Synthesis and detection method of N-trityl-l-serine

Abstract

The application discloses a kind of synthesis and detection method of N-triphenylmethyl-L-serine, mainly solve the technical problem of safety and purity detection in production process.The present application comprises the following steps: one, the chemical synthesis process of product;Two, the reagent used for preparation is prepared;Three, carry out high performance liquid chromatography determination, chromatograph is Agilent 1260 chromatograph, using VWD ultraviolet detector, chromatographic column Shim-pack GIS CN chromatographic column, mobile phase is mass percentage concentration 0.025% formic acid aqueous solution (A) and mass percentage concentration 0.025% formic acid acetonitrile solution (B);Through the present application, the effect of safety, low cost, high purity is improved for N-triphenylmethyl-L-serine chemical synthesis.

Description

Technical Field

[0001] This invention relates to methods for the synthesis and detection of non-natural amino acids, and particularly to a method for the synthesis and detection of N-triphenylmethyl-L-serine. Background Technology

[0002] N-Triphenylmethyl-L-serine, starting material: Based on L-serine, with trimethylchlorosilane as a protective agent, N-triphenylmethyl-L-serine is generated by dropwise addition of a solution of triphenylchloromethane and dichloromethane under alkaline conditions and low temperature. Strict temperature control is required during the reaction to avoid racemization. Inorganic citric acid solution is used instead of dilute hydrochloric acid for washing and extraction, as hydrochloric acid is mostly a byproduct, and washing with a dilute hydrochloric acid solution can easily introduce unknown impurities. Summary of the Invention

[0003] The purpose of this invention is to provide a method for synthesizing N-triphenylmethyl-L-serine, which mainly solves the technical problems of low safety and low product purity in existing production processes.

[0004] This invention employs the following technical solution: a method for the synthesis and detection of N-triphenylmethyl-L-serine, comprising the following steps: Step 1, Chemical Synthesis of the Product: L-serine and dichloromethane were added to a reaction vessel. At room temperature, with stirring, trimethylchlorosilane was added dropwise. After the addition was complete, the temperature was raised to 60°C and refluxed for 2 hours. After this, the temperature was lowered to 0°C. Triethylamine was added, followed by dropwise addition of a mixed solution of triphenylchloromethane and dichloromethane (1.0 eq / 1 L). After the addition was complete, the temperature was raised to 25°C and the reaction was carried out for 24 hours. The reaction was confirmed by TLC. After the reaction was complete, the reaction solution was concentrated to dryness. Ethyl acetate was added to completely dissolve the product, and a pH 5 citric acid aqueous solution was added with stirring to wash and extract. This process was repeated twice. Finally, the organic phase was evaporated to dryness to obtain the product. Reaction formula:

[0005] Step 2: Prepare the reagents for testing: acetonitrile - chromatographic grade, deionized water, formic acid - chromatographic grade.

[0006] Step 3: Perform high performance liquid chromatography (HPLC) determination. Chromatographic conditions: sample chamber temperature is room temperature, autosampler is used, and the chromatographic column is a Shim-pack GISCN (cyanopropyl) column with a column temperature of 40 ℃ and a flow rate of 1.0 mL / min. Mobile phase A is a 0.025% formic acid aqueous solution, and mobile phase B is a 0.025% formic acid acetonitrile solution. Gradient elution program is used.

[0007] The chromatograph used was an Agilent 1260 liquid chromatograph, which uses a VWD ultraviolet detector.

[0008] The chromatographic column has dimensions of 4.6 mm × 250 mm and 5.0 μm.

[0009] The gradient elution program is as follows: 0-12 min, the volume ratio of mobile phase B increases from 70% to 100%; 12-12.1 min, the volume ratio of mobile phase B decreases from 100% to an initial ratio of 70%; 12.1-18 min, the volume ratio of mobile phase B decreases from 70% to the equilibrium system.

