Synthesis method of N-methyl chiral Fmoc amino acid
The three-step synthesis of N-methyl chiral Fmoc amino acids avoids the use of hazardous materials and strong acids, solves the safety hazards and limited applicability of existing technologies, and achieves high-yield, high-purity product synthesis, which is suitable for the production of peptide drugs.
Patent Information
- Application Number
- CN202511584655.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-02-06
AI Technical Summary
Existing methods for synthesizing N-methyl chiral Fmoc amino acids have safety concerns and limited applicability. In particular, the use of sodium hydrogen can easily cause combustion and explosion, and trifluoroacetic acid is destructive to acid-sensitive groups, failing to meet the needs of diverse substrates.
A three-step synthetic method is adopted, which involves trifluoroacetyl protection, methylation, and hydrolysis to introduce Fmoc groups. Low-toxicity weak bases such as triethylamine and potassium carbonate are used to avoid the use of hazardous materials and strong acids. Methyl groups are introduced and Fmoc groups are hydrolyzed by reacting amino acid methyl esters with ethyl trifluoroacetate.
It reduces safety risks in the synthesis process, broadens the scope of application, improves product yield and purity, is suitable for substrates containing acid-sensitive groups, meets the synthesis requirements of peptide drugs, and is suitable for large-scale production.
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Abstract
Description
Technical Field
[0001] This application relates to the field of non-natural amino acid synthesis technology, and more specifically, it relates to a method for synthesizing N-methyl chiral Fmoc amino acids. Background Technology
[0002] N-methyl chiral Fmoc amino acids, as an important class of organic synthesis intermediates, play an irreplaceable role in the research and development and production of peptide drugs. Their unique structure can effectively regulate the conformational stability, biological activity and metabolic half-life of peptides, and they are widely used in the synthesis of various peptide drugs such as antitumor, antiviral and antibacterial drugs.
[0003] There are two main methods for synthesizing N-methyl chiral Fmoc amino acids using existing technologies: Method 1 uses Boc-protected amino acids as starting materials, which undergo methylation with iodomethane (CH3I) in the presence of sodium hydrogen (NaH). The Boc protecting group is then removed, and an Fmoc protecting group is introduced to obtain the target product. The specific synthetic route is shown below: Method 2 utilizes Fmoc-protected amino acids to generate a five-membered ring intermediate with paraformaldehyde under the catalysis of p-toluenesulfonic acid, followed by reduction with triethylsilane in the presence of trifluoroacetic acid (TFA) to obtain N-methyl chiral Fmoc amino acids. The specific synthetic route is shown below:
[0004] Regarding the aforementioned technologies, the inventors discovered that Method 1 has significant safety hazards. Sodium hydrogen is a highly reactive hazardous material that reacts violently with water or moisture in the air, releasing hydrogen gas. In large-scale factory production or laboratory operations, improper control can easily lead to combustion or even explosion, posing a serious threat to operators and production equipment, and also increasing the cost of safety management during the production process. Method 2 is limited by the reaction conditions. The trifluoroacetic acid used in the reaction is a strong acid that can damage acid-sensitive groups (such as ester groups, acetal groups, etc.) in the substrate, making this method only applicable to substrates that do not contain acid-sensitive groups, greatly limiting its scope of application and failing to meet the demand for diverse substrates in peptide synthesis. Summary of the Invention
[0005] To improve the safety, expand the scope of application, increase production efficiency, and scale up production of N-methyl chiral Fmoc amino acids, this application provides a method for synthesizing N-methyl chiral Fmoc amino acids.
