N < 2 >-[fluorenylmethoxycarbonyl]-L-lysine allyl ester monohydrochloride synthesis and chiral detection method

Abstract

The invention discloses a synthesis and chiral detection method of N2-[fluorenylmethoxycarbonyl]-L-lysine allyl ester monohydrochloride, and mainly solves the technical problems of complex conventional chiral detection and difficult isomer separation. The synthesis and chiral detection method comprises the following steps: 1, preparing a reagent for preparation; 2, synthesizing a sample; 3, high performance liquid chromatography determination is carried out, an Agilent 1260 chromatographic instrument is adopted, a DAD ultraviolet detector is adopted, CHIRALPAKR AD-H is adopted as a chromatographic column, and a normal hexane solution (A) with the mass percentage concentration being 100% and an acetic acid-ethanol solution (B) with the mass percentage concentration being 0.1% are adopted as mobile phases; the invention provides a reliable and convenient means for improving the N < 2 >-[fluorenylmethoxycarbonyl]-L-lysine allyl ester monohydrochloride chiral detection method.

Description

Technical Field

[0001] The present invention relates to the synthesis of lysine allyl ester monohydrochloride and the method for chiral detection, and particularly relates to a method for the synthesis of N2 - [9 - fluorenylmethoxycarbonyl] - L - lysine allyl ester monohydrochloride and chiral detection. Background Art

[0002] In the process of drug research and development, amino acid derivatives are often used to modify the structure of drug molecules to improve the pharmacokinetic properties of drugs, reduce toxicity, increase bioavailability, etc.; N2 - [9 - fluorenylmethoxycarbonyl] - L - lysine allyl ester monohydrochloride is a lysine derivative, which is used as a key intermediate for constructing drug molecules to synthesize drugs with specific biological activities. If the biological activity is low, it will affect the secretion of anabolic hormones. In chemical research, N2 - [9 - fluorenylmethoxycarbonyl] - L - lysine allyl ester monohydrochloride is widely used in the synthesis of various complex organic molecules. Its application in organic synthesis is very extensive, especially in the synthesis of polypeptides and proteins containing lysine residues. Summary of the Invention

[0003] The present invention provides a method for the synthesis of N2 - [9 - fluorenylmethoxycarbonyl] - L - lysine allyl ester monohydrochloride and chiral detection, which mainly solves the technical problems of the existing synthesis temperature being affected by seasons, the conventional chiral detection being complex, and the separation of isomers being difficult.

[0004] The present invention adopts the following technical solutions: A method for the synthesis of N2 - [9 - fluorenylmethoxycarbonyl] - L - lysine allyl ester monohydrochloride and chiral detection, comprising the following steps: Step 1, prepare the reagents for preparation: n - hexane - chromatographic grade, acetic acid - analytical pure, ethanol - chromatographic grade.

[0005] Step 2: Synthesis reaction The first step: Add FMOC - LYS(BOC) - OH, potassium carbonate, and anhydrous ethanol to the reaction vessel in sequence, dissolve them, stir and react fully, concentrate, dissolve the residue with THF, dropwise add allyl tribromide at 5 - 10 °C, after the addition is completed, raise the temperature to 20 - 25 °C for reaction, monitor by TLC, after the reaction ends, filter, wash the filter cake with DCM, add water to the filtrate, acidify with HCl to pH = 5 - 6, extract with DCM, combine the organic phases, wash with 1N hydrochloric acid, wash with saturated brine, concentrate to obtain the product, slurry with PE, filter and dry to obtain the intermediate; the reaction formula is as follows: .

[0006] The second step: Add the intermediate and isopropyl acetate into the reaction vessel, and dropwise add the hydrogen chloride solution of isopropyl acetate at 0-5 °C. After the addition is completed, react at 20-25 °C, monitor by TLC, end the reaction, filter by suction, and dry to obtain a yellowish solid; the reaction formula is as follows: 。

[0007] Step 3, perform high performance liquid chromatography determination: The chromatographic conditions are that the sample chamber temperature is at room temperature, an automatic injector is used, and the chromatographic column is: The chromatographic column uses CHIRALPAKR AD-H, column temperature: 35 °C, flow rate: 0.8 mL / min, and the mobile phase is a 100% hexane solution (A) with a mass percentage concentration and a 0.1% acetic acid ethanol solution (B) with a mass percentage concentration, and an isocratic elution program is used.