[0010] This invention offers the following advantages: It provides a method for the synthesis and detection of N-triphenylmethyl-L-serine, primarily addressing the issue of strictly controlling the reaction process by adding a mixed solution of triphenylchloromethane and dichloromethane (1.0 eq / 1L) and then heating the mixture to 25°C for 24 hours. This strict temperature control reduces the formation of racemic mixtures due to temperature variations. Furthermore, the use of a citric acid aqueous solution (pH=5) instead of dilute hydrochloric acid for extraction effectively controls the generation of unknown impurities, ensuring product purity. The replacement of hydrochloric acid with citric acid also increases process safety. After obtaining the product, precise quantification is achieved using the autosampler of a high-performance liquid chromatograph (HPLC). The VWD ultraviolet detector provides a good response value for N-triphenylmethyl-L-serine, enabling efficient detection of the product's purity, which can reach over 99%. This method is simple, rapid, accurate, and exhibits good stability. Attached Figure Description

[0011] Figure 1 The sample used in Example 1 is a qualified product already marketed by Jier Biochemical Co., Ltd., batch number GLS250912-J16202, with a chemical purity of ≥98.0%. The HPLC chromatogram was obtained using a 0.025% (w / w) formic acid aqueous solution as mobile phase B and a 0.025% (w / w) formic acid acetonitrile solution as mobile phase B. The detection result was 99.66%. Figure 2 The HPLC chromatogram for the self-made product in Example 1 using a 0.025% (w / w) formic acid aqueous solution and a 0.025% (w / w) formic acid acetonitrile solution as mobile phase B is shown; the chemical purity was determined to be 99.74%. Figure 3 The standard sample purchased from Bid Pharmaceuticals in Example 1, coded BD582885, has a chemical purity of ≥95.0%. The HPLC chromatogram shows the results when using a 0.025% (w / w) formic acid aqueous solution as mobile phase B and a 0.025% (w / w) formic acid acetonitrile solution as mobile phase B; the detection result is 99.25%. Figure 4 The HPLC chromatogram is shown in Example 2 when the injection concentration is 0.2 µg / mL. Figure 5 The HPLC chromatogram is shown in Example 2 when the injection concentration is 0.4 µg / mL. Figure 6 The HPLC chromatogram is shown in Example 2 when the injection concentration is 0.6 µg / mL. Figure 7 The HPLC chromatogram is shown in Example 2 when the injection concentration is 0.8 µg / mL. Figure 8 The HPLC chromatogram is shown in Example 2 when the injection concentration is 1.0 µg / mL. Figure 9 This is the HPLC chromatogram of the reproducibility-1 test sample in Example 4; Figure 10 This is the HPLC chromatogram of the reproducibility-2 test sample in Example 4; Figure 11 This is the HPLC chromatogram of the reproducibility-3 test sample in Example 4; Figure 12 This is the HPLC chromatogram of the reproducibility-4 test sample in Example 4; Figure 13 This is the HPLC chromatogram of the reproducibility-5 test sample in Example 4; Figure 14 A graph showing the linear relationship between injection volume (X) and average peak area (Y); Figure 15 NMR spectra used to confirm the product structure of Jier Biochemical Company for market launch; Figure 16 NMR spectra used to confirm the structure of the self-made product; Figure 17 Mass spectra of molecular weights of products launched into the market by Jier Biochemical Company; Figure 18 Mass spectra of the molecular weight of the self-made product; Figure 19 For product chirality testing (test spectrum of mixed L-type and D-type samples); Figure 20 For product chirality testing (L-type single sample test spectrum). Detailed Implementation

[0012] Main instruments: The system used was an Agilent 1260 high-performance liquid chromatograph equipped with a VWD UV detector, a Shimadzu Shim-pack GISCN (cyanopropyl) column (4.6 mm × 250 mm, 3.5 μm), an ultrasonic extractor (KQ5200E, Zhengzhou Kete Experimental Equipment Co., Ltd.), a 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instruments Co., Ltd.), and an electronic analytical balance (CPA225D, Sartorius Scientific Instruments (Shanghai) Co., Ltd.). Main reagents: The reagents used in the measurements included deionized water; acetonitrile (preparative grade, Titan Chemicals), formic acid (chromatographic grade, Shanghai Guoyao Company); and deionized water in its standard form.

[0013] Sample pretreatment: Weigh a certain amount of the sample and place it in a 10ml volumetric flask. Add 8ml of acetonitrile and dissolve it by ultrasonication at room temperature. Then add deionized water. Finally, filter the sample through a polypropylene organic filter membrane before testing. Example 1

[0014] Chemical synthesis process of the product: L-serine and dichloromethane were added to a reaction vessel. At room temperature, with stirring, trimethylchlorosilane was added dropwise. After the addition was complete, the temperature was raised to 60°C and refluxed for 2 hours. After completion, the temperature was lowered to 0°C. Triethylamine (433.5 g 4.5 eq) was added, followed by dropwise addition of a mixed solution of triphenylchloromethane and dichloromethane (1.0 eq / 1 L). After the addition was complete, the temperature was raised to 25°C and the reaction was carried out for 24 hours. The reaction was confirmed by TLC. After the reaction was complete, the reaction solution was concentrated to dryness. Ethyl acetate was added to completely dissolve the product, and a pH 5 citric acid aqueous solution was added with stirring for washing and extraction. This process was repeated twice. Finally, the organic phase was evaporated to dryness to obtain the product; purity 99.1%, yield 90.5%.