[0006] In a first aspect, this application provides a method for synthesizing N-methyl chiral Fmoc amino acids, employing the following technical solution: A method for synthesizing an N-methyl chiral Fmoc amino acid includes the following steps: S1: Amino acid methyl ester reacts with ethyl trifluoroacetate under the action of a base to form compound 1; S2: Compound 1 reacts with iodomethane under the action of a base to introduce a methyl group, generating compound 2; S3: Compound 2 was hydrolyzed under alkaline conditions, followed by the introduction of an Fmoc group to obtain the target product, an N-methyl chiral Fmoc amino acid. The synthetic route is as follows:
[0007] By adopting the above technical solution, a three-step method of trifluoroacetyl protection, methylation followed by hydrolysis to introduce Fmoc groups is used. This avoids the use of hazardous raw materials during the reaction process and also avoids the damage to acid-sensitive groups caused by dependence on strong acids. This greatly reduces the safety hazards of the synthesis process and is also applicable to substrates containing acid-sensitive groups, significantly expanding the application scenarios.
[0008] The synthesis method described in this application is simple to operate, uses inexpensive and readily available raw materials, and has high yields in all three steps. It is universally applicable, suitable for large-scale production, and meets the requirements of low cost and high reliability in the field of peptide drug synthesis.
[0009] Optionally, the base in step S1 is selected from triethylamine or DIPEA.
[0010] Optionally, the base is triethylamine.
[0011] By adopting the above technical solution, triethylamine and DIPEA are both low-toxicity and low-reactivity organic amines. Compared with the use of sodium hydrogen in the prior art, on the one hand, there is no risk of hydrogen generation and no need for special safety control, which significantly reduces the safety cost of the laboratory or factory. On the other hand, using organic amines as weak bases can selectively promote the acylation reaction of amino groups of amino acid methyl esters with ethyl trifluoroacetate, reduce the occurrence of side reactions such as ester hydrolysis, and improve the yield of compound 1.
[0012] Furthermore, the use of triethylamine compared to DIPEA optimizes proton capture efficiency and prevents the reaction from slowing down due to excessive steric hindrance of DIPEA, resulting in a higher yield for compound 1.
[0013] Optionally, the reaction in step S1 is carried out in a solvent, which is selected from methanol or ethanol.
[0014] Optionally, the solvent in step S1 is methanol.
[0015] By adopting the above technical solution, methanol and ethanol are both inexpensive and readily available conventional solvents, which are more environmentally friendly. They also have good solubility for amino acid methyl ester, ethyl trifluoroacetate and triethylamine / DIPEA, which can ensure the uniformity of the reaction system, avoid side reactions caused by excessive local concentration, and improve the purity of compound 1.
[0016] Optionally, the alkali in step S2 is selected from potassium carbonate or sodium carbonate.
[0017] Optionally, the alkali is potassium carbonate.
[0018] By adopting the above technical solution, potassium carbonate and sodium carbonate are weakly basic inorganic salts with high reaction stability, mild reaction and no risk of hydrogen release. They can effectively activate the amino group (trifluoroacetyl-protected amino group) of compound 1 with simple stirring, and react efficiently with iodomethane to achieve N-methylation, reduce side reactions such as O-methylation, and do not require strict temperature control or inert gas protection. The operation is simple and safe.
[0019] Optionally, the reaction in step S2 is carried out in a solvent selected from DMF or acetonitrile.
[0020] Optionally, the solvent in step S2 is DMF.
[0021] By adopting the above technical solution, DMF and acetonitrile have moderate polarity and boiling point, and the products can be quickly separated by adding water after reaction, simplifying the operation process and improving industrial efficiency.
[0022] Optionally, the reaction temperature in step S2 is 25-35℃.
[0023] Optionally, the reaction temperature in step S2 is 25°C.
[0024] By adopting the above technical solution, the decrease in methylation selectivity caused by temperature fluctuations can be effectively avoided within this temperature range, ensuring zero inversion of the chiral center and guaranteeing the yield and chiral purity of compound 2. The yield and chiral purity of compound 2 are optimal at 25°C, meeting the requirements for optical purity of peptide drugs.
[0025] Optionally, the base in step S3 is selected from sodium hydroxide or lithium hydroxide.
[0026] Optionally, the alkali is lithium hydroxide.
