A method for synthesizing l-gamma-glutamyl-p-nitroaniline
Patent Information
- Application Number
- CN202611424315.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-15
- Publication Date
- 2026-11-03
AI Technical Summary
The existing synthesis methods for L-gamma-glutamyl-p-nitroaniline require high reaction conditions, are difficult to purify, are cumbersome to operate, and are costly, making large-scale production impossible.
N,N'-bis(4-nitrophenyl)phosphorylimide amide was generated by reacting p-nitroaniline and phosphorus trichloride in pyridine. Then, it was reacted with Boc-L-Glu-OtBu in DMF to generate the intermediate N-Boc-L-Glu(PNA)-OtBu. The intermediate was then treated with dichloromethane and trifluoroacetic acid, neutralized, and purified by reflux filtration with methanol, avoiding the use of high-temperature microwave and easily explosive reagents.
It enables the simple preparation of high-purity (over 99%) products, which is suitable for large-scale production and reduces production costs and operational complexity.
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Figure CN123026533A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for synthesizing L-gamma-glutamyl-p-nitroaniline. Background Technology
[0002] L-gamma-glutamyl-p-nitroaniline is a glutamate derivative with the structure shown below. It is a specific chromogenic substrate for γ-glutamyl transpeptidase (GGT) and is primarily used for quantitative detection of enzyme activity and research on biochemical mechanisms.
[0003]
[0004] Currently, the main synthetic methods are as follows: The journal *Bioorganic and Medicinal Chemistry Letters* (2015, vol. 25, #1, p. 113-116) reported a condensation reaction of Boc-L-Glu-OtBu and p-nitroaniline in DMF, HATU, and DIEA systems under microwave radiation at 120°C, which is not suitable for large-scale production. The *Journal of Biological Chemistry* (1965, vol. 240, p. 338-347) reported the reaction of N-phthaloyl-L-glutamic anhydride and p-nitroaniline in acetic acid at 60-65°C to generate N-phthaloyl-L-glutamic acid-p-nitroaniline, followed by deprotection with hydrazine hydrate to obtain the target product. However, due to the use of easily explosive hydrazine hydrate, post-processing is difficult and unsuitable for large-scale production. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing L-gamma-glutamyl-p-nitroaniline that is simple to operate, easy to purify, and readily applicable for industrial production. It mainly addresses the technical problems of existing methods, such as demanding reaction conditions, difficulty in purification, cumbersome operation, high cost, and inability to achieve large-scale production.
[0006] The technical solution of this invention: A method for synthesizing L-gamma-glutamyl-p-nitroaniline, characterized by comprising the following steps: (1) In the first step, p-nitroaniline and phosphorus trichloride are reacted in pyridine at room temperature to generate N,N'-bis(4-nitrophenyl)phosphineimide amide. Then, a Boc-L-Glu-OtBu solution dissolved in DMF is added to the above reaction solution and the reaction is carried out at 40-50°C. The resulting reaction solution is washed successively with 10% citric acid solution, saturated sodium bicarbonate solution, tap water, and dried with anhydrous sodium sulfate. The solution is concentrated to obtain crude N-Boc-L-Glu(PNA)-OtBu intermediate of the first step. It does not need to be purified and can be used directly in the next step of the reaction.
[0007] (2) In the second step, the crude N-Boc-L-Glu(PNA)-OtBu product is reacted in a solution of dichloromethane and trifluoroacetic acid, then concentrated, tap water is added to the residue, and the residue is neutralized and filtered in sequence with potassium carbonate solution. The resulting filter cake is refluxed with methanol to make pulp, filtered, and dried to obtain the target product.
[0008] The reaction formula is as follows:
[0009] (1) In the first step, the molar ratio of Boc-L-Glu-OtBu, p-nitroaniline, and phosphorus trichloride was 1:2:1, the volume ratio of pyridine to Boc-L-Glu-OtBu was 5:1, and the volume ratio of DMF to Boc-L-Glu-OtBu was 5:1. The reaction of p-nitroaniline and phosphorus trichloride in pyridine was at room temperature for 1 to 2 hours. The reaction temperature of the resulting reaction solution and the Boc-L-Glu-OtBu solution dissolved in DMF was 40 to 50 °C for 3 to 4 hours.
[0010] (2) In the second step, the volume ratio of N-Boc-L-Glu(PNA)-OtBu (crude product), dichloromethane, and TFA is 1:2.5:2.5, and the reaction is carried out at room temperature for 2-3 hours. In the post-treatment, the volume ratio of N-Boc-L-Glu(PNA)-OtBu and tap water is 1:2.5. The alkali used for neutralization is potassium carbonate or sodium carbonate, the solvent used for reflux pulping is methanol, the drying temperature is 50-55℃, and the drying time is 8-12 hours.