[0015] Table 1 Raw Materials and Reagents

[0016] Chromatographic conditions: The sample chamber temperature was room temperature. The autosampler injection volume was 10 µL. A Shimadzu Shim-pack GISCN (cyanopropyl) column was used. The column temperature was 40 °C, the flow rate was 1.0 mL / min, and the mobile phase consisted of a 0.025% formic acid aqueous solution (A) and a 0.025% formic acid acetonitrile solution (B). A gradient elution program was employed. The gradient elution program was as follows: 0–12 min, the volume ratio of mobile phase B increased from 70% to 100%; 12–12.1 min, the volume ratio of mobile phase B decreased from 100% to an initial ratio of 70%; 12.1–18 min, the volume ratio of mobile phase B decreased from 70% to the equilibrium system.

[0017] Selection and optimization of sample pretreatment methods: Currently, N-triphenylmethyl-L-serine is available in the market as a powdered solid. The sample tested in this invention is a self-made product, and a product manufactured and marketed by Jier Biochemical (Shanghai) Co., Ltd. was also purchased. Batch number: GLS250912-J16202. The product purity is 99.66%, and the HPLC chromatogram is shown below. Figure 1 As shown, the NMR spectrum of the product is shown below. Figure 15 The molecular weight mass spectra of the product are shown in [reference needed]. Figure 17 .

[0018] The sample chamber temperature of this invention is set to room temperature, which is beneficial for the preservation of acetonitrile solvent in the sample chamber. The column oven temperature is set to 40°C in this invention.

[0019] The HPLC chromatogram of the self-made product after purification is shown below. Figure 2 As shown, the NMR spectrum of the self-made product is shown below. Figure 16 The molecular weight mass spectra of the self-made products are shown in [reference needed]. Figure 18 Chirality testing of self-made products (the test chromatogram of the mixed L-type and D-type samples is shown in...) Figure 19 For the chiral testing of the self-made product (L-type single-sample test spectrum), see [link to relevant documentation]. Figure 20 The method was used to detect N-triphenylmethyl-L-serine products. The results showed that the production process was safe, no racemic mixture was generated, the retention time of the main peak was basically consistent with that of qualified products on the market, and the purity could reach over 99%. It has practical application value. Example 2

[0020] 1. Linearity verification Using acetonitrile solution, the self-made sample stock solution from Example 1 was serially diluted to 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, and 10 μg / mL. The HPLC chromatograms of the samples at these concentrations are shown in the figures below. Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 The high-performance liquid chromatography (HPLC) conditions in Example 1 were followed for detection. Three injections were performed at each concentration point, and the average peak area of ​​the three injections was calculated as the average peak area corresponding to that concentration point. The injection concentration, retention time, target peak corresponding value, theoretical number of column pedals for the target peak, resolution, and detection purity are shown in Table 2 below. Table 2. Injection concentration, injection volume (X), and average peak area (Y)

[0021] A linear regression was performed with the injection volume (X, μg) on ​​the x-axis and the average peak area (Y, mAU×min) on the y-axis. The resulting regression equation was Y = 850.3X + 713.1, with R² = 0.997. (Details are as follows...) Figure 14 As shown.

[0022] from Figure 14 As can be seen, N-triphenylmethyl-L-serine exhibits an excellent linear relationship between injection volume and peak area (R²=0.997) within the injection concentration range of 0.2-1.0 mg (corresponding to concentrations of 200-1600 μg / mL), with a detection limit as low as 0.01 μg, meeting the requirements for trace detection.

[0023] 2. Accuracy Table 3. Self-made products and commercially available products purchased from the market

[0024] The deviation between the detected purity and the theoretical purity is ≤0.01%, proving that the method is highly accurate and can be used as an authoritative testing basis. Example 3

[0025] Furthermore, when the injection volume of N-triphenylmethyl-L-serine was only 0.001 μg (concentration 0.01 μg / mL), the response value of the main peak was approximately 1.7 mAU, and the signal-to-noise ratio (S / N) was 3, meeting the detection limit requirement; this indicates that the method has a low detection limit and can detect low-concentration samples. Specifically, as follows... Figure 4 As shown. Example 4

[0026] 3. Precision verification A 1 mg / mL sample of self-prepared N-triphenylmethyl-L-serine was taken and analyzed under the high-performance liquid chromatography (HPLC) conditions described in Example 1. The HPLC chromatograms of samples at various concentrations are shown in the table below. Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 The sample to be tested was injected five times consecutively, and the test results are shown in Table 4 below. The results of the precision test of 1 mg / mL of self-made N-triphenylmethyl-L-serine were obtained.