[0027] By adopting the above technical solution, sodium hydroxide or lithium hydroxide can selectively hydrolyze ester groups and protecting groups. The reaction is mild and effectively avoids the breakage of the amino acid backbone. The hydrolysis and Fmoc introduction are completed in one pot, reducing the purification steps and resulting in a higher yield.
[0028] Optionally, the reaction in step S3 is carried out in a solvent selected from dioxane or tetrahydrofuran.
[0029] Optionally, the solvent in step S3 is tetrahydrofuran.
[0030] By adopting the above technical solution, dioxane and tetrahydrofuran have good solubility for compound 2 (trifluoroacetyl-protected N-methyl amino acid ester) and base (such as lithium hydroxide), which can ensure that the hydrolysis reaction proceeds uniformly, avoid side reactions caused by excessive local concentration, provide a suitable environment for the subsequent reaction of FmocOSu with amino groups, and improve the yield of the target product.
[0031] Secondly, this application provides an N-methyl chiral Fmoc amino acid, synthesized by the synthesis method of the N-methyl chiral Fmoc amino acid of this application.
[0032] In summary, this application has the following beneficial effects: 1. The synthesis method of this application adopts a three-step reaction to synthesize N-methyl chiral Fmoc amino acids by protecting with trifluoroacetyl, methylating and then hydrolyzing to introduce Fmoc groups. This avoids the use of hazardous materials and strong acid raw materials, and there is no risk of residual hazardous reagents in the product. Moreover, the chiral center is stable, and the product yield and purity are both high and stable, which meets the synthesis requirements of peptide drugs.
[0033] 2. The synthesis method of this application avoids the destruction of acid-sensitive groups caused by dependence on strong acid raw materials. It is applicable to substrates containing protecting groups such as OtBu, Boc, and Trt, and has universality, which greatly expands the application scenarios.
[0034] 3. The synthetic method of this application uses inexpensive and readily available raw materials, has short steps and simple operation, and the product yield and purity of each reaction step are high, making it suitable for large-scale production. Attached Figure Description
[0035] Figure 1 This is a liquid chromatogram of the target product of Example 1 of this application; Figure 2 This is the chiral liquid chromatogram of the target product in Example 1 of this application; Figure 3 This is the mass spectrum of the target product in Example 1 of this application; Figure 4 This is the NMR spectrum of the target product in Example 1 of this application; Figure 5 This is the liquid chromatogram of the target product in Example 2 of this application; Figure 6 This is the chiral liquid chromatogram of the target product in Example 2 of this application; Figure 7This is the mass spectrum of the target product in Example 2 of this application; Figure 8 This is the NMR spectrum of the target product in Example 2 of this application. Detailed Implementation
[0036] The present application will be further described in detail below with reference to embodiments and comparative examples. Example
[0037] Example 1 A method for synthesizing an N-methyl chiral Fmoc amino acid includes the following steps: S1: L-Tyr(OtBu)-OMe (125 g, 0.50 mol) was dissolved in methanol (1 L), and ethyl trifluoroacetate (80 g, 0.56 mol) and triethylamine (51 g, 0.5 mol) were added. The mixture was stirred overnight at room temperature, concentrated to remove the solvent, and the concentrate was dissolved in ethyl acetate. After acid washing, the concentrate was washed with saturated sodium bicarbonate, dried, and evaporated to dryness to give compound 1, a white solid, with a yield of 81.7%. S2: Compound 1 (140 g, 0.57 mol) was dissolved in DMF (0.7 L), iodomethane (143 g, 1.02 mol) and