[0011] The beneficial effects of this invention are as follows: By setting a reasonable process route, this invention avoids the situation where the substrate Boc-L-Glu-OtBu and p-nitroaniline react at high temperatures under microwave radiation in DMF, which is detrimental to large-scale production. It also avoids the difficult post-processing problem of using N-phthaloyl-L-glutamic anhydride as a substrate to react with p-nitroaniline, followed by deprotection with easily explosive hydrazine hydrate. The key solution of this invention is that the first step involves reacting p-nitroaniline and phosphorus trichloride in pyridine to generate N,N'-bis(4-nitrophenyl)phosphineimide amide, which is then reacted with Boc-L-Glu-OtBu. This allows the reaction to proceed smoothly at room temperature, and the product prepared from the obtained intermediate can achieve a purity of over 99%. The operation is simple, the reaction cycle is short, the cost is low, and it is suitable for large-scale production. Attached Figure Description
[0012] Figure 1 This is the MS spectrum of the product.
[0013] Figure 2 The product's HNMR spectrum. Detailed Implementation
[0014] The present invention will be further described with reference to specific embodiments, but the present invention is not limited to these embodiments. Example
[0015] Step 1: Preparation of intermediate N-Boc-L-Glu(PNA)-OtBu
[0016] In a 5 L three-necked reaction flask, 1.5 L of pyridine, 135.8 g of phosphorus trichloride, and 273.0 g of p-nitroaniline were added sequentially. The mixture was stirred at room temperature for 1 h. Then, 300 g of Boc-L-Glu-OtBu dissolved in 1.5 L of DMF was added. The mixture was heated to 40 °C and reacted for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into 4 L of 10% citric acid solution and 4 L of DCM. The phases were separated. The organic phase was washed sequentially with 2 L of saturated sodium bicarbonate solution and 2 L of tap water. The solution was dried over anhydrous sodium sulfate and concentrated to obtain 790 g of crude intermediate, which was directly used in the next reaction step.
[0017] Step 2: Preparation of the target product L-gamma-glutamyl-p-nitroaniline 790 g of the crude intermediate obtained in step 1 was dissolved in 1.98 L of DCM and then transferred to a 10 L three-necked flask. 1.98 L of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 3 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure until no solvent evaporated. 1.98 L of tap water was added to the residue, and the pH was adjusted to -6 to 7 with potassium carbonate. The mixture was filtered, and the filter cake was transferred to a 5 L three-necked flask. 1.98 L of methanol was added, and the mixture was heated to reflux and maintained at that temperature for 30 min. The temperature was then slowly lowered to room temperature, filtered, and dried under reduced pressure at 50 °C for 12 h to obtain 166.5 g of the product. The overall yield of the two steps was 63.0%.
[0018] The product L-gamma-glutamyl-p-nitroaniline was analyzed by HPLC. The purity of L-gamma-glutamyl-p-nitroaniline was 99.1%. MS and ¹H NMR were correct. (See attached image) Figure 1 and Figure 2 .
[0019] Example 2 Step 1: Preparation of intermediate N-Boc-L-Glu(PNA)-OtBu
[0020] In a 5 L three-necked reaction flask, 1.5 L of pyridine, 135.8 g of phosphorus trichloride, and 273.0 g of p-nitroaniline were added sequentially. The mixture was stirred at room temperature for 2 h. Then, 300 g of Boc-L-Glu-OtBu dissolved in 1.5 L of DMF was added. The mixture was heated to 50 °C and reacted for 3 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into 4 L of 10% citric acid solution and 4 L of DCM. The phases were separated. The organic phase was washed sequentially with 2 L of saturated sodium bicarbonate solution and 2 L of tap water. The solution was dried over anhydrous sodium sulfate and concentrated to obtain 760 g of crude intermediate, which was directly used in the next reaction step.
[0021] Step 2: Preparation of the target product L-gamma-glutamyl-p-nitroaniline
[0022] 760 g of the crude intermediate obtained in step 1 was dissolved in 1.9 L of DCM and then transferred to a 10 L three-necked flask. 1.9 L of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure until no solvent evaporated. 1.9 L of tap water was added to the residue, and the pH was adjusted to -6 to 7 with potassium carbonate. The mixture was filtered, and the filter cake was transferred to a 5 L three-necked flask. 1.9 L of methanol was added, and the mixture was heated to reflux and held at that temperature for 30 min. The mixture was then slowly cooled to room temperature, filtered, and dried under reduced pressure at 55 °C for 8 h to obtain 171.8 g of the product. The overall yield of the two steps was 65.0%.
[0023] The product L-gamma-glutamyl-p-nitroaniline was analyzed by HPLC. The purity of L-gamma-glutamyl-p-nitroaniline was 99.3%. MS and ¹H NMR were correct. Figure 1 and Figure 2 .