[0008] The chromatograph used is an Agilent 1260 liquid chromatograph, and this instrument uses a VWD ultraviolet detector.

[0009] The size of the chromatographic column is 4.6mm × 250mm, 5.0μm.

[0010] The isocratic elution program is: 0-25 min, and the volume ratio of the mobile phase B is kept at 20%.

[0011] The present invention has the following beneficial effects: The present invention provides a method for synthesizing and chiral detection of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride, which mainly solves the temperature control of room temperature reactions in summer and winter during production, and the importance of controlling the reaction temperature at 20-25 °C for some special process reactions. At the same time, by controlling the proportion of hexane in the normal phase system and superimposing the control of the precise injection concentration, the chiral compounds of the product are used to accurately separate the isomers by the response value of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride in the DAD detector, so as to efficiently detect the chiral purity of the product, achieving the effects of simple operation, rapidity, accuracy, and good stability. Description of the Drawings

[0012] Figure 1 The HPLC chromatogram of the product of the present invention and the mixture of isomers.

[0013] Figure 2 The chiral HPLC chromatogram of the product of the present invention.

[0014] Figure 3 The chiral HPLC chromatogram of the isomers of the present invention. Detailed Embodiments

[0015] Main instruments: Agilent 1260 high performance liquid chromatograph, equipped with DAD ultraviolet detector, CHIRALPAKR AD-H (4.6 mm × 250 mm, 5.0 μm) from the United States; ultrasonic extractor (KQ5200E, Zhengzhou Kete Experimental Equipment Co., Ltd.), 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instruments Co., Ltd.), electronic analytical balance (CPA225D, Sartorius Scientific Instruments (Shanghai) Co., Ltd.).

[0016] Step 1: Prepare the reagents for preparation: n-hexane - chromatographic grade, acetic acid - analytical pure, ethanol - chromatographic grade.

[0017] Step 2: Synthesis reaction The first step: Add FMOC-LYS(BOC)-OH (200 g), potassium carbonate (35.4 g), and anhydrous ethanol (10V) to a 5L reaction flask in sequence, stir for 1h, concentrate, dissolve the residue with THF (12V), add allyl tribromide (56.8 g) dropwise at 5 - 10 °C, after the addition is complete, raise the temperature to 20 - 25 °C and react for 18h, monitor by TLC (PE:EA = 2:1, Rf = 0.5), after the reaction is completed, filter, wash the filter cake with DCM (2L x 2), add 1L of water to the filtrate, acidify with 1N HCl to pH = 5 - 6, extract with 2L of DCM, combine the organic phases, wash with 1N hydrochloric acid (2L x 2), wash with saturated brine (1L x 2), concentrate to obtain the product, slurry with 5L of PE, filter and dry to obtain the intermediate; The second step: Add the intermediate (145 g) and isopropyl acetate (5V) to a 3L reaction flask, add a hydrochloric acid solution of isopropyl acetate (1160 ml, 3 mol / L) dropwise at 0 - 5 °C, after the addition is complete, react at 20 - 25 °C for 6h, monitor by TLC (PE:EA = 2:1, Rf = 0.5), after the reaction is completed, filter and dry at 30 °C to obtain a yellowish solid.

[0018] Step 3: Perform high performance liquid chromatography determination: The chromatographic conditions are that the sample chamber temperature is at room temperature, with an auto sampler, and the chromatographic column is: The chromatographic column uses CHIRALPAKR AD-H (4.6 mm × 250 mm, 5.0 μm) from the United States, column temperature: 35 °C, flow rate: 0.8 mL / min, the mobile phase is a 100% n-hexane solution (A) with a mass percentage concentration and a 0.1% acetic acid ethanol solution (B) with a mass percentage concentration, and an isocratic elution program is used. The isocratic elution program is: 0 - 25 min, the volume ratio of the mobile phase B is maintained at 20%.