potassium carbonate (117 g, 0.85 mol) were added, and the mixture was stirred overnight at 25 °C. The solid was filtered off, and water (1.5 L) was added to the filtrate. The mixture was extracted with methyl ether, dried, and evaporated to dryness to give compound 2 as a white solid with a yield of 96.1%. S3: Compound 2 (190 g, 0.53 mol) was dissolved in tetrahydrofuran (0.95 L), and 2N LiOH (1 L) was added dropwise. After stirring at room temperature for 3 h, the pH was adjusted to 8 with 1N HCl. Then, FmocOSu (168.5 g, 0.5 mol) was added, and the pH was controlled at (8 ± 0.2). The mixture was stirred overnight to obtain a reaction solution. The reaction solution was acidified and extracted with ethyl acetate to obtain an extract. The extract was then acid-washed twice, dried, and concentrated to obtain a white solid Fmoc-N-Me-Tyr(OtBu)-OH, with a yield of 84.9%. The synthetic route is as follows:
[0038] Example 2 A method for synthesizing an N-methyl chiral Fmoc amino acid includes the following steps: S1: L-Ser(OtBu)-OMe (87.5 g, 0.50 mol) was dissolved in methanol (0.5 L), and ethyl trifluoroacetate (80 g, 0.56 mol) and triethylamine (51 g, 0.5 mol) were added. The mixture was stirred overnight at room temperature, concentrated to remove the solvent, and the concentrate was dissolved in ethyl acetate. After acid washing, the concentrate was washed with saturated sodium bicarbonate, dried, and evaporated to dryness to give compound 1, with a yield of 85.7%. S2: Compound 1 (110 g, 0.41 mol) was dissolved in DMF (0.6 L), iodomethane (104 g, 0.74 mol) and potassium carbonate (117 g, 0.85 mol) were added, and the mixture was stirred overnight at 25 °C. The solid was filtered off, and water (1.3 L) was added to the filtrate. The mixture was extracted with methyl ether, dried, and evaporated to dryness to obtain the oily compound 2 with a yield of 94.3%. S3: Compound 2 (100 g, 0.35 mol) was dissolved in tetrahydrofuran (0.5 L), and 2N LiOH (0.5 L) was added dropwise. The mixture was stirred at room temperature for 3 h, and the pH was adjusted to 8 with 1N HCl. Then, FmocOSu (101.1 g, 0.3 mol) was added, and the pH was controlled at (8 ± 0.2). The mixture was stirred overnight. The reaction solution was acidified and extracted with ethyl acetate to obtain the extract. The extract was then acid-washed twice, dried, and concentrated to obtain a white solid Fmoc-N-Me-Ser(OtBu)-OH with a yield of 78.9%. The synthetic route is as follows:
[0039] Example 3 A method for synthesizing an N-methyl chiral Fmoc amino acid includes the following steps: S1: L-Tyr(OtBu)-OMe (125 g, 0.50 mol) was dissolved in ethanol (1 L), and ethyl trifluoroacetate (80 g, 0.56 mol) and triethylamine (51 g, 0.5 mol) were added. The mixture was stirred overnight at room temperature, concentrated to remove the solvent, and the concentrate was dissolved in ethyl acetate. After acid washing, the concentrate was washed with saturated sodium bicarbonate, dried, and evaporated to dryness to give a white solid compound 1. S2: Compound 1 (140 g, 0.57 mol) was dissolved in acetonitrile (0.7 L), iodomethane (143 g, 1.02 mol) and potassium carbonate (117 g, 0.85 mol) were added, and the mixture was stirred overnight at 25 °C. The solid was filtered off, and water (1.5 L) was added to the filtrate. The mixture was extracted with methyl ether, dried, and evaporated to dryness to obtain a white solid, compound 2. S3: Compound 2 (190 g, 0.53 mol) was dissolved in dioxane (0.95 L), and 2N LiOH (1 L) was added dropwise. After stirring at room temperature for 3 h, the pH was adjusted to 8 with 1N HCl. Then, FmocOSu (168.5 g, 0.5 mol) was added, and the pH was controlled at (8 ± 0.2). The mixture was stirred overnight to obtain a reaction solution. The reaction solution was acidified and extracted with ethyl acetate to obtain an extract. The extract was then washed twice with acid, dried, and concentrated to obtain a white solid Fmoc-N-Me-Tyr(OtBu)-OH.