[0024] Example 3 Step 1: Preparation of intermediate N-Boc-L-Glu(PNA)-OtBu
[0025] In a 20 L three-necked flask, 7.5 L of pyridine, 0.67 kg of phosphorus trichloride, and 1.36 kg of p-nitroaniline were added sequentially. The mixture was stirred at room temperature for 1.5 h. Then, 1.5 kg of Boc-L-Glu-OtBu dissolved in 7.5 L of DMF was added. The mixture was heated to 45 °C and reacted for 3.5 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into 20 L of 10% citric acid solution and 20 L of LDCM. The phases were separated. The organic phase was washed sequentially with 10 L of saturated sodium bicarbonate solution and 10 L of tap water. The solution was dried over anhydrous sodium sulfate and concentrated to obtain 4.0 kg of crude intermediate, which was directly used in the next reaction step.
[0026] Step 2: Preparation of the target product
[0027] The crude intermediate obtained in step 1 was dissolved in 10 L of DCM and then transferred to a 50 L reactor. 10 L of TFA was added, and the reaction was carried out at room temperature for 2.5 h. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure until no solvent evaporated. 10 L of tap water was added to the residue, and the pH was adjusted to -6 to 7 with potassium carbonate. The mixture was filtered, and the filter cake was transferred to a 20 L three-necked flask. 10 L of methanol was added, and the mixture was heated to reflux and held at that temperature for 30 min. The temperature was then slowly lowered to room temperature, filtered, and dried under reduced pressure at 52 °C for 10 h to obtain 0.9 kg of the product. The overall yield of the two steps was 68.1%.
[0028] The product L-gamma-glutamyl-p-nitroaniline was analyzed by HPLC. The purity of L-gamma-glutamyl-p-nitroaniline was 99.7%. MS and ¹H NMR were correct. Figure 1 and Figure 2 .
Claims
1. A method for synthesizing L-gamma-glutamyl-p-nitroaniline, characterized in that, Includes the following steps: (1) In the first step, p-nitroaniline and phosphorus trichloride are reacted in pyridine at room temperature to generate N,N'-bis(4-nitrophenyl)phosphineimide amide. Then, a Boc-L-Glu-OtBu solution dissolved in DMF is added to the above reaction solution and the reaction is carried out at 40-50°C. The resulting reaction solution is washed successively with 10% citric acid solution, saturated sodium bicarbonate solution, tap water, and dried with anhydrous sodium sulfate. The solution is then concentrated to obtain crude N-Boc-L-Glu(PNA)-OtBu intermediate from the first step, which is directly used in the next step. (2) In the second step, the crude N-Boc-L-Glu(PNA)-OtBu product is reacted in a solution of dichloromethane and trifluoroacetic acid, then concentrated, and tap water is added to the residue. The residue is then neutralized with potassium carbonate or sodium carbonate solution and filtered. The resulting filter cake is refluxed with methanol or ethanol, filtered, and dried to obtain the target product. The reaction formula is as follows: 。 2. The method for synthesizing L-gamma-glutamyl-p-nitroaniline according to claim 1, characterized in that: (1) In the first step, the molar ratio of Boc-L-Glu-OtBu, p-nitroaniline and phosphorus trichloride is 1:2:1, the volume ratio of pyridine to Boc-L-Glu-OtBu is 5:1, and the volume ratio of DMF to Boc-L-Glu-OtBu is 5:
1. (2) In the second step, the volume ratio of crude N-Boc-L-Glu(PNA)-OtBu, dichloromethane, and TFA is 1:2.5:2.5, and the volume ratio of post-treatment N-Boc-L-Glu(PNA)-OtBu and tap water is 1:2.
5.
3. The method for synthesizing L-gamma-glutamyl-p-nitroaniline according to claim 1, characterized in that: (1) In the first step, the reaction of p-nitroaniline and phosphorus trichloride in pyridine is carried out at room temperature for 1 to 2 hours. The resulting reaction solution is then reacted with a Boc-L-Glu-OtBu solution dissolved in DMF at a temperature of 40 to 50°C for 3 to 4 hours. (2) The second step is to react at room temperature for 2 to 3 hours.
4. The method for synthesizing L-gamma-glutamyl-p-nitroaniline according to claim 1, characterized in that: The second step involves using potassium carbonate as the alkali for neutralization.
5. The method for synthesizing L-gamma-glutamyl-p-nitroaniline according to claim 1, characterized in that: In the second step, the reflux pulping solvent is methanol.
6. The method for synthesizing L-gamma-glutamyl-p-nitroaniline according to claim 1, characterized in that: The drying temperature in the second step is 50-55℃, and the time is 8-12 hours.