages in industrial production and research.
[0065] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing an amino acid having a protected tetrapeptide, characterized in that: The amino acid is Boc-Gly-Gly-Phe-Gly-OH, and the preparation method comprises the following steps: Step 1: Dissolve Boc-Phe-Gly-OH in N,N-dimethylformamide, then add dimethyl sulfate dropwise under base catalysis to obtain BOC-Phe-Gly-OMe; Step 2: Deprotect BOC from BOC-Phe-Gly-OMe in HCl / ethyl acetate solution to obtain Phe-Gly-OMe.HCl; Step 3: Dissolve Boc-Gly-Gly-OH in dichloromethane, and quickly add a dichloromethane solution of oxalyl chloride dropwise under reflux. After the addition is complete and the reaction is continued for a period of time, a Boc-Gly-Gly-Cl acyl chloride product is obtained; Step 4: dissolve Phe-Gly-OMe.HCl in dichloromethane, add triethylamine as an acid-binding agent, cool with ice water, add the reaction solution of step 3 to react, and after the addition is complete, naturally warm to room temperature to react for one hour, and then treat to obtain pure BOC-Gly-Gly-Phe-Gly-OMe; Step 5: Dissolve the pure BOC-Gly-Gly-Phe-Gly-OMe in a mixture of tetrahydrofuran and methanol, saponify with liquid alkali, and obtain BOC-Gly-Gly-Phe-Gly-OH.
2. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 1, the base is one of potassium carbonate, sodium carbonate and triethylamine, preferably sodium carbonate.
3. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 1, the ratio of Boc-Phe-Gly-OH to sodium carbonate is 1:1.2-3.0, preferably 1:
2.
4. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 2, the BOC protection is removed by one of concentrated hydrochloric acid, TFA / DCM, HCL / EA, and HCl / MeOH, preferably HCL / EA.
5. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 3, the solvent used is one of DCM, THF and EA, preferably DCM.
6. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 3, the acyl chloride reagent is one of SOCl2, PCl3, and (COCl)2, preferably (COCl)2.
7. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 3, the temperature of the dichloromethane to which oxalyl chloride is added dropwise is 0°C to 40°C, preferably 40°C.
8. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 4, the reaction liquid of step 3 is added dropwise into the reaction system of step 4 to carry out reaction.
9. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 4, the system temperature must be maintained at 5-10° C. when the acyl chloride solution is added dropwise.
10. The method for preparing an amino acid having a protected tetrapeptide according to claim 1, characterized in that: In the step 4, the acid binding agent is one of triethylamine, DIPEA and sodium carbonate, preferably triethylamine.