[0040] Example 4 A method for synthesizing an N-methyl chiral Fmoc amino acid differs from Example 1 only in that step S1 includes the following steps: L-Tyr(OtBu)-OMe (125 g, 0.50 mol) is dissolved in methanol (1 L), ethyl trifluoroacetate (80 g, 0.56 mol) and DIPEA (65 g, 0.5 mol) are added, the mixture is stirred overnight at room temperature, the solvent is removed by concentration, the concentrate is dissolved in ethyl acetate, acid-washed, washed with saturated sodium bicarbonate, dried, and evaporated to dryness to obtain a white solid compound 1. All other steps are the same as in Example 1.
[0041] Example 5 A method for synthesizing an N-methyl chiral Fmoc amino acid differs from Example 1 only in that step S2 includes the following steps: dissolving compound 1 (140 g, 0.57 mol) in DMF (0.7 L), adding iodomethane (143 g, 1.02 mol) and sodium carbonate (90 g, 0.85 mol), stirring overnight at 25 °C, filtering off the solid, adding water (1.5 L) to the filtrate, extracting with methyl ether, drying, and evaporating to obtain a white solid compound 2. All other steps are the same as in Example 1.
[0042] Example 6 A method for synthesizing an N-methyl chiral Fmoc amino acid differs from Example 1 only in that step S2 includes the following steps: dissolving compound 1 (140 g, 0.57 mol) in DMF (0.7 L), adding iodomethane (143 g, 1.02 mol) and potassium carbonate (117 g, 0.85 mol), stirring overnight at 30 °C, filtering off the solid, adding water (1.5 L) to the filtrate, extracting with methyl ether, drying, and evaporating to obtain a white solid compound 2. All other steps are the same as in Example 1.
[0043] Example 7 A method for synthesizing an N-methyl chiral Fmoc amino acid differs from Example 1 only in that step S2 includes the following steps: dissolving compound 1 (140 g, 0.57 mol) in DMF (0.7 L), adding iodomethane (143 g, 1.02 mol) and potassium carbonate (117 g, 0.85 mol), stirring overnight at 35 °C, filtering off the solid, adding water (1.5 L) to the filtrate, extracting with methyl ether, drying, and evaporating to obtain a white solid compound 2. All other steps are the same as in Example 1.
[0044] Example 8 A method for synthesizing an N-methyl chiral Fmoc amino acid differs from Example 1 only in that step S3 includes the following steps: Compound 2 (190 g, 0.53 mol) is dissolved in tetrahydrofuran (0.95 L), 2N NaOH (1 L) is added dropwise, and the mixture is stirred at room temperature for 3 h. The pH is then adjusted to 8 with 1N HCl. FmocOSu (168.5 g, 0.5 mol) is added, and the pH is controlled at (8 ± 0.2). The mixture is stirred overnight to obtain a reaction solution. The reaction solution is acidified and extracted with ethyl acetate to obtain an extract. The extract is then washed twice with acid, dried, and concentrated to obtain a white solid Fmoc-N-Me-Tyr(OtBu)-OH. All other steps are the same as in Example 1.
[0045] Performance testing Test Example 1 The Fmoc-N-Me-Tyr(OtBu)-OH obtained by the synthesis method of N-methyl chiral Fmoc amino acids in Example 1 and the Fmoc-N-Me-Ser(OtBu)-OH obtained by the synthesis method of N-methyl chiral Fmoc amino acids in Example 2 were subjected to HPLC, MS and other analytical methods, respectively. 1 H NMR detection, results are attached. Figure 1-8 .
[0046] according to Figure 1-2 and Figure 5-6 The results under different HPLC detection conditions show that a single main peak is present, the area ratio of impurity peaks is extremely low, the purity of the target product Fmoc-N-Me-Tyr(OtBu)-OH is consistently above 98.6%, and the purity of the target product Fmoc-N-Me-Ser(OtBu)-OH is consistently above 99.9%. This indicates that the product obtained by the three-step reaction method of synthesizing N-methyl chiral Fmoc amino acids through trifluoroacetyl protection, methylation, and hydrolysis to introduce Fmoc groups has high purity and can meet the synthesis requirements of peptide drugs.