[0019] Main reagents: The reagents used for measurement include deionized water; n-hexane (chromatographic grade, Titan Chemicals Co., Ltd.), acetic acid (analytical grade, Shanghai Sinopharm Chemical Reagent Co., Ltd.); ethanol (chromatographic grade, Merck KGaA).

[0020] Pretreatment of the test samples: Weigh approximately 10 mg of the N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride sample and place it in a 5 mL volumetric flask. Add pure ethanol and dissolve it by ultrasonic treatment at room temperature for 3 min. Finally, filter the sample through a 0.22 µm polypropylene organic filter membrane and perform HPLC analysis. The injection volume must be precisely controlled at 5 µL to ensure that the main peak is around 1500 mAu, so as to achieve baseline separation between the product and its isomers; Selection and optimization of the sample pretreatment method: Currently, N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride on the market is a powdery solid. The sample measured in this invention is a product produced by Gil Biochemical (Shanghai) Co., Ltd.

[0021] The temperature of the sample chamber in this invention is set at room temperature, which is beneficial to the preservation of the ethanol solvent in the sample chamber. The temperature of the column oven is set at 35°C in this invention.

[0022] The HPLC chromatogram of the product and its isomer mixture is as Figure 1 shown. The chiral HPLC chromatogram of the purified product is as Figure 2 shown. The chiral HPLC chromatogram of the product isomer is as Figure 3 shown. By using this method to synthesize and perform chiral detection on the N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride product, the results show that the chiral detection results after synthesizing the N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride product by this method indicate that this method is fast, accurate, highly sensitive, the main peak has a symmetric peak shape, achieves baseline separation from the isomer peak, has good stability, and has practical application value.

Claims

1. A method for synthesizing and chiral detection of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride; characterized in that: Including the following steps: Step 1: Prepare the reagents for preparation: n-hexane - chromatographic grade, acetic acid - analytical pure, ethanol - chromatographic grade Step 2: Synthesis reaction of the sample The first step: Add FMOC-LYS(BOC)-OH, potassium carbonate, and anhydrous ethanol to the reaction vessel in sequence, dissolve, stir fully for reaction, concentrate, dissolve the residue with THF, add allyl tribromide dropwise at 5 - 10 °C, after dropping, raise the temperature to 20 - 25 °C for reaction, monitor by TLC, after the reaction ends, filter, wash the filter cake with DCM, add water to the filtrate, acidify with HCl to pH = 5 - 6, extract with DCM, combine the organic phases, wash with hydrochloric acid, wash with saturated brine, concentrate to obtain the product, slurry with PE, filter and dry to obtain the intermediate; the reaction formula is as follows: ； The second step: Add the intermediate and isopropyl acetate to the reaction flask, add the hydrochloric acid solution of isopropyl acetate dropwise at 0 - 5 °C, after dropping, react at 20 - 25 °C, monitor by TLC, after the reaction ends, filter and dry to obtain a yellowish solid; the reaction formula is as follows: ； Step 3: Perform high performance liquid chromatography determination: The chromatographic conditions are that the sample chamber temperature is at room temperature, with an automatic sampler, the chromatographic column is: CHIRALPAKR AD-H, column temperature: 35 °C, flow rate: 0.8 mL / min, mobile phase A is a 100% n-hexane solution by mass percentage concentration, mobile phase B is a 0.1% acetic acid ethanol solution by mass percentage concentration, and an isocratic elution program is adopted.

2. The synthesis and chiral detection method of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride according to claim 1, wherein: The chromatograph used in the liquid chromatography determination is an Agilent 1260 liquid chromatograph, and this instrument uses a DAD ultraviolet detector.

3. The synthesis and chiral detection method of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride according to claim 1, characterized in that: The size of the chromatographic column is 4.6mm × 250mm, 5.0μm.

4. The synthetic and chiral detection method of N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine allyl ester monohydrochloride according to claim 1, characterized in that: The isocratic elution program is: 0 - 25 min, the volume ratio of mobile phase B is kept at 20%.

Images