[0027] Table 4. Detection results of Example 4

[0028] Wherein, RSD (%) = (average of the five peak areas / variance of the five peak areas) × 100%; A linear regression was performed with the injection volume (X, μg) on ​​the x-axis and the average peak area (Y, mAU×min) on the y-axis. The resulting regression equation was Y=1908X, where R0 2 =1.0, specifically as follows Figure 14 As shown.

[0029] from Figure 14 As can be seen, N-triphenylmethyl-L-serine exhibits an excellent linear relationship between injection volume and peak area (R²=1.0) within the injection concentration range of 20-100 μg (corresponding to a concentration of 20-100 μg / mL). Furthermore, when the injection amount of N-triphenylmethyl-L-serine was only 0.1 μg (concentration 10 μg / mL), the response value of the main peak was 2.7 mAU, and the signal-to-noise ratio S / N=3, which met the detection limit requirement; indicating that the method has a low detection limit and can detect low concentration samples.

[0030] 2. Precision verification Take 1 mg / mL of self-prepared N-triphenylmethyl-L-serine as the test sample and detect it according to the high performance liquid chromatography conditions in Example 4. The test sample was injected 5 times consecutively, and the detection results are shown in Table 5 below. Table 5. Detection results of N-triphenylmethyl-L-serine precision.

[0031] Wherein, RSD (%) = (average of the five peak areas / variance of the five peak areas) × 100%; The results in the table above show that the peak area RSD of five consecutive injections is only 0.16% (RSD≤1% is excellent), indicating that the peak area is stable, which proves that the method has high precision and good repeatability.

Claims

1. A method for the synthesis and detection of N-triphenylmethyl-L-serine; characterized in that: Includes the following steps: Step 1, Chemical Synthesis of the Product: L-serine and dichloromethane were added to a reaction vessel. At room temperature, with stirring, trimethylchlorosilane was added dropwise. After the addition was complete, the temperature was raised to reflux. After this, the temperature was lowered to 0°C. Triethylamine was added, followed by the dropwise addition of a mixed solution of triphenylchloromethane and dichloromethane. After the addition was complete, the temperature was raised to 25°C and the reaction was carried out for 24 hours. The reaction was confirmed by TLC. After the reaction was complete, the reaction solution was concentrated to dryness. Ethyl acetate was added to completely dissolve the product, and then citric acid aqueous solution was added with stirring for washing and extraction. This process was repeated twice. Finally, the organic phase was evaporated to dryness to obtain the product. Reaction formula: ; Step 2: Prepare the reagents for testing: acetonitrile - chromatographic grade, deionized water, formic acid - chromatographic grade; Step 3: Perform high performance liquid chromatography (HPLC) determination: Chromatographic conditions: sample chamber temperature at room temperature, autosampler, chromatographic column: Shim-pack GIS CN column, column temperature: 40 ℃, flow rate: 1.0 mL / min, mobile phase A: 0.025% formic acid aqueous solution; mobile phase B: 0.025% formic acid acetonitrile solution, using a gradient elution program.

2. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The reflux reaction was carried out at 60 degrees Celsius for 2 hours.

3. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The citric acid aqueous solution has a pH of 5.

4. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The volume of the triphenylchloromethane and dichloromethane mixed solution is 1.0 eq / 1L.

5. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The chromatograph used in the liquid chromatography determination was an Agilent 1260 liquid chromatograph, which used a VWD ultraviolet detector.

6. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The chromatographic column has dimensions of 4.6 mm × 250 mm and 5.0 μm.

7. The method for synthesizing and detecting N-triphenylmethyl-L-serine according to claim 1, characterized in that: The gradient elution program is as follows: 0-12 min, the volume ratio of mobile phase B increases from 70% to 100%; 12.0-12.1 min, the volume ratio of mobile phase B decreases from 100% to an initial ratio of 70%; 12.1-18 min, the volume ratio of mobile phase B decreases from 70% to the equilibrium system.

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