[0047] according to Figure 3-4 and Figure 7-8 The detection results show that the mass spectrometry fragment peaks match the target product Fmoc-N-Me-Tyr(OtBu)-OH and Fmoc-N-Me-Ser(OtBu)-OH fragments, with no obvious structural isomerism or incorrect modification. The chemical shift and peak shape of the proton nuclear magnetic resonance spectrum are completely matched with those of the target product Fmoc-N-Me-Tyr(OtBu)-OH and Fmoc-N-Me-Ser(OtBu)-OH, confirming that the functional group connections in the molecule are correct and the chiral purity is good.
[0048] Test Example 2 The yields of compound 1, compound 2, the target product, and the purity of the target product synthesized by the methods for synthesizing N-methyl chiral Fmoc amino acids in Examples 1-8 were detected and calculated, and the detection results were recorded in Table 1, where yield = mass of reagent reaction product / theoretical product mass × 100%.
[0049] Table 1 As can be seen from the performance test results in Table 1, the synthesis method of N-methyl chiral Fmoc amino acids of this application can efficiently synthesize N-methyl chiral Fmoc amino acids, with a yield of 78.9-84.9% of the target product and a purity of ≥98.6%. It avoids the use of hazardous materials and strong acid raw materials, avoids the damage to acid-sensitive groups caused by dependence on strong acid raw materials, has universality, has no residual risk of hazardous reagents in the product, and the chiral center is stable, meeting the synthesis requirements of peptide drugs.
[0050] According to the performance test results of Examples 1 and 3-8, when methanol is used as the solvent and triethylamine is used as the weak base in step S1, it can more selectively promote the acylation reaction of amino groups of amino methyl esters with ethyl trifluoroacetate, reduce the occurrence of side reactions such as ester hydrolysis, and improve the yield of compound 1 compared with DIPEA.
[0051] When DMF is used as the solvent and potassium carbonate is used as a weakly basic inorganic salt in step S2, the yield of compound 2 is higher. When tetrahydrofuran is used as the solvent and lithium hydroxide is used as the base in step S3, it can be more effectively mixed with compound 2, ensuring the uniformity of the hydrolysis reaction, avoiding side reactions caused by excessively high local concentrations, providing a suitable environment for the subsequent reaction of FmocOSu with amino groups, and improving the yield of the target product.
[0052] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A method for synthesizing an N-methyl chiral Fmoc amino acid, characterized in that, Includes the following steps: S1: Amino acid methyl ester reacts with ethyl trifluoroacetate under the action of a base to form compound 1; S2: Compound 1 reacts with iodomethane under the action of a base to introduce a methyl group, generating compound 2; S3: Compound 2 was hydrolyzed under alkaline conditions, followed by the introduction of an Fmoc group to obtain the target product, an N-methyl chiral Fmoc amino acid. The synthetic route is as follows:
2. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The base in step S1 is selected from triethylamine or DIPEA.
3. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The reaction in step S1 is carried out in a solvent, which is selected from methanol or ethanol.
4. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The alkali in step S2 is selected from potassium carbonate or sodium carbonate.
5. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The reaction in step S2 is carried out in a solvent selected from DMF or acetonitrile.
6. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The reaction temperature in step S2 is 25-35℃.
7. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The alkali used in step S3 is selected from sodium hydroxide or lithium hydroxide.
8. The method for synthesizing N-methyl chiral Fmoc amino acids according to claim 1, characterized in that, The reaction in step S3 is carried out in a solvent selected from dioxane or tetrahydrofuran.
9. An N-methyl chiral Fmoc amino acid, synthesized by the method for synthesizing N-methyl chiral Fmoc amino acids according to any one of claims 1-8.